T E C H N I Q U E S IN T H E B E H A V I O R A L A N D N E U R A L S C I E N C E S V O L U M E 15 HANDBOOK OF STRESS AND THE BRAIN Part 1" The Neurobiology of Stress
Previously published in TECHNIQUES IN THE BEHAVIORAL AND NEURAL SCIENCES Volume 1: Feeding and Drinking, by F. Toates and N.E. Rowland (Eds.), 1987, ISBN 0-444-80895-7 Volume 2: Distribution-free Statistics: Application-oriented Approach, by J. Krauth, 1988, ISBN 0-444-80934-1, Paperback ISBN 0-444-80988-0 Volume 3: Molecular Neuroanatomy, by F.W. Van Leeuwen, R.M. Buijs, C.W. Pool and O. Pach (Eds.), 1989, ISBN 0-444-81014-5, Paperback ISBN 0-444-81016-1 Volume 4: Manual of Microsurgery on the Laboratory Rat, Part 1, by J.J. van Dongen, R. Remie, J.W. Rensema and G.H.J. van Wunnik (Eds.), 1990, ISBN 0-444-81138-9, Paperback ISBN 0-444-81139-7 Volume 5: Digital Biosignal Processing, by R. Weitkunat (Ed.), 1991, ISBN 0-444-81140-0, Paperback ISBN 0-444-98144-7 Volume 6: Experimental Analysis of Behavior, by I.H. Iversen and K.A. Lattal (Eds.), 1991, Part 1, ISBN 0-444-81251-2, Paperback ISBN 0-444-89160-9, Part 2, ISBN 0-444-89194-3, Paperback ISBN 0-444-89195-1 Volume 7: Microdialysis in the Neurosciences, by T.E. Robinson and J.B. Justice, Jr. (Eds.), 1991, ISBN 0-444-81194-X, Paperback ISBN 0-444-89375-X Volume 8: Techniques for the Genetic Analysis of Brain and Behavior, by D. Goldowitz, D. Wahlsten and R.E. Wimer (Eds.), 1992, ISBN 0-444-81249-0, Paperback ISBN 0-444-89682-1 Volume 9: Research Designs and Methods in Psychiatry, by M. Fava and J.F. Rosenbaum (Eds.), 1992, ISBN 0-444-89595-7, Paperback ISBN 0-444-89594-9 Volume I0: Methods in Behavioral Pharmacology, by F. van Haaren (Ed.), 1993, ISBN 0-444-81444-2, Paperback ISBN 0-444-81445-0 Volume 11: Methods in Neurotransmitter and Neuropeptide Research, by S.H. Parvez (Eds.), 1993, Part 1, ISBN 0-444-81369-1, Paperback ISBN 0-444-81674-7, Part 2, ISBN 0-444-81368-3, Paperback ISBN 0-444-81675-5 Volume 12: Neglected Factors in Pharmacology and Neuroscience Research, by V. Claassen (Ed.), 1994, ISBN 0-444-81871-5, Paperback ISBN 0-444-81907-X Volume 13: Handbook of Molecular-Genetic Techniques for Brain and Behavior Research, by W.E. Crusio and R.T. Gerlai (Eds.), 1999, ISBN 0-444-50239-4 Volume 14: Experimental Design. A Handbook and Dictionary for Medical and Behavioral Research, by J. Krauth (Ed.), 2000, ISBN 0-444-50637-3, Paperback ISBN 0-444-50638-1
Cover image by Tim Teebken/Getty Images.
T E C H N I Q U E S IN THE B E H A V I O R A L A N D N E U R A L SCIENCES
Series Editor J.P. H U S T O N Dtisseldorf
V O L U M E 15
H A N D B O O K OF STRESS A N D THE B R A I N Part l" The Neurobiology of Stress Edited by T. S T E C K L E R Johnson & Johnson Pharmaceutical Research & Development, A Division of Janssen Pharmaceutica N.V., Turnhoutseweg 30, 2340 Beerse, Belgium
N.H. K A L I N Department of Psychiatry and Health Emotions Research Institute, University of Wisconsin Medical School, 6001 Research Park Boulevard, Madison, WI 53719-1176, USA
REUL
J.M.H.M.
Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, The Dorothy Hodgkin Building, University of Bristol, Whitson Street, Bristol BS1 3NY, UK
ELSEVIER AMSTERDAM
-
BOSTON-
HEIDELBERG-
LONDON-
NEW YORK-
PARIS - SAN D I E G O - SAN F R A N C I S C O - S I N G A P O R E 2005
SYDNEY-
OXFORD TOKYO
ELSEVIER B.V. Radarweg 29 P.O. Box 211,1000 AE Amsterdam, The Netherlands
ELSEVIER Inc. 525 B Street Suite 1900, San Diego CA 92101-4495, USA
ELSEVIER Ltd The Boulevard Langford Lane, Kidlington, Oxford OX5 1GB, UK
ELSEVIER Ltd 84 Theobalds Road London WC1X 8RR UK
9 2005 Elsevier BV. All rights reserved. This work is protected under copyright by Elsevier BV, and the following terms and conditions apply to its use: Photocopying Single photocopies of single chapters may be made for personal use as allowed by national copyright laws. Permission of the Publisher and payment of a fee is required for all other photocopying, including multiple or systematic copying, copying for advertising or promotional purposes, resale, and all forms of document delivery. Special rates are available for educational institutions that wish to make photocopies for non-profit educational classroom use. Permissions may be sought directly from Elsevier's Rights Department in Oxford, UK: phone: (+44) 1865 843830, fax: (+44) 1865 853333, e-mail:
[email protected]. Requests may also be completed on-line via the Elsevier homepage (http://www.elsevier.corn/locate/permissions). In the USA, users may clear permissions and make payments through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA; phone: (+ 1) (978) 7508400, fax: (+ 1) (978) 7504744, and in the UK through the Copyright Licensing Agency Rapid Clearance Service (CLARCS), 90 Tottenham Court Road, London W1P 0LP, UK; phone: (+44) 20 7631 5555; fax: (+44) 20 7631 5500. Other countries may have a local reprographic rights agency for payments. Derivative Works Tables of contents may be reproduced for internal circulation, but permission of the Publisher is required for external resale or distribution of such material. Permission of the Publisher is required for all other derivative works, including compilations and translations. Electronic Storage or Usage Permission of the Publisher is required to store or use electronically any material contained in this work, including any chapter or part of a chapter. Except as outlined above, no part of this work may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without prior written permission of the Publisher. Address permissions requests to: Elsevier's Rights Department, at the fax and e-mail addresses noted above. Notice No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made. First edition 2005 Second impression 2005 British Library Cataloguing in Publication Data A catalogue record is available from the British Library. Library of Congress Cataloging in Publication Data A catalog record is available from the Library of Congress.
Handbook of stress and the brain. - (techniques in the behavioural and neural sciences, V 15) 1. Brain - Effect of stress on I. Steckler, T. II Kalin, N. H. III Reul J. M. H. M. 612.8'2 ISBN: 0-444-51173-3 (Part 1) ISBN: 0-444-51823-1 (part 2) ISBN: 0-444-51822-3 (Volume 15 Two-Part Set) Series ISSN: 0921-0709
Working together to grow libraries in developing countries www.elsevier.com ] www.bookaid.org I www.sabre.org ~[ ~x/t Ft] '.L.~I~.VI E(X. "l
I~()OK AID IHtcr~atiouiil
e- ~ t ~ . ~-~C]l)l'C [ ' O t l l ] ( I C ] [
I01
@) The paper used in this publication meets the requirements of ANSI/NISO Z39.48-1992 (Permanence of Paper). Printed in The Netherlands
List of Contributors, Part 1
K.-B. Abel, Division of Endocrinology, Children's Hospital Boston, Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA D. Adams, Laboratory of Molecular Psychiatry, Departments of Psychiatry and Pharmacology, Yale University School of Medicine, 34 Park Street, New Haven, CT 06508, USA L Akirav, Department of Psychology and, The Brain and Behavior Research Center, University of Haifa, Haifa 31905, Israel O.F.X. Almeida, Max Planck Institute of Psychiatry, Kraepelinstrasse 2-10, D-80804 Munich, Germany B. Bali, Laboratory of Molecular Neuroendocrinology, Institute of Experimental Medicine, Hungarian Academy of Science, Szigony u. 43, Budapest H-1083, Hungary C.W. Berridge, Departments of Psychology and Psychiatry, University of Wisconsin, 1202 W. Johnson Street, Madison, WI 53706, USA J.J. Cerqueira, Life and Health Science Research Institute, Health Science School, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal K.C. Chambers, Department of Psychology, University of Southern California, Seely G. Mudd Bldg. # 501, Los Angeles, CA 90089-1061, USA G.P. Chrousos, Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 10 Room 9D42, 10 Center Drive MSC 1583, Bethesda, MD 20892-1583, USA O. Cohen, Departments of Biological Chemistry and Psychology, The Hebrew University of Jerusalem, Jerusalem, Israel W.E. Cullinan, Department of Biomedical Sciences, Marquette University, Milwaukee, WI 53233, USA B. CzOh, Clinical Neurobiology Laboratory, German Primate Center, Kellnerweg 4, 37077 Gottingen, Germany F.M. Dautzenberg, Johnson & Johnson Pharmaceutical Research & Development, A Division of Janssen Pharmaceutica NV, Turnhoutseweg 30, 2340 Beerse, Belgium E.R. De Kloet, Division of Medical Pharmacology, LACDS-LUMC, University of Leiden, P.O. Box 9502, 2300 RA Leiden, The Netherlands A.J. Douglas, Section of Biomedical Sciences, DBCLS, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK G. Drolet, Centre de Recherche en Neurosciences, CHUL, RC-9800, 2705 Boulevard Laurier, Ste-Foy G1V 4G2 QC, Canada S.K. Droste, Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, The Dorothy Hodgkin Building, University of Bristol, Whitson Street, Bristol, BS1 3NY, UK J. Du, Laboratory of Molecular Pathophysiology, National Institute of Mental Health, Building 49, Room B1EE16, 49 Convent Drive, Bethesda, MD 20892-4405, USA
vi R.S. Duman, Laboratory of Molecular Psychiatry, Departments of Psychiatry and Pharmacology, Yale University School of Medicine, 34 Park Street, New Haven, CT 06508, USA Y. Dwivedi, Psychiatric Institute, Department of Psychiatry, University of Illinois at Chicago, 1601 W. Taylor St, Chicago, IL 60612, USA W.C. Engeland, Departments of Surgery and Neuroscience, University of Minnesota, Mayo Mail Code 120, 420 Delaware St SE, Minneapolis, MN 55455, USA N. Farzad, Laboratory of Molecular Pathophysiology, National Institute of Mental Health, Building 49, Room B1EE16, 49 Convent Drive, Bethesda, MD 20892-4405, USA H. Figueiredo, Department of Psychiatry, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH 45267-0559, USA E. Fuchs, Clinical Neurobiology Laboratory, German Primate Center, Kellnerweg 4, 37077 Gottingen, Germany A.J. Fulford, Department of Anatomy, University of Bristol, Southwell Street, Bristol, BS2 8EJ, UK D. Glick, Department of Biological Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel E. Gould, Department of Psychology, Princeton University, Princeton, NJ 08544, USA T.D. Gould, Laboratory of Molecular Pathophysiology, National Institute of Mental Health, Building 49, Room B 1EEl 6, 49 Convent Drive, Bethesda, MD 20892-4405, USA A. Gratton, McGill University, Douglas Hospital Research Center, Montreal, H4H 1R3 QC, Canada N.A. Gray, Laboratory of Molecular Pathophysiology, National Institute of Mental Health, Building 49, Room B1EE16, 49 Convent Drive, Bethesda, MD 20892-4405, USA M.S. Harbuz, University Research Centre for Neuroendocrinology, Bristol Royal Infirmary, Marlborough Street, Bristol, BS2 8HW, UK U.L. Hayes, University of Massachusetts, Amherst, MA, USA A.L.O. Hebb, Department of Pharmacology, Faculty of Medicine, Dalhousie University, Sir Charles Tupper Medical Building, 5850 College Street, Halifax, NS B3H 1X5, Canada S.C. Heinrichs, Boston College, Department of Psychology, McGuinn Hall, 140 Commonwealth Avenue, Chestnut Hill, MA 02467, USA J.P. Herman, Department of Psychiatry, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH 45267-0559, USA M.C. Holmes, Endocrinology Unit, Molecular Medicine Centre, Edinburgh University, Western General Hospital, Edinburgh EH4 2XU, UK S. Y.T. Hsu, Department of Obstetrics and Gynecology, Division of Reproductive Biology, Stanford University School of Medicine, Pasteur Drive, Room A344E, Stanford, CA 94305-5317, USA C.D. Ingram, Psychobiology Research Group, School of Neurology, Neurobiology and Psychiatry, University of Newcastle, Royal Victoria Infirmary, Newcastle NE1 4LP, UK M. Joels, Swammerdam Institute for Life Sciences, Section Neurobiology, University of Amsterdam, Kruislaan 320, 1098 SM Amsterdam, The Netherlands N.H. Kalin, Department of Psychiatry and Psychology, University of Wisconsin, 6001 Research Park Blvd., Madison, WI 53719, USA
vii A.M. Karssen, Department of Psychiatry and Behavioral Sciences, School of Medicine, Stanford University, MSL5, P124, 1201 Welch Road, Palo Alto, CA 94304-5485, USA T. Kino, Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 10 Room 9D42, 10 Center Drive MSC 1583, Bethesda, MD 20892-1583, USA G.F. Koob, Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA 92037, USA K.J. Kovdcs, Laboratory of Molecular Neuroendocrinology, Institute of Experimental Medicine, Hungarian Academy of Science, Szigony u. 43, Budapest H-1083, Hungary H.J. Krugers, Swammerdam Institute for Life Sciences, Section Neurobiology, University of Amsterdam, Kruislaan 320, 1098 SM Amsterdam, The Netherlands S. Laforest, Centre de Recherche en Neurosciences, CHUL, RC-9800, 2705 Boulevard Laurier, Ste-Foy G1V 4G2 QC, Canada M. Le Moal, INSERM U588, Institut Fran~;ois Magendie, 1 rue Camille St Saans, 33077 Bordeaux, France V. Lemaire, INSERM U588, Institut Francois Magendie, 1 rue Camille St Sa6ns, 33077 Bordeaux, France S. Levine, Department of Psychiatry, Center for Neuroscience, University of California at Davis, Davis, CA 95616, USA A.C.E. Linthorst, Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, The Dorothy Hodgkin Building, Whitson Street, Bristol BS1 3NY, UK J. Lu, Max Planck Institute of Psychiatry, Kraepelinstrasse 2-10, D-80804 Munich, Germany S.J. Lupien, Laboratory of Human Psychoneuroendocrine Research, Douglas Hospital Research Center, 6875 Bldg. Lasalle, Verdun, H4H-1RS QC, Canada F.S. Maheu, Department of Psychology, University of Montreal, 6875 Bld Lasalle, Montreal, H4H 1R3 QC, Canada J.A. Majzoub, Division of Endocrinology, Children's Hospital Boston, Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA H.K. Manji, Laboratory of Molecular Pathophysiology, National Institute of Mental Health, Building 49, Room B 1EEl 6, 49 Convent Drive, Bethesda, MD 20892-4405, USA C.A. Marsden, School of Biomedical Sciences, Institute of Neuroscience, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH, UK O.C. Meijer, Division of Medical Pharmacology/LACDR-LUMC, Leiden/Amsterdam Center for Drug Research, Leiden University Medical Center, P.O. Box 9502, 2300 RA Leiden, The Netherlands I.H. Mikl6s, Laboratory of Molecular Neuroendocrinology, Institute of Experimental Medicine, Hungarian Academy of Science, Szigony u. 43, Budapest H-1083, Hungary N.K. Mueller, Department of Psychiatry, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH 45267-0559, USA Z. NOmethy, Max Planck Institute of Psychiatry, Kraepelinstrasse 2-10, D-80804 Munich, Germany M. P(tez-Pereda, Department of Endocrinology, Max Planck Institute of Psychiatry, Kraepelinstr 10, 80804 Munich, Germany G.N. Pandey, Department of Psychiatry, Psychiatric Institute, University of Illinois at Chicago, 1601 W. Taylor St, Chicago, IL 60612, USA
viii J.M. Pego, Life and Health Science Research Institute, Health Science School, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal J.D. Peters, Department of Psychology, Princeton University, Princeton, NJ 08544, USA P.V. Piazza, INSERM U-588, Universit6 de Bordeaux 2, Institut Frangois Magendie, 1 Rue Camille Saint-Satins, 33077 Bordeaux Cedex, France J. Prickaerts, Johnson & Johnson Pharmaceutical Research & Development, a Division of Janssen Pharmaceutica NV, Turnhoutseweg 30, B-2340 Beerse, Belgium J.M.H.M. Reul, Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, The Dorothy Hodgkin Building, University of Bristol, Whitson Street, Bristol, BS1 3NY, UK G. Richter-Levin, Department of Psychology, The Brain and Behavior Research Center, University of Haifa, Haifa 31905, Israel M.A. Riva, Department of Pharmacological Sciences, Center for Neuropharmacology, University of Milan, Via Balzaretti 9, 20133 Milan, Italy P.H. Roseboom, Department of Psychiatry and Pharmacology, 6001 Research Park Blvd., Madison, WI 53719, USA R. Rupprecht, Department of Psychiatry, Nussbaumstr. 7, 80336 Munich, Germany M. Schmidt, Division of Medical Pharmacology/LACDR-LUMC, University of Leiden, P.O. Box 9502, 2300 RA Leiden, The Netherlands J.R. Seckl, Endocrinology Unit, Molecular Medicine Centre, Edinburgh University, Western General Hospital, Edinburgh EH4 2XU, UK B.B. Simen, Laboratory of Molecular Psychiatry, Departments of Psychiatry and Pharmacology, Yale University School of Medicine, 34 Park Street, New Haven, CT 06508, USA H. Soreq, Department of Biological Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel N. Sousa, Life and Health Science Research Institute, Health Science School, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal G.K. Stalla, Department of Endocrinology, Max Planck Institute of Psychiatry, Kraepelinstr. 10, 80804 Munich, Germany S. Stanford, Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK T. Steckler, Johnson & Johnson Pharmaceutical Research & Development, A Division of Janssen Pharmaceutica N.V., Turnhoutseweg 30, 2340 Beerse, Belgium R.M. Sullivan, Centre de Recherche Fernand-S6guin, Universit6 de Montr6al, Montreal, H4H 1R3 QC, Canada Y.M. Ulrich-Lai, Department of Psychiatry, Albert Sabin Way, University of Cincinnati, Cincinnati, OH 45267-0559, USA R.J. Valentino, The Children's Hospital of Philadelphia, 402C Abramson Pediatric Research Center, 34th and Civic Center Blvd, Philadelphia, PA 19104, USA E.J. Van Bockstaele, Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, 1020 Locust St, Philadelphia, PA 19107, USA J.M. Verkuyl, Swammerdam Institute for Life Sciences, Section Neurobiology, University of Amsterdam, Kruislaan 320, 1098 SM Amsterdam, The Netherlands N. Weekes, Department of Psychology, Pomona College, 550 N. Harvard Avenue, Claremont, CA 91711, USA J.L.W. Yau, Endocrinology Unit, Molecular Medicine Centre, Edinburgh University, Western General Hospital, Edinburgh EH4 2XU, UK
R. Yirmiya, Department of Psychology, The Hebrew University of Jerusalem, Jerusalem, Israel P.-X. Yuan, Laboratory of Molecular Pathophysiology, National Institute of Mental Health, Building 49, Room B 1EEl 6, 49 Convent Drive, Bethesda, MD 20892-4405, USA R. Zhou, Laboratory of Molecular Pathophysiology, National Institute of Mental Health, Building 49, Room B1EE16, 49 Convent Drive, Bethesda, MD 20892-4405, USA E.P. Zorrilla, Department of Neuropharmacology, The Scripps Research Institute, CVN-7, La Jolla, CA 92037, USA
This Page Intentionally Left Blank
Preface
Stress is a phenomenon being all around us, but seemingly being too well known and too little understood at the same time, despite the fact that the field has advanced enormously over recent years. We have learned that stress can shape various types of behaviour in the individual long after exposure to the stressor itself has terminated. Exposure to a stressful stimulus during the perinatal period, for example, can have long-term consequences over weeks and months, well into adulthood. This is accompanied by a variety of characteristic neurochemical, endocrine and anatomical changes in the brain, leading, for example, to changes in neural plasticity and cognitive function, motivation and emotionality. We have started to discover the differentiated effects of various stressors in the brain and how expression of a wide variety of gene products will be altered in the CNS as a function of the type and duration of the stressor. Activity in higher brain areas in turn will shape the response to acute and chronic stress and there are intricate interactions with, for example, immune functions. Cytokines will access the brain and affect its function at various levels. It has become increasingly clear that stress serves as one of the main triggers for psychiatric and non-psychiatric disorders, including depression, anxiety, psychosis, drug abuse and dementia. Recognizing these intricate relationships has initiated a wealth of research into the development of novel animal models and novel treatment strategies aiming at influencing stress responsivity in patients suffering from these diseases. Moreover, novel technologies, such as molecular techniques, including gene targeting methods and D N A microarray methods start to unravel the cellular events taking place as a consequence of stress and facilitate the understanding of how stress affects the brain. Thus, the topic of stress, the brain and behaviour gains increasing relevance, both from a basic scientific and clinical perspective, and spans a wide field of expertise, ranging from the molecular approach to in-depth behavioural testing and clinical investigation. This book aims at bringing these disciplines together to provide an update of the field and an outlook to the future. We think these are exciting times in a rapidly developing area of science and hope that the reader will find it both useful as an introductory text as well as a detailed reference book. The Handbook of Stress and the Brain is presented in two parts, i.e. Part 1: The Neurobiology of Stress, and Part 2: Stress: Integrative and Clinical Aspects. This part, Part 1, addresses basic aspects of the neurobiology of the stress response including the involvement of neuropeptide, neuroendocrine and neurotransmitter systems, and its corollaries regarding gene expression and behavioural processes such as cognition, motivation and emotionality. Thomas Steckler Ned Kalin Hans Reul
xi
A Memorial for David de Wied (1925-2004)
It is almost an eerie coincidence that this volume, dedicated to the subjects of Stress and Behavior, should be published at a time when the field has lost one of its giants and the man whose work has inspired much of what is written here. On February 21, 2004 Professor David de Wied died. David had just celebrated his 79th birthday. For me not only did the field lose one of its founding fathers but I lost a dear friend. Professor de Wied was born on January 12, 1925. His life prior to embarking on his professional career was marked by a period of several years when he went underground and was in hiding during the German occupation of Holland. Following the war he decided to attend the University of Groningen to study medicine. This involved a tremendous effort since he had lost many precious academic years. He did receive his medical degree in 1955. I shall not document the details of his remarkable academic achievements. These are presented in detail in a volume dedicated to David on his 75th birthday (Smelik and Witter, 2000) and more recently by de Kloet (2004). In my chapter on the history of stress research I devoted several pages to David de Wied and his importance to the field, but his impact on the field was of such significance that it is worth repeating. David was in every sense a pioneer and a visionary. I have often wondered how one defines a visionary. Perhaps the critical dimension is the ability to see relationships between events that are not immediately apparent to normal mortals. He is best known for his formulation of the "neuropeptide concept" although throughout his career he made many other major contributions. In its simplicity the neuropeptide concept postulated that there were peptides produced in the brain and pituitary that directly influenced brain function, and of particular importance, behavior. The field of hormones and behavior at the time when David began to work on the effects of peptides on behavior was almost exclusively dedicated to studying the effects of gonadal steroids on sexual behavior. There were a few scattered reports of effects of thyroid and adrenal steroid compounds but they had little impact. The demonstration that neuropeptides could influence complex behavioral processes such as learning and memory was indeed revolutionary and met with a great deal of skepticism when first introduced. However, the skeptics were silenced when he continued to demonstrate the powerful influence of these molecules on behavior. It was primarily based on this work that fundamental behavioral processes were integrated into the general rubric of neuroendocrinology and new dimensions of the effects of the hormones of the hypothalamic-pituitary-adrenal axis on behavior were introduced. The neuropeptide concept pre-dated the characterization of the "releasing hormones" synthesized in the hypothalamus. That these hormones have been shown to have a profound influence on behavior is one of the legacies of David's work. In 1963 he became Professor of Medical Pharmacology at the University of Utrecht which in 1968 became the Rudolf Magnus Institute of Pharmacology in honor of the Dutch pharmacologist Rudolf Magnus. This institute rapidly became the Mecca for the study of hormones and behavior. It was the place to visit and study if your field of interest xiii
xiv encompassed neuroendocrinology and behavior. Investigators came from every part of the world to study at the institute. On the numerous occasions that I lectured in the institute I was always prepared to be challenged by David and his students. The discussions were vigorous, animated and sometimes heated, but always stimulating and provocative. David's legacy extends well beyond his scientific contributions. There are multitudes of Ph.D. and post-doctoral students as well as collaborators who are indebted to him. They were privileged to share his scientific rigor, and perhaps of more importance, his unique intellect. On the occasion of his 75th birthday celebrations the room was filled with many of these students and colleagues. What was impressive is that many of them are now the current leaders in the field. Although he is best known for his life as a scientist there was much more to the man. He had other passions that made up his life. He was an avid art collector and loved music. Until his death he continued to play the violin and take music lessons. We shared a common love for both music and art. One of our secret ambitions was to perform the famous tenor and baritone duet from Bizet's "The Pearl Fisher." There were two problems, first neither of us could qualify as a tenor and second we really did not sing very well. This did not prevent us from singing opera at any occasion whether it be a dinner at a congress or a party in Hungary. David was a complex man. He received numerous prestigious honors throughout his career and yet in his later years he did not feel he had achieved the recognition he deserved. Perhaps the problem with being the originator of a concept that gains universal acceptance is that its origin is often forgotten. David had the most unusual sense of humor I have encountered. On one occasion my youngest daughter spent a weekend with us and David and his wife Lie on the Italian Riviera. She spent the first day terrified of him until she realized that he was indeed one of the funniest people she had ever met. She spent the next few days in almost constant laughter. It was my dream and hope that David and I would have a grand celebration for our joint 80th birthdays in 2005. We were both overjoyed when we experienced the millennium. That dream has now been shattered. I will continue to revere and respect him for all the contributions he has made to biology and to the quality of all our lives. We shared many adventures, many avid scientific discussions and the pleasure of watching the growth of our science. These memories are always present and it was indeed a privilege to have shared these with him over many years. Seymour Levine Center for Neuroscience University of California, Davis
References De Kloet, E.R. (2004) In honour of David de Wied. Psychoneuroendocrinology (in press). Smelik, P.G. and Witter, A. (2000) David de Wied a biographical sketch. In: David de Wied (Honorary Editor), Neuropeptides, Basics and Perpectives. Elsevier, Amsterdam.
Contents, Part 1
List of Contributors, Part 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preface
v
......................................................
xi
A Memorial for David de Wied (1925-2004) . . . . . . . . . . . . . . . . . . . . . . . . . . . .
xiii
Section 1. Concepts of Stress 1.1.
Stress: an historical perspective S. Levine (Davis, CA, USA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2. The neuropsychology of stress T. Steckler (Beerse, Belgium)
............................
1.3. An introduction to the HPA axis A.J. Fulford and M.S. Harbuz (Bristol, UK) . . . . . . . . . . . . . . . . . 1.4.
1.5.
1.6.
25
43
Hormones of the pituitary M. Pfiez-Pereda and G.K. Stalla (Munich, Germany) . . . . . . . . . . .
67
Molecular biology of the HPA axis K.-B. Abel and J.A. Majzoub (Boston, MA, USA)
79
............
The hypothalamic-pituitary-adrenal axis as a dynamically organized system: lessons from exercising mice J.M.H.M. Reul and S.K. Droste (Bristol, UK) . . . . . . . . . . . . . . . .
95
Section 2. Hypothalamic Hormones Involved in Stress Responsivity 2.1.
2.2.
2.3.
Novel C R F family peptides and their receptors: an evolutionary analysis S.Y.T. Hsu (Stanford, CA, USA) . . . . . . . . . . . . . . . . . . . . . . . . .
115
Molecular regulation of the C R F system P.H. Roseboom, N.H. Kalin, T. Steckler and F.M. Dautzenberg (Madison, WI, USA and Beerse, Belgium) . . . . . . . . . . . . . . . . . . .
133
Behavioral consequences of altered corticotropin-releasing factor activation in brain: a functionalist view of affective neuroscience S.C. Heinrichs (Chestnut Hill, MA, USA) . . . . . . . . . . . . . . . . . . .
155
XV
xvi 2.4. The roles of urocortins 1, 2, and 3 in the brain E.P. Zorrilla and G.F. Koob (La Jolla, CA, USA) . . . . . . . . . . . . .
179
2.5. Vasopressin and oxytocin A.J. Douglas (Edinburgh, UK) . . . . . . . . . . . . . . . . . . . . . . . . . . .
205
2.6. The role of vasopressin in behaviors associated with aversive stimuli K.C. Chambers and U.L. Hayes (Los Angeles, CA and Amherst, MA, USA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
231
Section 3. Stress and the HPA Axis
3.1. Corticosteroid receptors and HPA-axis regulation E.R. de Kloet, M. Schmidt and O.C. Meijer (Leiden, The Netherlands)
265
3.2. Glucocorticoid effects on gene expression T. Kino and G.P. Chrousos (Bethesda, MD, USA)
295
............
3.3. The role of 11 [3-hydroxysteroid dehydrogenases in the regulation of corticosteroid activity in the brain J.R. Seckl, J.L.W. Yau and M.C. Holmes (Edinburgh, UK) . . . . . .
313
3.4. Corticosteroids and the blood-brain barrier A.M. Karssen, O.C. Meijer and E.R. de Kloet (Leiden, The Netherlands) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
329
3.5. Glucocorticoids and motivated behaviour V. Lemaire, P.V. Piazza and M. Le Moal (Bordeaux, France) . . . . .
341
3.6. Effects of glucocorticoids on emotion and cognitive processes in animals J. Prickaerts and T. Steckler (Beerse, Belgium) . . . . . . . . . . . . . . . .
359
3.7. Glucocorticoids: effects on human cognition S.J. Lupien, F.S. Maheu and N. Weekes (Montreal, QC, Canada and Claremont, CA, USA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
387
Section 4. Neurotransmitter Systems Involved in Stress Responsivity
4.1.
4.2.
Neurocircuit regulation of the hypothalamo-pituitary-adrenocortical stress response- an overview J.P. Herman, N.K. Mueller, H. Figueiredo and W.E. Cullinan (Cincinnati, OH and Milwaukee, WI, USA) . . . . . . . . . . . . . . . . . .
405
Sympatho-adrenal activity and hypothalamic-pituitary-adrenal axis regulation Y.M. Ulrich-Lai and W.C. Engeland (Minneapolis, MN, USA) . . . .
419
4.3. The locus coeruleus-noradrenergic system and stress: modulation of arousal state and state-dependent behavioral processes C.W. Berridge (Madison, WI, USA) . . . . . . . . . . . . . . . . . . . . . . .
437
xvii 4.4. Functional interactions between stress neuromediators and the locus coeruleus-norepinephrine system R.J. Valentino and E.J. Van Bockstaele (Philadelphia, PA, USA)
465
4.5. Regional specialisation in the central noradrenergic response to unconditioned and conditioned environmental stimuli S.C. Stanford and C.A. Marsden (London, UK and Nottingham, UK)
487
4.6. Stress, corticotropin-releasing factor and serotonergic neurotransmission A.C.E. Linthorst (Bristol, UK) . . . . . . . . . . . . . . . . . . . . . . . . . . .
503
4.7. Modulation of glutamatergic and GABAergic neurotransmission by corticosteroid hormones and stress M. Joels, H.J. Krugers and J.M. Verkuyl (Amsterdam, The Netherlands) . . . . . . . . . . . . . . . . . . . . . . . . . . .
525
4.8. Neuroactive steroids R. Rupprecht (Munich, Germany)
545
4.9.
........................
Endogenous opioids, stress, and psychopathology A.L.O. Hebb, S. Laforest and G. Drolet (Ste-Foy, QC, Canada) . . .
561
4.10. Acetylcholinesterase as a window onto stress responses H. Soreq, R. Yirmiya, O. Cohen and D. Glick (Jerusalem, Israel) ..
585
4.11. Pathways and transmitter interactions mediating an integrated stress response C.D. Ingram (Newcastle, UK) . . . . . . . . . . . . . . . . . . . . . . . . . . . .
609
Section 5. Neuroplasticity and Stress 5.1. The intracellular signaling cascade and stress Y. Dwivedi and G.N. Pandey (Chicago, IL, USA) . . . . . . . . . . . . .
643
5.2. The role of neurotrophic factors in the stress response M.A. Riva (Milan, Italy) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
665
5.3. Transcription factors as modulators of stress responsivity R.S. Duman, D.H. Adams and B.B. Simen (New Haven, CT, USA)
679
5.4. Experience, structural plasticity and neurogenesis J.D. Peters and E. Gould (Princeton, NJ, USA) . . . . . . . . . . . . . . .
699
5.5. Adult neurogenesis in rodents and primates: functional implications E. Fuchs and B. Cz6h (G6ttingen, Germany) . . . . . . . . . . . . . . . . .
711
5.6. Cellular and molecular analysis of stress-induced neurodegeneration - methodological considerations J. Lu, Z. N~methy, J.M. Pego, J.J. Cerqueira, N. Sousa and O.F.X. Almeida (Munich, Germany and Braga, Portugal) . . . . . . . . . . . . . .
729
xviii 5.7.
Enhancing resilience to stress: the role of signaling cascades P.-X. Yuan, R. Zhou, N. Farzad, T.D. Gould, N.A. Gray, J. Du and H.K. Manji (Bethesda, MD, USA)
.......
751
6.1. Psychological and physiological stressors K.J. Kovfics, I.H. Mikl6s and B. Bali (Budapest, Hungary) . . . . . . .
775
6.2. Involvement of the amygdala in the neuroendocrine and behavioral consequences of stress I. Akirav and G. Richter-Levin (Haifa, Israel) . . . . . . . . . . . . . . . .
793
6.3. Role of prefrontal cortex in stress responsivity A. Gratton and R.M. Sullivan (Montreal, QC, Canada) . . . . . . . . .
807
Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
819
Section 6. The Stressed Brain
SECTION 1
Concepts of Stress
This Page Intentionally Left Blank
T. Steckler, N.H. Kalin and J.M.H.M. Reul (Eds.) Handbook of Stress and the Brain, Vol. 15 ISBN 0-444-51173-3 Copyright 2005 Elsevier B.V. All rights reserved
CHAPTER 1.1
Stress" an historical perspective Seymour Levine Department of Psychiatry, Center for Neuroscience, University of California, Davis, California 95616, USA
Abstract: Some of the major landmarks in the history of neuroendocrinology, glucocorticoid physiology and psychoneuroendocrinology are discussed in this chapter. The primary emphasis is on the evolution of the major theories and their experimental underpinnings on the regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Initially an attempt was made to deal with the issues concerning the definitions of stress. The origins of the stress concept, the neural control of the pituitary, the history of the search for corticotrophin-releasing factor (CRF), and the developments that resulted in shaping the current views of the action of the adrenal hormones are elaborated. Further, the role of environment and behavior on the regulation of the HPA axis and the effects of specific neuropeptides on behavior were also covered. The purpose of this chapter is to provide a perspective on the major events that were crucial in the history of stress research that shaped the directions of the field. "The past is never dead. It's not even the past" William Faulkner
Introduction During the course of my career, which now spans over five decades, I could not begin to count the number of conferences, workshops, and symposia related to stress that I have attended. I will not attempt to describe the number of times during these meeting that at least one, if not several, of the participants had championed the notion that we discard the concept for a more precise definition. The absolute failure of these attempts is attested to by my most recent visit to one of my favorite biomedical computer searches. As of this moment Pub-Med listed 209,744 references that in one way or another had some reference to the term stress. It would be difficult to predict what this number will be at the time this chapter is published. These staggering figures are at best an underestimate since computer searches rarely go beyond the late 1970s and publications in this field began long before this time. Further, the particular search that was used lists mostly articles and ignores the extensive list of ,.
Tel.: + 1530 752 1887; Fax: + 1530 757 8827; E-mail:
[email protected] books dedicated to this subject. What is also evident from the information obtained from the computer is that the number of publications is accelerating. Over 60,000 papers have been published since the beginning of the new millennium. A close examination of only a small sample of these references made it abundantly clear that the term stress was used in so many different ways that it would be necessary to determine, for each article listed, the precise manner the term was used and in what context. It would be further difficult to specify all the different disciplines that have in some way found the concept of stress useful, though each discipline will define stress in its own idiosyncratic manner.
Defining stress After the completion of my last effort to define stress (Levine and Ursin, 1991), I made myself the promise that I would never again engage in what I consider a futile exercise. One of the more recent definitions was presented by McEwen (2000). "Stress may be defined as a real or interpreted threat to the physiological
or psychological integrity of an individual that results in physiological and/or behavioral responses. In biomedicine, stress often refers to situations in which adrenal glucocorticoids (GCs) and catecholamines are elevated because of an experience." Chrousos and Gold (1992) state "we define stress as state of disharmony, or threatened homeostasis. The adaptive response can be specific or can be generalized and non specific." At the core of these definitions is the concept of homeostasis. Thus, some disturbance of homeostasis results in a cascade of physiological and/or behavioral responses that presumably are required to reinstate the ideal homeostatic balance. However, these definitions as well as most others are problematic. As stated by Levine and Ursin (1991) "The major problem with the concept of stress is that we are confronted with a composite, multidimensional concept. All existing definitions include some components. We can identify three main subclasses. These subclasses can be identified as the input (stress stimuli), the processing systems, including the subjective experience of stress and the output (stress responses). One basic difficulty is that these subclasses interact. The essential picture we want to convey is one of a complex system with feedback and control loops, no less but no more complicated than any other of the body's selfregulated systems. This system affects many other biological processes and may function as a common alarm and drive system, whenever there is a real or apparent challenge to the self-regulating systems of the organism." Steptoe (2000) has suggested that "the effects of stress are manifest in four distinct domains; physiology, behavior, subjective experience, and cognitive function. The physiological effects of stress include alterations in neuroendocrine, autonomic nervous system and immune function." If one were to isolate only the physiological effects of stress, the history of three major areas of investigation would need to be covered and this would not include the relationships or lack of relationship among these systems. This historical perspective will focus primarily on the neuroendocrine system and more specifically with the hypothalamic-pituitary-adrenal (HPA) axis. For this discussion we will use the more traditional designation HPA, although more recently we have seen a trend to label the axis as LHPA.
The "L" stands for limbic system and is intended to indicate that the regulation of the hormonal cascade caused by exposure to stress involves extrahypothalamic structures. The aim of this chapter is to provide a glimpse into the historical events that have provided the framework, the insights, and the theories that in many ways still guide our current research. Insofar as several papers will appear in subsequent volumes that deal extensively with many different aspects of the HPA axis, I will provide only a very simple description of some of the key elements in the neuroendocrine cascade that results in an increased secretion of adrenocorticotropic hormone (ACTH), and that ultimately results in elevations of the levels of GCs. Some environmental event which involves either physical demands or is psychologically challenging or a combination of both, induces an increase in the release of corticotrophin-releasing factor (CRF) and arginine vasopressin (AVP) into the portal circulation. CRF/AVP then activates the corticotrophs in the pituitary to release A C T H into the general circulation. ACTH acts upon the adrenal cortex to induce synthesis and increased secretion of the GCs. Under normal circumstances these elevated levels of GC activate the GC receptors which serve to terminate the release of CRF and ACTH, thus returning the organism to its basal state. Traditionally, a discourse on the history of this or any other field would trace the evolution of the critical components along some time dimension. If I were to take this approach the result would simply be a compilation of the information contained in many other sources. Since history does unfold through time it is difficult to avoid using a temporal framework. However, I intend to trace this history as it unfolded in my academic lifetime and how it influenced and shaped the field as we know it today. This will be an historical perspective, but the perspective will be autobiographical.
Not so ancient history As was stated earlier I will not attempt to present all the details of the variety of thought and experimental evidence that eventually leads us to the 21st century views of stress. The earliest references to the concept of homeostasis come from the Greek philosophers
and physicians, in particular, Hippocrates. This is best summarized by Chrousos et al. (1988). What is important is that the concepts of harmony and disharmony (homeostasis?) of man and animals with both the external and internal environment have long been a concern of serious thinkers. The concept of stress was originally taken from the dynamics of physics to describe the relationship between stress and strain in an elastic body. "The term stress is applied to the mutual actions which take place across any section of a body to which a system of forces is applied. The term strain is applied to any changes occurring in the dimension or shape of a body when forces are applied" (Duncan and Starling, 1959). However, our current interest in and views on the physiology and psychology of stress are of more recent vintage and can be traced primarily to the contributions of Walter B. Cannon (1914, 1915, 1932) and Hans Selye (1950, 1956). The overarching principles that emerged from the studies by Cannon and Selye were: (1) there was a physiology that was specific to stress and (2) that an integral part of this physiology was related to some function of the adrenal. Although both emphasized the role of the adrenal, there was a clear distinction between them. (1) Cannon focused primarily on the sympathetic nervous system, including the adrenal medulla, and the role of the adrenal medullary hormones, epinephrine also called adrenaline and noradrenaline, in the response to emergency situations. Selye, in contrast, emphasized the hormones of the adrenal cortex, primarily the GCs. (2) Canon was describing the responses to an acute threat, whereas Selye was concerned with the adaptation of the organism to chronic challenges. (3) For Canon, stress was defined in terms of the stimulus required to elicit these responses. Selye described a triad of responses that he hypothesized constituted stress: hypertrophy of the adrenal, stomach ulceration, and involution of the thymus gland. This triad of responses implicated the endocrine, autonomic, and immune systems. Stress was defined in terms of the response. Selye in fact turned the original physical definition of stress on its head. Instead of strain being produced by stress, he did not use the term strain, and assumed that stress was produced by stimuli he referred to as stressors. There is some controversy over who first
used the term stress in a biological context. Although it is a common belief that Selye was responsible for introducing the term (Medvei, 1982), Sapolsky (1994) contends that it was indeed Cannon (1914) who was responsible. However, Selye was clearly responsible for popularizing the concept and bringing it to the attention of the biomedical community and the general public. The history of science is replete with examples of serendipity. Sapolsky (1994), in what I consider one of the best written popular books on stress, describes the origins of Selye's observations as follows: "To be only a bit facetious, stress physiology exists only because this man (Selye) was both a very insightful scientist and somewhat inept at handling rats." Originally Selye was attempting to discover the function of some extract of ovarian tissue. However, "he attempted to inject his rats daily, but apparently with not a great display of dexterity. Selye would try to inject the rats, miss them, drop them, spend half the morning chasing the rats around the room or visa versa, and so on. At the end of a number of months of this, Selye examined the rats and discovered something extraordinary: the rats had peptic ulcers, greatly enlarged adrenals, and shrunken immune tissues. He was delighted; he had discovered the effects of the mysterious ovarian extract." However, following several subsequent presumably control experiments the physiological manifestations of these procedures continued to be evident. This led Selye (1936) to postulate that the response to stress was nonspecific. Thus, a wide array of stimuli (stressors) resulted in a similar set or responses and that eventually exposure to stress will result in illness. Selye (1949) defined the General Adaptation Syndrome (GAS) as the "Physiological mechanism which raises the resistance to damage as such." The GAS consisted of a three-stage reaction to stress, an alarm reaction, a stage of resistance, and a final stage of exhaustion. It remains one of the more robust theories in the stress field, although it was in fact incorrect in many of its assumptions. I should state that I come to praise Selye not to bury him. The impact of Selye cannot be measured in the specific details of his work and theories. He pioneered and launched a field of investigation that has had an enormous influence on biology and medicine and is still growing at an exponential rate.
There were a number of developments in the field that followed shortly after Selye's early publications that cast doubt on some of the fundamental aspects of Selye's theories and led to a decline in the interest in GC physiology. One of Selye's claims was that the excess secretion of adrenocortical hormone would cause arthritis, allergies, and collagen-related disorders. However, Hench et al. (1949) demonstrated that the GCs had profound anti-inflammatory activity and could be used therapeutically to treat some of these pathologies. One of the coauthors on this paper was Kendall, who was awarded the Nobel Prize for determining the structure of the steroid hormones. These findings were paradoxical to what Selye had proposed. That there is a relationship between stress and illness is now extensively documented, as is the relationship between stress and GCs. What is not clear is the relationship between the GCs and illness. The history of science is a history of great ideas and advances in technologies. The techniques that were available to the stress researcher during the embryonic phase of the field were at best crude and often laborious. There were no known methods for directly measuring the levels of circulating hormones. The physiology of the stress response was based initially on changes in weight of the adrenal and counts made of the circulating lymphocytes and eosinophils in blood. The first direct biochemical measure was the depletion of ascorbic acid in the adrenal. It had been observed that, following an injection of ACTH or stress, there was a drop in the content of adrenal ascorbic acid within the adrenal. This observation was to be the basis for an ACTH bioassay developed by Sayers (1950). There were many limitations to these indices of adrenocortical activity. At best they were crude and only by inference could they be related to the output of the GCs. It was difficult to obtain a time course since only the measures of eosinophils did not require sacrificing the animals, and therefore the dynamics of the adrenal response were difficult to ascertain. It was not until the 1950s (Silber et al., 1958) that the first direct measurement of the adrenal steroids was available. However, despite these limitations, many of the initial observations, hypotheses, and theories have held up remarkably well, which is a testimony to the brilliance and insightfulness of the founders of this field.
Although the Selye's definition of stress was to dominate the thinking and direction of stress research for many years, there were other groups of investigators that had a very different perspective. In the late 1940s and early 1950s, one of the bastions of biological psychiatry was located at the Michael Reese Hospital in Chicago. This group had undertaken a large study of normal human subjects who were volunteers in the US Army undergoing airborne training. The definition of stress used in the context of this study was "Any stimulus may in principle arouse an anxiety response because of the particular meaning of threat it may have acquired for that particular individual. However, we distinguish a class of stimuli which are more likely to produce disturbances in most individuals. The term stress is applied to this class of conditions, thus, we can conceive of a continuum of stimuli differing in meaning to the organism and in their anxietyproducing consequences. At one end are such stimuli or cues, often highly symbolic, which have meaning only to a single or limited number of persons and which to the observer appear as innocuous or trivial. At the other end are such stimuli, here called stress, which by their explicit threat to vital functioning and their intensity are likely to overload the capacity of most organisms coping mechanisms. Anxiety has been defined in terms of an affective response; stress is the stimulus condition likely to arouse such responses. Ultimately, we can truly speak of a stress situation only when a given response occurs, but for schematic purposes as well as consistency with common usage, we may use the term stress to designate certain kinds of stimulating conditions without regard to response." (Basowitz et al., 1955). This definition stands in direct contrast to the one proposed by Selye. In this instance stress is defined almost exclusively in terms of the stimulus with expectations that these events will evoke some response. For Selye, stress is only stress if the triumvirate of physiological responses is elicited. Thus far none of the definitions of stress have focused on or even mentioned the brain although inherent in the psychological approach to stress was the notion that stimuli could be interpreted differentially based on a host of experiential factors, which implies some process by which the stimuli are evaluated.
The brain: the birth of neuroendocrinology In 1948 Geoffrey W. Harris published a paper entitled "Neural Control of the Pituitary Gland." This was followed in 1955 by an expanded monograph with the same title (Harris, 1955). At the time this book was published, Harris was the Fitzmary Professor of Physiology at the Institute of Psychiatry, Maudsley Hospital, London. He was to become the Professor of Anatomy at Oxford University until his very untimely death at the age of 57. I was privileged to have been a student of Harris from 1960 to 1962 and continued my relationship with him until his death. I cannot recall any time that was more exhilarating in the course of my professional career. Every discovery was major advance, every important scientist at the time came through the laboratory, and it was a new way to look at the world. Geoffrey Harris was many things to me. He was my hero, my mentor, my friend, and my squash instructor. An excellent description of him as a scientist and as a man is contained in the book by Nicholas Wade "The Nobel Duel" (1981). At the time the "Neural Control" book was published, I was a young faculty member in a new research unit that has just been opened in the psychiatry department at Ohio State University. I had been there only a few years when I encountered the Harris volume. For me this was a revelation, a new approach, whereby there was at last a way to link behavior with endocrinology. I applied for a postdoctoral position in his laboratory in London, was accepted, and packed my wife, three very small children, and one large dog and off we went. We had numerous adventures along the way, but the fateful day arrived in March 1960 when I first encountered the laboratory. After meandering through the grounds of the Maudsley Hospital I finally came upon two rather shabby looking temporary structures that was the Laboratory of Neuroendocrinology. I was both surprised and depressed. My vision based upon my image of the greatness of the man in charge led me to expect a physical plant that would be commensurate with his stature. One could not swing a fat cat or even a skinny one in the laboratory that was assigned to me. To add further to my depression, Harris wanted me to work on a problem related to sexual differentiation, a far cry from my passion for stress and anxiety.
As I discovered years later Geoffrey had little regard for psychology and/or psychologists. The only useful activity for an experimental psychologist was the study of sex behavior, and since I had been the first postdoctoral applicant with behavioral credentials it was obvious that I knew how to examine sex behavior. After several months watching rats with a well-worn description of rat sexual behavior written by Frank Beach, I did learn how. In the end what began as a nightmare turned out to be a fulfillment of a dream far beyond my expectations and was to shape the remainder of my scientific career. It would be erroneous to imply that neuroendocrinology was born with the publication of Harris' extremely influential paper and monograph. There were numerous suggestions and hypotheses that postulated a relationship between the brain and the endocrine system. Harris did not readily embrace the concept of neuroendocrinology. For him the brain was an endocrine organ and therefore neuroendocrinology was a redundancy. Hippocrates in his discourse on the glands anticipated this perspective many centuries ago when he wrote "The flesh of the glands is different from the rest of the body, being spongy and full of veins; they are found in moist parts of the body where they receive humidity.., and the brain is a gland as well as the mammae" (Medvei, 1982). In 1936 Francis Marshall concluded in his discussion of periodicity in reproduction that "the primary periodicity is a function of the gonad, the anterior pituitary, acting as a regulator, and the internal rhythms adjusted to the environment, by the latter acting on the pituitary, partly or entirely, through the intermediation of the nervous system" (Marshall, 1936). The role of the brain in the regulation of reproductive processes was critical in the development of neuroendocrinology. Implicating the central nervous system (CNS) with regards to the regulation of the adrenal cortex did not occur until several years later. One of the early models concerning the regulation of adrenocorticotropin (ACTH) secretion and the secretion of GCs from the adrenal proposed that ACTH secretion is regulated by the systemic blood levels of epinephrine. Epinephrine or adrenaline, as it was then commonly called, was proposed as the corticotrophin-releasing factor (CRF). The principle proponent of this view was Long (1947, 1952).
In response to stress, the increased secretion of adrenaline by the adrenal medulla acts directly on the pituitary gland to induce the secretion of ACTH. an alternative view of the regulation of ACTH was presented by Sayers (1950). He proposed that ACTH secretion is regulated by the blood levels of GCs. Under conditions of stress the peripheral tissues utilize these hormones more rapidly. The blood content of the GCs falls rapidly as a consequence, and this in turn directly stimulates anterior pituitary to secrete increased amounts of ACTH. According to this view, the anterior pituitary and the adrenal cortex form a self-regulating system, the balance of which is broken mainly by variations in the activity of the peripheral tissue. The position held by Long and coworkers would not withstand the scrutiny of subsequent experimental examination. There was no question that injections of adrenaline could stimulate ACTH secretion. It did not appear, however, that it was absolutely essential. Amongst the experiments that challenged the adrenaline hypothesis, perhaps the most damaging was an experiment conducted by Marthe Vogt (1952), who also stands as one of my heroes. She determined that enucleating the adrenal medulla reduced circulating levels of adrenaline to close to zero. However, in response to emotional stress there was a fall in adrenal ascorbic acid in the adrenal demedullated rat that followed an identical time course to those of intact animals. Similar results were reported by Hodges (1953), effectively dismissing the hypothesis that adrenaline was the CRF. Regulation of ACTH secretion based upon the level of circulating adrenal hormones was to suffer a similar fate. Although the evidence that supported Sayers indicated that blood levels of the adrenal hormones do in part regulate the output of ACTH, there were many facts that could not be explained by this theory. One example is that this theory could not explain why adrenal atrophy occurs when the pituitary is transplanted to a site remote from the sella turcica. As long as the pituitary was properly vascularized it should maintain a normal functional adrenal cortex. The history of stress research in some ways reads like a chamber of horrors. Animals were subjected to a wide variety of stress inducing stimuli that were to say the least massively invasive. They were boiled,
frozen, surgically insulted, subjected to electric shock, injected with toxic agents, irritants, hemorrhaged, and this does not come close to an exhaustive list. That the peripheral responses to this wide variety of stimuli were similar supported Selye's view of the nonspecificity of the stress response. Studies were beginning to appear that challenged the view that all stress was equal, and at the same time implicated the CNS as critical for the regulation of the pituitary. Fortier (1951) transplanted pituitary tissue to the anterior chamber of eye. This was a favorite transplantation site for the obvious reason that this area is heavily endowed with a rich blood supply, though I have commented that the real purpose was to better visualize the pituitary. Fortier exposed the animal with the transplanted pituitary to several stress-inducing stimuli. He divided them into two groups: (a) systemic stress which included injection of adrenaline or histamine, exposure to cold and (b) neurotropic stress, which included exposure to loud sounds and immobilization. Fortier reported that systemic stress was able to elicit an ACTH response in animals bearing a transplanted pituitary, but that in the case of neurotropic stress there was no indication of ACTH release. Fortier argued that the so-called neurotropic stress required the mediation of the CNS in order to release ACTH, whereas the response to systemic stress may be mediated by other blood-borne factors. There were other studies that suggested that stressinducing stimuli could be differentiated. Dallman (1979) summarized a number of experiments and classified the different stimuli into those in which the adrenal response was blocked by the pretreatment with a single dose of GCs (GC sensitive) and those that still elicited a response after pretreatment with large doses of GCs (GC insensitive). Feldman (Feldman et al., 1970; Feldman, 1985) reported that in rats with hypothalamic islands, the GC response to peripheral neural stimulation was inhibited. The responses to systemic stress remained intact. That there are differences in the types of stress has been demonstrated by at least three distinctly different procedures, transplants, GC inhibition, and hypothalamic deafferentation. Recent methodological advances have made it possible to examine the neural pathways in the CNS in response to different stimuli that induce increased secretion of corticosterone and
ACTH. Sawchenko (1991) has described at least five different pathways that converge on the PVN and stimulate the release of CRF. Herman and Cullinan (1997) have presented the most recent version of the stress dichotomy. They state "Stressors involving an immediate physiologic threat ('systemic stressors') are relayed directly to the PVN, probably via brainstem catcholaminergic projections. By contrast, stressors requiring interpretation by higher brain structures ('processive stressors') appear to be channeled through limbic forebrain structures." At the core of Harris' theory was the hypothesis that the pituitary gland was regulated by blood-borne chemical substances that were transported via the portal vessels from the hypothalamus to the pituitary. The growth of neuroendocrinology was slowed down initially by the description, by Popa and Fielding (1930), that the blood flowed from the pituitary to the hypothalamus. If the blood flow was from south to north, then the question of how the hypothalamic hormones could be transported to the pituitary gland was an enigma. Subsequently, it was demonstrated (Wisloski, 1937; Harris, 1947) that the blood flow through the portal vessels could indeed flow from the hypothalamus to the pituitary, thus making it possible for the brain hormones to directly communicate with the pituitary. Harris postulated a specific releasing factor for each of the pituitary hormones. The term "releasing factor," to describe the substances in the hypothalamus that could induce secretion of the pituitary hormones, had been already proposed by Schally. Harris used two different approaches to prove that the regulation of the pituitary was a consequence of hormones being transported via the portal circulation. These were (1) pituitary transplants and (2) pituitary stalk sections. That endocrine organs could be removed from their original anatomical location and remain viable had history in experimental endocrinology, dating back to the work of John Hunter (1728-1793). Harris utilized these techniques to demonstrate that in order for the pituitary to function normally the communication between the hypothalamus and the pituitary via the portal vessels had to be intact. When the pituitary was transplanted to a site remote from the hypothalamus (Harris and Jacobsohn, 1952), as long as it was adequately supplied with blood,
it remained viable though largely nonfunctional. The experiment by Fortier (1951) described previously is but one example of the importance of the transplant experiments in proving the neuroendocrine hypothesis. A more direct approach intended to show the importance of the portal system was that of sectioning the portal vessels. Under these conditions the pituitary remained in situ, but was deprived of the blood flow from the hypothalamus. One of the goals I had set for myself when I went to the Harris laboratory was to learn the technique of sectioning the portal vessels. It did not take long to realize that the surgical skills involved were far beyond my abilities. Harris' surgical skills were legendary. In the early stalk-section experiments although there was unambiguous evidence that the ACTH secretion was seriously impaired, after a relatively short period of time the pituitary once again became functional. The reasons for this appeared to be the capacity for the portal vessels to show remarkable regeneration. If regeneration was prevented by placing a wax paper barrier between the cut ends of the stalk (Harris, 1950), then the release of ACTH was continually impaired. I continue, even though many years have elapsed, to be impressed by the simplicity and elegance of these studies. By using basic physiological logic and with some surgical magic, the major tenants of neuroendocrinology were established. In addition to the experiments which disrupted the blood-borne communication between the hypothalamus and the pituitary, there were other lines of investigation that presented convincing evidence that the brain was directly involved in regulating ACTH. (1) Hypothalamic lesions." Numerous experiments (Porter, 1953; McCann, 1953) were reported using animals bearing lesions in the hypothalamus. These studies indicated that hypothalamic lesions prevented the ACTH response to most stressors. Many of these lesion studies failed to support the conclusions of Fortier that the response to systemic stress did not require CNS involvement. Lesions which obliterated the hypothalamus prevented the release of ACTH to all stimuli. I use the term obliterate to indicate the state of the art for making lesions. Most of the early lesions studies usually produced large, massive lesions making it difficult to specify which if any of the specific nuclei in the hypothalamus was responsible for the secretion
10 of the putative CRF. (2) Electrical stimulation of the hypothalamus: On my first tour of the laboratory in London I was given the "grand" tour. I was ushered into a large room which contained a large circular arena. Seated in this arena was a rabbit with something implanted in its skull. The outer part of this contraption appeared to be something resembling an electrical coil. I was informed that this apparatus (I use this term kindly) was presenting electrical stimulation directly to the hypothalamus on the assumption that the electrical pulses would elicit a response from the hypothalamus that would result in the release of the tropic hormones for the pituitary. Despite its Rube Goldberg appearance this procedure produced some of the most convincing data that the hypothalamus was intimately involved in the regulation of pituitary hormones in general and ACTH specifically (deGroot and Harris, 1950).
The CRF quest Given this background and the growing acceptance of the role of the brain in regulating ACTH, it is not surprising that during the period of time when neuroendocrinology was blossoming there should also be a growing interest in attempting to find the chemical substance in the hypothalamus that was responsible for activating ACTH. The names most associated with the first attempts to describe CRF were Roger Guillemin and Andrew Schally, who were awarded the Noble Prize in Medicine in 1977 for their identification of TRH and LH-RH. The relationship between these two men on the way to Stockholm is legendary and well documented in Nicholas Wade's book "The Nobel Duel." In addition to describing much of the persona and background of Guillemin and Schally and the intensity of the animus between them, it is perhaps the best written and most entertaining history of neuroendocrinology. Since most of the evidence indicated that the communication between the hypothalamus and pituitary was by some blood-borne factor, it should be possible to find the compound and characterize its structure. Previously it was mentioned that the first proposed CRF was adrenaline secreted by the adrenal medulla. The search for the new secretagogue
responsible for activating ACTH focused on the hypothalamus. During the early 1950s several investigators independently reported that the extracts of hypothalamic tissue could stimulate the release of ACTH from anterior pituitary cell in vitro. Ironically, although the initial attempts to isolate a releasing factor centered on CRF, it proved to be one of the most difficult and elusive of all of the major hypophysiotropic hormones in the CNS. The pitfalls on the way to the CRF were many. There were problems with obtaining sufficient hypothalamic tissue needed to extract and analyze adequate quantities of material. My introduction to Guillemin was in 1961 during my first visit to Paris. I traveled to Paris with a fellow postdoc from London, who had been an associate of Guillemin at Baylor. Guillemin had recently arrived in Paris and had established his laboratory at the very prestigious College de France. At first glance he had achieved the prominence and all that went with it that a young scientist could ever hope for. He lived in part of a lovely chateau with beautiful grounds on the outskirts of Paris. Unlike the laboratory in London, this lab was sparkling and replete with all the latest technology, but what was most impressive was the very large freezer which contained thousands and thousands of sheep hypothalami that Guillemin had obtained from the slaughter houses of France. There were numerous problems that were encountered in attempting to isolate and characterize CRF. Hypothalamic extracts often contained other, weaker secretagogues of ACTH such as vasopressin, catecholamines, etc. Another major obstacle was the claim (McCann and Brobeck, 1954) that in fact the CRF was in reality vasopressin and there was little need to try to identify a novel substance. The similarities in the size of ACTH (39 amino acids) and what ultimately turned out to be the size of CRF (41 amino acids) was not conducive to easy separation of the peptides by the existing methods. It also turned out to be the case that the structure of CRF was much more complex than other hypophysiotropic hormones. Thyrotrophic-releasing hormone (TRH), for example, contains only three amino acids. Concentrating on the identification of CRF proved to be a quagmire in the hormone-releasing enterprise. In his conclusions to the chapter describing the quest
for CRF, Wade wrote "To this day, CRF has not been found, and may not even exist as such." However, at almost the identical time this statement was uttered, Vale et al. (1981) at the Salk Institute reported the isolation, characteristics, synthesis, and in vitro and in vivo biological activity of a 41-amino acid ovine hypothalamic CRF. This was a testimony once again to perseverance, but also to the major advances in the available technology, amongst them being the development of the radioimmunoassay (RIA) for peptide hormones, advances in molecular biology, ion exchange, and liquid chromatographic techniques. In one way the quest for CRF was finally over and yet in another way it has just begun. Several years after presenting the structure of CRF, Vale reported the cloning of the CRF1 receptor and further the identification of additional CRF2 receptors which has resulted in the discovery of at least three new and distinct CRF-like p e p t i d e s - Urocortin 1, 2, or stress copin-related peptide and Urocortin 3 also known as stresscopin (Hsu and Hsueh, 2001). The functional significance of these peptides is only beginning to be described (Dautzenberg and Hauger, 2002). CRF, however, has proven to be a remarkable molecule. Not only is it found in abundance in the paraventricular nuclei (PVN) of the hypothalamus, but it is widely distributed throughout the brain and the periphery. CRF is also involved in the regulation of a broad range of physiological and psychological processes. Amongst these is the modulation of the autonomic system, gastrointestinal activity, behavior, and immune function. CRF has also been implicated in a variety of psychiatric and other neurological disorders. Thus, what began as the search for a hypothalamic hypophysiotropic hormone may eventually extend into many areas of pathophysiology that are not immediately associated with the regulation of the pituitary-adrenal axis. I stand in awe of many of the intellectual giants that were involved in the ontogeny of neuroendocrinology. Although the specifics of the hypotheses and theories may not have been entirely correct, they each in some way contained elements that have proven to be prophetic. Peripheral adrenaline was not the CRF, but the catecholamines are clearly part of the neuroendocrine cascade that activates the release of the peripheral stress hormones. Harris postulated
a specific releasing factor for each of the pituitary hormones. We now know that there are hypothalamic inhibiting as well as releasing hormones. Vasopressin may not have been the CRF but it is an active cosecretagogue. Once it became doctrine that the regulation of the pituitary tropic hormones was via some blood-borne substance synthesized in the hypothalamus and released into the portal circulation, there were other investigators who attempted to characterize the structure of these hormones. However, only Guillemin and Schally were willing to take the big gamble and devote all of their time and resources that resulted in the identification of the first releasing factors and ultimately to the discovery of CRF and the CRF-like peptides.
GCs We have thus far discussed some of the milestones that were crucial in the establishment of neuroendocrinology as a discipline. However, when we trace the history of stress research it began with the adrenal cortex, and although the peptides in the brain appear to have multiple functions, at least one of the major functions of CRF is to regulate the secretion of ACTH and GCs. Conversely, the adrenal hormones in turn regulate the synthesis of CRF by the way of a negative-feedback mechanism. Any basic textbook of endocrinology presents a detailed description of most, if not all, of the known physiological actions of the GCs and the dire consequences of either under or overproduction of these hormones. Previously we discussed the developments that focused on the response of the adrenal as a central component of the stress response. However, Sapolsky et al. (2000) state that "Few contemporary endocrinologists view the GC actions as part of a coherent physiological picture, or see the need to. Today the focus is on the molecular and cell biology of GC action, e.g., GC receptors as ligand-activated transcription factors or GC-induced apoptosis in lymphocytes." What has been, and still is to some extent, one of the important questions that plague the stress researcher is what the adaptive significance of increased GC secretion is? It is not difficult to understand the importance of GC when the organism
12 is confronted with acute life-threatening and physically invasive events. Under these circumstances the HPA axis is an exquisite adaptive mechanism. However, in our contemporary view of stress there is as much, if not more focus, on psychological and social stress as on the physical. The question of how GCs influence the stress response continues to be an issue that has still not been completely resolved. Historically there have been several critical hypotheses that have guided the way we view this problem. Originally Selye speculated that GCs facilitate or mediate the ongoing stress response. One of the pioneers in GC physiology was Dwight Ingle. Ingle was an important member of the group at the Mayo clinic and contributed the bioassay that was used to test the purified fractions of the adrenal cortical hormones that were eventually characterized by this group. He also was amongst the first to demonstrate negative feedback when he observed that administration of adrenal cortical extracts or purified GCs caused atrophy of the adrenal gland that could be reversed by simultaneous administration of pituitary extracts. One of his major contributions was to propose a new and unique function for the GCs. The assumption that prevailed in the stress literature was that the damaging effects of stress were due to hypersecretion of the adrenal cortex. Ingle (1952) showed that the characteristic damaging effects of stress persisted when the GCs were supplied to the adrenalectomized animals at a constant, but not excessive rate of administration. From these observations he deduced that the role of the GCs in stress appears to be due to a subtle "permissive" or supporting role rather than as the primary mediator of the stress reaction. The most recent definition of the permissive action is "permissive actions are exerted by GCs present before the stressor and prime the defense mechanisms by which an organism responds to stress. Their consequences are first manifested during the initial stress response and occur whether or not there is a stress-induced increase in GC concentrations" (Sapolsky et al., 2000). Thus the GCs were now relegated to an essential, but a more supportive role. Around the same time a Dutch physiologist/ pharmacologist, Marius Tausk, proposed another role for the GCs. He suggested that GCs exerted a suppressive action. However, Tausk's view of GC
actions did not receive much attention due to the fact that it was originally published in the house organ of Organon Pharmaceuticals. In a now well-quoted metaphor he viewed stress as a fire and that the function of the GCs was to limit the water damage created by the fire fighters. This view of the role of GCs in the stress response remained obscure until many years later when Alan Munck wrote a classic paper that independently proposed that GCs role in the stress response was to exert a suppressive action. Munck et al. (1984) wrote "We propose that: (a) the physiological function of stress induced increases in GC levels is to protect not against the source of stress itself, but against the normal defense reactions that are activated by stress; and (b) the GCs accomplish this function by turning off those defense reactions, thus preventing them from overshooting and themselves threatening homeostasis." Thus we begin to see an integrated set of functions that are all part of the complex actions of the GCs in the stress response. Recently, Sapolsky et al. (2000) have attempted once again to answer the question "How do glucocorticoids influence the stress response." In this chapter the previously proposed actions of the GCs, stimulatory, permissive, and suppressive, are integrated along with another newly proposed function of the GCs, "preparative." It has been over 30 years since McEwen et al. (1968) injected adrenalectomized rats with radioactive GCs and observed that the GC was retained in high levels by the hippocampus as well as other regions of the limbic forebrain. Although it is beyond the scope of this chapter to cover the molecular biology of the GCs, one of the most influential developments in our attempts to understand the physiology of the GCs was the discovery of the GC receptors, their anatomical distributions, structure, and functions. Steroid hormones are a privileged class of molecules that have direct access to the brain and the brain is a target organ for these hormones. One of the surprises occasioned by the McEwen et al.'s results was the hippocampal localization of the GC. There was little evidence at the time that the hippocampus was associated with neuroendocrine function. The presence of GC receptors in areas of the CNS other than the hypothalamic and preoptic areas, which were known to have direct neuroendocrine functions, indicated that the effects of the GC on
13 the CNS may extend beyond its presumed role in the regulation of the stress response and that GC may influence those functions, spatial learning and memory, that have been ascribed to the hippocampus. Since the original observation there has been considerable progress in GC receptor research. A further significant development was the findings of Reul and deKloet (1985) that there were two distinct types of GC receptors. Based primarily on studies which examined the cytosol-binding properties of these receptors, there appeared a high-affinity receptor which was occupied by low levels of GCs and a lowaffinity receptor which required levels of GCs that are present only at the peak of the circadian rhythm or when elevated due to stress. Originally these two receptors were designated as type 1 and type 2, but in recent years type 1 is referred to more frequently as mineralo-corticoids (MR) and type 2 as glucocorticoids (GR). Since McEwen used tracer amounts of radiolabeled GC, the receptors that were found in the hippocampus were most likely the high affinity MRs and not GR. Although both types of receptors can be found in the hippocampus, the MR is more localized anatomically whereas the GR is more widely distributed throughout the brain. The development of specific steroid agonists and antagonists firmly established that the receptors located in the hippocampus were indeed MR. The cloning of these receptors demonstrated that each form was the product of a distinct gene. de Kloet et al. (1998) present a table indicating the "milestones" in GC receptor research. Although their last entry is 1996, I doubt that we have seen the end of new contributions to this field. What makes the discovery of two distinct GC receptors in the brain important is that it created a different approach to our view of the actions of GCs. de Kloet et al.'s (1998) hypothesis is that the "tonic influences of corticosterone are exerted via hippocampal MRs, while the additional occupancy of GRs with higher levels of corticosterone mediate feedback actions aimed at restoring disturbances in homeostasis" (de Kloet and Reul, 1987). de Kloet et al. (1998) further posited two modes of negative feedback which are implied in his hypothesis. (1) "proactive" negative feedback mediated via the MR maintains normal variations due to circadian rhythms and (2) "reactive" feedback operating via the GR serves to inhibit further secretion of ACTH
and facilitate the return to basal levels. Well before the discovery of the GC receptors there was considerable work that described the different aspects of GC negative feedback (Jones et al., 1972; Dallman and Jones, 1973; Dallman, 1979) and postulated that reactive negative feedback occurred in three different modes, fast, intermediate, and slow. According to Dallman (2000) "fast effects occurs in milliseconds and must be exerted at the cell membrane, 'intermediate feedback has its onset at about 30min after a pulse or continuous exposure to steroid and last about for a period of hours', slow feedback is found in conditions in which supraphysiological levels of exogenous GC have been provided for days or weeks." The unique anatomical distribution of the MRs has caused the field to examine a much broader range of possible effects of GCs on brain function including cognition. One example is the studies that suggest that the ratio of M R / G R appear to be involved in depression (Young et al., 2003). As we begin to determine the many ways that the GCs influence the brain, these hormones have and will continue to move beyond their simple designation as stress hormones. The history of the stress and the HPA system is, when thought of in real time, relatively recent. It should not come as surprise that many of the individuals that have been mentioned as historical figures are now the leaders in the field. Notably amongst these are Mary Dallman, Bruce McEwen, E. Ron de Kloet, and Wylie Vale.
Psychoneuroendocrinology In 1970 I attended the inaugural meeting in New York City of a newly formed society, which had been named the International Society of Psychoneuroendocrinology. The name was indeed daunting, but there was a group of people who believed that the relationship between psychology, psychiatry, and endocrinology had advanced to the point where a new forum for the dissemination of information regarding these relationships was necessary. Psychoneuroendocrinology is a broad-based discipline that examines the relationship between brain, behavior, and numerous endocrine and immune systems. The psychobiology of the HPA axis is
14 an integral part of psychoneuroendocrinology. Once again it is almost impossible to determine any event or group of studies that could be considered the catalyst for including psychological factors within the rubric of HPA physiology. John Mason (1968, 1975) pointed out that the most potent stimuli for activating the pituitaryadrenocortical system were psychological. Mason's position was a direct attack on doctrine presented by Selye. Mason emphasized the crucial role for what he called "the psychological approaches involved in the emotional or arousal reactions to threatening or unpleasant factors in the life situation as a whole." He argued that the so-called nonspecificity of the endocrine responses to stress occurs because of the emotional component surrounding the experience associated with exposure to stressinducing stimuli. Thus, the nonspecific responses described by Selye are primarily behavioral or psychological in nature and "the interpretive processes underlying the nonspecific bodily responses probably involves a higher level of CNS function than was previously realized." For Mason there was only one type of stress, that involving some emotional component. This doctrine has become known as the "Mason Principle." Hennessy and Levine (1979) hypothesized that the HPA axis was a sensitive indicator of emotional arousal and therefore its response was a reflection of heightened emotional arousal. These views certainly reflect a parochial approach to the psychobiology of stress. We now know there are many different pathways that can activate the CRF neuron. However, the hypotheses presented by Mason and others were very influential in the growth of HPA psychoneuroendocrinology. Once it was accepted that the psychological factors play an important role in the activation of the HPA axis, it became theoretically possible that the psychological processes can also modulate the HPA response. The psychobiology of stress has many faces and one cannot presume to cover all of the clinical and experimental data that are germane to this topic. The focus in the discussion will emphasize: (1) some of the psychological factors that modulate the HPA response, and (2) the effects of some of the hormones that comprise the HPA axis on behavior.
Early experiences That the psychological factors were important in the expression of the HPA response to stress long predated the views expressed by Mason. In 1952 I went as a postdoctoral fellow to the Institute of Psychiatry and Psychosomatic Research at the Michael Reese Hospital in Chicago. Although my espoused purpose was to be trained as a clinical psychologist, the fellowship turned out to be very different than I had bargained for. For the first time I was exposed to the area of stress and also to the endocrinology of the HPA axis. It was during that period we conducted the initial experiment (Levine et al., 1956) that demonstrated long-term behavioral consequences as a result of early postnatal manipulations. This first experiment examined the effects of postnatal exposure to electric shock as a model for early exposure to trauma on a behavior presumed to be a measure of anxiety, the conditioned avoidance response. In addition to pups that were exposed to a brief electric shock (3 min daily starting on day 1 of life) we included two control groups. One group was removed from the dam placed in the shock apparatus for the identical period of time (3min), but not shocked. The second control group remained in the nest totally undisturbed until weaning. The results were completely paradoxical to what we had predicted. The group that was most emotionally disturbed was the totally undisturbed (nonhandled) pups. Thus, the postnatal experience of being removed and separated briefly from the dam, with or without shock, appeared to permanently modify the behavioral responses to subsequent traumatic events well into adulthood. The first indication that brief separations from the dam which became known as early handling also influenced the HPA system was a study (Levine, 1957) that exposed early handled and nonhandled adults to an injection of glucose and examined adrenal weight 24 h after the injection. Although basal adrenal weights did not differentiate between the two early experience groups, adrenal weight was significantly increased only in the nonhandled animals that had received the 20% glucose injection. These results were surprising since adrenal hypertrophy following stress usually takes considerably more time to develop than the 24 h that elapsed between the injection and removal of the adrenal.
15 Between 1957 and 1960 we conducted a series of experiments that indicated these early experiences also altered the developmental trajectory of the HPA axis. These data will be discussed in the chapter (Vazquez and Levine, this volume) on the development of the HPA axis. These initial studies did strongly suggest that early experiences have a long-term influence on the activity of the adrenal and the mechanisms that regulate this organ system. Research in this area has continued for four decades and although many other biological processes have been investigated, the original findings have held up remarkably well. It was not until almost ten years had passed that the effects of early experience the HPA axis were revisited. The impetus was the development of a reliable biochemical assay for examining the levels of circulating corticosterone. This fluoremetric assay made it possible to examine the dynamics of the release of corticosterone and to obtain repeated measures on the same animal. In the years following the introduction of the fluoremetric assay, which was modified to assay corticosterone in small quantities of plasma (Glick et al., 1964), numerous studies emanating from many laboratories studied adrenocortical activity in animals subjected to different early experiences using a multitude of different paradigms. In our laboratory we published numerous studies on the effects of early handling in rats and mice. The effects of early handling on the dynamics of the adrenal response to stress have proved to be an extremely robust phenomenon. The initial experiments (Levine et al., 1967) demonstrated that the corticosterone response following exposure to an open field was significantly reduced in early handled animals. This study was conducted in collaboration with Victor Denenberg whose contributions were vital to the growth of developmental psychobiology. These findings have been robust and reproduced under a variety of different conditions (Meaney et al., 1993). The adrenocortical responses to novelty, restraint, shock, conditioned taste aversion, etc. all have been shown to be significantly reduced in early handled animals. Further, early handled animals appear to have a more efficient negative feedback regulation. Early handling also modifies the response to neonatal malnutrition (Wiener and Levine, 1978) and fetal alcohol syndrome (Weinberg et al., 1995).
It has always been assumed that the effects of early handling were to modify those aspects of the CNS that regulated the response of the HPA axis. There were numerous reasons for this assumption. The evidence indicated that there were no differences between handled and nonhandled animals in basal levels of corticosterone and ACTH. No differences were observed between the different early experience groups on adrenal sensitivity to ACTH, pituitary response to exogenous CRF, or clearance of corticosterone and ACTH. Adult early handled and nonhandled rats have similar levels of corticosteronebinding globulin. Insofar as none of the indices of the peripheral aspects of the HPA axis differ as a consequence of early handling, the origins of the differences on the response to stress must be a function of some change in programming of the central regulatory mechanisms. At least two components of the central regulatory mechanisms have been reported to differ between the early experience groups. Plotsky and Meaney (1993) reported that the resting levels of CRF mRNA in the PVN were significantly higher in nonhandled compared to handled adult animals. Median eminence levels of CRF and AVP are also higher in nonhandled rats. These differences in CRF gene expression and protein levels are apparent under resting conditions, although there are no differences in circulating corticosterone levels. More recently we have shown that increased CRF gene expression following restraint is much more rapid in nonhandled rats (Gordon and Levine 1999). Early handling also markedly changes the GR receptor density in the hippocampus of adult rats. In general, early handled animals have increased hippocampal GR sites and also show an increased gene expression for the GR receptor. The MR receptors do not appear to change with early handling. Bhatnagar et al. (1996) suggest that "it seems that the increase in GR sites is a critical feature of neonatal handling on HPA function. This increased receptor density appears to increase the sensitivity of the hippocampus to circulating GCs, enhancing the efficacy of negative feedback inhibition on HPA activity, and serving to reduce post-stress secretion of ACTH and GC in handled animals." Although there have been several hypotheses proposed to account for the effectiveness of early handling, one hypothesis that seems to have received
16 the most attention and support from the existing data is the "maternal mediation" hypothesis (Smotherman and Bell, 1980). Intuitively, it was difficult to understand why the seemingly innocuous manipulation of very brief bouts of maternal separation could have such permanent and pervasive influence on behavior and the HPA axis. The first suggestion that the effects of early handling could be maternally mediated was an experiment by Denenberg and Whimbey (1963). These investigators reared pups with mothers who had no prior early interventions and compared them to offspring of mothers who had been handled as infants. Elaborate cross-fostering procedures were included in an attempt to tease out postnatal from prenatal effects. The results indicated that, on some measures of emotionality, the early experience of the mother significantly altered the behavioral outcome measure. On other indices of emotionality the effects were a result of the interaction between pre- and postnatal influences. Levine (1967) demonstrated that the maternal early experience could influence the adrenocortical response of the pups. The corticosterone response to novelty of pups reared by handled dams was reduced compared to pups reared by nonhandled dams. Handling the pups resulted in a reduction of the response when reared by a nonhandled dam. In contrast, handled pups from handled dams did not differ from their nonhandled counterparts if the dam had been handled as a pup. There is a direct evidence that handling altered maternal behavior (Smotherman et al., 1977). They observed maternal behavior in dams when reunited with pups that were handled or shocked. Several aspects of maternal behavior were intensified when the treated pups were returned to the dams. Clearly, manipulation of the pup altered and increased dam-pup interactions. For those of us who have had the opportunity to observe the field over an extended period of time, the cyclic quality of science is apparent. Questions that were posed decades ago are revisited often with new approaches, new techniques, and insights. Recently, Meaney and coworkers (Liu et al., 1997; Francis et al., 1999) have used a naturalistic approach to the question of maternal mediation. They observed maternal behavior in undisturbed females. They reported that variations in maternal behavior influence the development of behavioral and
endocrine responses to stress in the offspring. In particular, increased licking/grooming and arched back nursing are correlated with a reduction in HPA activity and less-fearful behavior in the offspring. They further demonstrated that variations in maternal care serve as the basis for nongenomic behavioral transmission of individual differences in stress reactivity. In recent years new paradigms have been introduced in an attempt to investigate the effects of adverse early experiences on the neurobiology of stress. Several laboratories have begun to explore the consequences of more prolonged periods of maternal separation (Plotsky and Meaney 1993; Patchev et al., 1997). Although there are only a limited number of published papers using these paradigms, it does appear as though these longer periods of separation can reverse the effects of brief separations (early handling). In some instances these animals as adults exhibit hyperreactive HPA activity in contrast to the well-established hyporeactivity that results from early handling. Although the ultimate outcome of these postnatal manipulations depends upon a number of different variables, there is little question that one major source of individual differences in the neuroendocrine regulation of the HPA axis is based on the environment during development and that these effects are pervasive and difficult to reverse.
Stress and coping One issue that was confronted early in this chapter was the definition of stress. There was no attempt to arrive at a consensus definition. Also discussed was the pervasive issue that all stress-inducing events do not fall into simple distinct categories and that there are different neural pathways that are activated in response to different types of stress-inducing events. What is now abundantly clear is that events that can be viewed as purely psychological, and do not involve any immediate threat to homeostasis, are potent activators of the HPA axis. With the advent of a simple noninvasive procedure for measuring cortisol in saliva, the literature demonstrating that the activation of the HPA axis in response to psychological stimuli has been extensive (Kirschbaum and Hellhammer, 1994). Further, there is evidence that in animals and man the anticipation of an event is as
17 potent an activator of the HPA axis as the event itself. Phobic patients show the highest elevation of cortisol on the day prior to being exposed to the phobic stimulus (Wiedenfeld et al., 1990). What has also emerged from studies in this area is that there are large individual differences in response to these stimuli. Although some of these individual differences may be attributable to early experiences, there are other behavioral processes that modulate the responses to psychological stimuli. There is abundant evidence that unambiguously supports the hypothesis that psychological factors are important in determining the endocrine responses to stress. The field is indebted to Weiss (1972) for his innovative research on stress and coping. What was demonstrated in these studies was that a physiological response (stomach ulceration), in response to a well-defined stimulus (electric shock), can be modified: if the animal is permitted to exert control which regulates in some manner the duration and/or intensity of the shock, and/or is presented with information concerning the onset or offset of the shock, predictability, or is given information concerning the efficacy of the response, feedback. Weiss further postulated that the amount of stress an animal actually experiences when exposed to noxious stimuli depends on two variables: the number of coping attempts (responses) and the amount of relevant feedback these responses produce. Some of these psychological principles are directly applicable to the regulation of the HPA axis. Evidence that control is a potent modulator of HPA activity is found in studies using a variety of species. Coover et al. (1973) examined corticosterone levels during active avoidance in rats. Plasma samples were obtained following the first training session during which time the rats received shock on the majority of the trials, after the seventh training session when the animals have achieved asymptotic performance, and ten days later. There was a decline in plasma corticosterone levels from the first to the seventh training session, which was attributed to the absence of shock. However, as the training continued there was a further decline in corticosterone levels, although performance of the avoidance task did not differ from the early training. This decline was interpreted as evidence for the effects of control and predictability on the response of the HPA axis. Other
studies (Weinberg and Levine 1977) reported similar findings. Davis et al. (1977) found declines in adrenocortical activity using a lever-press escape paradigm that permitted escape but not avoidance of the aversive stimulus. The avoidance component of the shuttle box task appears to be less important than the ability to make an active response (control) that terminates the noxious event. Dess et al. (1983) examined the issues of control and predictability in dogs. The results revealed that control reduced the cortisol response to shock, whereas predictability in the absence of control has no discernable immediate effect. Increased circulating levels of cortisol induced by shock occurred whether or not the shock was predictable. Hanson et al. (1976) presented a clear demonstration that monkeys who could control the duration of a noxious sound reduce the cortisol response to these loud aversive noise levels. These are but a few of the many examples of importance of control in modulating the GC and presumably the neural components of the HPA axis. The influence of predictability on HPA activity is more problematic. Davis and Levine (1982) and Dess et al. (1983) failed to show that predictability in the absence of control exerted any effect on the HPA axis. However, these investigators pointed out the interaction between control and predictability. Whereas prediction may occur with or without control, control in the absence of predictability is not necessarily true. The very act of making a stimulus (or its offset) response contingent dictates that the stimulus will also be predictable. As we have documented the absence of control results in an exacerbated GC response. There is further evidence that the loss of control can induce increased HPA activity. In the study by Coover et al. (1973), a procedure was introduced that prevented the animal from making the now well-learned avoidance response. During this "forced extinction" period a locked door was placed between the compartments. Although the conditioned stimulus was presented and shock was omitted, corticosterone levels were again elevated compared to the plasma levels of corticosterone during avoidance conditioning. These data were seen to indicate that preventing the rat from making its response-contingent avoidance response represented a loss of control. Another example of the effects of loss of control is the rise in
18 corticosterone following extinction of an appetitive response. Rats trained to press a lever for water or food on a continuous reinforcement schedule show an elevation of corticosterone levels as a consequence of reinforcement being withdrawn during extinction (Coover et al., 1971). These data were interpreted as suggesting that "frustration," defined as the absence of reinforcement occurring in a context where reinforcement is expected, does result in activation of the HPA axis. Frustration, however, can also be viewed as loss of control. This notion is supported by a study by Davis et al. (1976). Extinction of an instrumental response can be achieved in several ways. The traditional procedure is to permit the animal to respond and omit the reward. Another extinction paradigm is to permit the subject to continue to respond and receive reward, but to make obtaining the reinforcement no longer response contingent. Under this extinction procedure no change in corticosterone levels occurs. The concept of loss of control implies that there is an accompanying loss of predictability. This would suggest that HPA activation would also be observed under circumstances where predictability of obtaining reward is altered from high to low predictability, and conversely that a shift from low to high predictability should result in a reduction of arousal and therefore a decrease in HPA activity. Goldman et al. (1973) trained rats to bar press for water on either a continuous reinforcement or a variable interval schedule. When the variable interval-trained rats were shifted to continuous reinforcement their corticosterone levels decreased. In contrast, when rats are shifted from a predictable schedule of reward to a more unpredictable schedule, corticosterone levels invariably elevate (Levine et al., 1972). That the HPA axis is bidirectional has been well established. The presentation of food or water to deprived rats results in a decline in the levels of ACTH and GC (Gray et al., 1978; Romero et al., 1995). Perhaps even more impressive is that simply presenting cues signaling the occurrence of food and water also produce declines in circulating levels of GCs (Levine and Coover, 1976; Coover et al., 1977). Is there an overall set of assumptions that can account for the data just presented? Several attempts have been made to provide a theoretical basis to account for the activation and inhibition of the HPA
axis (Hennessy and Levine, 1979; Levine and Ursin, 1991). The underlying principle that pervades most psychobiological approaches to stress invokes cognitive processes. The primary cognitive operation is one of the comparison between the immediate external event and some cognitive representation based on prior experiences. When discrepancies occur between the event and the cognitive representation, arousal is increased. The neurophysiological basis for this concept is derived from Sokolov's (1963) theory of the orienting response. Insofar as stress and arousal appear to be analogous concepts (Hennessey and Levine, 1979), those events that serve to increase arousal should activate the HPA axis and those, which would reduce arousal, should result in inhibition. Thus, such notions as uncertainty, expectancies, response outcomes, etc. have all been invoked to deal with the manner in which psychological events can influence the HPA system. What is clear is that the psychological processes can exert profound influences on the magnitude and direction of the responses of the HPA axis. Over the years there has been a concerted effort to delineate the neurobiology of affect and emotions. It should be noted that the CNS structures that have been implicated are critical in the regulation of fear and anxiety. The amygdala and other limbic system structures have been shown to be activated by the psychological stress. What has been presented in this section is far from an exhaustive view of the psychobiology of stress. There is an extensive literature that social isolation can exacerbate and that social support can reduce the responses to stress (Levine, 1993). Dysregulation of the HPA axis has been demonstrated to occur in a number of mental diseases. One of the earliest examples was the studies by Sachar (1980) that patients with depression were hypercortisolemic. Caroll et al. (1976) reported deficiencies in negativefeedback regulation in some cases of depression using the dexamethasone suppression test. Throughout this paper the major focus has been on the activation of the HPA axis and the factors that modulate this activation. In recent years there has been an evergrowing body of evidence that stress can result in a persistent hypocortisolemia (Yehuda, 1998; Gunnar and Vazquez, 2001). The mechanisms that are involved in the persistent down regulation of the HPA axis are still unknown.
19
The neuropeptide concept No discourse on the history of stress research would be even mildly comprehensive if the pioneering work of David deWied were not included. I met David when we were still young Turks trying to convince the world that our then somewhat offbeat research findings were of some importance in the complex universe of stress, deWied and I are of the same generation (our birthdays are 12 days apart). Whereas I was engaged in research that was attempting to delineate some of the factors that determine individual differences in the HPA response, his mission was to show that the peptides of the pituitary were active molecules that influenced behavior not through their regulation of some remote hormone but acted directly on the neural substrates that regulate specific behavioral outcomes. His early work was concerned with the pituitary response to stress. However, critical to the development of his future interests was the time he spent with I. Arthur Mirsky in Pittsburgh in 1957-1958. It was during that time de Wied collaborated with R.E. Miller, an experimental psychologist. These studies demonstrated that the effects of ACTH on shuttle-box performance were not a function of the steroidogenic action of ACTH, and in fact ACTH and prednisone had paradoxical effects in the shuttle box. That the effect of ACTH was not mediated by the adrenal cortex suggested that the brain was another target organ for ACTH. There was emerging a body of work around this time that suggested that ACTH may have behavioral effects that could be isolated from the behavioral consequences of the GCs. Much of this work came from the group of researchers at the University of Pecs in Hungary which included Endroczi, Koryani, and Bohus. Bela Bohus later left Hungary and joined with deWied's group in Utrecht. He went on to become a Professor at Groningen and an influential figure in the area of stress and behavior. Upon his return to the Netherlands, de Wied and a very notable cohort of coworkers concentrated their research efforts on demonstrating what is often referred to as the "Neuropeptide Concept" (for a review, see de Wied, 1990). The grand hypothesis of this work was that peptides emanating from the pituitary had actions on the brain that were a consequence of these peptides acting directly on the
neural substrates that regulates specific behaviors. Under deWied's guidance, numerous ingenious experiments were conducted. In his laboratory in Groningen they were able to successfully surgically remove either the posterior or anterior pituitary. This was accomplished by a laboratory technician J. Melchior. As far as I can determine he may have been the only man on earth who successfully performed this surgery. However, the ability to isolate either lobe of the pituitary made it possible to examine the role of specific anterior or posterior pituitary hormones. He discovered that not only ACTH but also vasopressin had observable effects on learning and memory. In his laboratory in Utrecht, where he had assumed the professorship of pharmacology in 1963, he demonstrated that the complete sequence of these peptides was not required for the behavioral effects to be manifested. He initiated a structure-activity program that eventually showed that the complete sequence of the peptide was not essential and that fragments of the ACTH molecule have profound behavioral effects in the absence of any influence on the secretion of GCs. The shortest of these fragments contained only four amino acids ACTH 4-7, though the most behaviorally potent fragment was ACTH 4-10. I could present a litany of experiments which were conducted not only on ACTH fragments but also on fragments of vasopressin. Over the years deWied has convincingly made the case for the neuropeptide concept. There is now complete acceptance of this doctrine to the point where its origin has almost been forgotten. Peptides derived from the gut, fat cells, pituitary, and brain have all been shown to exert their effects on specific behaviors, deWied's accomplishments extend well beyond the limits of this discussion. What makes him an important figure in the history of stress is that he was able to "push the envelope" and demonstrate that the so-called pituitary stress hormones can act directly on the brain to influence behaviors relevant to the stress response.
Conclusions While writing this brief history of research on stress I have been constantly aware of some of the sage comments regarding history. Santayana wrote
20 "history is a pack of lies, about events that never occurred, written by people who were never there." In my defense of this particular history I can say that the events described did happen and that I was, in some instances, there when the events unfolded. I can also say with pride that I was acquainted with most of the names that are mentioned in this chapter with of course some notable exceptions. However, what is also true is that it is impossible to present an historical account of anything that is not biased by the historian. Thus the history of this field, as presented, is what I believe to have been the critical milestones, the important figures, and the concepts that altered the course of how we understand stress. The most glaring omission in this history is the absence of a discussion concerning the impact of molecular biology on stress research. The year 2003 is the 50th anniversary of the Watson and Crick's classic paper on the structure of DNA. Since that time the face of biology has been dramatically transformed. It is difficult to communicate to the student of today what research in biology was like without the vast array of techniques that have become commonplace. Much of what has been discovered through molecular biology was beyond the wildest dreams of those of us who began their careers prior to the molecular biology revolution. Much of what we have learned about neural circuitry, receptors, gene expression, and genetic regulation of the biological systems involved in the stress response has come about through the use of the tools provided by molecular biology. Many of the chapters that will follow will focus on the molecular biology of stress. What I have described is my perspective on the milestones in thinking and experimentation that dictated the directions of the field. The techniques provided by the molecular biologist have built upon this foundation. The early stress researchers were not unlike the early explorers. They used the scientific equivalent of wooden sailing ships to make major discoveries. We are no longer earth bound and are light-years way from where we started. But clearly we have not solved the mysteries that surround the exquisite adaptive capacity of living organisms and the consequences of the failure of the adaptive functions. In 1960 1 attended a lecture by the eminent physiologist Sir A.S. Parkes. This lecture occurred at the time the Russians had launched the first space
craft. He commented that for him inner space was as exciting and engrossing as outer space. I have had the privilege and joy of having observed and participated in the most exciting period in the history of biology. I suspect that if I were able to see the face of stress research 50 years from now it would be as unrecognizable to me as the contemporary face of biology would be to Charles Darwin.
Acknowledgments I would like to express my appreciation to the National Institutes of Health, in particular N I C H D and N I M H for the many years of support that enabled me to pursue much of the research contained in the chapter. I would like to thank Drs. F. Robert Brush, Robert Murison, and Juan Lopez for their advice and critical comments. Finally, my deepest gratitude to the postdoctoral fellows, graduate, and undergraduate students who made my life as a scientist far more gratifying and rewarding and who contributed immeasurably to the research in my laboratories at Stanford University and the University of Delaware. Many of these students are now the leaders in the field.
References Basowitz, H., Persky, H., Korchin, S. and Grinker, R. (1955) Anxiety and Stress. McGraw-Hill, New York. Bhatnagar, S. and Meaney, M.J. (1995) Hypothalamicpituitary-adrenal function in chronic intermittently coldstressed neonatally handled and non handled rats. J. Neuroendocrinol, 7: 97-108. Bhatnagar, S., Shanks, N., Plotsky, P.M. and Meaney, M.J. (1996) Hypothalamic-pituitary-adrenal responses in neonatally handled and nonhandled rats: differences in faciltatory and inhibitory neural pathways. In: McCarthy, R., Agulara, G., Sabba, E. and Kvetnansky, R. (Eds.), Stress; Molecular and Neurobiological Advances. Gordon and Breach, New York, pp. 1-24. Cannon, W.B. (1914) The interrelations of emotions as suggested by recent physiological researches. Am. J. Psychology, 25: 256. Cannon, W.B. (1915) Bodily Changes in Pain, Hunger, Fear and Rage. Applegate and Co., New York, London. Cannon, W.B. (1932) Wisdom of the Body. Norton, New York. Carroll, B.J., Curtis, G.C. and Mendels, J. (1976) Neuroendocrine regulation in depression: II. Discrimination
21 of depressed from nondepressed patients. Arch. Gen. Psychiatry, 33: 1039-1051. Chrousos, G.P. and Gold, P.W. (1992) The concepts of stress systems disorders: overview of physical and behavioral homeostasis. JAMA, 267: 1244-1252. Chrousos, G., Loriaux, L.D. and Gold, P.W. (1988) The concept of stress and its historical development. Adv. Exp. Med. Biol., 245: 3-7. Coover, G.D., Goldman, L. and Levine, S. (1971) Plasma corticosterone increases produced by extinction of operant behavior in rats. Physiol. Behav., 6: 261-263. Coover, G.D., Ursin, H. and Levine, S. (1973) Plasmacorticosterone levels during active-avoidance learning in rats. J. Comp. Physiol. Psychol., 82: 170-174. Coover, G.D., Sutton, B.R., and Heybach, J.P (1977) Conditioning decreases in plasma corticosterone levels in rats by pairing stimuli with daily feedings. J. Comp. Physiol. Psychol., 91 (4): 716-726. Dallman, M.F. (1979) Adrenal feedback on stress induced corticoliberin (CRF) and corticotropin (ACTH). In: Jones, M.T., Gillham, B., Dallman, M.F. and Cahttopadhyay, S. (Eds.), Interactions within the Brain-Pituitary-Adrenocortical System. Academic Press, London, pp. 149-162. Dallman, M.F. (2000) Glucocorticoid negative feedback. In: Fink, G. (Ed.), Encyclopedia of Stress, Vol. 2. Academic Press, San Diego, pp. 224-228. Dallman, M.F. Jones, M.T. (1973) Corticosteroid feedback control of stress-induced ACTH secretion. In: Brodish, A. and Redgate, W.S. (Eds.), Brain-Pituitary-Adrenal Interrelationships. S. Karger, Basel, pp. 176-196. Dautzenberg, F.M. and Hauger, R.L. (2002) The CRF peptide family and their receptors: yet more partners discovered. Trends Pharmacol. Sci., 23: 71-77. Davis, H. and Levine, S. (1982) Predictability, control, and the pituitary-adrenal axis. J. Comp. Physiol. Psych., 96: 393-404. Davis, H., Memmot, J., MacFadden, L. and Levine, S. (1976) Pituitary-adrenal activity under different appetitive extinction procedures. Physiol. Beh., 17: 687-690. Davis, H., Porter, J.W., Livingston, J., Hermann, T., MacFadden, L. and Levine, S. (1977) Pituitary-adrenal activity and lever press escape behavior. Physiol. Beh., 17: 280-284. deGroot, J. and Harris, G.W. (1950) Hypothalamic control of the anterior pituitary and blood lymphocytes. J. Physiol., 111: 335-356. de Kloet, E.R. and Reul, J.M. (1987) Feedback action and tonic influence of corticosteroids on brain function: a concept arising form the heterogeneity of brain receptor systems. Psychoneuroendocrinology, 12: 83-105. de Kloet, E.R., Vreugdenhil, E., Oitzl, M. and Joels, M. (1998) Brain glucocorticoid receptors in health and disease. Endocr. Rev., 19: 269-301.
Denenberg, V.H. and Whimbey, A.E. (1963) Behavior of adult rats is modified by the experiences the mother had as infants. Science, 142:1192-1193. Dess, N.K., Linwick, D., Patterson, J., Overmier, J.B. and Levine, S. (1983) Immediate and proactive effects of controllability and predictability on plasma cortisol responses to shocks in dogs. Behav. Neurosci., 97: 1005-1016. de Wied, D. (1990) Effects of peptides hormones on behavior. In: de Wied, D. (Honorary Ed.), Neuropeptides: Basic and Perpectives. Elsevier, Amsterdam, pp. 1-35. Duncan, J. and Starling, S.G. (1959) A Textbook of Physics, Macmillan, London. Feldman, S. (1985) Neural pathways mediating adrenocortical responses. Fed. Proc., 44: 169-175. Feldman, S.C., Conforti, N., Chowers, I. and Davidson, J.M. (1970) Pituitary-adrenal activation in rats with medial basal hypothalamic islands. Acta Endocrinol., 63(3): 405-414. Fortier, C. (1951) Dual control of adrenocorticotropin release. Endocrinology, 49: 782-788. Francis, D.D., Champagne, F.A., Liu, D. and Meaney, M.J. (1999) Maternal care, gene expression, and the development of individual differences in stress reactivity. Ann. N.Y.. Acad Sc., 896: 66-84. Glick, D., von Redlich, D. and Levine, S. (1963) Flouremetric determination of corticosterone and cortisol in 0.02-0.05 milliliters of plasma or submilligrams of adrenal tissue. Endocrinology, 74: 653-655. Goldman, L., Coover, G.D. and Levine, S. (1973) Bidirectional effects of reinforcement shifts on pituitary adrenal activity. Physiol. Behav., 10:209-214. Gordon, M.K. and Levine, S. (1999) Behavioral and neuroregulatory patterns in rats that experienced maternal separation as pups. Soc. Neurosci. Abstracts, 270. Gray, G.D., Bergfors, A.M., Levin, R. and Levine, S. (1978) Comparison of the effects of restricted morning or evening water intake on the adrenocortical activity in female rats. Neuroendocrinology, 25: 236-246. Gunnar, M.R. and Vazquez, D.M. (2001) Low cortisol and a flattening of daytime rhythm: potential indices of risk in human development. Dev. Psychopathol., 3(3): 515-538. Hanson, J.D., Larson, M.E. and Snowdon, C.T. (1976) The effects of control over high intensity noise on plasma cortisol levels in rhesus monkeys. Beh. Biol., 16: 333-340. Harris, G.W. (1947) The blood-vessels of the rabbit's pituitary gland and the significance of the pars and zona tuberalis. J. Anat. Lond., 81: 343-351. Harris, G.W. (1948) Neural control of the pituitary gland. Physiol. Rev., 28: 139-179. Harris, G.W. (1950) Oestrus rhythm, pseudopregnancy, and the pituitary stalk in the rat. J. Physiol., 111: 347-360. Harris, G.W. (1955) Neural Control of the Pituitary Gland. Edward Arnold LTD, London. Harris, G.W. and Jacobsohn, D. (1952) Functional grafts of the anterior pituitary gland. Proc. Roy. Soc. B., 139: 263-276.
22 Hench, P.S., Kendall, E.C., Slocumb, C.H. and Polley, H.F. (1949) The effect of a hormone of the adrenal cortex (17-hydroxy-11-dehydrocorticosterone); compound E and of pituitary adrenocorticotropic hormone on rheumatoid arthritis. Proc. Mayo Clinic, 24: 181-197. Hennessy, J.W. and Levine, S. (1979) Stress, arousal and the pituitary-adrenal system: a psychoendocrine model. In: Sprague, J.M. and Epstein, A.S. (Eds.), Progress in Psychobiology and Physiological Psychology. Academic Press, New York, pp. 133-178. Herman, J.P. and Cullinan, W.E. (1997) Neurocircuitry of stress, central control of the hypothalamic-pituitary-adrenal axis. Trends Neurosci., 20: 78-84. Hodges, J.R. (1953) The function of adrenaline in the production of pituitary adrenocorticotropic activity. J. Endocrinol., 9: 342-350. Hsu, S.Y. and Hsueh, A.J. (2001) Human stresscopin and stress copin-related peptide are selective ligands for the type 2 corticotropin-releasing hormone receptor. Nat. Med., 7:605-611. Ingle, D.J. (1952) The role of the adrenal cortex in homeostasis. J. Endocrinol., 8: 23-37. Jones, M.T., Brush, F.R. and Neame, R.L.B. (1972) Characteristics of fast feedback control of corticotrophin release by glucocorticoids. J. Endocrinol., 55: 489-497. Kirschbaum, C. and Hellhammer, D.H. (1994) Salivary cortisol on psychoendocrine research: recent developments and applications. Psychoneuroendocrinology, 19(4): 313-333. Levine, S. (1957) Infantile experience and resistance to stress. Science, 126:405 Levine, S. (1967) Maternal and environmental influences on adrenocortical responses to stress in weanling rats. Science, 156: 258-260. Levine, S. (1993) The influence of social factors on the response to stress. Psychotherapy and Psychosomatics, 60: 33-38. Levine, S. and Coover, G.D. (1976) Environmental control of suppression of the pituitary-adrenal system. Physiol. Behav., 17: 35-37. Levine, S. and Ursin, H. (1991) What is stress? In: Brown, M.R. and Koob, G.F. (Eds.), Stress Neurobiology and Neuroendocrinology. Rivier Marcel Dekker, Inc, New York, pp. 3-21. Levine, S., Chevalier, J.A. and Korchin, S.J. (1956) The effects of early handling and shock on later avoidance behavior. J. Pers., 24: 475-493. Levine, S., Haltmeyer, G.C., Karas, G.G. and Denenberg, V.H. (1967) Physiological and behavioral effects of infantile stimulation. Physiol. Behav., 2: 55-59. Levine, S., Goldman, L. and Coover, G.D. (1972) Expectancy and the pituitary-adrenal system. In: Porter, R. and Knight, J. (Eds.), Physiology, Emotion and Psychosomatic
Illness. Ciba Foundation Symposium, Elsevier, Amsterdam, pp. 281-291. Liu, D., Diorio, J., Tannenbaum, B., Caldji, C., Francis, D., Freedman, A., Sharma, S., Pearson, D., Plotsky, P.M. and Meaney, M.J. (1997) Maternal care, hippocampal glucocorticoid receptors, and hypothalamic-pituitary-adrenal responses to stress. Science, 277: 1659-1662. Long, C.N.H. (1947) The conditions associated with the secretion of the adrenal cortex. Fed. Proc., 6: 461-471. Long, C.N.H. (1952) Regulation of A.C.T.H. Secretion. Rec. Prog. in Horm. Res., 7: 75-97. Marshall, F.H.A. (1936) Sexual periodicity and the causes which determine it. Phil. Trans. R. Soc. London, 226: 423-456. Mason, J.W. (1968) A review of psychoendocrine research on the pituitary adrenal system. Psychosom.Med., 30: 576-607. Mason, J.W. (1975) A historical view of the stress field. J. Hum. Stress, 16:11-22. McCann, S.M. (1953) Effect of hypothalamic lesions on the adrenocortical response to stress. Amer. J. Physiol., 175: 13-20. McCann, S. and Brobeck, J.R. (1954) Evidence for the role of the supraoptochypophyseal system in the regulation of adrenocorticotrophin secretion. Proc. Soc. Experimental Biol. and Med., 87: 318-324. McEwen, B. (2000) Stress, definition and concepts of. In: Fink, G. (Ed.), Encyclopedia of Stress, Vol. 3. Academic Press, San Diego, pp. 508-509. McEwen, B.s., Weiss, J. and Schwartz, L. (1968) Selective retention of corticosterone by limbic system structures in the rat brain. Nature, 220:911-912. Meaney, M.J., Aitken, D.H., Viau, V., Sharma, S. and Sarrieau, A. (1993) Individual differences in the hypothalamic-pituitary-adrenal stress response and the hypothalamic CRF system. In: Tache, Y. and Rivier, C. (Eds.), Corticotropin-Releasing Factor and Cytokines: The Role of the Stress Responses. New York Academy of Science, New York, pp. 70-85. Medvei, V.C. (1982) A History of Endocrinology. MTP Press, Lancaster. Munck, A.U., Guyre, P.M. and Holbrook, N.J. (1984) Physiological functions of glucocorticoids in stress and their relation to pharmacological actions. Endocr. Rev, 5: 25-44. Patchev, V.K., Montkowski, A., Rouskova, D., Koranyi, L., Holsboer, F. and Almeida, O.F. (1997) Neonatal treatment of rats with the neuroactive steroid tetrahydrodeoxycorticosterone (THDOC) abolishes the behavioral and neuroendocrine consequences of adverse early life events. J Clin. Invest., 99: 962-966. Plotsky, P.M. and Meaney, M.J. (1993) Early, postnatal experience alters hypothalamic corticotropin-releasing factor (CRF) mRNA, median eminence CRF content and stress-induced release in adult rats. Brain Res. Mol. Brain Res., 18: 195-200.
23 Popa, G.T. and Fielding, U. (1930) A portal circulation from the pituitary to the hypothalamus. J. Anat., 65: 227-232. Porter, R.W. (1953) Hypothalamic involvement in the pituitary adrenocortical response to stress stimuli. Amer. J. Physiol., 172: 515-519. Reul, J.M. and de Kloet, E.R. (1985) Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation. Endocrinology, 117:2505-2511. Romero, L.M., Levine, S., and Sapolsky, R. (1995) Adrenocorticotropin secretagog release: stimulaion by frustration and paradoxically by reward presentation. Brain Res. 676(1): 151-156. Sachar, E.J. (1980) Hormonal changes in stress and mental illness. In: Krieger, D.T. and Hughes, J.C. (Eds.), Neuroendocrinology. Sinauer Associates, Sunderland, pp. 177-184. Sapolsky, R.M. (1994) Why Zebras Don't Get Ulcers. W.H. Freeman and Co., New York. Sapolsky, R.M., Romero, L.M. and Munck, A.U. (2000) How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparatory actions. Endrocr. Rev., 21: 55-89. Sawchenko, P.E. (1991) The final commom path: issues concerning the organization of central mechanisms controlling corticotropin secretion. In: Brown, M.R., Koob, G.F. and Rivier, C. (Eds.), Stress: Neurobiology and Neuroendocrinology. Marcel Dekker, Inc, New York, pp. 55-72. Sayers, G. (1950) The adrenal cortex and homeostasis. Physiol. Rev., 30: 241-320. Selye, H. (1936) A syndrome produced by diverse nocuous agents. Nature, 38: 32. Selye, H. (1949) General adaptation syndrome. In: Textbook of Endocrinology, 2nd ed. Acta Endocrinol., Montreal, pp. 837-839. Selye, H. (1956) The Stress of Life. McGraw-Hill, New York. Silber, R.H., Busch, R.D. and Oslaps, R. (1958) Practical procedure for the estimation of corticosterone and hydrocortisone. Clin. Chem., 4: 278-284. Smotherman, W.P. and Bell, R.W. (1980) Maternal mediation of experiences. In: Smotherman, W.P. and Bell, R.W. (Eds.), Maternal Influences and Early Behavior. Spectrum Publications, New York, pp. 201-210.
Smotherman, W.P., Brown, C.P. and Levine, S. (1977) Maternal responsiveness following differential pup treatment and mother-pup interactions. Dev. Psychobiol., 10: 242-253. Sokolov, E.N. (1963) Perception and the Conditioned Reflex. Pergamon, Oxford. Steptoe, A. (2000) Stress effects, overview. In: Fink, G. (Ed.), Encyclopedia of Stress, Vol. 3. Academic Press, San Diego, pp. 510-511. Vale, W.S., Speiss, J., Rivier, C. and Rivier, J. (1981) Characterization of a 41-residue ovine hypothalamic peptide that stimulates the secretions of corticotropin and betaendorphin. Science, 213(4514): 1394-1397. Vogt, M. (1952) Plasma adrenaline and release of A.C.T.H. in normal and demedullated rats. J. Physiol., 118: 588-594. Wade, N. (1981) The Nobel Duel; Two Scientists 21-year Race to Win the Worlds most Coveted Research Prize. Anchor Press, Doubleday, Garden City. Weinberg, J. and Levine, S. (1977) Early handling influences on behavioral and physiological responses during active avoidance. Dev. Psychobiol., 10: 1661-1669. Weinberg, J., Kim, C.K. and Yu, W. (1995) Early handling can attenuate adverse effects of fetal alcohol exposure. Alcohol, 12: 317-327. Weiss, J.M. (1972) Influences of psychological variables on stress induced ulcers. In: Porter, R. and Knight, J. (Eds.), Physiology, Emotions and Psychosomatic Illness. Ciba Foundation Symposium, Elsevier, Amsterdam, pp. 253-264. Wiedenfeld, S.S., O'Leary, A., Bandura, A., Brown, S., Levine, S. and Raska, K. (1990) Impact of self-efficacy in coping with stressors on components of the immune system. J. Pers. Psychol., 59: 1082-1094. Wiener, S.G. and Levine, S. (1978) Perinatal malnutrition and early handling: interactive effects on the development of the pituitary-adrenal system. Dev. Psychobiol., 11: 251-259. Wislocki, G.B. (1937) The vascular supply of the hypophysis cerebri of the cat. Anat. Rec., 69: 361-387. Yehuda, R. (1998) Psychoneuroendocrinology of posttraumatic stress disorder. Psychiatr. Clin. North Am., 21: 359-379. Young, E.A., Lopez, J.F., Murphy-Wienberg, V., Watson, S.J. and Akil, H. (2003) Mineralocorticoid receptor function in major depression. Arch. Gen. Psychiatry, 60: 24-28.
This Page Intentionally Left Blank
T. Steckler, N.H. Kalin and J.M.H.M. Reul (Eds.) Handbook of Stress and the Brain, Vol. 15
ISBN 0-444-51173-3 Copyright 2005 Elsevier B.V. All rights reserved
CHAPTER 1.2
The neuropsychology of stress Thomas Steckler Johnson & Johnson Pharmaceutical Research & Development, A Division of Janssen Pharmaceutica N.V., Turnhoutseweg 30, 2340 Beerse, Belgium
Abstract: This chapter focuses on the psychological processes which govern the stress response. After an introduction aiming to define the terminology used in the area of stress research and some of the related concepts, such as emotions, the stress response will be discussed within the framework of cognitive functions, including learning theory. It will be demonstrated how situational appraisal and anticipation, predictability and controllability, and differences in coping style will affect stress responsivity. In the final parts of the chapter, these concepts will be related to the behavioural inhibition theory as defined by Gray and McNaughton (The Neuropsychology o f Anxiety, 2nd ed. Oxford University Press, Oxford, 2000), and how this could be mediated by various areas in the brain.
adrenal (HPA) axis, 1 activation of peripheral catecholaminergic systems, and of various neurotransmitter changes in the brain. More chronically, physiological stress responses can consist of the development of gastric ulcers, chronic changes in H P A axis and neurotransmitter activity, hypertrophy of the adrenal cortex, atrophy of the thymus, and loss of body weight, amongst other effects. At the behavioural level, exposure to stress has been reported to lead to a decrease in food and water intake (Pare, 1964), to inhibit exploratory activity (Weiss et al., 1980), to suppress appetitively motivated responses (Annau and Kamin, 1961), to increase anxiety-related behaviour (File, 1980), and to enhance or to impair both aversive and appetitive learning under certain conditions (Overmier and Seligman, 1967; Rosellini, 1978; Shors, 2001).
Stressors and the stress response
Stress can be defined as any challenge to homeostasis of an individuum that requires an adaptive response of that individuum (Newport and Nemeroff, 2002). Conceptually, stress consists of three components, that is the input of a stimulus, the evaluation of this information, and a response output. Aversive exteroceptive (e.g., electric shock, cold, social dominance in animals, but also several psychosocial aversive situations in humans, such as public speech) or interoceptive (e.g., pain) stimuli are referred to as stressors. A stressor can be defined as a change in the environment that is sensed by an organism, is aversive and potentially harmful to that organism and elicits acute and/or chronic responses (Ottenweller, 2000). A stress response in turn consists of a complex pattern of physiological, behavioural, cognitive, and/ or emotional components. Physiological processes can acutely comprise of, for example, piloerection, increases in heart rate, modulation of intestinal motility, activation of the hypothalamic-pituitary-
1One of the main players regulating HPA axis activity is corticotropin-releasing factor (CRF). CRF is released from the parvocellular part of the hypothalamic paraventricular nucleus (PVN) into portal vessels, subsequently activating the HPA axis by stimulation of release of adrenocorticotropic hormone (ACTH) from the anterior pituitary. ACTH in turn triggers the release of glucocorticoids (corticosterone or cortisol, respectively) from the adrenal cortex.
Fax: + 32 11 460 6121; E-mail:
[email protected] 25
26 In other words, there is a prioritisation of stress responses over other types of behaviour, and the stressor-induced behavioural responses serve the goal to reduce or eliminate the negative effects of a stressor.
Types of stressors A stressor can be any unpleasant intrusion to the external or internal environment (e.g., a social encounter, noise, an electric footshock, exposure to extreme temperatures, or an infection), or a withdrawal from the environment (e.g., starvation, social isolation, or separation of an infant from its mother). The stressor can be presented once only for a short time (e.g., administration of a single footshock of a few milliseconds duration), for longer times (e.g., exposure to cold temperatures for several hours), repeatedly (e.g., exposure to a series of repeated footshocks spaced over time, or repeated social defeat by a dominant subject, with the two subjects being fully separated in-between), or chronically/enduring (e.g., exposure to cold temperatures over days, or constant exposure of a defeated subject to the dominant subject). Moreover, some internal stimuli, such as anxiety and fear, can constitute a component of a stressor (e.g., in case of a patient suffering from an anxiety disorder) but, as will be discussed below, can also be part of a stress response (Young and Liberzon, 2002). A stress response (e.g., an increase in HPA-axis activity) can be induced in a relatively simple, reflexlike manner, in which case it does not necessarily require an evaluation of the situation by the subject. Alternatively, a stress response can also entail inputs from higher brain areas, i.e., a stress response can involve the evaluation of the stressor as being stressful. In particular, so-called psychological stress (for example, novelty stress and social defeat) has been suggested to activate these higher systems (Herman et al., 1996; Herman and Cullinan, 1997). Psychological stress can be defined as involving a reaction to an aversive stimulus in an individual's external environment (Kollack-Walker et al., 2000), and has been viewed as an asymmetry between the motivational systems of reward and punishment (Walker, 1987). This already implies that cognitive
functions play an important role in the processing of this type of stress-related information. However, as mentioned above, not every stressor may involve cognitive processing and a stress response can be induced directly by the exposure to an aversive stimulus. This second type of stressors has been called a physical stressor and can be defined as disturbing an individual's internal milieu, leading to activation of regulatory mechanisms that serve to restore homeostasis (Kollack-Walker et al., 2000). Such stressors would include, for example, starvation, noise, cold exposure, or haemorrhage. Besides covering different baskets with stressors, which involve cognitive processes to different degrees, the distinction between psychological and physical stressors gains relevance by the finding that these two types of stressors seem to activate different parts of the brain. Physical stress seems to be relayed directly to the PVN of the hypothalamus, part of the HPA axis, by ascending viscero- and somatosensory pathways (Sawchenko and Swanson, 1983; Kovacs and Makara, 1990; Sawchenko et al., 1996), rather than to higher brain areas. Psychological stress, on the other hand, seems to involve a number of higher brain areas, including various neocortical areas, the hippocampus, and the septal complex, presumably because it needs to be identified and evaluated first, and the PVN serves as relay point between the higher brain areas and the HPA axis. What exactly constitutes a physical stressor rather than a psychological stressor can, however, sometimes be a matter of debate. Although KollackWalker and colleagues (2000) provide a clear definition, which unambiguously distinguishes between stressors based on their primary location, i.e., whether they originate internally or externally of the subject, it is obvious that most stressors must be seen as compound stressors, affecting the subject both from its external and internal milieu. Swim stress, for example, includes both an external component (e.g., a situation where the subject faces the threat of drowning) and an internal component (e.g., hypothermia due to swimming in cold water). Therefore, it could be argued that it may be more appropriate to see the two definitions of physical and psychological stressors as two extremes of a continuous scale, with most stressors laying in-between. According to this view, it is the relative balance between
27 psychological and physical components that varies between stressors. Indeed, some stressors, originally considered as physical stressors (e.g., swim stress), are now increasingly considered psychological stress. Thus, there is some intuitive blurring of the dichotomy and there appears to be a gradual difference rather than an absolute distinction. On the functional neuroanatomical level, however, it becomes increasingly clear that different stressors activate different pathways, and it has been suggested that one way to distinguish between psychological and physical stressors is by directly looking at the pathways activated by these stressors, i.e., the brain itself would be the best tool to categorise complex stressors (Dayas et aI., 2001). Inherent to the concept of psychological and physical stressors is the fact that the subject is exposed to (external or internal) aversive stimuli. To fully appreciate the role of stress systems, it is however important to realise that not only aversive stimuli, but also reinforcement and withdrawal from an appetitive situation affect the activity of the HPA axis (withdrawal from reward may of course be considered as representing an aversive stimulus in its own right). Thus, reinforcement has been shown to decrease plasma corticosterone level in rats trained on an operant task, while extinction, i.e., operant responding followed by withdrawal of the expected reward, increases plasma corticosterone level (Coe et al., 1983; De Boer et al., 1990). In the following parts of this chapter, I will focus on the processes which take place during the evaluation of the information provided by a stressor. Before going into further detail, it is however important to first clarify a concept central to this discussion, that is the question of what is meant when talking about an emotion.
Emotions related to stress exposure
The stress-induced behavioural changes mentioned above can be, but do not have to be, part of what is considered an emotional response. James (1890) suggested that emotion-provoking stimuli induce bodily changes, and that the feeling of these changes would be the emotion. Others extended this view and associated emotions with changes in the brain
and the body, i.e., considered emotions to consist of behavioural, autonomic, and endocrine responses, which can differ according to the nature of the emotion. For example, there is evidence that negative emotions increase heart rate and change skin temperature more than pleasant emotions (Ekman et al., 1983). Behaviourally, emotions can be viewed as central states elicited by reinforcing stimuli (Plutchik, 1967). Especially secondary punishing stimuli have been associated with negative emotions such as fear (Mowrer, 1960), which can be viewed as a state anticipating primary punishment. Rolls (1999) defined emotions as 'a state elicited by rewards or punishers, leading to changes in rewards or punishers' (p. 60). He considers emotions as a cognitive process which results in a decoded signal that an environmental event (at the time of presentation or as a remembrance) is reinforcing or punishing, together with the affective state produced as a result. Within that definition, an affective state differs from an emotion in that the former on its own lacks an external sensory input and the cognitive decoding, i.e., there is no present or past environmental event towards which the affective state is directed, which would then lead to a goal-directed behaviour (Rolls, 1999). It is clear from these definitions that emotions form an integral part of the response to a (psychological) stressor. Functionally, it has been suggested that the emotional responses are of relevance for coping reactions to short-term aversive events (a topic which will be covered in more detail below) and for the initiation of fight-flight-freezing reactions, which serve the subject to deal effectively with a source of danger and to return into a state of safety. Interestingly, there seems to be a more rapid target detection for emotional stimuli such as fear-related (but also positive emotional) stimuli in humans (Ohman et al., 2001), and such target stimuli seem to be processed even though the stimuli are not perceived, as can be evidenced by alterations in skin conductance responses (Esteves et al., 1994), i.e., these emotionally relevant stimuli will even be detected under conditions of limited attentional resources (pre-attentive processing), which makes sense in that those stimuli to which a value has been ascribed, e.g., a stressor, will be of high relevance to the individuum.
28 The stress r e s p o n s e - a cognitive view
It is obvious from the points raised above that cognitive processes play an important role for the adequate reaction to stress exposure. The importance of such processes appears evident by the simple notion that subjects should learn to repeat responses that lead to reward or prevent punishment, and should learn to inhibit responses that prevent or truncate rewards or lead to punishment, i.e., it is learned that the appropriate action should prevent the (re-)occurrence of a certain stressor. As an extreme example, a subject should remember the appearance, smell, sound, and environmental location of a predator to predict its next occurrence to maximize the likelihood for survival. Under daily laboratory conditions, a rat will use related information about its cage mates to adhere to hierarchical orders for food and water access. This concept is not new, but the relevance of cognitive processes for the response to a stressor has been noted as early as 1926 by Freud in the psychoanalytical theory of defence mechanisms, suggesting that denial and intellectualisation are fundamental ways to reduce anxiety. Thus, changes in cognitive function can result in changes in the way a stressor is perceived, remembered, or how a stressful situation is solved.
paradigm, and Roman Low Avoidance rats were faster than Roman High Avoidance rats (Koene and Vossen, 1991). In a runway situation, Wistar Kyoto rats were faster in solving a conflict than randomly bred Wistar Wu rats and Brown Norway rats were faster than Wistar Wu rats (Koene and Vossen, 1991). The value of the defensive distance is composed of the objective physical or temporal distance between subject and stressor, and the subjective assessment of threat originating from the stressor (Blanchard and Blanchard, 1990). This in turn implies that in the absence of a stressor, there should be no behavioural inhibition. In the presence of a moderate stressor, there may be behavioural inhibition of non-defensive behaviour (e.g., a decrease in food and water intake, inhibition of exploratory activity, and suppression of appetitively motivated responses), but not of defensive behaviour (fight or flight; risk assessment), while under conditions of high stress, both defensive and non-defensive types of behaviour would be inhibited (freezing). In this respect, it is interesting to note that Takahashi (1996) suggested that glucocorticoids, which are released under stressful conditions, may also influence freezing during development by actions at the septohippocampal level, thereby modulating the individual's levels of stress-induced arousal and attention to threat. This is an interesting idea in the context of Gray's theory on behavioural inhibition, which will be discussed below.
The concept of defensive distance In order to cope with a stressful situation, a subject can have two major strategies: it can try to avoid the stressor (defensive avoidance) or to approach the stressor (defensive approach), the latter of which includes risk assessment and behavioural inhibition. It has been proposed that the relative importance of the two competing types of behaviour (avoidance and approach) will depend on the so-called defensive distance between the subject and the stressor (Blanchard and Blanchard, 1990). Moreover, the speed with which such conflict can be resolved in an approach-avoidance situation seems to be related to genetic factors and underlying stress responsivity. For example, Tryon Maze Bright rats have been reported to be faster in speed of conflict resolution than Tryon Maze Dull rats in an operant-conditioned punishment
Appraisal and anticipation In order to choose between approach and avoidance most effectively, the subject must be able to appraise the situation it has to deal with. The concept of defensive distance suggests that the subject is able to appraise its position relative to the stressor, i.e., there is a quantitative aspect. However, a subject must also be able to appraise a stressor in a qualitative way, i.e., whether a given stimulus is a severe stressor, a mild stressor, or no stressor at all. In 1968, Mason suggested that the appraisal of a stimulus determines whether it is perceived as a stressor, i.e., that the subject must discriminate between threatening and non-threatening situations. In other words, the meaning of a stimulus is, at least in part, determined
29 by the representations held by the subject, which can be innate or acquired. This can be exemplified by the condition of novelty stress. Exposure to novelty alters both HPA axis and behavioural activities (Lemaire et al., 1999; Hall et al., 2000). Novelty, however, is not an inherent characteristic of a stimulus, but an attribution given to the stimulus by the subject. In addition to the appraisal of a situation, it is important to acknowledge that a subject's responses are not only driven by reactive demands, but also by anticipation. Arthur (1987), for example, demonstrated that anticipation of an aversive stimulus, such as an electric shock, may result in an even greater stress response in rats (as measured in activation of the HPA-axis) than confrontation with the aversive stimulus itself. Similar findings were reported by others and extended to other measures, including anticipatory changes in pain threshold (Przewlocka et al., 1990; Yamamotova et al., 2000). Likewise, anticipatory stress responses can be observed in humans, for example, when facing a public speech, and this stress response can be altered by manipulation of cognitive processes (Rohrmann et al., 1999): the degree to which individuals are stressed anticipating public speech (as measured by HPA-axis activation, changes in heart rate, subjective arousal, and state anxiety) depends on manipulative feedback (reassuring or arousing) given during that period (Rohrmann et al., 1999), but also on the level of control that subjects experience over the situation (see below).
Stress coping It is evident that cognitive appraisal of a situation will determine the strategy chosen by an individual to deal with, or to cope with a stressor. In this context, it is common knowledge derived from everyday experience, but also from sophisticated and wellcontrolled studies, that there are very clear individual differences in stress coping. As already pointed out, these differences between individuals seem to be in part based on genetic factors (Koene and Vossen, 1991), which interact with developmental and situational influences (see below). The importance of the concept of coping is highlighted by the fact that the ability to cope is the
integral part of some definitions of stress. Lazarus (1966), for example, conceptualised stress as the interaction between the demands of the situation and the individuals ability to cope. Hence, coping is an active response to resolve a stressful situation. It encompasses cognitive and other behavioural efforts to reduce or adapt to a stressor. In principle, a subject can appraise two components of a situation: First, it can assess whether a situation is irrelevant, appetitive, or aversive, as was discussed above (so-called primary appraisals). Secondly, it can make an assessment of the relevance and availability of its own coping strategies (so-called secondary appraisals). The success of a given coping strategy is situation specific and its appropriateness will, at least in part, depend on defensive distance, as the defensive distance will determine whether approach or avoidance are more likely to be successful. Differences in stress coping have been implicated in stress resiliance and hence different vulnerability to psychiatric disease. The constitutional vulnerability of individuals to stress is named diathesis. As already mentioned, such individual differences are based on genetic factors, physiological developmental factors, and lifetime experience. We have already seen that different strains of rats exhibit different speeds of conflict resolution. Another example derives from the simple reaction to novelty, which has been shown to differ between inbred strains of rats, suggesting a genetic base (Gilad and Shiller, 1989). Conversely, differences in reaction to novelty can also be observed within a population of rats (Lemaire et al., 1999; Piazza et al., 1991), suggesting epigenetic factors also play a role. The importance of epigenetic factors is also illustrated by the findings that prenatally stressed animals are more impaired in coping with stress compared to non-stressed controls (Weinstock, 1997), and that rat pups, which differ in the level of maternal care (maternal licking and grooming) while being nursed, will show differences in the response to novelty stress later in life (Liu et al., 1997). Interestingly, these high- and lowreacting rats do not only differ in behavioural and neuroendocrine measures, but also in neuroanatomical features, and show a reduced neurogenesis in the dentate gyrus of the hippocampus (Lemaire et al., 1999).
30 As an interim summary, we can conclude so far that different stressors will lead to different patterns of brain activation. Some, but not all, stressors will involve a cognitive component, including appraisal and anticipation of a situation, leading to an emotional response and a variety of different coping strategies, which are under control of genetic and epigenetic factors. From that, it can easily be seen that behavioural and affective changes can even precede the presentation of a primary stressor and are under control of the laws of classical- and operant-conditioning processes, where stimuli presented to the subject have been associated with the stressor before.
Learning processes modulating s t r e s s responsivity Next, the role of learning processes in the modulation of stress responsivity will be discussed in more detail. Associating a given stimulus with a stressor is equivalent to meaning that a certain stimulus will have acquired the qualities of a secondary stressor (or a conditioned stimulus). Once the subject has made such an association, it can respond in a way to the aversive situation similar to a response that has led to a successful outcome in the past, i.e., it can elicit a prepared response (Phillips, 1989). One model which would allow a subject to predict the occurrence of a stressor would be related to classical conditioning procedures, whereby the subject has to associate the stressor with a discrete cue, which could be simple (such as a tone or a light) or complex (such as the environmental context in which this stressor appears). Such classical conditioning procedures are frequently used in animals and have also been reported in humans (Watson and Rayner, 1920). The conditional stimulus in turn can be associated with an emotional tone, such as fear. Alternatively, the subject could learn an instrumental or so-called operant response, i.e., the subject acquires a behavioural pattern that has the capacity to alter the frequency of the subject's exposure to certain events. Within this framework, two types of learning have been distinguished, namely escape and avoidance learning, essential forms of behavioural reactions
in stressful environments. In escape learning, the aversive stimulus occurs conspicuously before each response, while in avoidance learning, the stressor, if learning is successful, is rarely, if ever, seen. These avoidance responses can be very persistent under conditions of extinction, i.e., in the absence of the primary stressor, and well-learned avoidance responses may be sustained for some time purely as an automatic habit (Mackintosh, 1983). By analogy, presentation of a secondary stressor, followed by presentation of a primary stressor and escape, is likely to generate anxiety, while absence of the primary stressor due to avoidance after presentation of the secondary stressor, should result in diminished anxiety and merely lead to a habitual response. It is of course also possible to extinguish an avoidance response. Interestingly, extinction of avoidance learning has been suggested to be under control of the activity of the HPA axis. More specifically, extinction of passive avoidance has been suggested to be modulated via activation of the mineralocorticoid receptor (MR), while extinction of active avoidance has been suggested to be modulated via activation of the glucocorticoid receptor (GR) 2 (Korte, 2001). Such learned responses can be assessed in the animal in procedures such as conditioned suppression or conflict procedures, conditioned emotional response (CER), or fear-potentiated startle. Conditioned suppression or conflict procedures entail the suppression of a consummatory or exploratory behaviour in the presence of a stimulus (the conditioned stimulus) previously associated with an aversive stimulus (the unconditioned stimulus) and could, for example, involve the suppression of a lick for water or a freezing response (the latter being mediated by the periaqueductal grey; Graeff, 1994). These behavioural responses can occur within parts of a second, faster than any neuroendocrine changes, but are still under neuroendocrine control. The freezing response, for example, already takes place
2Two corticosteroid receptors have been identified in the brain, the MR and the GR, which differ in expression patterns and binding properties (GR binds corticosteroids with a 10-fold lower affinity than MRs). It has been suggested that MRs are involved in the maintenance of the activity of the stress system, while GRs, in conjunction with MRs, may mediate the recovery from stress (Reul and De Kloet, 1985; De Kloet et al., 1998).
31 before activation of the HPA axis, but has been suggested to be acutely modulated by M R activation (Korte, 2001). Korte argues further that the first reaction of an animal (such as a rat or a mouse) visiting a dangerous location for which an aversive memory has been formed will be conditioned freezing behaviour, modulated by a permissive role of the MR. Although it is evident thatthe other factors will contribute to such response as well, this example again nicely illustrates the interrelationship between behavioural processes and stress systems, which goes both ways. If the environment turns out to be safe, extinction of passive and active avoidance will take place. In contrast, a CER often involves suppression of an instrumental response, for example, of lever pressing for food, upon presentation of an aversive CS. In fear-potentiated startle (mediated by the pontine reticular nucleus; Davis, 1992b), the magnitude of the startle response is increased in the presence of an aversive CS, the state retrieved by the CS is fear. Fear-potentiated startle has been suggested to be a sensitive measure of anticipatory anxiety (Davis et al., 1993). The examples given above highlight the importance of first-order conditioning processes in the response of a subject to a stressor, but this can of course also be extended to second-order conditioning. For example, a rat might have learned to stop responding in the presence of a CS (e.g., a light) in a conditioned suppression paradigm in the absence of an UCS. If the CS is now preceded by another neutral stimulus (e.g., a tone), the presentation of the second stimulus can eventually also lead to suppression of the ongoing behaviour after a few pairings. The relevance of second-order conditioning lays in the fact that it helps to explain how anxiety can generalise from one anxiogenic event to a number of other, more or less closely related, originally neutral stimuli. However, conditioning is not restricted to behavioural responses, but extends to autonomic and neuroendocrine functions as well. For example, it has been shown that increases in plasma corticosterone levels can be conditioned to stimuli associated with a poison (Adler, 1976). Conversely, decreases in plasma corticosterone levels have been conditioned to stimuli associated with daily feeding and drinking (Coover
et al., 1977). Thus, our stress systems are capable of learning and we can a d a p t - or m a l a d a p t - to a stressful situation, with all the consequences associated with stress-related psychiatric disorders. In the context of learning theory, adaptation and maladaptation are better referred to as habituation and sensitization. Habituation, an active-learning process not to respond to irrelevant situations, governs the decline of the stress response when the subject is repeatedly exposed to the stressor. During habituation, the orienting response towards the stressor is diminished. This can be seen, for example, by the fact that a novel stimulus, which induces a stress response upon initial exposure, becomes a habituated stimulus after repeated exposure. Behaviourally, such stimulus will elicit exploration, which is reduced upon repeated exposure. Sensitization refers to an exaggeration of the stress response and is seen, for example, if animals are exposed to a mild stressor (e.g., a novel stimulus) after exposure to a strong aversive stimulus. Rats pre-exposed to inescapable footshock, for example, have been demonstrated to display progressive and long-lasting increases in anxiety-related behaviour (Van Dijken et al., 1992), and to develop autonomic (Bruijnzeel et al., 2001) and neuroendocrine changes (Van Dijken et al., 1993), which last for weeks. Likewise, social defeat of one rat by another rat can induce long-lasting behavioural changes in the defeated animal, such as an increase in immobility (Koolhaas et al., 1990). During this stage, the orienting response towards the stressor would be enhanced. At the neuroendocrine level, it has been shown that a single exposure to electric footshock can induce vasopressin (AVP) levels in corticotropinreleasing factor (CRF) terminals in the median eminence for at least 11 days (Schmidt et al., 1996), and it has been suggested that the sensitized response is due to enhanced AVP release, which potentiates the effect of CRF on ACTH release (Van Dijken et al.,
1993). Sensitization has also been reported in humans, leading to greater hormonal stress response over time and an increase in baseline cortisol levels (Young and Akil, 1985; Dallman, 1993). Moreover, it has been shown that pre-exposure to stress sensitizes the release of noradrenaline at various brain levels
32 (Tsuda et al., 1986; Pacac et al., 1995), which in turn can activate the HPA axis by stimulation of CRF release by parvocellular PVN neurons (Plotsky et al., 1989; Plotsky, 1991; Pacak et al., 1995). More recently, Bruijnzeel et al. (1999) demonstrated longterm activation of brain areas involved in the mediation of anxiety-related behaviour, autonomic, and neuroendocrine responses for at least two weeks :n the pre-shocked rats, as measured by an upregulation of Fos-immunoreactivity. These alterations are not non-specific changes, but seem to affect certain stages of information processing more than others. Shors (2001), for example, has shown that exposure to a stressor enhances the formation of new associations rather than affecting retention or performance of the conditioned response. In this study, acute exposure to intermittent tail shock or acute inescapable swim stress, but not inescapable noise stress or the unconditioned stimulus of periorbital eyelid stimulation, enhanced classical eyeblink conditioning in male rats when trained 24h after stress cessation (Shors, 2001). This facilitating effect of the stressor occurred within 30 min of stressor cessation, but did not occur once the response had been acquired (Shors, 2001). Thus, the response to stress will be shaped by prior learning experience, but at the same time the ability to acquire new information will also be affected by the stressor. However, it remains to be shown that this is a direct effect on memory and not due to changes in other types of behaviour.
Arousal and attention One non-mnemonic type of behaviour which is affected by stress is arousal. It is a condition sine qua non that subjects ought to be alerted to orient, process, and respond adequately to a stressful stimulus. An increase in arousal prepares the subject to change behavioural responses within parts of a second to alter between approach and avoidance. As already discussed, this processing of emotionally relevant stimuli is not only rapid, but can occur in a pre-attentive state, i.e., without conscious perception (Esteves et al., 1994). Arousal is therefore important in face of threatening situations, as it alters the stimulus-processing
capacities of the individuum, it lowers the sensory threshold, and allows more rapid processing of sensory stimuli (Lynn, 1966). Various neurotransmitter systems have been implicated in the modulation of arousal. The noradrenergic projections of the locus coeruleus to higher brain areas, including various cortical areas, the hippocampus, and the basal forebrain, seem to play an important role in the mediation of arousal (Berridge, 2004). This is of particular interest as exactly these projections are also activated under stressful conditions, suggesting that this could be one mechanism via which the organism ensures proper information processing to relevant stimuli. However, too much arousal can also be detrimental: according to the arousal hypothesis of Yerkes and Dodson (1908), arousal will affect performance in a bell-shaped manner, i.e., medium levels of stress will lead to increased arousal and improve stress reactivity, while performance is lower under conditions of low stress and low arousal. This can explain the improved stimulus-processing capacity under conditions of low to medium stress level. However, when arousal is increasing further, e.g., due to high stress levels, performance is likely to drop again. This could be one mechanism explaining the sometimes controversial findings of cognition enhancing or impairing effects of stress exposure. Let me illustrate this point with a few examples derived from the animal and the human literature: we have already seen in one of the studies discussed above that an anticipatory stress response can be manipulated externally by giving arousing feedback to humans facing public speech (Rohrmann et al., 1999; see above). Thus, the level of arousal can determine the degree of stress exposure. The same holds true for non-human animals. In the experiment by Shors (2001; see above), for example, an interaction between arousal and stress could also perfectly well explain the results obtained, as stress exposure would have led to an increase in arousal, which could have led to improved learning, i.e., the effects of the stressor on conditioning could have been indirect, rather than on learning per se. This then would suggest that arousal influences the formation of new memories. Another example clearly demonstrating that arousal processes affect not only the sensory-perceptual
33 systems, but also the cognitive systems involved (Pribram and McGinnes, 1975) derives from a classical study by Schachter and Singer (1962). In this study, human volunteers were pharmacologically aroused with adrenaline and placed in a stressful situation, which led to both subjective and objective evidence of emotional states. In contrast, pharmacological treatment alone did not alter emotional states. Likewise, stress-induced emotional changes were less pronounced when the subjects were not pharmacologically aroused. Thus, the combination between an increase in arousal and the exposure to stress had synergistic effects, leading to a higher appraisal of a situation as being stressful. According to Easterbrook (1959), a stress-induced increase in arousal serves the purpose to narrow attention to central details of a situation, leading to better recall of details central to the event. This is in agreement with Mandler (1984), showing that memory for the emotionally arousing event is enhanced (presumably up to a certain stress level and hence a certain level of arousal, as the descending part of the bell-shaped curve will be reached at a certain point). Narrowing of attention towards relevant stimuli seems to be one of the prerequisites for enhanced mnemonic processing under stressful conditions and attention must be regarded as another preparatory response altered in such situation: an increase in attention leads to increased scanning of the environment for relevant stimuli and/or display of risk assessment behaviours, which would enhance acquisition of information associated with the stressor. Moreover, it is under these conditions of increased attention to a stressor that there may also be an increasing retrieval of memories associated with the stressor. Under conditions of sensitization, attention/ orientation towards the stressor is enhanced, while habituation will lead into reduced attention/orientation to the stressor, as mentioned above. As with arousal and the appraisal of a signal as being stressful (the subject becoming aroused by a stressor and the level of arousal in turn will determine the appraisal of the stressor), there are intricate relationships between stress and attention. In humans, an increase of attention to bodily sensations has been shown to exacerbate anxiety and physiological reactivity (Epstein et al., 1978; Pennebaker,
1982). However, not only will an appropriate level of attention be required for detection of a stressor, but the stress systems also influence attentional mechanisms. Hypocortisolaemic patients, for example, have been reported to be impaired in their ability to recognize and integrate sensory information, although they can detect sensory stimuli at lower levels than control subjects (Henkin, 1970). Wolkowitz et al. (1990) suggested that the glucocorticoids affect selective attention by raising arousal to sub-optimal level. Thus, the hormones of the HPA axis seem to play an important role in the direct or indirect modulation of attention.
Stress predictability, control, and helplessness From the preceding paragraphs it is evident that appraisal and anticipation are important psychological processes involved in the processing of stressful information. In procedures such as conditioned suppression or conflict procedures, CER, or fearpotentiated startle, the subject is able to make a prediction about the likelihood for the occurrence of a stressor (the unconditioned stimulus) by presentation of a warning stimulus (the conditioned stimulus). The behavioural response to the warning stimulus is an anticipatory response to prevent occurrence of the primary stressor. This is called behavioural control. On the other hand, the subject can just have the sense that it is or is not in control of an aversive situation, which is called perceived control. Under this condition, the subject has the perception that the stressor is potentially controllable by making the correct response. Behavioural and perceived control are two different phenomena. For example, a subject may perceive itself out of control when, in fact, objective behavioural control is given. As such, perceived control is the appraisal the subject gives to its ability to control the situation. While behavioural control should allow a rat (or in fact any other subject) to perform appropriate in tasks such as CER (we do not really know about an animal's degree of perceived control), lack of control should have opposite effects. Therefore, it can be expected that lack of control has anxiogenic-like effects and, for example, potentiates fear responses. Exposure of rats to a yoked control design of shock exposure,
34 where half of the animals have control over the shock (i.e., they are able to make a behavioural response that terminates the shock) and the other half has no control (i.e., animals receive identical shocks, but are unable to control it, irrespective of responses made), leads to increased freezing in the rats that previously had no control when they are tested for conditioned fear 24 h later (Maier, 1990). Along similar lines, it has been shown that the level of plasma corticosterone and the degree of fear conditioning are higher after exposure to an inescapable stressor than following exposure to an escapable and, hence, better controllable, but otherwise comparable, stressor (Mineka et al., 1984). This goes as far that the degree of control over a stressor has been shown to be inversely related to levels of plasma corticosterone (Sandi et al., 1992), and it has been suggested that in particular in those situations where the future demands are uncertain, there is a strong activation of the HPA axis (Toates, 1995). Other behavioural and physiological responses are also affected by controllability of the stressful situation. Seligman (1968), for example, showed that shock produced more extensive gastric ulcerations and suppression of food-rewarded lever presses in rats if the shock was unpredictable rather than signalled. Moreover, lack of stress control interfered with subsequent learning (Seligman, 1975). From this one would assume that the ability to control a situation is the preferred state over an uncontrollable situation. Indeed, rats will choose signalled and predictable over unsignalled and unpredictable shocks (Lockard, 1963; Badia et al., 1979). One reason could be that the perception of a stimulus as a stressor will be stronger when it is uncontrollable or unpredictable (Weiss, 1970). Thus, the ability of a subject to predict the occurrence of a stressor and hence its ability to control its own environment has a major impact on the appraisal of the stressor by the subject. How does the degree to which a stressful situation can be controlled by an individual affect cognitive function? In the previous paragraphs it was shown that the learning processes are influenced by the degree to which a stressor can be controlled: if subjects are pre-exposed to unpredictable shock, they show an impairment in learning a subsequent escape response, a phenomenon called learned helplessness (Seligman, 1975). Helplessness refers to the inability
of a subject to control a (stressful) situation even when it has the opportunity to do so. A number of concepts have been proposed to explain this phenomenon, ranging from general emotional exhaustion leading to inhibition of active learning, over adoption of inappropriate, passive, response habits, to more cognitive explanations, suggesting that the subjects learn to be helpless and give up trying. The latter concept again relates to the idea that the attribution given to a stimulus will interfere with subsequent performance. This could then be viewed as a special case of classically conditioned learned irrelevance (Gray and McNaughton, 2000). The degree to which the aversive situation can be controlled by the subject will also determine whether the subject regards its coping skills as sufficient. This serves as a model to explain why some subjects may perceive a situation as stressful, while others may not. Being in control of a stressful situation entails that a subject can learn that the consequence of its action is termination of an aversive situation, and this knowledge should reduce an individual's stress response. For example, it has been shown that monkeys being able to terminate loud noise have lower plasma cortisol level than yoked monkeys exposed to the same noise, but having no control (Hanson et al., 1976). Comparable effects of control on stress coping have been reported in rats (Overmier and Seligman, 1967; Weiss, 1968; Rosellini, 1978). Thus, exposure to uncontrollable stress not only results in higher glucocorticoid plasma levels, but also in behavioural alterations, such as impaired learning of a controllable avoidance response (Seligman, 1975).
Gray's behavioural inhibition system Having discussed some of the cognitive processes which play a role in the modulation of stress responsivity, the next question would be whether one can fit this into a contemporary psychological theory. Gray (1976) proposed a behavioural inhibition system, which controls ongoing behaviour whenever the subject wants to achieve a goal which requires to move towards a source of danger, i.e., when there are conflicting aims. Under these conditions, there is an increase in vigilance (the
35 subject orients towards threat, i.e., attention is directed towards the stressor) and in arousal, which can be induced by the presentation of a stressor, such as by secondary (conditioned)punishing or painful stimuli, and by secondary frustrative stimuli, such as failure or loss of reward, but also by innate aversive stimuli, such as novelty or uncertainty. Important to this concept is that the subject has to experience conflict to activate the behavioural inhibition system, i.e., it is not sufficient to just present an aversive stimulus to the subject, but the subject has to know of a variety of stimuli and contingencies between them as to engender a conflict between mutually incompatible appetitive and aversive goals. The behavioural inhibition system has been proposed to oppose, to a certain degree, the so-called fight-flight system, a defence system which serves the purpose to remove the subject from a source of danger (Gray and McNaughton, 2000). Thus, activation of the fight-flight system leads to avoidance of anxiety-arousing stimuli and their consequences and, hence, should ultimately result into a reduction of exposure to a stressful stimulus, i.e., activation of the fight-flight system should direct attention away from the stressor. In contrast, activation of the behavioural inhibition system could lead to even enhanced stress exposure. Gray and McNaughton (2000) propose a third system, which deals with approach in appetitive conditions, the behavioural approach system. However, for the purpose of the present review, the focus will be on the behavioural inhibition and the fight-flight systems. According to Gray and McNaughton, the distinction between these two systems is not only behaviourally, but also neuroanatomically relevant, as will be discussed below.
Brain regions involved in the processing of the stress response A hierarchical defence system has been proposed by Graeff (1994), where progressively more brain areas will be involved with progressively more complex aspects of defence against stressful stimuli (also discussed in Gray and McNaughton, 2000). The hierarchical defence system proposes that the
periaqueductal grey at the lowest level coordinates undirected escape, resulting in freezing, flight or fight. The medial hypothalamus at the next level coordinates directed escape. The amygdala coordinates simple active avoidance, and the cingulate cortex at the highest level coordinates more complex active avoidance. This has been extended more recently by Gray and McNaughton (2000), suggesting that the periaqueductal grey, the medial hypothalamus, the amygdala and the anterior cingulate cortex are involved in defensive avoidance, while the septohippocampal system and the posterior cingulate are more relevant for defensive approach, including risk assessment and behavioural inhibition (Fig. 1). Gray and McNaughton (2000) further propose that the posterior cingulate cortex plays a role in the generation of innate anxiety plans, while the prefrontal cortex is involved in working memory processes and motor programming functions required for acquired anxiety plans. Under certain conditions, automated responding will be advantageous over more slow cognitive processing of stimuli. Noradrenaline release increases in the prefrontal cortex under stressful conditions (Feenstra, 2000). This increase in noradrenergic activity in the prefrontal cortex may lead to activation of postsynaptic ~l adrenoceptors, which causes a decline in prefrontal functioning. An inhibition of prefrontal cortex functioning under stressful conditions in turn may lead to more rapid habitual subcortical modes of responding, adding to survival (Arnsten, 1998). Indeed, stress can impair working memory and this effect can be attenuated by infusion of an czl adrenoceptor antagonist directly into the prefrontal cortex (Birnbaum et al., 1999), suggesting that it is the stress-induced release of noradrenaline at this site which favours rapid automatic processing over slower declarative processes. Moreover, changes in prefrontal dopaminergic activity have been demonstrated following exposure to stress. Activation of the prefrontal dopaminergic system has been shown to occur preferentially after exposure to mild stressors and has been suggested to be of relevance for experiencing anxiety states, possibly in an attempt to cope with the stressful situation (Deutch and Roth, 1990). Central to the behavioural defence theory by Gray and McNaughton (2000) is the septohippocampal
36 signals of signals of. novel innate fear punishment non-reware stimuli stimuli
signals of non- signals of punishment rewara
Fight-Flight-Freezing System ~
novel innatesafety stimuli stimuli
BehaviouralApproach System
Behavioural [ / InhibitionSysteT.~_~/" ~ ConflictDetector~ ' Anxiety
4, +
?
Avoidance
Attention
environmental 9 scanning external 9 scanning
Arousal
Approach
(risk assessment)
internal 9 scanning (memory)
Fig. 1. The behavioural inhibition system (modified after Gray and McNaughton, 2000): the behavioural inhibition system can be activated by simultaneous activation of the fight-flight-freezing system and the behavioural approach system, leading to conflict. The consequence will be an increase in arousal and attention (scanning of the environment, risk assessment, and retrieval of relevant memories) and concurrent inhibition of simple approach and simple avoidance behaviour.
system, which according to these authors forms an essential part of the behavioural defence system. In this context, the septohippocampal system should act to detect conflict between concurrently available goals and to resolve conflict between these goals by increasing bias towards affectively negative information. Under normal conditions, the system has been suggested to act as a comparator and to play a role in interrupting ongoing behaviour to allow information gathering, which in the animal is seen as risk assessment. As mentioned before, Takahashi (1996) suggested that glucocorticoids may influence the development of the freezing response by actions at the septohippocampal level. Given that, according to Gray and McNaughton, the septohippocampal system is central to behavioural inhibition, the ideas of Takahashi may also have implications for the manifestation of the behavioural inhibition system of Gray and McNaughton: it is tempting to speculate that glucocorticoid action during development may
affect the behavioural inhibition system over and above just freezing by influencing the development of the septohippocampal system, thereby modulating the individual's levels of stress-induced arousal and attention to threat. Both cholinergic and monoaminergic mechanisms have been suggested to play important roles in the modulation of stressor-induced increases of arousal and attention at hippocampal level: various stressors have been reported to increase hippocampal acetylcholine release (Dudar et al., 1979; Gilad et al., 1985; Imperato et al., 1991; Acquas et al., 1996; Day et al., 1998). The hippocampal cholinergic system has been suggested to be involved in the mechanisms underlying the arousal that is associated with fear and anxiety-provoking stimuli (Hess and Blozovski, 1987; Smythe et al., 1992) and one of the possible roles of the cholinergic system could be to ensure that the animal is appropriately responsive to its environment, being able to monitor and amend its behaviour
37 in an appropriate manner when exposed to a fearful or anxiety-provoking stimulus (Bhatnagar et al., 1997). Likewise, stress exposure increases noradrenergic and serotonergic activity at the hippocampal level (Abercombie et al., 1988; Vahabzadeh and Fillenz, 1994; Linthorst et al., 2000). These stress-induced increases in hippocampal monoamine release have been suggested to facilitate information processing by hippocampal circuits and hence bias towards affectively negative information (Gray and McNaughton, 2000). Another brain area closely involved in the processing of stress-related information and in the modulation of an individual's responses to stress is the amygdala. Within the framework proposed by Gray and McNaughton (2000), the projection from the septohippocampal system to the amygdala is viewed as being crucial for generating arousal and the autonomic components of anxiety, which is part of the stress response. For example, the amygdala plays an important role in the conditioning of emotional responses. Amygdaloid activation has been demonstrated using fMRI during fear stimuli and fear conditioning (LaBar et al., 1998; Morris et al., 1999). Information from the thalamus is relayed in the lateral nucleus of the amygdala to the central nucleus of the amygdala (CeA). There are both direct and indirect (via the basolateral amygdaloid nucleus) projections from the lateral nucleus of the amygdala to the CeA. In that function, the lateral nucleus has been suggested to represent the gateway into the amygdala (Le Doux, 1996). The basolateral amygdala in turn seems to control the activity of the central nucleus of the amygdala (CeA), which then affects hypothalamic and brainstem function (Davis, 1992a; Le Doux, 2000), leading to increases of adrenaline and plasma corticosterone (Roozendaal et al., 1991, 1997). The CeA also affects the periaqueductal grey and the pontine reticular nucleus, thereby modulating freezing and fear-potentiated startle, respectively. More specifically, it has been suggested that the CeA modulates stimulus-specific fear (related to specific tones, visual signals, etc.), while more complex information such as, for example, provided by exposure to a threatening environment for several minutes, may activate another structure, the bed
nucleus of the stria terminalis (Davis, 1998). The basolateral amygdaloid nucleus, on the other hand, has been viewed as being responsible for emotional classical conditioning and seems to be critical for the memory-enhancing effects of stress hormones such as adrenaline and glucocorticoids (McGaugh et al., 2000; Prickaerts and Steckler, 2004). Cardinal et al. (2002) suggested that the basolateral amygdala is of particular relevance for the re-evaluation of the motivational significance of a stimulus. These authors have argued that the basolateral amygdala will be activated under conditions when a subject has to assess the affective value of a stressor predicted by a conditioned stimulus. The basolateral nucleus has also been suggested to provide information about affect to the frontal and cingulate cortices, and to the striatum, and in return to receive projections providing information about current plans from cortical areas (Gray and McNaughton, 2000). This learned information of the affective value of a stimulus from the basolateral nucleus to the striatum seems to be relayed to the core of the nucleus accumbens, where its effects may be potentiated by dopamine (Cardinal et al., 2002). Conversely, unconditioned aversive stimuli have been reported to increase dopamine in the shell of the accumbens but not in the core (Deutch and Cameron, 1992), opening the possibility that dopamine plays a similar role in the accumbens shell for unconditioned stimuli as it does in the core for conditioned stimuli. More specifically, the accumbens shell has been suggested to be a high-level control system which allows the subject to switch between behavioural patterns based on primary motivational states, such as to override feeding and instead to engage in a more appropriate behaviour, such as fleeing, freezing, or fighting, in the face of a stressor such as a predator (Kelley, 1999). The CRF and the noradrenergic projections innervating the shell of the nucleus accumbens have been proposed to signal this stress-related information to the accumbens (Kelley, 1999). But how does this information from higher brain areas affect HPA axis activity? Earlier on, it has been mentioned that the hypothalamic PVN serves as relay point between the higher brain areas and the HPA
38 axis (the limbic input into the parvocellular PVN is extensively reviewed by Herman et al., 2004). It has been proposed that the indirect projection from the hippocampus to the PVN (from the subiculum via the BNST and GABAergic relay neurons in the hypothalamus) communicates learned information, including spatial information to the PVN and hence to the H P A axis, while the amygdala relays input to the P V N on perceived danger (Herman et al., 2004). Furthermore, and as pointed out by these authors, there are interconnections between amygdala and hippocampus, which may subserve complementary roles in assessing the relative safety of an environment, which can then be integrated into an appropriate activity state of the H P A axis.
Conclusion We have seen that the effects of aversive stimuli do not only depend on the nature of the stimt:lus, but also on the circumstances in which the stimulus is presented, the individual perception, the past experience, and cognitive abilities of the individuum. These processes are mediated by a network of brain regions comprising of the hippocampal formation and amygdala, prefrontal and cingulate areas, basal ganglia, hypothalamic areas, periaqueductal grey, and other subcortical structures. An impaired capacity to discriminate between relevant (threatening) and irrelevant (non-threatening) information, be it genetically or environmentally determined, will increase reactivity to stress. This cognitive model of stress helps to explain the susceptibility of individuals suffering from certain psychiatric disorders, which will be discussed in more detail in other parts of this book: impaired ability to discriminate threatening and non-threatening stimuli leads to increased reactivity to stress, which in turn can lead to more rapid symptom development of, for example, depression or psychosis. For example, it has been suggested that depressed patients have a diminished behavioural approach system in conjunction with a high activation of the behavioural inhibition system, a pattern which remains stable over time and clinical state (Meyer et al., 2001; Kasch et al., 2002). This in turn leads to lower responsiveness to reward and higher
responsiveness to punishment, i.e., stressful situations. Consequently, therapeutic approaches which improve stress coping, be it pharmacologically or by other means, should be beneficial in these conditions.
References Abercombie, E.D., Keller, R.W., Jr. and Zigmand, M.J. (1988) Characterization of hippocampal noradrenaline release as measured by microdialysis perfusion: pharmacological and behavioral studies. Neuroscience, 27: 897-904. Acquas, E., Wilson, C. and Fibiger, H.C. (1996) Conditioned and unconditioned stimuli increase frontal cortical and hippocampal acetylcholine release: effects of novelty, habituation, and fear. J. Neurosci., 16: 3089-3096. Adler, R. (1976) Conditioned adrenocortical steroid elevations in the rat. J. Comp. Physiol. Psychol., 90: 1156-1163. Annau, Z. and Kamin, L.J. (1961) The CER as a function of the intensity of the U.S.J. Comp. Physiol. Psychol., 54: 428-432. Arnsten, A.F.T. (1998) Catecholamine modulation of prefrontal cortical cognitive function. Trends Cognit. Sci., 2: 436-447. Arthur, A.Z. (1987) Stress as a state of anticipatory vigilance. Percept. Motor Skills, 64: 75-85. Badia, P., Harsh, J. and Abbott, B. (1979) Choosing between predictable and unpredictable shock conditions: data and theory. Psychol. Bull., 86:1107-1131. Berridge, C.W. (2005) The locus coeruleus-noradrenergic system as a modulator of arousal state and state-dependent behavioural processes. In: Steckler, T., Kalin, N. and Reul, J.M.H.M. (Eds.), Handbook of Stress and the Brain, Part 1, Elsevier, Amsterdam, pp. 437-464. Bhatnagar, S., Costall, B. and Smythe, J.W. (1997) Hippocampal cholinergic blockade enhances hypothalamicpituitary-adrenal responses to stress. Brain Res., 766: 244-248. Birnbaum, S., Gobeske, K.T., Auerbach, J., Taylor, J.R. and Arnsten, A.F. (1999) A role for norepinephrine in stressinduced cognitive deficits: alpha-1-adrenoceptor mediation in the prefrontal cortex. Biol. Psychiatry, 46: 1266-1274. Blanchard, D.C. and Blanchard, R.J. (1990) An ethoexperimental analysis of defense, fear and anxiety. In: McNaughton, N. and Andrews, G. (Eds.), Anxiety. Otago University Press, Dunedin, pp. 124-133. Bruijnzeel, A.W., Stam, R., Croiset, G. and Wiegant, V.M. (2001) Long-term sensitization of cardiovascular stress responses after a single stressful experience. Physiol. Behav., 73: 81-86. Brujnzeel, A.W., Stam, R., Compaan, J.C., Croiset, G., Akkermans, L.M.A., Olivier, B. and Wiegant, V.M. (1999) Long-term sensitization of Fos-responsivity in the rat central nervous system after a single stressful experience. Brain Res., 819: 15-22.
39 Cardinal, R.N., Parkinson, J.A., Hall, J. and Everitt, B.J. (2002) Emotion and motivation: the role of the amygdala, ventral striatum, and prefrontal cortex. Neurosci. Biobehav. Rev., 26: 321-352. Coe, C.L., Stanton, M.E. and Levine, S. (1983) Adrenal responses to reinforcement and extinction: role of expectancy versus instrumental responding. Behav. Neurosci., 97: 654-657. Coover, B.D., Sutton, B.R. and Heybach, J.P. (1977) Conditioning decreases plasma corticosterone levels in rats by pairing stimuli with daily feedings. J. Comp. Physiol. Psychol., 91: 716-726. Dallman, M.F. (1993) Stress update: adaptation of the hypothalamic-pituitary-adrenal axis to chronic stress. Trends Endocrinol. Metab., 4: 62069. Davis, M. (1992a) The role of the amygdala in conditioned fear. In: Aggleton, J.P. (Ed.), The Amygdala: Neurobiological Aspects of Emotion, Memory, and Mental Dysfunction. Wiley, New York, pp. 255-306. Davis, M. (1992b) The role of the amygdala in fear-potentiated startle: implications for animal models of anxiety. Trends Pharmacol. Sci., 13: 35-41. Davis, M. (1998) Are different parts of the extended amygdala involved in fear versus anxiety? Biol. Psychiatry, 44: 1239-1247. Davis, M., Falls, W.A., Champeau, S. and Kim, M. (1993) Fear-potentiated startle: a neural and pharmacological analysis. Behav. Brain Res., 58: 175-198. Day, J.C., Koehl, M., Deroche, V., Le Moal, M. and Maccari, S. (1998) Prenatal stress enhances stress- and corticotropin-releasing factor-induced stimulation of hippocampal acetylcholine release. J. Neurochem., 18: 1886-1892. Dayas, C.V., Buller, K.M., Crane, J.W. and Day, T.A. (2001) Stressor categorization: acute physical and psychological stressors elicit distinctive recruitment patterns in the amygdala and medullary noradrenergic cell groups. Eur. J. Neurosci., 14:1143-1152. De Boer, S.F., De Beun, R., Slangen, J.L. and Van der Gugten, J. (1990) Dynamics of plasma catecholamine and corticosterone concentrations during reinforced and extinguished operant behaviour in rats. Physiol. Behav., 47:691-698. De Kloet, E.R., Vreugdenhil, E., Oitzl, M.S. and Joels, M. (1998) Brain corticosteroid receptor balance in health and disease. Endocrine Rev., 66: 183-201. Deutch, A.Y. and Roth, R.H. (1990) The determinants of stress-induced activation of the prefrontal cortical dopamine system. Prog. Brain Res., 85: 367-403. Deutch, A.Y. and Cameron, D.S. (1992) Pharmacological characterization of dopamine systems in the nucleus accumbens core and shell. Neuroscience, 46: 49-56. Dudar, J.D., Whishaw, I.Q. and Szerb, J.C. (1979) The role of the septal nuclei in the release of acetylcholine from the
hippocampus of freely moving rats during sensory stimulation and running. Neuropharmacology, 18: 673-678. Easterbrook, J.A. (1959) The effect of emotion on cue utilization and the organization of behavior. Pychol. Rev., 66:183-201. Ekman, P., Levenson, R.W. and Friesen, W.V. (1983) Autonomic nervous system activity distinguishes emotions. Science, 221: 1208-1210. Epstein, S., Rosenthal, S. and Szpiler, J. (1978) The influence of attention upon arousal, habituation, and reactivity to a noxious stimulus. J. Res. Pers., 12: 30-40. Esteves, F., Parra, C., Dimberg, U. and Ohman, A. (1994) Nonconscious associative learning: Pavlovian conditioning of skin conductance responses to masked fear-relevant facial stimuli. Psychophysiology, 31: 375-385. Feenstra, M.G. (2000) Dopamine and noradrenaline release in the prefrontal cortex in relation to unconditioned and conditioned stress and reward. Prog. Brain Res., 126: 133-163. File, S.E. (1980) The use of social interaction as a method for detecting anxiolytic activity of chlordiazepoxide-like drugs. J. Neurosci. Meth., 2: 219-238. Freud, S. (1926) Hemmung, Symptom und Angst. Internationaler Pychoanalytischer Verlag, Leipzig. Gilad, G.M. and Shiller, I. (1989) Differences in open-field behavior and in learning tasks between two rat strains differing in their reactivity to stressors. Behav. Brain Res., 32: 89-93. Gilad, G.M., Mahon, B.D., Finkelstein, Y., Koffler, B. and Gilad, V.H. (1985) Stress induced activation of the hippocampal cholinergic system and the pituitary adrenocortical axis. Brain Res., 347: 404-408. Graeff, F.G. (1994) Neuroanatomy and neurotransmitter regulation of defensive behaviors and related emotions in mammals. Brazil. J. Med. Biol. Res., 27: 67-70. Gray, J.A. (1976) The behavioural inhibition system: a possible substrate for anxiety. In: Feldman, M.P. and Broadhurst, A.M. (Eds.), Theoretical and Experimental Bases of Behaviour Modification. Wiley, London, pp. 3-41. Gray, J.A. and McNaughton, N. (2000) The Neuropsychology of Anxiety, 2nd ed. Oxford University Press, Oxford. Hall, F.S., Huang, S., Fong, G.W., Sundstrom, J.M. and Pert, A. (2000) Differential basis of strain and rearing effects on open-field behavior in Fawn Hooded and Wistar rats. Physiol. Behav., 71: 525-532. Hanson, J.D., Larson, M.E. and Snowdon, C.T. (1976) The effects of control over high intensity noise on plasma cortisol levels in rhesus monkeys. Behav. Biol., 16: 333-340. Henkin, R.I. (1970) The effects of corticosteroids and ACTH on sensory systems. Prog. Brain Res., 32: 270-294. Herman, J.P. and Cullinan, W.E. (1997) Neurocircuitry of stress: central control of the hypothalamo-pituitary-adrenocortical axis. Trends Neurosci., 20: 78-84.
40 Herman, J.P., Prewitt, C.M. and Cullinan, W.E. (1996) Neuronal circuit regulation of the hypothalamo-pituitaryadrenocortical stress axis. Crit. Rev. Neurobiol., 10:371-394. Herman, J.P., Mueller, N.K., Figueiredo, H. and Cullinan, W.E. (2005) Neurocircuit regulation of the hypothalamo-pituitary-adrenocortical stress response - an overview. In: Steckler, T., Kalin, N. and Reul, J.M.H.M. (Eds.), Handbook of Stress and the Brain, Part 1, Elsevier, Amsterdam, pp. 405-418. Hess, C. and Blozovski, D. (1987) Hippocampal muscarinic cholinergic mediation of spontaneous alternation and fear in the developing rat. Behav. Brain Res., 24: 203-214. Imperato, A., Puglisi-Allegra, S., Casolini, P. and Angelucci, L. (1991) Changes in brain dopamine and acetylcholine release during and following stress are independent of pituitaryadrenocortical axis. Brain Res., 538:111-117. James, W. (1890) The Principles of Psychology. Holt, New York. Kasch, K.L., Rottenberg, J., Arnow, B.A. and Gotlib, I.H. (2002) Behavioral activation and inhibition systems and the severity and course of depression. J. Abnorm. Psychol., 111: 589-597. Kelley, A.E. (1999) Neural integrative activities of nucleus accumbens subregions in relation to learning and motivation. Psychobiology, 27: 198-213. Koene, P. and Vossen, J.M. (1991) Strain differences in rats with respect to speed of conflict resolution. Behav. Genet., 21: 21-33. Kollack-Walker, S., Day, H.E.W. and Akil, H. (2000) Central stress neurocircuits. In: Fink, G. (Ed. in chief), Encyclopedia of Stress, Vol. 1. Academic Press, San Diego, pp. 414-423. Koolhaas, J.M., Hermann, P.M., Kempermann, C., Bohus, g., Van den Hoofddakker, R.H. and Beersma, D.G.M. (1990) Single social defeat in male rats induces a gradual but longlasting behavioural change: a model of depression? Neurosci. Res. Comm., 7: 35-41. Korte, S.M. (2001) Corticosteroids in relation to fear, anxiety and psychopathology. Neurosci. Biobehav. Rev., 25: 117-142. Kovacs, K.J. and Makara, G.B. (1990) Partial deafferentiation of the hypothalamic paraventricular nucleus: effect on the stress or adrenalectomy-induced ACTH secretion. Neuroendocrinol. Lett., 12: 383-389. LaBar, K.S., Gatenby, J.C., Gore, J.C., LeDoux, J.E. and Phelps, E.A. (1998) Human amygdala activation during conditioned fear acquisition and extinction: a mixed trial fMRI study. Neuron, 20: 937-945. Lazarus, R.S. (1966) Psychological Stress and the Coping Process. McGraw-Hill, New York. Le Doux, J.E. (1996) The Emotional Brain. Simon & Schuster, New York. Le Doux, J.E. (2000) Emotion circuits in the brain. Ann. Rev. Neurosci., 23: 155-184.
Lemaire, V., Aurousseau, C., Le Moal, M. and Abrous, D.N. (1999) Behavioural trait of reactivity to novelty is related to hippocampal neurogenesis. Eur. J. Neurosci., 11: 4006-4014. Linthorst, A.C., Flachskamm, C., Barden, N., Holsboer, F. and Reul, J.M.H.M. (2000) Glucocorticoid receptor impairment alters CNS responses to a psychological stressor: an in vivo microdialysis study in transgenic mice. Eur. J. Neurosci., I2: 283-291. Liu, D., Diorio, J., Tannenbaum, B., Caldji, C., Francis, D., Freedman, A., Sharma, S., Pearson, D., Plotsky, P.M. and Meaney, M.J. (1997) Maternal care, hippocampal glucocorticoid receptors, and hypothalamic-pituitary-adrenal responses to stress. Science, 277: 1659-1662. Lockart, J.A. (1963) Choice of a warning signal or no warning signal in an unavoidable shock situation. J. Comp. Physiol. Psychol., 56: 526-530. Lynn, R. (1966) Attention, Arousal and the Orientation Reaction. Pergamon, Oxford. Mackintosh, N.J. (1983) Conditioning and Associative Learning. Clarenndon, Oxford. Maier, S.F. (1990) The role of fear in mediating the shuttle escape learning deficit produced by inescapable shock. JEP ABP, 16:137-150. Mandler, G. (1984) Mind and Body, the Psychology of Emotion and Stress. W.W. Norton & Company, New York. Mason, J.W. (1968) A review of psychoendocrine research on the pituitary-adrenal cortical system. Psychosom. Med., 30: 576-607. McGaugh, J.L., Ferry, B., Vazdarjanova, A. and Roozendaal, B. (2000) Amygdala: role in modulation of memory storage. In: Aggleton, J.P. (Ed.), The Amygdala: A Functional Analysis, 2nd ed. Oxford University Press, New York, pp. 391-423. Meyer, B., Johnson, S.L. and Winters, R. (2001) Responsiveness to threat and incentive in bipolar disorder: relations of the BIS/BAS scales with symptoms. J. Psychopathol. Behav. Assess., 23: 133-143. Mineka, S., Cook, M. and Miller, S. (1984) Fear conditioned with escapable and inescapable shock: the effects of a feedback stimulus. J. Exp. Psychol., 10: 307-323. Morris, J.S., Ohman, A. and Dolan, R.J. (1999) A subcortical pathway to the right amygdala mediating "unseen" fear. Proc. Natl. Acad. Sci. USA, 96: 1680-1685. Mowrer, O.H. (1960) Learning Theory and Behavior. Wiley, New York. Newport, D.J. and Nemeroff, C.B. (2002) Stress. In: (Ed. in chief), Encyclopedia of the Human Brain, Vol. 4. Elsevier, pp. 449-462. Ohman, A., Flykt, A. and Esteves, F. (2001) Emotion drives attention: detecting the snake in the grass. J. Exp. Psychol., 130: 466-478. Ottenweller, J.E. (2000) Animal models (nonprimate) for human stress. In: Fink, G. (Ed. in chief), Encyclopedia of Stress, Vol. 1. Academic Press, San Diego, pp. 200-205.
41 Overmier, J.B. and Seligman, M.E.P. (1967) Effects of inescapable shock upon subsequent escape and avoidance responding. J. Comp. Physiol. Psychol., 63: 28-33. Pacac, K., Palkovits, M., Kopin, I.J. and Goldstein, D.S. (1995) Stress-induced norepinephrine release in the hypothalamic paraventricular nucleus and pituitary-adrenocortical and sympathoadrenal activity: in vivo microdialysis studies. Front. Neuroendocrinol., 16: 89-150. Pare, W.P. (1964) The effects of chronic environmental stress and stomach ulceration, adrenal function and consummatory behavior in the rat. J. Psychol., 57: 143-151. Pennebaker, J.W. (1982) The Psychology of Physical Symptoms. Springer, New York. Phillips, K. (1989) Psychophysiological consequences of behavioural choice in aversive situations. In: Steptoe, A. and Appels, A. (Eds.), Stress, Personal Control and Health. John Wiley, Chichester, pp. 239-256. Piazza, P.V., Maccari, S., Deminiere, J.M., Le Moal, M., Mormede, P. and Simon, H. (1991) Corticosterone levels determine individual vulnerability to amphetamine selfadministration. Proc. Natl. Acad. Sci. USA, 88: 2088-2092. Plotsky, P.M. (1991) Pathways to the secretion of adrenocorticotropin: a view from the portal. J. Neuroendocrinol., 3: 1-9. Plotsky, P.M., Cunningham, E.T. and Widmaier, E.P. (1989) Catecholaminergic modulation of corticotropin-releasing factor and adrenocorticotropin secretion. Endocr. Rev., 10: 437-458. Plutchik, R. (1962) The Emotions: Facts, Theories and a New Model. Random House, New York. Pribram, K.H. and McGuinnes, D. (1975) Arousal, activation and effort in the control of attention. Psychol. Rev., 182: 116-145. Prickaerts, J. and Steckler, T. (2005) Effects of glucocorticoids on emotion and cognitive processes in animals. In: Steckler, T., Kalin, N. and Reul, J.M.H.M. (Eds.), Handbook of Stress and the Brain, Part 1, Elsevier, Amsterdam, pp. 359-386. Przewlocka, B., Sumova, A. and Lason, W. (1990) The influence of conditioned fear-induced stress on the opioid systems in the rat. Pharmacol. Biochem. Behav., 37: 661-666. Reul, J.M.H.M. and De Kloet, E.R. (1985) Two receptor systems for corticosterone in the rat brain: microdistribution and differential occupation. Endocrinology, 117: 2505-2512. Rohrmann, S., Henning, J. and Netter, P. (1999) Changing psychobiological stress reactions by manipulating cognitive processes. Int. J. Psychophysiol., 33: 149-161. Rolls, E.T. (1999) The Brain and Emotion. Oxford University Press, Oxford. Roozendaal, B., Koolhaas, J.M. and Bohus, B. (1991) Attenuated cardiovascular, neuroendocrine, and behavioral responses after a single footshock in central amygdaloid lesioned rats. Phyisol. Behav., 50: 777-781.
Roozendaal, B., Koolhaas, J.M. and Bohus, B. (1997) The role of the central amygdala in stress and adaptation. Acta Physiol. Scand. Suppl., 640: 51-54. Rosellini, R.A. (1978) Inescapable shock interferes with the acquisition of an appetitive operant. Anita. Learn. Behav., 6: 155-159. Sandi, C., Borrell, J. and Guaza, C. (1992) Behavioral, neuroendocrine, and immunological outcomes of escapable and inescapable shocks. Physiol. Behav., 51: 651-656. Sawchenko, P.E. and Swanson, L.W. (1983) The organization of forebrain afferents to the paraventricular and supraoptic nuclei of the rat. J. Comp. Neurol., 218: 121-144. Sawchenko, P.E., Brown, E.R., Chan, R.K.W., Ericsson, A., Li, H.Y., Ronald, B.L. and Kovacs, K.J. (1996) The paraventricular nucleus of hypothalamus and the functional neuroanatomy of visceromotor responses to stress. Prog. Brain Res., 107: 201-222. Schachter, S. and Singer, J.E. (1962) Cognitive, social and physiological determinants of emotional state. Psychol. Rev., 69: 379-399. Schmidt, E.D., Binnekade, R., Janszen, A.W.J.W. and Tilders, F.J.H. (1996) Short stressor induced long-lasting increases in vasopressin stores in hypothalamic corticotropinreleasing hormone (CRH) neurons in adult rats. J. Neuroendocrinol., 8: 703-712. Seligman, M. (1968) Chronic fear produced by unpredictable electric shock. J. Comp. Physiol. Psychol., 66:402-411. Seligman, M. (1975) Helplessness. W.H. Freeman, San Francisco. Shots, T.J. (2001) Acute stress rapidly and persistently enhances memory formation in the male rat. Neurobiol. Learn. Mere., 75: 10-29. Smythe, J.W., Colom, L.V. and Bland, B.H. (1992) The extrinsic modulation of hippocampal theta depends on the coactivation of cholinergic and GABA-ergic medial septal inputs. Neurosci. Biobehav. Rev., 16: 289-308. Takahashi, L.K. (1996) Glucocorticoids and the hippocampus. Developmental interactions facilitating the expression of behavioural inhibition. Mol. Neurobiol., 13: 213-226. Toates, F. (1995) Stress: Conceptual and Biological Aspects. Wiley, Chichester. Tsuda, A., Tanaka, M., Ida, Y., Tsujimaru, S., Ushijima, I. and Nagasaki, N. (1986) Effects of preshock experience on enhancement of rat brain noradrenaline turnover induced by psychological stress. Pharmacol. Biochem. Behav., 24: 115-119. Vahabzadeh, A. and Fillens, M. (1994) Comparison of stressinduced changes in noradrenergic and serotonergic neurons in the rat hippocampus using microdialysis. Eur. J. Neurosci., 6, 1205-1212. Van Dijken, H.H., Van der Heyden, J.A.M., Mos, J. and Tilders, F.J.H. (1992) Inescapable footshocks induce progressive and long-lasting behavioural changes in male rats. Physiol. Behav., 51: 787-794.
42 Van Dijken, H.H., De Groeij, D.C.E., Sutano, W., Mos, J., De Kloet, E.R. and Tilders, F.J.H. (1993) Short inescapable stress produces long-lasting changes in the brain-pituitaryadrenal axis of adult male rats. Neuroendocrinology, 58: 57-64. Walker, S. (1987) Animal Learning: An Introduction. Routledge and Kegan Paul, London. Watson, J.B. and Rayner, R. (1920) Conditioned emotional reactions. J. Exp. Psychol., 3: 1-14. Weinstock, M. (1997) Does prenatal stress impair coping and regulation of hypothalamic-pituitary-adrenal axis? Neurosci. Biobehav. Rev., 21: 1-10. Weiss, J.M. (1968) Effects of coping responses on stress. J. Comp. Physiol. Psychol., 65: 251-260. Weiss, J.M. (1970) Somatic effects of predictable and unpredictable shock. Psychosom. Med., 32: 397-408. Weiss, J.M., Baley, W.H., Pohorecky, L.A., Koreniowski, D. and Grillone, G. (1980) Stress-induced depression of motor activity correlates with regional changes in brain norepinephrine but not in dopamine. Neurochem. Res., 5: 9-22.
Wolkowitz, O.M., Reus, V.I., Weingartner, H., Thompson, K., Breier, A., Doran, A., Rubinow, D. and Pickar, D. (1990) Cognitive effects of corticosteroids. Am. J. Psychiatry, 147: 1297-1303. Yamamotova, A., Starec, M., Holecek, V., Racek, J., Trefil, L., Raskova, H. and Rokyta, R. (2000) Anticipation of acute stress in isoprenaline-sensitive and -resistant rats: strain and gender differences. Pharmacol. Toxicol., 87: 161-168. Yerkes, R.M. and Dodson, J.D. (1908) The relation of strength of stimulus to rapidity of habit-information. J. Comp. Neurol. Psychol., 18: 459-482. Young, E.A. and Akil, H. (1985) CRF stimulation of ACTH/ [3-endorphine release: effect of acute and chronic stress. Endocrinology, 117: 23-30. Young, E.A. and Liberzon, I. (2002) Stress and anxiety disorders. In: Pfaff, D.W., Arnold, A.P., Etgen, A.M., Fahrbach, S.E. and Rubin, R.T. (Eds.), Hormones, Brain and Behavior, Vol. 5. Academic Press, Amsterdam, pp. 443-465.
T. Steckler, N.H. Kalin and J.M.H.M. Reul (Eds.) Handbook of Stress and the Brain, Vol. 15
ISBN 0-444-51173-3 Copyright 2005 Elsevier B.V. All rights reserved
CHAPTER 1.3
An introduction to the H P A axis Allison J. Fulford l'* and Michael S. H a r b u z 2 lDepartment o[ Anatomy, University off Bristol, Southwell Street, Bristol, BS2 8E J, UK 2University Research Centre for Neuroendocrinoiogy, Bristol Royal Infirmary, Marlborough Street, Bristol, BS2 8HW, UK
Abstract: Integrity of the hypothalamo-pituitary-adrenal (HPA) axis is essential to survival of vertebrate species. This neuroendocrine axis functions to coordinate neural, endocrine and immune responses to diverse stressful stimuli that threaten homeostasis. The final products of activation of the HPA axis are the glucocorticoids that exert widespread effects on body functions, including cellular metabolism and immune function. Inappropriate secretion of endogenous glucocorticoids is potentially damaging and may predispose to disease. Homeostatic regulation of the HPA axis is complex and involves coordination of multiple systems of the body, in part mediated by the bi-directional communication network between the brain, endocrine and immune systems. Health and integrity of the individual relies on the appropriate integration of stress signals, including pro-inflammatory messages, generated at central and peripheral sites. Functional balance between pro- and anti-inflammatory mediators is fundamental to the appropriate control of the HPA axis and the prevention of dysregulation in its activity, a characteristic of numerous stress-related disorders including chronic inflammatory disease.
that the adrenocorticotrophic hormone (ACTH), released from the pituitary, was associated with increased production of glucocorticoids from the adrenal glands that exerted profound and widespread effects on metabolism and lymphoid organ activity. These seminal research findings laid the foundations for our understanding of how neuroendocrine systems respond to stress. In addition to activation of the pituitary-adrenalcortical system, endocrine responses to stress include activation of the sympathoadreno-medullary system. Sympathetic nerves innervate the adrenal glands directly. In response to stress sympathetic activation enhances secretion of the adrenal catecholamines, principally the hormone adrenaline (epinephrine), which is important in the regulation of the autonomic response to stress. Thus the stress response is a complex phenomenon encompassing autonomic, physical and behavioural changes in addition to neuroendocrine changes. For appropriate adaptation to an acute stress challenge, a coordinated response involving widespread systems of the body is required. Changes are necessary to
Introduction
The body's ability to adapt to external and internal factors that challenge the self-regulation of biological systems, or homeostasis, is essential to survival. An inappropriate response to any factor that impinges on homeostasis may result in a stress response. Stress is a term, originally defined by Hans Selye, to describe the pathophysiological state associated with specific physiological changes that could be induced by diverse physical and psychological stimuli (Selye, 1936). If exposure to a stressful stimulus persists or is intensified, the consequences for the animal may be severe, leading to disease or even death. The research pursued by Selye in the 1940s lead to further characterisation of the 'general adaptation syndrome' and the proposal that the pituitary gland was critical for the control of endocrine secretions, including those of the adrenal glands (Selye, 1946). He proposed
*Corresponding author. Tel.: + 44 117 928 8692; Fax: +44 117 925 4794; E-mail: A.J.Fulford(abris.ac.uk 43
44 promote arousal and attention to salient stimuli. Non-essential vegetative behaviours are inhibited, such as feeding and sexual behaviour, so that energy can be conserved for the 'fight-flight' response. The latter term, coined by Cannon (1939), describes those non-specific rapid autonomic and physiological changes necessary for mounting an acute stress response, including an increase in heart rate, blood pressure, respiration rate and liver glycogenolysis. Individuals vary in their response to stress and several factors contribute to this inherent variability. Genetic and environmental factors exert significant influence over human stress responsiveness. Human studies suggest that the genetic variables contribute strongly to an individual's ability to respond to a stressful stimulus. However, it is now widely appreciated that environmental influences, especially during development, exert significant effects on the neural pathways controlling emotional responses and behaviour (see Sanchez et al., 2001; Lapiz et al., 2003). The contribution of both genes and environment shape the neural mechanisms subserving stress responses and in doing so, govern the vulnerability of an individual to stress.
The hypothalamo-pituitary axis The endocrine system encompasses the pituitary gland, the peripheral endocrine organs and the hormones the two produce. The pituitary gland, or hypophysis, is responsible for the production of a range of hormones, which exert strong regulatory control over a wide range of bodily functions, including behaviour, growth and development, metabolism, salt and water balance, reproduction and immunity. Harris (1948) first proposed the functional link between the brain and the adenohypophysis (anterior pituitary). He suggested that the hypothalamus was responsible for the release of factors into the hypophysial blood that could directly influence the control of adenohypophysial hormone secretion. Indeed, the pituitary gland is subject to afferent control, via the actions of specific hypothalamic peptide 'releasing' or 'inhibiting factors' that act on the discrete populations of pituitary cells to regulate synthesis and release of pituitary hormones.
Thus the hypothalamus represents the neural control centre whereby the brain can coordinate endocrine activity. Stress influences the neuroendocrine regulation of a number of pituitary hormones including A CTH, prolactin, growth hormone, luteinising hormone, thyrotrophin, vasopressin and oxytocin. The neuroendocrine regulation of stress is a major focus of research interest, since dysregulated hormone activity may contribute to major life illnesses including cancer, metabolic, cardiovascular, autoimmune and psychiatric disorders. This chapter aims to provide an overview of the hypothalamo-pituitary-adrenal (HPA)-axis and its regulation. In addition, we will describe the significance of the changes in HPA-axis regulation associated with acute and chronic stress and the importance of the bi-directional communication network between the HPA axis and the immune system.
The hypothalamo-pituitary-adrenal axis In vertebrates, appropriate functioning of the HPA axis is absolutely vital for species survival. Upon release into the hypophysial blood supply, the hypothalamic-releasing factors, corticotrophin-releasing factor (CRF) and the nonapeptide, arginine vasopressin (A VP), are transported to the adenohypophysis where they activate pituitary corticotrophs to synthesise and release ACTH into the general circulation (see Fig. 1). A corticotrophin-releasing factor, which was able to stimulate secretion of ACTH, was first identified in the 1950s but was not characterised until many years later as the 41 amino acid residue CRF (Vale et al., 1981). In the blood ACTH passes to the adrenal glands where it binds to receptors on cells of the zona fasciculata of the adrenal cortex to promote the conversion of cholesterol esters into free cholesterol and stimulate the steroidogenic pathway. The rapid enzymatic conversion of the precursor cholesterol into steroid intermediates ultimately results in the formation of the end product of the steroid pathway, the glucocorticoids (cortisol in humans and most mammals, corticosterone in rodents) (see James and Few, 1985). Only small amounts of these hormones are stored in the adrenal gland, therefore HPA activation results in rapid secretion of nascent glucocorticoids
45
PVN
Stress 1~ ACTH secretagogue release
PITUITARY anterior
Anterior pituitary POMC W & ACTH secretion
lobe posterior lobe ACTH ADRENAL GLAND
ACTH 1~ synthesis & release of corticosteroJds from adrenal cortex medulla (seoretes adrenaline)
Fig. 1. Diagramatic representation of the hypothalamo-pituitary-adrenal axis in the rat. Following stimulation by a range of stressful stimuli, CRF- and AVP-containing parvocellular neurones of the medial PVN release their contents into the hypophysial portal blood. Once transported to the adenohypophysis, CRF and AVP increase synthesis of the ACTH precursor, POMC, which is cleaved into bioactive ACTH at the pituitary corticotrophs. ACTH is released from the pituitary and circulates to the adrenal glands, where it promotes synthesis and release of the glucocorticoids from the adrenal cortex. M, magnocellular division; Pm, medial parvocellular division; V, 3rd ventricle.
into the systemic circulation. In a normal human, cortisol secretion rates (8-25mg/day) and plasma concentration (40-180ng/ml) are maintained within close limits, although the plasma concentration will vary depending on the time of day, and in women, on the stage of the menstrual cycle. In conditions associated with chronic ACTH secretion, cortisol release may increase several fold resulting in secretion rates of up to 200-250mg/day. In the rat, corticosterone is the principal glucocorticoid product and a small amount of this steroid is also secreted in humans (1-4mg/day). Circulating ACTH is the major factor regulating glucocorticoid release; however, additional hormones from the adrenal medulla are involved but to a far lesser extent (EhrhartBornstein et al., 2000). The role of the effector glucocorticoids is to promote homeostatic adaptation to stress and this is achieved through catabolic actions that mobilise
energy resources necessary for appropriate adaptive responses. The secretion of glucocorticoids during adversity promotes survival and the integrity of the HPA axis is critical, since homeostatic dysregulation may culminate in immunosuppression, neuroendocrine/autonomic dysfunction and tissue atrophy (McEwen and Stellar, 1993).
The parvocellular paraventricular nucleus is the apex of the H P A axis The peptides, CRF and AVP are synthesised in the tuberoinfundibular parvocellular cells of the paravenlricular nucleus (PVN) that evoke release of ACTH via their synergistic actions on pituitary corticotrophs. The axon terminals of the parvocellular neurons terminate in the external zone of the median
46 eminence adjacent to the capillaries of the hypophysial portal blood supply where they secrete their contents into the portal blood. Parvocellular AVP, in contrast to magnocellular AVP, is involved with regulation of pituitary ACTH release and does not contribute to osmotic balance regulation. CRF is the principal ACTH secretagogue, whereas AVP colocalises with CRF in approximately 50% of the CRF-containing neurones of resting animals and humans (Whitnall, 1993). The two peptides act synergistically on ACTH secretion in vitro (Gillies et al., 1982) and in vivo (Rivier and Vale, 1983); however, AVP alone has little ACTH secretagogue activity. In addition to evoking release of ACTH, CRF induces transcription of proopiomelanocortin (POMC) mRNA, the ACTH precursor protein (Lightman and Young, 1988). CRF is thought to be the only hypothalamic-releasing factor that can induce POMC gene expression. Thus, stressinduced HPA axis activation is highly reliant on neuroendocrine CRF. A population of parvocellular CRF-containing neurones project to extrahypothalamic sites including limbic nuclei and the brainstem (Sawchenko, 1987a). Therefore, in addition to coordinating the pituitary-adrenal system, CRF is directly involved in the orchestration of robust autonomic and behavioural responses to stress. During activation of the HPA axis, the synthesis and secretion of both secretagogues is increased leading to a direct increase in ACTH and glucocorticoid secretion. Thus, expression of CRF mRNA and AVP mRNA in the PVN is increased and POMC mRNA expression is increased in the adenohypophysis (Antoni, 1986; Harbuz and Lightman, 1992). The importance of dual peptide control of ACTH release by the pituitary corticotroph is not fully understood. Although CRF is the principal and most potent ACTH secretagogue, AVP appears to be involved in the regulation of stress-induced ACTH release (Scott and Dinan, 1998). Evidence suggests that during chronic stress, the CRF:AVP ratio may increase, possibly due to differential sensitivity of the secretagogues to negative-feedback regulation (Scott and Dinan, 1998). In addition to AVP, various neuropeptides are colocalised within the parvocellular CRF neurones including enkephalin, neurotensin, cholecystokinin, vasoactive intestinal peptide and galanin (Palkovits, 1988). In some cases
CRF-containing neurones express the inhibitory amino acid neurotransmitter, ~,-aminobutyric acid (GABA), instead of AVP (Meister et al., 1988). The coexistence of these peptides or transmitters with CRF provides a mechanism for subtle regulation of ACTH release.
Pulsatility in the H P A axis Across a variety of species, glucocorticoid secretion varies markedly throughout the day in a pulsatile fashion and is subject to circadian regulation (Kaneko et al., 1981). ACTH is also secreted in a pulsatile manner. Thus, circulating levels of glucocorticoids closely mirror the pulsatility exhibited by the ACTH release. Pulsatile control of HPA-axis hormone secretion facilitates the exquisitely sensitive and dynamic relationship between brain, adenohypophysis and adrenal glands. Peaks of glucocorticoid secretion are typically seen 15-30 min after an ACTH pulse in normal human subjects. A major pulse of ACTH occurs in the early hours of the morning. Following this major pulse, release of ACTH and glucocorticoids is stimulated by further pulses throughout the day, approximately once per hour. Precise information regarding the temporal profile of the peaks is subject to the resolution of sampling methodologies employed (Gudmundsson and Carnes, 1997). Following each brief pulse, rising glucocorticoid levels stimulate negative-feedback loops to inhibit further ACTH release. However, circulating glucocorticoid levels gradually decline to the setpoint level so that activation of the HPA axis is stimulated resulting in an additional pulse. This profile of episodic hormone release allows precise control over the HPA axis under normal conditions. Pulsatile release also ensures that receptor downregulation is prevented, as would most likely happen in the face of continuous exposure to an endogenous agonist. It is important to point out that ACTH does not always stimulate glucocorticoid secretion, although concordance is approximately 80% (see Gudmundsson and Carnes, 1997). The origins of pulsatility in the HPA axis are largely unknown; however, it has been suggested that the pulsatile ACTH release may originate at the level of the pituitary corticotroph, rather than at the level of
47 hypothalamic CRF and AVP release (Gambacciani et al., 1987). Concerted action of the corticotroph population leading to a pulsatile burst would presumably require coordinated signal transfer by efficient autocrine, paracrine or juxtacrine actions. The PVN subparaventricular zone receives dense afferent input consisting of vasopressin-containing neurones originating in the suprachiasmatic nucleus (SCN) (Buijs et al., 1998). The dorsal and lateral compartments of the parvocellular PVN also receive direct inputs from the SCN. The SCN is involved in the control of circadian rhythms of the body; however, it may also serve a role in the regulation of stress responses. The circadian regulation of the HPA axis is subserved by the SCN innervation of the PVN region (Kalsbeek et al., 1996). Studies in animals have demonstrated how activation of the SCN can stimulate evening secretion of ACTH (Cascio et al., 1987). There is some evidence for gender differences in the pulse pattern of ACTH secretion; however, there are inconsistencies in the human data, with reports of an increased number of pulses, but similar pulse amplitude, in males than females (Horrocks et al., 1990). In another study, males also differed in the pattern of their ACTH pulsatility, however, in this case the amplitude of the pulses was greater with the pulse frequency unchanged (Roelfsema et al., 1993). Abnormal pulsatility of the HPA axis or subtle alterations in the frequency or magnitude of the ACTH signal could have significant consequences for feedback regulation of the HPA axis. Such a mechanism may contribute to the apparent disturbances in neuroendocrine parameters characteristic of certain psychiatric conditions, such as major depression. Chronological decline in HPA-axis regulation has also been suggested to contribute to age-related illnesses; however, there is little evidence in direct support of this.
Negative-feedback regulation of the HPA axis The HPA axis functions as a closed-loop system involving tight negative-feedback control mediated by the glucocorticoids exerting multiple regulatory actions. Autoregulation of the HPA axis is essential for ensuring that the stress response is terminated,
preventing excessive activation in order for restoration of internal homeostasis. Regulatory feedback occurs at several sites and involves both rapid and delayed feedback in humans and rats (Keller-Wood and Dallman, 1984; Krishnan et al., 1991; Young and Vasquez, 1996). Rapid feedback occurs immediately following a rise in circulating glucocorticoids and lasts from 5 to 15 min, whereas delayed feedback emerges 1-2 h later, can persist for up to 4 h and is dependent on the glucocorticoid level. In the case of prolonged activation of the HPA axis, delayed feedback may continue for up to 24 h. This temporal profile suggests that delayed feedback relies on genomic actions of glucocorticoid receptors, whereas rapid feedback is presumably a consequence of nongenomic actions of glucocorticoids (see De Kloet et al., 1998). Rapid feedback is exerted primarily via an inhibitory action of glucocorticoids on the synthesis and release of ACTH at the hypothalamic level, by decreasing mRNA expression for CRF and AVP. Delayed feedback is also manifested at the level of the adenohypophysis where glucocorticoids decrease mRNA expression level of the ACTH precursor protein, pro-opiomelanocortin (POMC) (see Harbuz and Lightman, 1992). In addition, glucocorticoids can act centrally, at the hypothalamus and higher centres, principally the hippocampus, to exert delayed negative-feedback inhibition and thereby prevent continued activation of the HPA axis. The actions of the corticosteroids are mediated primarily through specific nuclear receptors of which there are two subtypes, the mineralocorticoid receptor (MR) and glucocorticoid receptor (GR). The steroid receptors are located intracellularly in the cytoplasm and bind steroids that can freely diffuse across the plasma membrane. Once bound, the receptor-ligand complex translocates to the nucleus and interacts with palindromic hormone response elements on the DNA molecule. Thus, activated steroid receptors function as transcription factors and influence transcription of target genes, ultimately leading to changes in protein synthesis. The MR subtype has high and equal affinity for aldosterone, corticosterone, cortisol and deoxycorticosterone, but lower affinity for the synthetic glucocorticoid, dexamethasone. The GR subtype can be distinguished by the following affinity profile:
48 dexamethasone > cortisol > corticosterone > deoxycorticosterone > aldosterone. Although, GR has lower affinity for the glucocorticoids than MR, its lack of affinity for aldosterone means that this subtype is effectively glucocorticoid selective. The two receptors are found densely expressed in the central nervous system, however, the distribution of both is generally quite distinct with overlap in a few areas. The distribution of MR is more restricted, being found in the hippocampus and sensory and motor nuclei outside the hypothalamus (Arriza et al., 1988; Reul et al., 2000). GRs, however, are more widely localised in the hypothalamic PVN, the brainstem catecholaminergic cell groups, amygdala and hippocampus, in addition to the pituitary gland (Fuxe et al., 1985). Research suggests that MR may be more important in regulating the basal expression of the ACTH secretagogues, CRF and AVP, at the nadir of diurnal ACTH secretion, and in the regulation of the peak ACTH release (Dallman et al., 1989). GR may be more critical for the termination of the HPA-axis response to stress. The hippocampus is an important component of the negative-feedback regulation of the neuroendocrine stress response. Both subtypes of corticosteroid receptor are expressed by hippocampal neurones. Lesions interrupting signals from the hippocampus to the PVN are associated with increased basal levels of circulating glucocorticoids and enhanced responsiveness to stress, highlighting the importance of this structure in the feedback regulation of the HPA axis (Herman et al., 1992). The importance of the adrenal glands to normal bodily function can be readily demonstrated by studying the effect of their removal. Excision of the adrenal glands is associated with enlargement of the thymus and impairments in the stress response. Adrenalectomised rats have excessive secretion of ACTH and enhanced expression of the POMC precursor gene in the adenohypophysis (Jingami et al., 1985; Marti et al., 1999). Adrenalectomy also increases immunoreactivity and mRNA expression for the ACTH secretagogues in the parvocellular neurones of the PVN (Wolfson et al., 1985; Sawchenko, 1987b). Interestingly, adrenalectomy appears to selectively affect those CRF/AVP-containing parvocellular neurones involved in the regulation of ACTH secretion. Replacement with exogenous steroids in drinking water, food or implanted pellets
normalises the ACTH secretion in rats (Akana et al., 1988), confirming the importance of glucocorticoids in the regulation of the HPA axis. However, adrenalectomised rats do not have maximal increases in basal plasma ACTH levels as evidenced by the fact that novel acute stress can still stimulate further ACTH release (Akana et al., 1988). This implicates an additional mechanism, aside from glucocorticoid feedback, in restraining HPA-axis activation and providing a tonic inhibitory input to the axis.
Afferent regulation of the HPA axis The parvocellular neurones are subject to close regulation from diverse afferent inputs. These neurones process excitatory and inhibitory inputs and function to coordinate secretion of CRF and AVP, thereby controlling the extent of ACTH stimulation of glucocorticoid secretion. Many brain regions are involved in the integration of responses to fear or stressful stimuli, including hypothalamic, septohippocampal and amygdaloid nuclei, cingulate and prefrontal cortex, brainstem catecholamine cell groups (A2/C2 cell bodies in the nucleus of the solitary tract (NTS); A1/C1 cell bodies in the ventrolateral medulla; A6 cell bodies in the locus coeruleus) and the dorsal raphe nucleus (see Pacak and Palkovits, 2001). Indeed, extensive neuroanatomical studies analysing the connections of the parvocellular PVN identify links with neural pathways concerned with homeostatic adaptation, cognition and affective behaviour (Silverman et al., 1981; Sawchenko and Swanson, 1983; Cunningham and Sawchenko, 1988). Parvocellular PVN afferents implicated in preserving homeostasis arise in the brainstem, hypothalamus and basal forebrain (see Fig. 2). Noradrenergic inputs, arising from the lower brainstem, innervate the medial parvocellular PVN and convey stress-related inputs to the parvocellular PVN for the coordination of endocrine, autonomic and behavioural output responses. These include strong visceral afferent inputs transmitting information directly from the NTS or via relays in the ventrolateral medulla that convey sensory stimuli (Sawchenko and Swanson, 1981; Cunningham and Sawchenko, 1988; Sawchenko et al., 2000).
49
(
// aN.
'\ \
~
*
9
"'~
..
.ippoc~p.~i
Ventral
~
Lateral
."
~-'-"
.....
subi 9
septum /
! I
! ( i
9-
:
~ \
a.,~,~
~ ,-"Y" ~! / ..... ~ \ / ADRENAL\
IMMUNOREGULATION
c.q
/
"--'-~.J~ t-
~"~ ..... ' ..........~ - ~ _
GLUCOCORTICOIDS ,. ~ L
i."
:
:;-~_ ~--" - ~
~
t, ~\
,
t__
~i
"
GLUCOCORTICOIDS ~'
~t"-
1
I
I
m ediators
Fig. 2. Schematic representation of the neuroendocrine-immune axis. Activation of the HPA axis results in secretion of immunoregulatory glucocorticoids. Immune cells respond to stimulation by the release of inflammatory mediators that modulate local inflammation and can communicate to the brain and pituitary. Regulation of HPA-axis activity involves integration at the hypothalamic PVN of diverse afferent inputs from other hypothalamic areas, limbic nuclei and brainstem nuclei. Solid lines represent stimulatory inputs; dashed lines represent inhibitory feedback loops.
Noradrenaline is vital for the response to some stressors and hypothalamo-pituitary-adrenal axis activation. It stimulates CRF-containing neurones in vitro and in vivo, providing supporting evidence for direct connections with the hypophysiotropic CRF-containing neurones (Sawchenko and Swanson, 1982; see Pacak and Palkovits, 2001). More recent research suggests that the majority of ascending noradrenergic afferents may relay in the hypothalamus with local excitatory glutamatergic neurones that innervate the PVN (Daftary et al., 2000). There are also reciprocal connections from the CRF neurones projecting to the brainstem locus coeruleus noradrenergic cell groups (Valentino et al., 1983; see Koob, 1999). In addition to these projection neurones, short-loop feedback mechanisms allow for close autoregulation of both CRF and noradrenergic neurones of the hypothalamus (Calogero et al., 1988). The importance of intact catecholaminergic inputs to the PVN for stress responses have been confirmed by selective lesioning studies of the PVN. Injection of the catecholaminergic neuronal toxin, 6-hydroxy-
dopamine, completely prevents the glucocorticoid response to conditioned fear or immobilisation stress (see Van de Kar and Blair, 1999). In contrast, ascending noradrenergic neurones do not appear to regulate AVP expression, suggesting that the two ACTH secretagogues are subject to differential regulation. The occurrence of dual mechanisms for maintenance of ACTH release from the adenohypophysis and their differential regulation highlights the complexity of the HPA axis and demonstrates the essence of its physiological importance under different stress conditions, as will be discussed later. The immediate vicinity of the PVN, but not the PVN itself, receives input from brainstem cholinergic (Ohmori et al., 1995) and serotonergic (Liposits et al., 1987) nuclei that are concerned with the control of arousal and wakefulness. Both ascending pathways are thought to mediate excitatory effects over HPAaxis drive. The PVN also receives afferents via the fimbria fornix and angular bundle that originate in the ventral hippocampus. These are important for the tonic regulation of the HPA axis and termination of
50 the stress response is thought to involve substance P arising in the arcuate nucleus (Nussdorfer and Malendowicz, 1998; Jessop et al., 2000b). Dense projections from the ventral subiculum directly innervate the subparaventricular zone, proximal to the PVN, and in addition transmit signals via the bed nucleus of the stria terminalis (BNST) and preoptic area to the medial PVN (Cullinan et al., 1993). These inputs from the ventral subiculum provide a strong influence over the PVN. The inputs via the medial preoptic area or BNST appear to exert both excitatory and inhibitory regulation over HPA-axis drive (Herman et al., 1994). These functional differences are probably due to discrete topographical organisation of inputs to the PVN from subdivisions of the nuclei. Neuronal afferents from the prefrontal cortex, lateral septum and paraventricular thalamus also terminate in the peri-PVN zone, providing an additional rich source of projections from limbic structures (see Herman et al., 2002). Furthermore, the amygdala can exert a stimulatory effect on the HPA axis, via some direct input to the peri-PVN zone from the medial nucleus and indirect input via the BNST and preoptic nucleus (Canteras et al., 1995). The paraventricular thalamus has links with the suprachiasmatic nucleus (SCN), which also innervates the peri-PVN zone, and these connections are thought to regulate the circadian rhythm of the HPA axis (Watts et al., 1987). Local interneurones in the vicinity of the PVN appear to directly modulate outgoing signals from the PVN, permitting fine integration of HPA-axis stimuli. The majority of these local circuit neurones express glutamic acid decarboxylase, the rate-limiting enzyme in the synthesis of the major inhibitory amino acid neurotransmitter, GABA (see Herman et al., 2002). Local excitatory inputs derived from glutamatergic neurones in the vicinity of the PVN may also contribute to its regulation, providing a balance between proximal inhibitory and excitatory influences that gate HPA-axis drive. In summary, in addition to the major inputs direct to the medial PVN, e.g. from brainstem noradrenergic afferents, the PVN is subject to complex regulation by a rich supply of limbic inputs which largely relay with local circuit neurones just proximal to the PVN. This additional level of regulation allows for concerted
integration of HPA-axis activity so that diverse stimuli are prioritised and can be responded to with appropriate intensity and urgency. Thus the complexity of HPA-axis input regulation allows for hierarchical integration of various stimuli, whether cognitive or physiological stimuli.
Immune-HPA axis interactions
The interaction between the immune system and HPA axis has been appreciated for many years. The most widely recognised effect of the communication between these two systems is demonstrated by the actions of the glucocorticoids, the end products of activation of the HPA axis. Glucocorticoids have potent anti-inflammatory effects with extensive effects on the immune system, including actions on every population of immune cell. Glucocorticoid effects are largely inhibitory, and encompass effects of cell growth, proliferation and differentiation, leukocyte trafficking, cytokine and eicosanoid production, antibody formation and cell death including receptor-mediated apoptosis (Munck and Naray Fejes-Toth, 1992; see Webster et al., 2002). The profound actions of the glucocorticoids require tight regulation, hence the importance of intact negative-feedback control of the HPA axis. Unchecked glucocorticoid secretion, due to prolonged inflammation, would have pathological consequences for host immunity, leading to hypercortisolaemia, immunosuppression and increased susceptibility to disease and infection (Munck and Naray Fejes-Toth, 1994). Integrity of the immune-neuroendocrine interactions is critically linked to physiological well being, since the ability of immune- or endocrine-derived cytokines to stimulate activation of the HPA axis represents a powerful defence mechanism against chronic inflammatory disease. Since the late 1980s, understanding of the extent of interaction between the immune and neuroendocrine systems has greatly increased. We now appreciate that the communication network between these two systems is bi-directional in nature (Blalock and Smith, 1985; see Besedovsky and del Rey, 2002) and encompasses a diverse collection of common chemical mediators including peptides, cytokines
51 and neurotransmitters that modulate activity of both systems by receptor-mediated actions (see Gaillard, 2001). Cytokines are polypeptides that are synthesised by immune cells and are key regulators of inflammatory responses. Cytokines are also synthesised by neuroendocrine tissues (Koenig, 1991) and by higher centres of the brain (e.g. hippocampus) where they contribute to local inflammation. Such complex interrelationships provide an important mechanism by which the immune system can modulate the activity of the HPA axis in order to preserve immune homeostasis and limit stress responses. The brain can influence the immune system by stimulation of the sympathetic nervous system that innervates the lymphoid organs. Thus, in conditions of stress, the brain can stimulate immune cell function by coordinated activation of the HPA axis and sympathetic nervous system. The neuroendocrine system synthesises hormones and neuropeptides that can influence immune function. These factors appear to act as paracrine or autocrine mediators, providing a system of discrete local regulation over inflammatory reactions. Many of the mediators are also synthesised locally by immune cells and their levels may be increased following immune activation (Weigent and Blalock, 1997). The immune-endocrine mediators are often characterised by having pleiotropic effects, including stimulatory or inhibitory actions, dependent on their relative local concentration or activation state of the immune system. Immune-derived peptides and hormones are believed to contribute to local inflammatory control mechanisms. Evidence suggests that sites of inflammation are associated with increased local concentrations of immunederived pro-inflammatory peptides, such as CRF and substance P (Jessop et al., 2001; Jessop, 2002). Levels of immune-derived opioid peptides, such as enkephalins and dynorphin, are also expressed at high levels during inflammation, although their actions are considered to be predominantly antiinflammatory (Cabot et al., 2001). A feature of the immune-derived peptide or hormone mediators and their receptors is that, although many appear to be largely similar to the classical brain or endocrine equivalents, in many cases the immune-expressed receptor may be a truncated version or express different pharmacologi-
cal characteristics (Sharp et al., 1998; Sharp, 2003; see Fulford and Jessop, 2001). The biologically active immune-derived peptides or hormones may also vary from the brain or endocrine equivalent. For example, immune cells express ACTH identical to that found in the pituitary in addition to truncated variants of ACTH (Smith et al., 1990). This highlights the potential importance of the immune-derived peptides as possessing functional significance. Furthermore, immune-derived peptides or hormones are expressed at very low levels suggesting roles as paracrine or autocrine regulators (Karalis et al., 1991; Aird et al., 1993; Sharp et al., 1998). POMC was one of the first endocrine proteins found expressed in the immune system. POMC, the precursor hormone for ACTH, is also cleaved into other biologically active peptides including the potent opioid peptide, 13-endorphin. Both ACTH and 13-endorphin are expressed in immune cells and are thought to contribute to local immunoregulatory control. As relatively low levels of these endogenous peptides and hormones are synthesised and expressed in the immune system, it seems unlikely that these would be able to interact directly with the endocrine system. Peptides are subject to rapid metabolism and so would be unlikely to remain biologically active following transportation in the circulation. However, in conditions associated with very high secretion of immune-derived peptide or hormone mediators, it is possible that these may be able to modulate peripheral endocrine secretions.
Stress-induced changes in the HPA axis We will now concentrate on the response of the HPA axis to the effects of (1) acute stress and (2) chronic or repeated stress. The majority of work underlying our understanding of stress mechanisms has arisen from preclinical research in animals despite the obvious limitations in extrapolating findings from animals to humans. However, these studies have undoubtedly made a major contribution to our current understanding of the mechanisms governing activation of the HPA axis in mammals. A wide range of behavioural paradigms have been developed in animals that closely correlate stress in humans
52 (Van de Kar and Blair, 1999; Pacak and Palkovits, 2001; Willner and Mitchell, 2002).
HPA-axis responsiveness to acute stress Systemic versus neurogenic stress
A wide range of acute stressors have been used in the study of HPA-axis regulation in animals. These stressors can be classified as either systemic or neurogenic stressors. Systemic stressors include physical stressors such as cold, ether, hypertonic saline challenge, insulin-induced hypoglycaemia, immune challenge such as by cytokine or endotoxin injection, formalin injection or surgical stress. Neurogenic stressors encompass those stressful stimuli bearing predominantly an emotional or psychological component. These include footshock, conditioned fear paradigms, forced swimming or restraint/immobilisation stress. Neurogenic stressors involve a strong somatosensory stimulus that requires cognitive or emotional interpretation. Exposure to these acute stress challenges results in enhanced secretion of ACTH and glucocorticoids, demonstrating acute activation of the HPA axis. The HPA axis responds to the intensity of the individual stressor, so that repeated or intensified stress results in enhanced secretion of the stress hormones. However, it is not possible to distinguish different stressors on the basis of simple measurement of circulating ACTH and glucocorticoid levels as these are common to all types of acute mild stress (Kant et al., 1982). However, studies at the central level have been important in the identification of stressorspecific neurocircuitry and neuroendocrine responses (see Pacak and Palkovits, 2001). Acute stress causes a short-lasting and rapid increase in CRF immunoreactivity in the median eminence (Buckingham, 1979). This is followed by increased synthesis of hypothalamic CRF, demonstrated using the technique of in situ hybridisation histochemistry to detect changes in mRNA expression (Lightman and Young, 1988). A range of acute stress paradigms have been demonstrated to increase CRF m R N A including footshock, insulin-induced hypoglycaemia, restraint and forced swim stress (see Lightman and Harbuz, 1993). However, the physical
stress of acute cold exposure does not alter CRF mRNA (Harbuz and Lightman, 1989). Acute stressors including cold, footshock, restraint and osmotic stress also increase expression of POMC mRNA in the anterior pituitary (Lightman and Young, 1988; Harbuz and Lightman, 1989; Wu and Childs, 1991). Differences between systemic and neurogenic stressors have been observed at the level of the hypothalamus. It appears that neurogenic stressors, including restraint or forced swim stress, only activate CRF mRNA, whereas physical stressors including osmotic stress or naloxone-precipitated opiate withdrawal increase proenkephalin A mRNA, in addition to CRF mRNA (Harbuz et al., 1991, 1994b). Neurogenic stressors that involve some physical component, e.g. footshock stress, also increase expression of proenkephalin A mRNA in the hypothalamus (Harbuz and Lightman, 1989). The stimulus-specific neuronal activation following stress has been further investigated by mapping stress-induced activation of immediate early gene products, such as c-fos (Chan et al., 1993). Immobilisation/restraint stress, a neurogenic stressor with a physical component, has been shown to increase immunostaining for fos in the hypothalamus, specifically in the medial and dorsal parvocellular PVN where most neurones are CRF positive (Kononen et al., 1992). c-Fos mRNA expression can be seen 30min after the start of physical restraint; however, maximal fos expression is observed 90min after stress onset (see Pacak and Palkovits, 2001). Acute footshock stress induces a similar pattern of fos activation to that seen following acute immobilisation stress. This is true for the main subdivisions of the PVN including the parvocellular CRFcontaining neurones. The wider neurocircuitry recruited in the processing of the footshock stress is essentially similar to the c-fos response observed following neurogenic stimuli (Sawchenko et al., 2000). These observations are suggestive of the existence of a neurogenic-stress circuit that becomes activated by phenotypically similar, acute stress challenges. Systemic stressors, which typically contain a strong physical component, appear to evoke a different pattern of fos activation to that shown for the neurogenic stressors. Whereas in the PVN the
53 parvocellular CRF neurones are activated, the wider circuitry involved in the processing of systemic stressors is contained within subcortical structures, a number of which are specifically involved in the integration of systemic stressors (Gaillet et al., 1991). Extensive research, using double-label immunohistochemical staining for tyrosine hydroxylase and c-los, has confirmed that in the case of both systemic and neurogenic stress, brainstem catecholaminergic cell groups are strongly activated. These neurones project directly to the PVN (Sawchenko and Swanson, 1982) and are likely to contribute to the robust neurohormonal responses seen following exposure to both types of stressors.
Immunological stress Of the diverse group of cytokines produced by the immune system, the effects of the inflammatory cytokines, interleukin-1 (IL-1), interleukin-6 (IL-6) and tumour-necrosisfactor-~ (TNF-~), have been the best characterised in terms of their ability to stimulate the HPA axis (see Turnbull and Rivier, 1999). HPAaxis activation can be induced by the cytokines alone or by concerted actions between them. These cytokines are able to act at the hypothalamus, pituitary and adrenal cortex to increase glucocorticoid secretion and so to suppress further immune/ inflammatory reactions. A single, cytokine injection (either central or peripheral) has been shown to cause potent activation of the HPA axis in rats and mice. For example, fosimmunostaining studies have shown how systemic injection of IL-1 causes activation of the PVN in an identical way to that seen for neurogenic stressors (Ericsson et al., 1994). In addition, ascending aminergic pathways appear to be involved in the neurohumoral response to IL-1 (Chuluyan et al., 1992), suggesting that for all types of acute stressors studied, activation of the HPA axis is strongly dependent on ascending aminergic inputs to the PVN. Many studies investigating the effect of cytokines on the HPA axis have studied the effect of injection of the endotoxin, lipopolysaccharide (LPS), a constituent of the bacterial cell wall (Tilders et al., 1994; Quan et al., 1998). Injection of this endotoxin causes
the release of a wide spectrum of cytokines, the exact profile of which is dependent on the concentration of endotoxin injected. The acute response involves predominantly IL-1 (principally, IL-l[3), IL-6 and TNF<x (Perlstein et al., 1993). If these cytokines are injected separately they also provoke a robust activation of the HPA axis, which confirms that during an acute inflammatory episode, cytokinemediated responses involve potent activation of the neuroendocrine stress axis (Sapolsky et al., 1987; Rivest et al., 1995; Turnbull and Rivier, 1999). In the hypothalamus, IL-l[3, TNFcx and IL-6 can all increase CRF production (Besedovsky et al., 1991). In the case of IL-1 [3, activation of the HPA axis occurs at the hypothalamic level and is not due to activation of pituitary corticotrophs at physiological concentrations of the cytokines. Pleiotropic cytokines, including IL-1 and IL-6, can act directly at the level of the adenohypophysis to promote differentiation and activation of corticotrophs at higher doses (see Buckingham et al., 1996; Navarra et al., 1997). This effect involves stimulation of POMC expression and hence ACTH formation. These in vitro studies suggest that the cytokines caused a slow release of ACTH. Evidence suggests that the folliculostellate cells of the pituitary may mediate the actions of cytokines on the corticotroph population, since the former are macrophage-like and likely to respond to cytokine stimulation. Indeed, the folliculostellate cells also synthesise cytokines that are able to contribute to local activation of the HPA axis at the level of the pituitary (Gloddek et al., 2001). If cytokines are administered in vivo, plasma ACTH levels rise rapidly demonstrating an acute activation of the HPA axis. Evidence suggests that the effects of peripherally administered cytokines are acting principally at the level of the hypothalamus via actions on the parvocellular CRF-containing neurones of the PVN. These effects can be blocked by central, but not peripheral, administration of an IL-1 receptor antagonist (Habu et al., 1998). The exact mechanism whereby cytokines are able to stimulate the HPA axis in vivo is incompletely understood; however, a number of potential sites of action have been proposed. Firstly, the presence of cytokine receptors in the brain and hypothalamus suggests that cytokines may be able to act locally to induce activation of hypophysiotropic parvocellular
54 PVN neurones (Farrar et al., 1987; Katsuura et al., 1988). A problem concerning the mechanism of action of cytokines on central activation of the HPA axis centres on the ability of peripheral cytokines to gain access to the brain. These large polypeptides are unable to traverse the blood-brain barrier alone; however, there is evidence for carrier-mediated transport mechanisms. Blood-borne cytokines may gain access to the brain via saturable transport systems. These have been described for a number of cytokines including IL-1 cz, IL-1 [3, IL-6 and TNFcz, but not IL-2 (Banks et al., 1995). Circulating cytokines may be able to penetrate the brain at the level of the circumventricular organs including the organum vasculosum of the lamina terminalis of the hypothalamus (OVLT), median eminence, the subfornical organ, choroid plexus and area postrema (Saper and Breder, 1994). These structures line the cerebral ventricles, where the blood-brain barrier is weak or absent, and therefore present sites whereby peripheral mediators may gain access to the CNS and stimulate the HPA axis. The circumventricular organs may alternatively represent sites where peripheral cytokine messages are communicated to the hypothalamus. Potential candidates for the signal communication molecule involved in the transduction of blood-borne cytokine effects to the brain include neurotransmitters, most notably 5-HT, prostaglandins, brain cytokines or nitric oxide (Van Dam et al., 1993; Rivier, 1995). Although, the circumventricular organs and transport carriers of the blood-brain barrier provide mechanisms whereby cytokines can gain access to the brain, these are unlikely to accumulate cytokines in the concentrations required to produce the profound behavioural and physiological effects typically seen following peripheral cytokine administration (Watkins et al., 1995). Cytokines also evoke pain pathways leading to activation of somatosensory networks in the brain that reflexly activate the HPA axis (Dantzer, 2001). Cytokine activation may additionally induce profound systemic effects including hypotension or hypoglycaemia that may activate vagal reflexes. In addition, secretion of inflammatory mediators will also influence C-fibre activity that will signal to the spinal cord and the brain. Systemic infection may also activate resident microglia, monocytes or
macrophages present in the CNS. These may also secrete IL-113 or TNFcz in response to infection that can signal to the HPA axis and cause direct stimulation of ACTH secretion (Hopkins and Rothwell, 1995). Of these mechanisms, stimulation of vagal afferent nerves is considered to be of major importance. Studies examining the effect of subdiaphragmatic vagotomy in rats have described complete abrogation of many of the effects of peripheral cytokines in the brain (see Maier and Watkins, 1998). This neural pathway may provide a vital link for the communication of peripheral immune signals to the CNS. The bi-directional communication network regulating the immune-neuroendocrine interface provides a dynamic link by which stress can impact on host immunity. The outcome of exposure to a stressor will depend on the interplay between psychological, neuroendocrine, behavioural and immunological factors. Thus, one must consider the response to stress as a phenomenon governed by adaptations in multiple systems of the body. From this holistic standpoint it becomes clear that in some clinical disorders, such as major depression, linked to stress, the breadth of symptoms displayed in some patients is consistent with dysregulation in neurobehavioural, immunological and neuroendocrine mechanisms. Now the pathways of communication between these physiological systems have been revealed, we will be better able to understand the possible mechanisms contributing to the aetiology of other diseases bearing a strong cognitive or emotional component.
HPA-axis responsiveness to repeated stress Regardless of the exact phenotypic profile of responses to the various stimuli that elicit an acute stress response, each is characterised by return of HPA-axis activity to baseline once the stressful stimulus is removed or is diminished. Studies of the impact of repeated acute stress, continued for several days, have been undertaken to establish the effect of long-term stress. However, models of repeated stress are generally poor correlates of long-term, persistent stress as repeated exposure to the same stress is often associated with habituation of the HPA-axis response and attenuation of the neurohormonal stress
55 response. Repeated footshock or restraint stress is associated with increased plasma glucocorticoid levels that remain above baseline for several days and then return to control levels (Kant et al., 1985). Similarly, plasma ACTH levels generally return to basal levels following repeated stress. Attenuation in the afferent regulatory control of the HPA axis is thought to underlie this habituation of the HPA axis with repeated stress. Interestingly, habituation to one type of acute stress appears to be specific as crosstolerance to other types of acute stress does not generally occur (Spencer and McEwen, 1990). This phenomenon may indicate differences in the neural processing of the various types of acute stressors. A problem with the use of experimental repeated stress paradigms is that they bear little physiological relevance to disorders associated with chronic dysfunction of the stress axis. To better understand the aetiology and progression of human disorders involving long-term changes in the activity of the HPA axis, a more successful strategy is to adopt chronic models with improved validity that provide better insight into the mechanisms governing adaptation to long-term stress.
HPA-axis responsiveness to chronic stress Studies of sustained chronic stress differ significantly from the effects of repeated stress, since a feature of the former condition is persistent elevated levels of circulating glucocorticoids. Inflammatory diseases may be considered as disorders associated with chronic stress as they are typically characterised by high circulating levels of glucocorticoids. The mechanistic changes that confer long-term upregulation of activity of the HPA axis and continuous secretion of stress hormones remain largely unknown; however, preclinical studies have furthered our understanding of the adaptations contributing to a state of chronic stress. It is apparent that the mechanisms essential to the maintenance of HPAaxis integrity, including negative-feedback control, have become dysregulated in the chronic inflammatory condition.
Experimental models of chronic inflammatory stress A particularly well-characterised animal model of inflammatory stress is adjuvant-induced arthritis (AA) in the rat, which is a T lymphocyte-dependent chronic inflammatory disease. AA has been used as a model with relevance for certain clinical inflammatory conditions including pain and rheumatoid arthritis. The arthritis can be induced in susceptible strains of rats following an intradermal injection of an oil suspension of ground, heat-killed Mycobacterium butyricum (10 mg/ml) into the base of the tail. Specific strains of rat will develop hindpaw inflammation within 12-14 days and other limbs are additionally affected by day 21 post-injection (Rook et al., 1994). The neuroendocrine effects of long-term inflammation in AA rats have been extensively studied. The objective of such studies has been to identify whether HPA-axis dysregulation is aetiologically relevant and essential for disease progression. The AA rat is characterised by similar pituitary and adrenal changes to that seen following acute and repeated stress, including raised circulating levels of ACTH and glucocorticoids and increased expression of POMC m R N A in the adenohypophysis (Harbuz et al., 1992). There is an apparent defect in the circadian regulation of the HPA axis resulting in the consistently high secretion rate of glucocorticoids that is elevated in the early hours of the morning, a time normally representing the nadir of the daily cycle (Sarlis et al., 1992). In AA, at the level of the hypothalamus, there is a paradoxical decrease in CRF m R N A in the parvocellular PVN and reduced release of CRF into the hypophysial blood (Harbuz et al., 1992). This effect is not entirely due to enhanced glucocorticoid feedback regulation of the CRF neurones, and the exact inhibitory mechanism responsible for the arthritis-induced CRF hypofunction is not completely understood although it may involve substance P (Jessop et al., 2000b). Timecourse studies have identified that the reduction in CRF m R N A is apparent when the first signs of inflammation appear (about day 11) and the maximal reduction in CRF is observed when inflammation is most severe (around day 21) (Harbuz et al., 1994a). Interestingly, the inhibition of parvocellular PVN CRF neurones is also seen in other chronic
56 immunological disease models in rodents, including the preclinical model for multiple sclerosis, experimental allergic encephalomyelitis (EAE) (Harbuz et al., 1993), and systemic lupus erythematosus (Shanks et al., 1997). This possibly also applies in human conditions (Harbuz, 2002). In contrast to the effect on CRF neurones, AA is associated with increased expression of AVP m R N A in the parvocellular PVN and AVP release into the portal circulation, indicating that the dominant ACTH secretagogue during chronic inflammatory stress is AVP (Chowdrey et al., 1995). However, how the increased activity of parvocellular AVP neurones contributes to increased synthesis of precursor POMC by the pituitary corticotrophs is unknown, although CRF may act in a permissive role. Evidently, there is marked derangement in the regulation of the HPA axis in association with chronic stress of immunological origin. The transition from the dominance of CRF in the stimulation of ACTH secretion to a major role for AVP has also been associated with repeated stress paradigms. Chronic exposure to immobilisation or other psychological stressors increases the proportion of parvocellular CRF PVN neurones that contain AVP and an increased ratio of AVP to CRF levels in the zona externa of the median eminence (de Goeij et al., 1991). It has been widely suggested that AVP of parvocellular origin is most important for maintaining HPA-axis responsiveness under conditions associated with defective CRF function, like chronic stress. The characteristic reduction in parvocellular PVN CRF neuronal activity associated with chronic immune-mediated stress impairs the ability of the HPA axis to respond to certain types of acute novel stress. Specifically, AA is associated with a blunted glucocorticoid response to psychological and physical stressors (Aguilera et al., 1997; Windle et al., 2001). In contrast, in AA, acute immunological stressors, such as LPS injection, elicit a robust neuroendocrine stress response that is equivalent to that seen in nonarthritic control rats (Harbuz et al., 1999). In this case, CRF neurones are activated following stimulation with peripheral endotoxin injection, albeit to a lesser extent than in non-arthritic controls. Clearly, there is differential feedback regulation of CRF neurones mediating ACTH release in response to
immunological versus other physical or psychological stressors, presumably reflecting the importance of responding to immune-mediated stimuli that directly threatens host survival. Of additional interest is the observation that, in contrast to males, AA female rats are unable to mount a robust corticosterone response to acute endotoxin treatment, possibly relating to significantly higher basal glucocorticoid secretion rates and impaired adrenal responsiveness (Harbuz et al., 1999). Studies in the late 1980s suggested that susceptibility to autoimmune disease may be linked to a defect in CRF regulation at the level of the PVN and a subsequent inability to mount an HPA-axis response (MacPhee et al., 1989; Sternberg et al., 1989). This inability to damp down the endogenous immune response could thus precipitate autoimmunity. Although a compelling hypothesis, subsequent studies have noted a number of exceptions to this. It is now believed that, although the HPA axis has a major role to play in determining severity of disease, susceptibility is more likely to reflect the balance of pro- and anti-inflammatory factors. Both neuroendocrine and immune factors have been implicated although the exact relationship remains to be determined (Harbuz, 2002).
HPA-axis activity and clinical inflammatory disease An inability to respond appropriately to novel stressful stimuli will influence host integrity and have serious implications for the long-term health of the individual. There are clear correlations between preclinical findings and clinical data as patients with rheumatoid arthritis (RA) experience defective glucocorticoid responses to the stress of surgery (Chikanza et al., 1992), although this is a contentious issue. A widely held hypothesis is that defective regulation of the HPA axis and the associated excessive secretion of powerful glucocorticoids will cause prolonged immunosuppression and dysregulation of immune cells, ultimately predisposing to autoimmune disease. However, alterations in HPAaxis activity in patients with RA, for example, are not reliably discernible. There is clear involvement of glucocorticoids in the disease process since treatment
57 of RA patients with a cortisol synthesis inhibitor, metyrapone, profoundly worsens symptoms of inflammation (Saldanha et al., 1986). This observation suggests that the HPA axis exerts inhibitory control over the disease process in RA and thereby regulates disease severity. Whether derangements in HPA-axis integrity contribute to disease progression in RA, however, is incompletely understood. There is some evidence in favour of aberrant HPA-axis activity in patients with chronic inflammatory conditions (Dekkers et al., 2000). Recent unpublished observations in our laboratory suggest that there may be sub-populations of patients in RA with altered glucocorticoid regulation. These sub-populations may explain some of the discrepancies in the literature. In one study RA patients did not show deficits in a CRF challenge test, indicating that pituitary ACTH and cortisol secretion were largely unaffected by the disease process. However, other studies have identified increased circulating levels of ACTH in RA, without change in plasma cortisol levels (see Morand and Leech, 2001). Evidence is in favour of an underactive HPA axis in RA, since the absence of elevated plasma glucocorticoids during persistent inflammation points towards a state of adrenal hyporesponsiveness. Importantly, studies of disease aetiology in man may be complicated by subject medication, such as prostaglandin synthesis inhibitors (Hall et al., 1994). Clearly, it is essential that adaptations to chronic inflammatory stress be considered in light of pre-existing therapeutic treatment since such adaptive changes can occur secondary to drug intervention. Additionally, in the case of clinical inflammatory disease, it is difficult to discern whether the HPA-axis dysfunction is of primary aetiological importance or is a state marker of the disease. Even during chronic stress, it is vital that the HPA-axis response to acute stressors is maintained. Pre-existing inflammatory disease in man may have serious consequences for the ability to appropriately respond to novel stressful situations. Indeed, a failure to respond to acute activation of the HPA axis may have implications for stress coping in individuals. Individuals vary in their ability to cope with stressful situations. In addition to genetic factors, a number of external factors influence propensity to stress, including childhood trauma, other early environ-
mental factors, major life events or infections (Chrousos, 1998; Sanchez et al., 2001). These can influence the development or adaptation of stress responses, in many cases exerting long-lasting effects. For example, in man, traumatic events may promote the onset of disease or exacerbate existing conditions, including RA (Marcenaro et al., 1999) and mutliple sclerosis (Mohr et al., 2000). Evidently, the factors regulating responses to stress are highly complex and changes in basal HPA activity alone cannot explain the phenomena of disease onset, progression and outcome. However, further advances through clinical and preclinical research will improve our understanding of the mechanisms driving dysregulation in the stress system and the consequences for disease.
Endogenous opioids and integrated stress responses In addition to CRF and noradrenaline, there are a number of other neuromediators strongly implicated in the regulation of responses to stress. The opioid peptides and more recently, certain opioid-like peptides, have been identified as specific peptide transmitter molecules that potently modulate both the HPA axis and the immune system and are therefore important regulators of the dynamic communication network between the two. Evidence that opioids are important regulators of the HPA axis has come from research in rats showing that acute administration of morphine or related opioid agonists induces activation of the HPA axis and increased levels of plasma ACTH and corticosterone (Ignar and Kuhn, 1990; Martinez et al., 1990; see Pechnick, 1993). Other opiate agonists, including kappa- and delta-opiate receptor ligands also stimulate the axis (Gonzalvez et al., 1991; Laorden and Milanes, 2000), suggesting that all major classes of opioid are able to stimulate this stress pathway. The effects of the opioids appear to involve stimulation of CRF-containing neurones by either a direct or indirect mechanism of activation (MartinezPinero et al., 1994b). In addition to exogenous opiate drugs, endogenous opioid peptides are also able to activate the HPA axis when administered into the CNS. Certain acute stressors are able to stimulate expression of mRNA for the opioid peptide,
58
200
7 0 - 8 0 % ) by circulating corticosterone (Reul and De Kloet, 1985; Reul et al., 1987b; for a review on MR, see Reul et al., 2000). Indeed, based on in vivo microdialysis experiments of Linthorst and Reul, at this time of the day, free corticosterone levels in the brain are estimated to be approximately 0.5nM (Linthorst et al., 1995, 1997; Reul et al., 2000). It should be noted that the synthetic glucocorticoid dexamethasone, at least in vitro, presents also a high-binding affinity for MRs. However, although the on-rate of dexamethasone to MR is comparable to that of corticosterone and aldosterone, yet, in contrast to that of corticosterone or aldosterone, the off-rate is extremely high (~ 20-fold higher than that of corticosterone and aldosterone), possibly explaining why the dexamethasone-MR complex is unstable and cannot be activated (Reul et al., 2000). These
observations explain why in early studies 3H-dexamethasone was not retained in hippocampal pyramidal and granular neurons (De Kloet et al., 1975; Warembourg, 1975), this in addition to recent observations that dexamethasone poorly penetrates the brain as it is a substrate for the multidrug resistance (mdr) pump (Meijer et al., 1998). Furthermore, the lack of stability of the dexamethasone-MR complex very likely explains the poor mineralocorticoid activity of dexamethasone in mineralocorticoid target tissues, such as the kidney and the colon. We have proposed that due to its unstable interaction with MRs, dexamethasone might even hamper the action of endogenous mineralocorticoids and glucocorticoids via MRs, thereby acting as an MR antagonist (Reul et al., 2000). Such MR antagonist activity of dexamethasone has been indeed reported (Bohus and De Kloet, 1981; Hoefnagels and Kloppenborg, 1983). Glucocorticoid receptors displaying a considerably lower affinity for natural glucocorticoids (Kd: 3-5 nM at 4~ are hardly occupied (_< 10%) under early morning baseline conditions. Occupancy of GRs rises as soon as glucocorticoid levels start to rise due to the increased circadian drive when time is moving to the dark, i.e. active, phase of the day for the rat or mouse (Reul and De Kloet, 1985; Reul et al., 1987b, 2000). After exposure of the animal to strong stressors such as forced swimming or restraint, GR occupancy is even higher than that at the diurnal peak of the circadian rhythm. Based on the differential occupancy pattern of MRs and GRs, the concept was formulated that apparently MRs mediate a tonic action of glucocorticoids whereas GRs are the receptors responsible for mediating the negative-feedback action of elevated levels of glucocorticoids as occurring after stress and during the activity phase (Reul and De Kloet, 1985; De Kloet and Reul, 1987; Reul et al., 2000).
Afferent control of the HPA axis
Preface To understand its physiological implications, the concept needs to be considered in the context of the neuroanatomical localisation of MRs and GRs.
98 Whereas MRs are most richly located in the hippocampus and to a much lesser extent in the lateral septum, central nucleus of the amygdala and the brainstem motor nuclei, GRs can be found in virtually all neurons (and glia) of the central nervous system with particularly high concentrations present in the PVN, neocortex and hippocampus (Fuxe et al., 1985; Reul and De Kloet, 1985, 1986; Reul et al., 1987b).
Hippocampus In view of the virtually exclusive localisation of MR in the hippocampus and its role in mediating tonic influences of corticosterone on various HPA axisrelated parameters (e.g. CRF and AVP expression in the PVN, early morning baseline ACTH and corticosterone levels), excitability of pyramical neurons and other parameters (see De Kloet and Reul, 1987; De Kloet, 1991; Joels and De Kloet, 1992; De Kloet et al., 1998), it was proposed that hippocampal MRs were involved in modulating the level of excitatory output of the hippocampus. Amongst other, this tonic excitatory output feeding into GABAergic interneurons located in the bed nucleus of the stria terminalis (BNST)/lateral septum area and known to inhibit PVN parvocellular neurons (Herman et al., 1995; Herman and Cullinan, 1997; see also Herman et al., in this volume) thereby would exert a tonic inhibitory influence on HPA activity. Potentially, the hippocampus can also affect HPA axis activity via its modulatory influence on autonomic output (Van den Berg et al., 1990, 1994) (see below) and its projections to the amygdala and frontal cortex (see below). It has been reported that in mice hippocampal MRs, but not GRs, are asymmetrically distributed with higher levels in the right hippocampus than in the left one (Neveu et al., 1998). The significance of the asymmetric distribution of hippocampal MRs for HPA axis regulation, autonomic output and other MR-regulated brain functions needs to be clarified. The localisation of GR throughout the brain and in the anterior pituitary is congruous to its role in regulating metabolism and negative-feedback action. GRs located in the PVN and anterior pituitary mediate negative feedback of elevated glucocorticoid
levels directly to the core structures of the HPA axis. GRs inhibit the synthesis and release of CRF and, to a lesser extent, AVP in parvocellular neurons of the PVN and the synthesis of proopiomelanocortin mRNA (POMC mRNA, the precursor molecule of ACTH) and release of ACTH from the corticotrophic cells in the anterior pituitary. In the hippocampus, GRs are substantially expressed in pyramidal (primarily CA1) and granular dentate gyrus neurons (Fuxe et al., 1985; Reul and De Kloet, 1986; Van Eekelen et al., 1988). Nevertheless, it seems that the GRs in this higher limbic brain structure with regard to exerting negative feedback on the HPA axis appear to be acting secondary to those in the PVN and anterior pituitary (Van Haarst et al., 1997). Reports suggesting a potent role of the hippocampus in the negative feedback control of the HPA axis have unfortunately mainly based their argument on indirect evidence. Therefore, until now, the exact role of hippocampal GRs in the regulation of HPA activity has not been exactly clarified yet. It seems clear, however, that GRs in the hippocampus play a prominent role in the behavioural adaptation to stressful events, as for instance in the forced swim test, also called the Porsolt swim test (Jefferys et al., 1985; De Kloet et al., 1986; Korte et al., 1996a) (J.M.H.M. Reul and S. Ulbricht, unpublished observations).
Central nucleus of the amygdala The central nucleus of the amygdala exerts via its projection to the PVN, and possibly also indirectly via a projection to the BNST, a stimulatory influence on the activity of the HPA axis under baseline conditions and after chronic stress (Beaulieu et al., 1986; Roozendaal et al., 1991; Van der Kar et al., 1991; Goldstein et al., 1996; Bhatnagar and Dallman, 1998). Both MRs and GRs are expressed in neurons of this nucleus, whereas GRs are also expressed in other nuclei of the amygdaloid complex (Reul and De Kloet, 1986). However, the role of amygdaloid MRs and GRs in HPA-axis regulation is still unknown. Interestingly, in contrast to their effect in the PVN, GRs exert a stimulatory effect on CRF expression in the central amygdaloid nucleus (and BNST)
99 (Schulkin et al., 1998), but its significance regarding the HPA axis is also unknown.
The medial prefrontal cortex Another important region of the brain playing an important role in the regulation of the HPA axis is the medial prefrontal cortex (for review, see Sullivan and Gratton, 2002). The role of the prefrontal cortex generally in stress-related processes is rather complex, as for instance the ventral region of the medial prefrontal cortex seems to act stimulatory and/or facilitatory on the HPA axis, whereas the dorsomedial region is thought to be responsible for inhibitory influences, at least partly through modulation of infralimbic outputs. Diorio et al. (1993) showed that GRs in the prefrontal cortex mediate negative-feedback effects of glucocorticoids on restraint stress-induced increases in circulating ACTH and corticosterone levels. In this study, baseline HPA hormone levels were not altered. Substantial amounts of GRs and, to a lesser extent, MRs have been found in the prefrontal cortex of rats, dogs and primates (Meaney and Aitken, 1985; Reul et al., 1990; Cintra et al., 1994; Sanchez et al., 2000). Interestingly, recent observations indicate that the influence of the prefrontal cortex on stress responses of the HPA axis and autonomic nervous system is lateralized. It was observed that the ibotenic acid lesions in the right or bilateral medial prefrontal cortex of rats affect baseline and stressinduced corticosterone levels and stress ulcer development, but not lesions in the left medial prefrontal cortex (Sullivan and Gratton, 1999). Thus, with regard to the influence of the frontal cortex on the HPA axis there seems to be a clearcut right-sided bias. The ventral medial prefrontal cortex most likely influences the activity of the PVN mainly indirectly as there are only few direct projections. However, regions in the direct vicinity of the PVN are heavily innervated as well as the brainstem (e.g. locus coeruleus, nucleus tractus solitarius (NTS), pontine (raphe nuclei) and limbic nuclei (Amygdala, BNST), which are known to directly affect the PVN activity (Terreberry and Neafsey, 1983, 1987; Hurley et al., 1991; Takagishi and Chiba, 1991; Jodo et al., 1998).
Thus, the ventral region of the medial prefrontal cortex, which is also been called the visceral motor cortex (Cechetto and Saper, 1990), is supremely positioned to modulate and coordinate neuroendocrine (and autonomic) responses to stressful and emotional stimuli. However, conversely, many of the mentioned nuclei have ascending projections to the medial prefrontal cortex thereby modulating its function. In addition, the hippocampus projects via the fimbriafornix to the ventral medial prefrontal cortex, very likely modulating its output. Hence, the sketched network underscores the complexity of the afferent control of the HPA axis.
Dynamic changes in HPA axis control due to acute stress The organisation of the HPA axis is not only complex in terms of space but also in terms of time. However, until recently, available evidence in the literature suggests that the overall control mechanisms of the HPA axis are mainly sensitive to long-term manipulations (i.e. time range: weeks to months). With overall control mechanism here is meant those driving and feedback mechanisms controlling the baseline activity and stress responsiveness of the HPA axis. The long-term manipulations observed to alter control processes within the HPA axis include aging (Meaney et al., 1987; Reul et al., 1988), perinatal manipulations (e.g. prenatal immune challenge, prenatal stress, maternal deprivation) (Levine, 1957, 1967; Meaney et al., 1985, 1987, 1989; Reul et al., 1994b; Vall6e et al., 1997), pharmacological treatments (e.g. antidepressant treatments) (Brady et al., 1991; Reul et al., 1993, 1994a) and endocrine extirpations (e.g. adrenalectomy) (Reul et al., 1987a, 1988; Spencer et al., 1990). Recently, we discovered that control mechanisms governing the HPA axis can be adjusted within hours after a stimulus. We found that hippocampal MRs are relatively quickly upregulated (i.e. within 8 h) by acute psychological stressors such as forced swimming and exposure to a novel environment (Gesing et al., 2001). A physical stressor such as cold exposure was ineffective. Interestingly, the effect of stress on M R levels could be blocked by pretreatment with a
100 CRF receptor antagonist and mimicked by intracerebroventricular injection of CRF. The effect of stress on MR levels was also observed in the amygdala and neocortex and lasted for up to 24 48 h (Gesing et al., 2001). Importantly, using a challenge test with the selective MR antagonist RU 28318, we observed that the rise in MR levels was associated with a stronger MR-mediated inhibitory tonus on the activity of the HPA axis. Thus, setpoints governing baseline activity and stress responsiveness of the HPA axis are rapidly adjustable allowing the system to adapt quickly to novel conditions. The exact functional significance of the transient rises in MR after psychological stress needs to be discerned. Possibly, the rises in MR levels are instigated to balance the impact of putative increases in HPA-driving processes, this in order to maintain "normal" HPA physiology. Given that MRs are known to be involved in several processes at the cellular and behavioural level, the stress-induced increases in MR levels are also expected to impact on these levels. Thus, given that MRs in the dentate gyrus granular neurons exert anti-apoptotic effects (Sloviter et al., 1989; Hassan et al., 1997; Almeida et al., 2000), an increased protection of these neurons after stress by elevated MR levels may be expected. Furthermore, in view of the involvement of hippocampal MRs in anxiety-related behaviour (Korte et al., 1996b; Smythe et al., 1997), increases in anxiety after the stressful experience may be anticipated. Increases in anxiety have indeed been observed after exposure to stressful stimuli (Owens and Nemeroff, 1991; Gutman et al., 2003). These MR-associated cellular, neuroendocrine and behavioural changes make sense from a physiological perspective as they act protectively, thereby maintaining a state of well-being and increasing the chances of survival for the organism.
Voluntary exercise exercises the HPA axis
inflicted upon animals (and sometimes upon humans). Such conditions also include disease states and aging, the latter not necessarily needing to be stressful. A problem is, however, defining what is "normal" (with respect to both animals and humans) and how to keep animals under "normal" conditions. Without even attempting to try and define what is normal and what are normal conditions (it would blast the scope of this book), we just wish to bring out that conditions can be altered in such a way that they are regarded as being "positive" to animals. Such positive effects have been observed in several animal species after enriching their environment with toys, burying material, etc. However, these studies have been focussing mainly on behavioural changes (Paylor et al., 1992; Chapillon et al., 1999; Williams et al., 2001) and, more recently, on neurogenesis (Kempermann et al., 1997; van Praag et al., 1999b) and spontaneous apoptosis (Young et al., 1999) in the adult dentate gyrus. In general, it seems though that relatively little attention has been payed to changes in stress-related systems such as the HPA axis. In view of the observation that mice held in an enriched environment show reduced anxiety-related behaviour (Chapillon et al., 1999), it would be apt to have a closer look at the HPA axis of such animals. Given that it has been recently reported that the presence of a running wheel as part of an enriched environment, at least regarding its effects on neurogenesis (van Praag et al., 1999b), seems to be the critical object, we decided a few years ago to conduct a detailed study on the changes in the HPA axis of mice allowed free access to a running wheel in their home cage (i.e. the voluntary exercise model). Before we report on our recent results regarding the effects of voluntary exercise on the mouse HPA axis, we provide briefly some background information on the voluntary exercise model.
Cellular and physiological correlates
of voluntary exercise Preface Until recently, the HPA axis has virtually always been studied under "normal" baseline conditions (in adulthood or during development) or during and after acutely or chronically stressful conditions
It is now widely accepted that the physical exercise has positive effects on a variety of biological systems, such as body composition, the cardiovascular system, the immune system and also the brain. Regarding body composition, the amount of peritoneal and
101 perirenal adipose tissue, which is indicative of the risk for cardiovascular pathology, is decreased in subjects regularly performing physical activity (Friedman et al., 1997; Lambert and Jonsdottir, 1998). A decreased heart rate, an enhanced oxidative capacity and a decreased blood pressure have been observed under resting conditions in exercised rats (Kramer et al., 2000) and humans (Gielen et al., 2001). Moderate training intensity also has been shown to enhance immune system function and to increase resistance to infections (Jonsdottir, 2000; Pedersen and Hoffman-Goetz, 2000). It has been shown in rodents that, at the level of the brain, voluntary exercise results in an enhanced performance in spatial learning and memory tasks (van Praag et al., 1999a). Moreover, voluntary exercise evokes in rats and mice increases in neurogenesis in the dentate gyrus of the hippocampus which is thought to be the result of an enhanced action of growth factors (e.g. IGF-1, BDNF) in the brain (van Praag et al., 1999b; Bilang-Bleuel et al., 2000; Carro et al., 2000; Russo-Neustadt et al., 2000; Trejo et al., 2001). In contrast to these stimulatory effects of exercise, a decrease in neurogenesis has been observed after exposing rats or mice to psychological stressors such as forced swimming or predators (van Praag et al., 1999b; Bilang-Bleuel et al., 2000). Thus, it appears that the regular physical exercise has effects on various biological systems which generally can be valued as positive. It has been suggested that, animals, including humans, show improved coping with stressful events after regular performance of moderate physical exercise (Steptoe et al., 1989; Byrne and Byrne, 1993; Salmon, 2001). The above-mentioned form of regular exercise is not to be mixed up with the high-demand endurance training (e.g. marathon running) in humans and forced exercise in rodents. Voluntary exercise yields many positive biological effects, whereas endurance training, due to its excessive (eccentric) physical demand, has been found to cause injuries (Warren and Stiehl, 1999; Proske and Morgan, 2001), reproductive disturbances (Chen and Brzyski, 1999; Warren and Stiehl, 1999), impaired immunity (Nieman, 2000) and accelerated wear of the movement apparatus (Warren and Stiehl, 1999; Feasson et al., 2002). Moreover, endurance training in humans has been shown to elicit chronic stress-like
changes in the HPA axis (Villaneueva et al., 1986; Luger et al., 1987; Duclos et al., 1998, 2001). In forced exercised rats, blunted ACTH and unchanged corticosterone responses to footshock and forced swimming have been found (Watanabe et al., 1991, 1992; Dishman et al., 1998). Furthermore, substantially enlarged adrenal medullas (in man called the socalled "sports adrenal medulla") have been observed in both man and animals after high-intensity exhaustive exercise (Man: Kj~er, 1998) and forced treadmill or swim exercise (Rats: Stallknecht et al., 1990; Schmidt et al., 1992).
HPA axis changes after long-term voluntary exercise: introduction We were prompted to investigate whether voluntary exercise would also affect the HPA axis, this in view of the many reported positive effects at the cellular, physiological and behavioural level. Studying the HPA axis is particularly apt as this is a neuroendocrine system which is highly involved in the coping response to metabolic and stressful challenges (for review, see Dallman et al., 1993; De Kloet et al., 1998; Reul et al., 2000). In our study published recently (Droste et al., 2003), male C57BL/6N mice were allowed to run voluntarily for a period of 4 weeks in a running wheel provided in their home cage. They ran approximately 7 km/day and this virtually exclusively during the first half of their active period, i.e. the night phase (Fig. 1A). As can be taken from Fig. 1B, the mean maximal distance accomplished by the animals per day was established within a few days. Figure 1C shows that the running capacity among mice can vary, but the variance in running within each individual animal is strikingly small. All in all, these observations are in agreement with other publications (Festing, 1977; Harriet al., 1999; Lancel et al., 2003). Mice run voluntarily when offered a running wheel. Thus, it seems that it complies with a natural urge of the animals (Brant and Kavanau, 1964, 1995) increasing physical fitness (Goodrick, 1978) and helping to control body weight (Leshner, 1971; Goodrick, 1978). Importantly, wheel running is not regarded as a form of stereotypic behaviour (Harri et al., 1999) because it is not expressed at the cost of
102 A 20 ..,~.~
w~
i5
. . . . 10
24 TiME (~..h intervals)
10000~ A
E
8000. 6000
u
c
4000
:~
2000.
1 2 3 4 5 6 7 8 9 1011 12131415161718192021 22
Days
12500 E 10000 >,, "o u
c
7500 5000 2500
1
2
3
4
Animal #
5
6
Fig. 1. Running performance in the voluntary exercise model. Male C57BL/6N mice were housed individually and were provided with a running wheel (diameter, 14cm) in their cage. The number of revolutions was recorded and thereof the distance run calculated. Figure 1A shows that mice run almost exclusively during the first half of the dark phase (the open and closed bar indicate the light and dark period, respectively). Figure 1B shows the establishment of the running performance during the first 22 days of running. It is clear that the full running performance is established within 4-5 days. Figure 1C shows the running performance of each individual mouse averaged over the 22-day period. Apparently, there is an inter-individual variance in running performance, but the inter-day variance within each animal is rather limited. Data presented in Figure 1A were taken with permission of the European Journal of Neuroscience (Publisher: Blackwell) from Lancel et al. (2003).
103 resting behaviour as is the case with other reported locomotor stereotypes (Cooper and Nicol, 1991, 1996).
HPA axis changes after long-term
voluntary exercise: physical changes Long-term voluntary exercise resulted in remarkable physical changes in the mice. Although body weight of exercised animals did not change, the abdominal fat mass was largely reduced (Droste et al., 2003), for which the reduction in weight was presumably compensated for by increases in muscle substance, heart weight and blood volume. Thymus weights were reduced, whilst total adrenal weight (i.e. the two added together) was increased, suggesting enhanced glucocorticoid action over an extended period of time. Increased adrenal weights after exercise is a long-known finding (Ingle, 1938; Riss et al., 1959; Kja~r, 1992). Using histological methods, however, we could demonstrate that the changes that had occurred in the adrenal glands were quite comprehensive. Similar to reports in the literature (Idelman, 1978; Coleman et al., 1998), we observed that the left adrenal gland is bigger than the right one. We could show that this is due to both a larger adrenal medulla and a larger adrenal cortex (Droste et al., 2003). Strikingly, after 4 weeks of voluntary exercise, the difference in size of the left versus the right adrenal gland was abolished. This was solely due to an enlargement of the right adrenal gland and, more specifically, mainly due to a substantial enlargement of the adrenal cortex and, to a lesser extent, to that of the adrenal medulla (Droste et al., 2003). An observation underscoring the tremendous impact of the voluntary exercise on the organism was that the overall shape of the adrenal gland was altered in that it had turned from an elongated form into a spherical organ (S.K. Droste, S. Ulbricht and J.M.H.M. Reul, unpublished observation). This observation also highlights that apparently a complex nervous/endocrine organ, such as the adrenal gland, is capable of significant organ restructuring as part of the complex of adaptational actions of the organism to cope with the demands pressed upon it by the high physical activity.
HPA axis changes after long-term voluntary
exercise: hormone secretion and the sympathoadrenomedullary system When considering the baseline HPA hormone secretion over the diurnal cycle, the most striking change in the exercised mice was a highly increased corticosterone level at the time of lights off (i.e. 18:00 h), thus at the start of the active phase (Droste et al., 2003). Actually, it appeared that the rise in glucocorticoid levels preceded the onset of the running behaviour (see Fig. 1A). Thus, the rise in glucocorticoids can be regarded as anticipatory, an observation which has been seen already several decades ago in other experimental paradigms (Levine et al., 1972; Goldman et al., 1973). In the exercised mice, the rise in corticosterone levels is most likely anticipatory to support the metabolic demand of the enhanced physical activity. The enhanced corticosterone secretion in the exercised mice at the crest of the diurnal cycle occurred in the absence of a concomitant enhancement in ACTH secretion. This change in adrenocortical sensitivity to ACTH is most likely associated with changes in the sympathoadrenomedullary system of the exercised mice. For an elaborate review about the role of the sympathoadrenomedullary system in adrenal function, see the chapter of W. Engeland in this volume. As mentioned above already, exercised animals do show enlargements of the right adrenal medulla. Moreover, we also discovered distinct changes in m R N A expression of tyrosine hydroxylase (TH; the rate-limiting enzyme in the catecholamine synthesis) in the adrenal medulla of exercised mice (Droste et al., 2003). The level of expression of this enzyme in the adrenal medulla is regarded as an indicator for the overall activity of the sympathoadrenomedullary system (Hamelink et al., 2002). However, first of all, we also found, in parallel to the asymmetry in size of the adrenal medullas in control mice, an asymmetry in the adrenomedullar TH m R N A expression in the control animals, i.e. the left medulla expressing higher levels than the right one (Fig. 2). Strikingly, after exercise the adrenomedullar TH m R N A expression had risen selectively in the right medulla, thereby abolishing the asymmetry in TH m R N A levels seen in the control mice (Fig. 2; Droste et al., 2003).
104 8000-
-o
§ *
~
6000
.c
v-o
§
4000-
I:: ~" 2000
7-
0
right
eft I
I control
~
exercise
Fig. 2. Changes in tyrosine hydroxylase (TH) mRNA levels in the adrenal medulla of control and (4 weeks) exercising mice (n = 10 for both groups). TH mRNA was detected by in situ hybridization histochemistry and autoradiograms were analyzed by computerized image analysis. TH mRNA levels are expressed as integrated optical density (i.e. nett grey values • square pixels/1000). Data are presented as means + SEM. Exercise evoked an overall increase in TH mRNA
expression in the adrenal medulla (analysis of variance (ANOVA): effect of exercise: F(1, 36) = 34.9, P < 0.0001). In addition, overall higher TH mRNA levels in the left adrenal medulla than in the right one were found (ANOVA: left vs. right: F(1,36)= 67.6, P < 0.0001). *, significant difference between exercise and control within the left or right adrenal medulla (post-hoc tests with contrasts); +, significant difference between left and right adrenal medulla within the same treatment group (post-hoc tests with contrasts). Data were taken from Droste et al. (2003) with permission of The Endocrine Society, Copyright 2003.
How do these observations fit together? First, sympathoadrenomedullary activity in control mice seems to be biased to the left side of the body. This observation is in line with the concept of a predominant involvement of the right brain hemisphere in the control of sympathetic activity and the higher levels of noradrenaline found in this side of the brain (Wittling et al., 1998; Wittling, 2001). However, until now asymmetry in the autonomic nervous system has been hardly studied and one should be careful drawing far-reaching conclusions. Nevertheless, our finding on the left-right difference in adrenomedullar size strengthens the concept on sympathetic asymmetry. Second, the increased adrenocortical sensitivity in exercised mice at the start of the active, i.e. running, phase may be explained by an increased sympathoadrenomedullary activity in these animals. However, such an increased
sympathoadrenomedullary activity is, for reasons presently unknown, virtually exclusively restricted to the right branch of the sympathoadrenomedullary input. Given that neural inputs to the adrenal glands are important growth-determining factors for the adrenal cortices (Engeland and Dallman, 1975, 1976; Dallman et al., 1976), the selective enlargement of the right adrenal cortex in the exercising mice may be due to an intensified rightsided sympathoadrenomedullary input. Thus, the enhanced glucocorticoid secretion at the start of the nocturnal phase appears to be due to an increased rightsided sympathoadrenomedullary activity in combination with an increased adrenocortical capacity. Nevertheless, despite the attractiveness of these suppositions, the exact causalities between the depicted interrelationships need to be further clarified in future research. The sympathoadrenomedullary changes in the exercised mice were found to be of relevance for the distinct responses of the HPA axis to stressful challenges seen in these animals (Droste et al., 2003). We observed that the plasma corticosterone responses, but not ACTH responses, were enhanced in exercising mice in response to stressors demanding or involving physical activity such as forced swimming or restraint. Previous reports have shown enhanced sympathoadrenomedullary activation in response to stressors demanding physical activity (Sothmann et al., 1996; Koolhaas et al., 1997; Kj~er, 1998) and to psychosocial stress (Sinyor et al., 1983). Our observation in exercising mice is in line with these reports as these mice show an increased sympathoadrenomedullary capacity as well as an enlarged (right) adrenal cortex. Thus, exercised mice respond to stressors containing a significant physical component with amplified glucocorticoid responses, most likely because their HPA and sympathoadrenomedullary axes have adapted to meet the enhanced metabolic demand during running. However, the exercised animals react in a novel environment with different HPA hormone responses. Overall, the plasma ACTH responses in the exercised mice were markedly lower than those in the sedentary mice, suggesting strongly that the exposure to novelty has lower impact in these animals. This observation dovetails with observations that exercised mice (Binder et al., 2004) and
105 humans (Steptoe et al., 1989; Byrne and Byrne, 1993; Salmon, 2001) show reduced anxiety. It also corresponds with the observed reduced expression of CRF mRNA in the PVN of exercised mice (Droste et al., 2003). Presently, the reason for the reduced CRF mRNA expression awaits further clarification, but could involve increased GR-mediated negativefeedback signalling as a result of the increased glucocorticoid levels during the first half of the dark phase and increased afferent inhibitory signals from as yet unknown sources. The plasma corticosterone response to novelty in our exercising mice depended decisively on the presence of a functional running wheel in the new cage (Droste et al., 2003). In the absence of the running wheel, the exercising animals produced the same glucocorticoid levels as the control mice which can be explained by the adrenocortical hyperresponsiveness generally observed in the exercising mice. However, if the exercising animals had access to a running wheel in the new cage, then the corticosterone responses were much lower. The animals indeed used the running wheel for a significant amount of the exposure time. Reversely, when the running wheel was made dysfunctional by blocking the turning mechanism, then plasma ACTH and corticosterone reponses of the exercised mice to the novelty stimulus was indistinguishable from that of the control animals (Droste et al., 2003). Thus, the use of the running wheel may be regarded as displacement behaviour, resulting in a further reduction of the emotional impact of the novel environment. A similar effect on corticosterone secretion has been observed when rats were given access to a running wheel after being exposed to a footshock paradigm (Starzec et al., 1983). The exact mechanism by which the glucocorticoid response is restrained under these conditions is presently unknown, but may involve reduced sympathoadrenomedullary outflow and local adrenomedullary inhibitory mechanisms. Summing up, long-term voluntary exercise elicits multiple changes in the HPA axis of mice which involves major alterations in the sympathoadrenomedullary system and central afferent control mechanisms. These changes are part of a network of physiological and behavioural changes effectuated in the exercised mice. The results obtained with this
model underscore the power of adaptive mechanisms capable of vastly altering the phenotype of an organism.
Concluding remarks In this chapter, it was our aim to point out that the HPA axis is a neuroendocrine system which is capable of adjusting its regulatory setpoints in a very dynamic fashion. It can do so irrespective of whether the adaptive measures are in response to adverse or positive challenges. As illustrated by our recent publication (Droste et al., 2003), long-term voluntary exercise has a huge impact on the HPA axis. In view of the increased amplitude in the diurnal cycle of plasma corticosterone, the increased glucocorticoid response to stressors entailing physical activity, the decreased responsiveness in HPA hormones to emotional stimuli, it is evident that the voluntary exercise increases the dynamic range of the HPA axis. Changes in the sympathoadrenomedullary system of the exercising mice turned out to be a principal factor substantially altering the responsiveness of the HPA axis in terms of glucocorticoid responses. The observations underscore that the final glucocorticoid output is actually steered by two pathways, being the neuroendocrine hypothalamic-hypophyseal pathway and the neural sympathoadrenomedullary pathway. We emphasize this view here, because it seems that the evidently prominent role of the sympathoadrenomedullary system in the control of adrenocortical glucocorticoid secretion has been, to put it mildly, somewhat neglected over the past 25 years. Importantly, these pathways do not interact only at the adrenal gland level, but more so at multiple levels including various levels within the central nervous system in addition to the adrenal gland and, possibly, the pituitary gland. One of the most striking observations in the exercised mice was the phenomenon that the increase in adrenal weight was solely the result of an enlargement of the right adrenal cortex and, to a lesser extent, right adrenal medulla. This enlargement appeared to be the result of a selective rise in the activity of the right branch of the sympathoadrenomedullary system; the reason for this selectivity is currently unknown and awaits further investigations.
106 A
8
E L
6
.C
~9
4-
.===
c
L qD t~
2 0
left
right control
I
suicide
Fig. 3. Left and right adrenal weights of suicide victims and controls. Data are presented as means • SEM. *, significant increase as compared to control (p = 0.0030, t = 3.22, df = 31; two-tailed t-test after analysis of covariance (ANCOVA)). There was no significant difference between suicides and controls in mean age, body weight, body height, of sex distribution. Data were taken from Szigethy et al. (1994) with permission of the Society of Biological Psychiatry (Publisher: Elsevier Science Inc.). The enlargement of the right adrenal gland abolished the asymmetry between the left and right adrenal gland seen in the control mice, in which the left adrenal is bigger than the right one. The asymmetry in the adrenal glands and the role of the sympathoadrenomedullary system therein may also be of pathophysiological interest in view of a post-mortem study on adrenal weights in suicide victims (Szigethy et al., 1994). It was reported that control subjects showed no difference in weight between the left and the right adrenal gland, whereas suicide victims showed a selectively increased weight of the left adrenal gland (Fig. 3; Szigethy et al., 1994). This increase appeared to be most likely due to an enlargement of the adrenal cortex. Clearly, it is impossible at this stage to make direct comparisons between the (patho)-physiological corollaries leading to adrenal gland asymmetries in control mice versus suicide victims, because the mechanisms underlying the asymmetries have not been clarified but also mere species differences could be playing a role. Nevertheless, the observed asymmetry in adrenal weight in the suicide victims is certainly of interest as it is associated with a pathological state. Speculatively, the asymmetry may be the result of an imbalanced sympathoadrenomedullary system. This may not even be that speculative given that vegetative
instability has often been reported in subjects with affective disorders (Steptoe et al., 1989; Byrne and Byrne, 1993; Salmon, 2001). Thus, the observation on voluntary exercise abolishing adrenal and sympathoadrenomedullary asymmetry can be regarded as an anti-stress phenomenon, similar to other reported anti-stress phenomena such as the effects of exercise on neurogenesis (van Praag et al., 1999b; Bilang-Bleuel et al., 2000; Carro et al., 2000; Russo-Neustadt et al., 2000; Trejo et al., 2001), sleep (Lancel et al., 2003) and anxiety-related behaviour (Steptoe et al., 1989; Byrne and Byrne, 1993; Salmon, 2001) (E. Binder, S.K. Droste, F. Ohl, and J.M.H.M. Reul, unpublished observations). Therefore, the asymmetry in the H P A axis and sympathoadrenomedullary system, including the central nervous system mechanisms controlling these neuroendocrine and neural output systems, should be the subject of future investigations.
Acknowledgments The authors are greatly indebted to Ms. Sabine Ulbricht who, due to her tremendous technical skills, contributed significantly to the mouse exercise project reviewed in this chapter.
References Almeida, O.F.X., Conde, G.L., Crochemore, C., Demeneix, B.A., Fischer, D., Hassan, A.H.S., Meyer, M., Holsboer, F. and Michaelidis, T.M. (2000) Subtle shifts in the ratio between pro- and antiapoptotic molecules after activation of corticosteroid receptors decide neuronal fate. FASEB Journal, 14: 779-790. Bale, T.L., Contarino, A., Smith, G.W., Chan, R., Gold, L.H., Sawchenko, P.E., Koob, G.F., Vale, W.W. and Lee, K.-F. (2000) Mice deficient for corticotropin-releasing hormone receptor-2 display anxiety-like behaviour and are hypersensitive to stress. Nature Genet., 24: 410-414. Barden, N., Stec, I.S.M., Montkowski, A., Holsboer, F. and Reul, J.M.H.M. (1997) Endocrine profile and neuroendocrine challenge tests in transgenic mice expressing antisense RNA against the glucocorticoid receptor. Neuroendocrinol., 66: 212-220. Beaulieu, S., Di Paolo, T. and Barden, N. (1986) Control of ACTH secretion by the central nucleus of the amygdala: implication of the serotoninergic system and its relevance to
107 the glucocorticoid delayed negative feedback mechanism. Neuroendocrinol., 44: 247-254. Bhatnagar, S. and Dallman, M. (1998) Neuroanatomical basis for facilitation of hypothalamic-pituitary-adrenal responses to a novel stressor after chronic stress. Neuroscience, 84: 1025-1039. Bhatnagar, S., Viau, V., Chu, A., Soriano, L., Meijer, O.C. and Dallman, M.F. (2000) A cholecystokinin-mediated pathway to the paraventricular thalamus is recruited in chronically stressed rats and regulates hypothalamic-pituitary-adrenal function. J. Neurosci., 20: 5564-5573. Bilang-Bleuel, A., Droste, S., Gesing, A., Rech, J., Linthorst, A.C.E. and Reul, J.M.H.M. (2000) Impact of stress and voluntary exercise on neurogenesis in the adult hippocampus: quantitative analysis by detection of Ki-67. Proc. 30th Ann. Meeting Soc. Neurosci., 26: 1534. Binder, E., Droste, S.K., Ohl, F., Reul, J.M.H.M. (2004) Regular voluntary exercise reduces anxiety-related behavior and impulsivity in mice. Behav. Brain Res., in press. Bohus, B. and De Kloet, E.R. (1981) Adrenal steroids and extinction behavior: antagonism by progesterone, deoxycorticosterone and dexamethasone of a specific effect of corticosterone. Life Sci., 28: 433-440. Brady, L.S., Whitfield, Jr., H.J., Fox, R.J., Gold, P.W. and Herkenham, M. (1991) Long-term antidepressant administration alters corticotropin-releasing hormone, tyrosine hydroxylase, and mineralocorticoid receptor gene expression in rat brain. Therapeutic implications. J. Clin. Invest., 87: 831-837. Brant, D.H. and Kavanau, J.L. (1964) Unrewarded exploration and learning of complex mazes by wild and domestic mice. Nature, 204: 267-269. Brant, D.H. and Kavanau, J.L. (1995) Exploration and movement patterns of the Canyon mouse Peromyscus crinitus. Ecology, 46: 452-461. Byrne, A. and Byrne, D.G. (1993) The effect of exercise on depression, anxiety and other mood states: a review. J. Psychosom. Res., 37: 565-574. Carro, E., Nufiez, A., Busiguina, S. and Torres-Aleman, I. (2000) Circulating insulin-like growth factor I mediates effects of exercise on the brain. J. Neurosci., 20: 2926-2933. Cechetto, D.F. and Saper, C.B. (1990) Role of the cerebral cortex in autonomic function. In: Loewy, A.D. and Spyer, K.M. (Eds.), Central Regulation of Autonomic Functions. Oxford University Press, Oxford, pp. 208-223. Chapillon, P., Manneche, C., Belzung, C. and Caston, J. (1999) Rearing environmental enrichment in two inbred strains of mice: 1. Effects on emotional reactivity. Behav. Genet., 29: 41-46. Chen, E.C. and Brzyski, R.G. (1999) Exercise and reproductive dysfunction. Fertil. Steril., 71: 1-6. Cintra, A., Zoli, M., Ros~n, L., Agnati, L.F., Okret, S., Wikstr6m, A.-C., Gustafsson, J.-A. and Fuxe, K. (1994) Mapping and computer assisted morphometry and microdensitometry of glucocorticoid receptor immunoreactive
neurons and glial cells in the rat central nervous system. Neuroscience, 62: 843-897. Coleman, M.A., Garland, T., Jr., Marler, C.A., Newton, S.S., Swallow, J.G. and Carter, P.A. (1998) Glucocorticoid response to forced exercise in laboratory house mice (Mus domesticus). Physiol. Behav., 63: 279-285. Cooper, J.J. and Nicol, C.J. (1991) Stereotypic behavior affects environmental preferences in bank voles, Chlethrionomys glareolus. Anim. Behav., 41: 971-977. Cooper, J.J. and Nicol, C.J. (1996) Stereotypic behavior in wild caught and laboratory bred bank voles (Clethrionomus glareolus). Anim. Welfare, 5: 245-257. Coste, S.C., Kesterson, R.A., Heldwein, K.A., Stevens, S.L., Heard, A.D., Hollis, J.H., Murray, S.E., Hill, J.K., Pantely, G.A., Hohimer, A.R., Hatton, D.C., Phillips, T.J., Finn, D.A., Low, M.J., Rittenberg, M.B., Stenzel, P. and Stenzel-Poore, M.P. (2000) Abnormal adaptions to stress and impaired cardiovascular function in mice lacking corticotropin-releasing hormone receptor-2. Nature Genet., 24: 403-409. Dallman, M.F., Engeland, W.C. and Shinsako, J. (1976) Compensatory adrenal growth: a neurally mediated reflex. Am. J. Physiol., 231: 408-4 14. Dallman, M.F., Akana, S.F., Cascio, C.S., Darlington, D.N., Jacobson, L. and Levin, N. (1987) Regulation of ACTH secretion: variations on a theme of B. Rec. Prog. Horm. Res., 43:113-173. Dallman, M.F., Strack, A.M., Akana, S.F., Bradbury, M.J., Hanson, E.S., Scribner, K.A. and Smith, M. (1993) Feast and famine: critical role of glucocorticoids with insulin in daily energy flow. Front. Neuroendocrinol., 14: 303-347. De Kloet, E.R. (1991) Brain corticosteroid receptor balance and homeostatic control. Front. Neuroendocrinol., 12: 95-164. De Kloet, E.R. and Reul, J.M.H.M. (1987) Feedback action and tonic influence of corticosteroids on brain function: a concept arising from the heterogeneity of brain receptor systems. Psychoneuroendocrinol., 12: 83-105. De Kloet, E.R., Wallach, G. and Mcewen, B. (1975) Differences in corticosterone and dexamethasone binding to rat brain and pituitary. Endocrinology, 96: 598-609. De Kloet, E.R., Reul, J.M.H.M., De Ronde, F.S.W. and Veldhuis, H.D. (1986) Brain corticosteroid receptor systems: heterogeneity, function and plasticity. Central Actions of ACTH and Related Peptides Fidia Research Series Symposia in Neuroscience IV. Liviana Press, Padova, pp. 115-129. De Kloet, E.R., Vreugdenhil, E., Oitzl, M.S. and JoWls, M. (1998) Brain corticosteroid receptor balance in health and disease. Endocr. Rev., 19: 269-301. Diorio, D., Viau, V. and Meaney, M.J. (1993) The role of the medial prefrontal cortex (cingulate gyrus) in the regulation of hypothalamic-pituitary-adrenal responses to stress. J. Neurosci., 13: 3839-3847. Dishman, R.K., Bunnell, B.N., Youngstedt, S.D., Yoo, H.S., Mougey, E.H. and Meyerhoff, J.L. (1998) Activity wheel
108 running blunts increased plasma adrenocorticotrophin (ACTH) after footshock and cage-switch stress. Physiol. Behav., 63: 911-917. Droste, S.K., Gesing, A., Ulbricht, S., Muller, M.B., Linthorst, A.C. and Reul, J.M.H.M. (2003) Effects of long-term voluntary exercise on the mouse hypothalamic-pituitaryadrenocortical axis. Endocrinology, 144:3012-3023. Duclos, M., Corcuff, J.B., Arsac, L., Moreau-Gaudry, F., Rashedi, M., Roger, P., Tabarin, A. and Manier, G. (1998) Corticotroph axis sensitivity after exercise in endurancetrained athletes. Clin. Endocrinol., 48: 493-501. Duclos, M., Corcuff, J.-B., Pehourcq, F. and Tabarin, A. (2001) Decreased pituitary sensitivity to glucocorticoids in endurance-trained men. Eur. J. Endocrinol., 144: 363-368. Edwards, C.R.W., Burt, D., McIntyre, M.A., De Kloet, E.R., Stewart, P.M., Brett, L., Sutanto, W.S. and Monder, C. (1988) Localisation of l l[3-hydroxysteroid dehydrogenasetissue specific protector of the mineralocorticoid receptor. Lancet, 2: 986--989. Engeland, W.C. and Dallman, M.F. (1975) Compensatory adrenal growth is neurally mediated. Neuroendocrinol., 19: 352-362. Engeland, W.C. and Dallman, M.F. (1976) Neural mediation of compensatory adrenal growth. Endocrinology, 99: 1659-1662. Feasson, L., Stockholm, D., Freyssenet, D., Richard, I., Duguez, S., Beckmann, J.S. and Denis, C. (2002) Molecular adaptations of neuromuscular disease-associated proteins in response to eccentric exercise in human akeletal muscle. J. Physiol., 543: 297-306. Festing, M.F.W. (1977) Wheel activity in 26 strains of mouse. Lab. Animals, 11: 257-258. Friedman, J.E., Ferrara, C.M., Aulak, K.S., Hatzoglou, M., McCune, S.A., Park, S. and Sherman, W.M. (1997) Exercise training down-regulates ob gene expression in the genetically obese SHHF/Mcc-fa(cp) rat. Horm. Metab. Res., 29: 214-219. Funder, J.W., Pearce, P.T., Smith, R. and Smith, A.I. (1988) Mineralocorticoid action: target tissue specifity is enzyme, not receptor, mediated. Science, 242: 583-585. Fuxe, K., Wikstr6m, A.-C., Okret, S., Agnati, L.F., H~irfstrand, A., Yu, Z.-Y., Granholm, L., Zoli, M., Vale, W. and Gustafsson, J.-A. (1985) Mapping of glucocorticoid receptor immunoreactive neurons in the rat Te 1 and diencephalon using a monoclonal antibody against rat liver glucocorticoid receptor. Endocrinol., 177: 1803-1812. Gesing, A., Bilang-Bleuel, A., Droste, S.K., Linthorst, A.C.E., Holsboer, F. and Reul, J.M.H.M. (2001) Psychological stress increases hippocampal mineralocorticoid receptor levels: involvement of corticotropin-releasing hormone. J. Neurosci., 21: 4822-4829. Gielen, S., Schuler, G. and Hambrecht, R. (2001) Exercise training in coronary artery disease and coronary vasomotion. Circulation, 103: el-e6.
Goldman, L., Coover, G.O. and Levine, S. (1973) Bidirectional effects of reinforcement shifts on pituitary adrenal activity. Physiol. Behav., 10:209-214. Goldstein, L.E., Rasmusson, A.M., Bunney, B.S. and Roth, R.H. (1996) Role of the amygdala in the coordination of behavioral, neuroendocrine, and prefrontal cortical monoamine responses to psychological stress in the rat. The Journal of Neuroscience, 16: 4787-4798. Goodrick, C.L. (1978) Effect of voluntary wheel exercise on food intake, water intake, and body weight for C57BL/6J mice and mutations which differ in maximal body weight. Physiol. Behav., 21: 345-351. Gutman, D.A., Owens, M.J., Skelton, K.H., Thrivikraman, K.V. and Nemeroff, C.B. (2003) The corticotropin-releasing factor1 receptor antagonist R121919 attenuates the behavioral and endocrine responses to stress. J. Pharmacol. Exp. Ther., 304: 874-880. Hamelink, C., Tjurmina, O., Damadzic, R., Young, W.S., Weihe, E., Lee, H.W. and Eiden, L.E. (2002) Pituitary adenylate cyclase-activating polypeptide is a sympathoadrenal neurotransmitter involved in catecholamine regulation and glucohomeostasis. Proc. Natl. Acad. Sci. USA, 99: 461-466. Harri, M., Lindblom, J., Malinen, H., Hyttinen, M., Lapvetel~iinen, T., Eskola, S. and Helminen, H.J. (1999) Effect of access to a running wheel on behavior of C57BL/6J mice. Laboratory Animal Science, 49: 401-405. Hassan, A.H.S., Von Rosenstiel, P., Patchev, V.K., Holsboer, F. and Almeida, O.F.X. (1997) Exacerbation of apoptosis in the dentate gyrus of the aged rat by dexamethasone and the protective role of corticosterone. Exp. Neurol., 140: 43-52. Herman, J.P. and Cullinan, W.E. (1997) Neurocircuitry of stress: central control of the hypothalamo-pituitary-adrenocortical axis. Trends Neurosci., 20: 78-84. Herman, J.P., Cullinan, W.E., Morano, M.I., Akil, H. and Watson, S.J. (1995) Contribution of the ventral subiculum to inhibitory regulation of the hypothalamo-pituitary-adrenocortical axis. J. Neuroendocrinol., 7: 475-482. Hoefnagels, W.H.L. and Kloppenborg, P.W.C. (1983) Antimineralocorticoid effects of dexamethasone in subjects treated with glycyrrhetinic acid. J. Hypertens., l(Suppl. 2): 313-315. Hurley, K.M., Herbert, H., Moga, M.M. and Saper, C.B. (1991) Efferent projections of the infralimbic cortex of the rat. J. Comp. Neurol., 308: 249-276. Idelman, S. (1978) The structure of the mammalian adrenal cortex. In: Jones, I.C. and Henderson, I.W. (Eds.), General, Comparative and Clinical Endocrinology of the Adrenal Cortex. Academic Press, New York, pp. 1-180. Ingle, D.J. (1938) The time for the occurrence of corticoadrenal hypertrophy in rats during continued work. Am. J. Physiol., 124: 627-630. Jefferys, D., Boublik, J. and Funder, J.W. (1985) A kappaselective opioidergic pathway is involved in the reversal of a
109 behavioural effect of adrenalectomy. Eur. J. Pharmacol., 107: 331-335. Jodo, E., Chiang, C. and Aston-Jones, G. (1998) Potent excitatory influence of prefrontal cortex activity on noradrenergic locus coeruleus neurons. Neuroscience, 83: 63-79. Joels, M. and De Kloet, E.R. (1992) Control of neuronal excitability by corticosteroid hormones. Trends Neurosci., 15: 25-30. Jonsdottir, I.H. (2000) Neuropeptides and their interaction with exercise and immune function. Immunol. Cell Biol., 78: 562-570. Kempermann, G., Kuhn, H.G. and Gage, F.H. (1997) More hippocampal neurons in adult mice living in an enriched environment. Nature, 386: 493-495. Kishimoto, T., Radulovic, J., Radulovic, M., Lin, C.R., Schrick, C., Hooshmaand, F., Hermanson, O., Rosenfeld, M.G. and Spiess, J. (2000) Deletion of Crhr2 reveals an anxiolytic role for corticotropin-releasing hormone receptor-2. Nature Genet., 24:415-4 19. Kja~r, M. (1992) Regulation of hormonal and metabolic responses during exercise in humans. Exerc. Sport Sci. Rev., 20: 161-184. Kja~r, M. (1998) Adrenal medulla and exercise training. Eur. J. Appl. Physiol., 77: 195-199. Koolhaas, J.M., De Boer, S.F., de Ruiter, A.J.H., Meerlo, P. and Sgoifo, A. (1997) Social stress in rats and mice. Acta Physiol. Scand., 161(Suppl. 640): 69-72. Korte, S.M., De Kloet, E.R., Buwalda, B., Bouman, S.D. and Bohus, B. (1996a) Antisense to the glucocorticoid receptor in hippocampal dentate gyrus reduces immobility in forced swim test. Eur. J. Pharmacol., 301: 19-25. Korte, S.M., Kortebouws, G.A.H., Koob, G.F., Dekloet, E.R. and Bohus, B. (1996b) Mineralocorticoid and glucocorticoid receptor antagonists in animal models of anxiety. Pharmacol. Biochem. Behav., 54: 261-267. Kramer, J.M., Plowey, E.D., Beatty, J.A., Little, H.R. and Waldrop, T.G. (2000) Hypothalamus, hypertension, and exercise. Brain Res. Bull., 53: 77-85. Lambert, G.W. and Jonsdottir, I.H. (1998) Influence of voluntary exercise on hypothalamic norepinephrine. J. Appl. Physiol., 85: 962-966. Lancel, M., Droste, S.K., Sommer, S. and Reul, J.M.H.M. (2003) Influence of regular voluntary exercise on spontaneous and social stress-affected sleep in mice. Eur. J. Neurosci., 17: 2171-2179. Leshner, A.I. (1971) The adrenals and the regulatory nature of running wheel activity. Physiol. Behav., 6: 551-558. Levine, S. (1957) Infantile experience and resistance to physiological stress. Science, 126: 405. Levine, S. (1967) Maternal and environmental influences on the adrenocortical response to stress in weanling rats. Science, 156: 258-260.
Levine, S., Goldman, L. and Coover, G.D. (1972) Expectancy and the pituitary-adrenal system. Ciba Found. Symp., 8: 281-291. Linthorst, A.C.E., Flachskamm, C., Mfiller-Preuss, P., Holsboer, F. and Reul, J.M.H.M. (1995) Effect of bacterial endotoxin and interleukin-1 beta on hippocampal serotonergic neurotransmission, behavioral activity, and free corticosterone levels: an in vivo microdialysis study. J. Neurosci., 15: 2920-2934. Linthorst, A.C.E., Flachskamm, C., Hopkins, S.J., Hoadley, M.E., Labeur, M.S., Holsboer, F. and Reul, J.M.H.M. (1997) Long-term intracerebroventricular infusion of corticotropin-releasing hormone alters neuroendocrine, neurochemical, autonomic, behavioral and cytokine responses to a systemic inflammatory challenge. J. Neurosci., 17: 4448-4460. Luger, A., Deuster, P.A., Kyle, S.B., Gallucci, W.T., Montgomery, L.C., Gold, P.W., Loriaux, D.L. and Chrousos, G.P. (1987) Acute hypothalamic-pituitaryadrenal response to the stress of treadmill exercise. N. Engl. J. Med., 316: 1309-1315. Meaney, M.J. and Aitken, D.H. (1985) (3H)Dexamethasone binding in rat frontal cortex. Brain Res., 328: 176-180. Meaney, M.J., Aitken, D.H., Bodnoff, S.R., Iny, L.J. and Tatarewicz, J.E. (1985) Early postnatal handling alters glucocorticoid receptor concentrations in selected brain regions. Behav. Neurosci., 99: 765-770. Meaney, M.J., Aitken, D.H. and Sapolsky, R.M. (1987) Thyroid hormones influence the development of hippocampal glucocorticoid receptors in the rat: a mechanism for the effects of postnatal handling on the development of the adrenocortical stress response. Neuroendocrinol., 45: 278-283. Meaney, M.J., Aitken, D.H., Viau, V., Sharma, S. and Sarrieau, A. (1989) Neonatal handling alters adrenocortical negative feedback sensitivity and hippocampal type II glucocorticoid receptor binding in the rat. Neuroendocrinol., 50: 597-604. Meijer, O.C., de Lange, E.C., Breimer, D.D., de Boer, A.G., Workel, J.O. and De Kloet, E.R. (1998) Penetration of dexamethasone into brain glucocorticoid targets is enhanced in mdrlA P-glycoprotein knockout mice. Endocrinology, 139:1789-1793. Neveu, P.J., Liege, S. and Sarrieau, A. (1998) Asymmetrical distribution of hippocampal mineralocorticoid receptors depends on lateralization in mice. Neuroimmunomodulation, 5: 16-21. Nieman, D.C. (2000) Exercise effects on systemic immunity. Immunol. Cell Biol., 78: 496-501. Owens, M.J. and Nemeroff, C.B. (1991) Physiology and pharmacology of corticotropin-releasing factor. Pharmacol. Rev., 43: 425-473. Patacchioli, F.R., Amenta, F., Ramacci, M.T., Taglialatela, G., Maccari, S. and Angelucci, L. (1989) Acetyl-L-carnitine
ll0 reduces the age-dependent loss of glucocorticoid receptors in the rat hippocampus- an autoradiographic study. J. Neurosci. Res., 23: 462-466. Paylor, R., Morrison, S.K., Rudy, J.W., Waltrip, L.T. and Wehner, J.M. (1992) Brief exposure to an enriched environment improves performance on the morris water task and increases hippocampal cytosolic protein kinase C activity in young rats. Behavioural Brain Research, 52: 49-59. Pedersen, B.K. and Hoffman-Goetz, L. (2000) Exercise and the immune, system: regulation, integration, and adaptation. Physiol. Rev., 80: 1055-1081. Proske, U. and Morgan, D.L. (2001) Muscle damage from eccentric exercise: mechanism, mechanical signs, adaptation and clinical application. J. Physiol., 537: 333-345. Reul, J.M.H.M. and De Kloet, E.R. (1985) Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation. Endocrinology, 117: 2505-2512. Reul, J.M.H.M. and De Kloet, E.R. (1986) Anatomical resolution of two types of corticosterone receptor sites in rat brain with in vitro autoradiography and computerized image analysis. J. Steroid Biochem., 24: 269-272. Reul, J.M.H.M. and Holsboer, F. (2002) Corticotropinreleasing factor receptors 1 and 2 in anxiety and depression. Curr. Opin. Pharmacol., 2: 23-33. Reul, J.M.H.M., Van den Bosch, F.R. and De Kloet, E.R. (1987a) Differential response of type I and type II corticosteroid receptors to changes in plasma steroid level and circadian rhythmicity. Neuroendocrinol., 45: 407-412. Reul, J.M.H.M., Van den Bosch, F.R. and De Kloet, E.R. (1987b) Relative occupation of type-I and type-II corticosteroid receptors in rat brain following stress and dexamethasone treatment: functional implications. J. Endocrinol., 115: 459-467. Reul, J.M.H.M., Tonnaer, J.A.D.M. and De Kloet, E.R. (1988) Neurotrophic ACTH analogue promotes plasticity of type I corticosteroid receptor in brain of senescent male rats. Neurobiol. Aging, 9: 253-260. Reul, J.M.H.M., De Kloet, E.R., Van Sluijs, F.J., Rijnberk, A. and Rothuizen, J. (1990) Binding characteristics of mineralocorticoid and glucocorticoid receptors in dog brain and pituitary. Endocrinology, 127: 907-915. Reul, J.M.H.M., Rothuizen, J. and De Kloet, E.R. (1991) Agerelated changes in the dog hypothalamic-pituitary-adrenocortical system: neuroendocrine activity and corticosteroid receptors. J. Steroid Biochem., 40: 63-69. Reul, J.M.H.M., Stec, I., S6der, M. and Holsboer, F. (1993) Chronic treatment of rats with the antidepressant amitriptyline attenuates the activity of the hypothalamic-pituitaryadrenocortical system. Endocrinology, 133: 312-320. Reul, J.M.H.M., Labeur, M.S., Grigoriadis, D.E., De Souza, E.B. and Holsboer, F. (1994a) Hypothalamic-pituitaryadrenocortical axis changes in the rat after long-term treatment with the reversible monoamine oxidase-A inhibitor moclobemide. Neuroendocrinol., 60:509-519.
Reul, J.M.H.M., Stec, I., Wiegers, G.J., Labeur, M.S., Linthorst, A.C.E., Arzt, E. and Holsboer, F. (1994b) Prenatal immune challenge alters the hypothalamic-pituitary-adrenocortical axis in adult rats. J. Clin. Invest., 93: 2600-2607. Reul, J.M.H.M., Gesing, A., Droste, S.K., Stec, I.S.M., Weber, A., Bachmann, C.G., Bilang-Bleuel, A., Holsboer, F. and Linthorst, A.C.E. (2000) The brain mineralocorticoid receptor: greedy for ligand, mysterious in function. Eur. J. Pharmacol., 405: 235-249. Riss, W., Burstein, S.D., Johnson, R.W. and Lutz, A. (1959) Morphologic correlates of endocrine and running activity. J. Comp. Physiol. Psychol., 52: 618-620. Roozendaal, B., Koolhaas, J.M. and Bohus, B. (1991) Attenuated cardiovascular, neuroendocrine, and behavioral responses after a single footshock in central amygdaloid lesioned male rats. Physiol. Behav., 50: 771-775. Rothuizen, J., Reul, J.M.H.M., Van Slujs, F.J., Mol, J.A., Rijnberk, A. and De Kloet, E.R. (1993) Increased Neuroendocrine reactivity and decreased brain mineralocorticoid receptor-binding capacity in aged dogs. Endocrinology, 132: 161-168. Russo-Neustadt, A.A., Beard, R.C., Huang, Y.M. and Cotman, C.W. (2000) Physical activity and antidepressant treatment potentiate the expression of specific brain-derived neurotrophic factor transcripts in the rat hippocampus. Neuroscience, 101: 305-312. Salmon, P. (2001) Effects of physical exercise on anxiety, depression, and sensitivity to stress: a unifying theory. Clin. Psychol. Rev., 21: 33-61. Sanchez, M.M., Young, L.J., Plotsky, P.M. and Insel, T.R. (2000) Distribution of corticosteroid receptors in the rhesus brain: relative absence of glucocorticoid receptors in the hippocampal formation. J. Neurosci., 20: 4657-4668. Sapolsky, R.M., Krey, E.C. and Mcewen, B.S. (1983) The adrenocortical stress-response in the aged male rat: impairment of recovery from stress. Experimental Gerontology, 18: 55-64. Schmidt, K.N., Gosselin, L.E. and Stanley, W.C. (1992) Endurance exercise training causes adrenal medullary hypetrophy in young and old Fischer 344 rats. Horm. Metab. Res., 24: 511-515. Schulkin, J., Gold, P.W. and Mcewen, B.S. (1998) Induction of corticotropin-releasing hormone gene expression by glucocorticoids: implication for understanding the states of fear and anxiety and allostatic load. Psychoneuroendocrinol., 23: 219-243. Seckl, J.R., Yau, J. and Holmes, M. (2002) llBeta-hydroxysteroid dehydrogenases: a novel control of glucocorticoid action in the brain. Endocr. Res., 28: 701-707. Shanks, N., Windle, R.J., Perks, P.A., Harbuz, M.S., Jessop, D.S., Ingram, C.D. and Lightman, S.L. (2000) Early-life exposure to endotoxin alters hypothalamic-pituitary-adrenal function and predisposition to inflammation. Proc. Natl. Acad. Sci. USA, 97: 5645-5650.
111
Sinyor, D., Schwartz, S.G., Peronnet, F., Brisson, G. and Seraganian, P. (1983) Aerobic fitness level and reactivity to psychosocial stress: physiological, biochemical, and subjective measures. Psychosom. Med., 45: 205-217. Sloviter, R.S., Valiquette, G., Abrams, G.M., Ronk, E.C., Sollas, A.L., Paul, L.A. and Neubort, S. (1989) Selective loss of hippocampal granule cells in the mature rat brain after adrenalectomy. Science, 243: 535-538. Smith, G.W., Aubry, J.-M., Dellu, F., Contarino, A., Bilezikijan, L.M., Gold, L.H., Chen, R., Marchuk, Y., Hauser, C., Bentley, C.A., Sawchenko, P.E., Koob, G.F., Vale, W. and Lee, K.-F. (1998) Corticotropin releasing factor receptor 1-deficient mice display decreased anxiety, impaired stress response, and aberrant neuroendocrine development. Neuron, 20:1093-1102. Smythe, J.W., Murphy, D., Timothy, C. and Costall, B. (1997) Hippocampal mineralocorticoid, but not glucocorticoid, receptors modulate anxiety-like behavior in rats. Pharmacol. Biochem. Behav., 56: 507-513. Sothmann, M.S., Buckmworth, J., Claytor, R.P., Cox, R.H., White-Welkey, J.E. and Dishman, R.K. (1996) Exercise training and the cross-stressor adaptation hypothesis. Exerc. Sport Sci. Rev., 24: 267-287. Spencer, R.L., Young, E.A., Choo, P.H. and McEwen, B.S. (1990) Adrenal steroid type-I and type-II receptor bindingestimates of in vivo receptor number, occupancy, and activation with varying level of steroid. Brain Res., 514: 3748. Stallknecht, B., Kjaer, M., Ploug, T., Maroun, L., Ohkuwa, T., Vinten, J., Mikines, K.J. and Galbo, H. (1990) Diminished epinephrine response to hypoglycemia despite enlarged adrenal medulla in trained rats. Am. J. Physiol., 259: R998-R1003. Starzec, J.J., Berger, D.F. and Hesse, R. (1983) Effects of stress and exercise on plasma corticosterone, plasma cholesterol and aortic cholesterol levels in rats. Psychosom. Med., 45: 219-226. Stenzel-Poore, M.P., Heinrichs, S.C., Rivest, S., Koob, G.F. and Vale, W.W. (1994) Overproduction of corticotropinreleasing factor in transgenic mice: a genetic model of anxiogenic behavior. J. Neurosci., 14: 2579-2584. Steptoe, A., Edwards, S., Moses, J. and Mathews, A. (1989) The effects of exercise training on mood and perceived coping ability in anxious adults from the general population. J. Psychosom. Res., 33: 537-547. Sullivan, R.M. and Gratton, A. (1999) Lateralized effects of medial prefrontal cortex lesions on neuroendocrine and autonomic stress responses in rats. J. Neurosci., 19: 2834-2840. Sullivan, R.M. and Gratton, A. (2002) Prefrontal cortical regulation of hypothalamic-pituitary-adrenal function in the rat and implications for psychopathology: side matters. Psychoneuroendocrinol., 27: 99-114.
Szigethy, E., Conwell, Y., Forbes, N.T., Cox, C. and Caine, E.D. (1994) Adrenal weight and morphology in victims of completed suicide. Biol. Psychiatry, 36: 374-380. Takagishi, M. and Chiba, T. (1991) Efferent projections of the infralimbic (area 25) region of the medial prefrontal cortex in the rat: an anterograde tracer PHA-L study. Brain Res., 566: 26-39. Terreberry, R.R. and Neafsey, E.J. (1983) Rat medial frontal cortex: a visceral motor region with a direct projection to the solitary nucleus. Brain Res., 278: 245-249. Terreberry, R.R. and Neafsey, E.J. (1987) The rat medial frontal cortex projects directly to autonomic regions of the brainstem. Brain Res. Bull., 19: 639-649. Timpl, P., Spanagel, R., Sillaber, I., Kresse, A., Reul, J.M.H.M., Stalla, G.K., Blanquet, V., Steckler, T., Holsboer, F. and Wurst, W. (1998) Impaired stress response and reduced anxiety in mice lacking a functional corticotropin-releasing hormone receptor 1. Nature Genet., 19: 162-166. Trejo, J.L., Carro, E. and Torres-Aleman, I. (2001) Circulating insulin-like growth factor I mediates exercise-induced increases in the number of new neurons in the adult hippocampus. J. Neurosci., 21: 1628-1634. Vall~e, M., Mayo, W., Dellu, F., Le Moal, M., Simon, H. and Maccari, S. (1997) Prenatal stress induces high anxiety and postnatal handling induces low anxiety in adult offspring: correlation with stress-induced corticosterone secretion. J. Neurosci., 17: 2626-2636. Van den Berg, D.T.W.M., De Kloet, E.R., Van Dijken, H.H. and De Jong, W. (1990) Differential central effects of mineralocorticoid and glucocorticoid agonists and antagonists on blood pressure. Endocrinology, 126:118-124. Van den Berg, D.T.W.M., De Kloet, E.R. and De Jong, W. (1994) Central effects of mineralocorticoid antagonist RU28318 on blood pressure of DOCA-salt hypertensive rats. Am. J. Physiol., 267: E927-E933. Van der Kar, L.D., Piechowski, R.A., Rittenhouse, P.A. and Gray, T.S. (1991) Amygdaloid lesions: differential effect on conditioned stress and immobilization-induced increases in corticosterone and renin secretion. Neuroendocrinol., 54: 89-95. Van Eekelen, J.A.M., Jiang, W., De Kloet, E.R. and Bohn, M.C. (1988) Distribution of the mineralocorticoid and the glucocorticoid receptor mRNAs in the rat hippocampus. J. Neurosci. Res., 21: 88-94. Van Haarst, A.D., Oitzl, M.S. and De Kloet, E.R. (1997) Facilitation of feedback inhibition through blockade of glucocorticoid receptors in the hippocampus. Neurochem. Res., 22: 1323-1328. van Praag, H., Christie, B.R., Sejnowski, T.J. and Gage, F.H. (1999a) Running enhances neurogenesis, learning and longterm potentiation in mice. Proc. Natl. Acad. Sci. USA, 96: 13427-13431.
112 van Praag, H., Kempermann, G. and Gage, F.H. (1999b) Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nature Neurosci., 2: 266-270. Vazquez, D.M., Van Oers, H., Levine, S. and Akil, H. (1996) Regulation of glucocorticoid and mineralocorticoid receptor mRNAs in the hippocampus of the maternally deprived infant rat. Brain Res., 731: 79-90. Villaneueva, A.L., Schlosser, C., Hopper, B., Liu, J.H., Hoffman, D.I. and Rebar, R.W. (1986) Increased cortisol production in women runners. J. Clin. Endo. Metab., 63: 133-136. Warembourg, M. (1975) Radioautographic study of the rat brain and pituitary after injection of 3H dexamethasone. Cell Tissue Res., 161: 183-191. Warren, M.P. and Stiehl, A.L. (1999) Exercise and female adolescents: effects on the reproductive and skeletal systems. J. Am. Med. Womens Assoc., 54:115-120. Watanabe, T., Morimoto, A., Sakata, Y., Wada, M. and Murakami, N. (1991) The effect of chronic exercise on the pituitary-adrenocortical response in conscious rats. J. Physiol., 439: 691-699. Watanabe, T., Morimoto, A., Sakata, Y., Tan, N., Morimoto, K. and Murakami, N. (1992) Running training attenuates the ACTH responses in rats to swimming and cage-switch stress. J. Appl. Physiol., 73: 2452-2456. Wiegers, G.J. and Reul, J.M.H.M. (1998) Induction of cytokine receptors by glucocorticoids: functional and pathological significance. Trends Pharmacol. Sci., 19:317-321.
Wiegers, G.J., Labeur, M.S., Stec, I.E.M., Klinkert, W.E.F., Holsboer, F. and Reul, J.M.H.M. (1995) Glucocorticoids accelerate anti-T cell receptor-induced T cell growth. J. Immunol., 155: 1893-1902. Wiegers, G.J., Stec, I.E.M., Klinkert, W.E.F. and Reul, J.M.H.M. (2000) Glucocorticoids regulate TCRinduced elevation of CD4: functional implications. J. Immunol., 164: 6213-6220. Wiegers, G.J., Stec, I.E.M., Klinkert, W.E., Linthorst, A.C.E., and Reul, J.M.H.M., (2001) Bidirectional effects of corticosterone on splenic T-cell activation: critical role of cell density and culture time. Neuroendocrinol., 73: 139-148. Williams, B.M., Luo, Y., Ward, C., Redd, K., Gibson, R., Kuczaj, S.A. and McCoy, J.G. (2001) Environmental enrichment: effects on spatial memory and hippocampal CREB immunoreactivity. Physiol. Behav., 73: 649-658. Wittling, W. (2001) Brain asymmetry in the control of the stress response. In: Gainotti, G. (Ed.), Emotional Behavior and its Disorders. Elsevier Science B.V., Amsterdam, pp. 207-233. Wittling, W., Block, A., Schweiger, E. and Genzel, S. (1998) Hemisphere asymmetry in sympathetic control of the human myocardium. Brain Cogn., 38: 17-35. Young, E.A., Akana, S. and Dallman, M.F. (1990) Decreased sensitivity to glucocorticoid fast feedback in chronically stressed rats. Neuroendocrinol., 51: 536-542. Young, D., Lawlor, P.A., Leone, P., Dragunow, M. and During, M.J. (1999) Environmental enrichment inhibits spontaneous apoptosis, prevents seizures and is neuroprotective. Nature Med., 5: 448-453.
SECTION 2
Hypothalamic Hormones Involved in Stress Responsivity
This Page Intentionally Left Blank
T. Steckler, N.H. Kalin and J.M.H.M. Reul (Eds.) Handbook of Stress and the Brain, Vol. 15 ISBN 0-444-51173-3 Copyright 2005 Elsevier B.V. All rights reserved
CHAPTER 2.1
Novel CRF family peptides and their receptors" an evolutionary analysis Sheau Yu Teddy Hsu* Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, 300 Pasteur Drive, Room A344E, Stanford, California 94305-5317, USA Abstract: During the last decade, the availability of genomic databases has provided a resource where biological
questions can be addressed in an unprecedented manner. Based on searches of homologous sequences, paralogous t genes from individual species or orthologous t genes from different species can be identified. The function of paralogous or orthologous genes can be inferred or predicted based on their degree of similarity to known genes, thereby augmenting our understanding of the gene functions that coevolved during evolution. Recent studies on human genomic sequences have led to the discovery of novel type 2 corticotropin-releasing factor (CRF) receptor-selective agonists that are related to CRF by structural and functional characteristics. In addition, analysis of vertebrate genomes showed that the CRF peptide family in mammals includes four distinct genes, CRF, Urocortin 1, Urocortin 3/stresscopin (SCP), and Urocortin 2/stresscopin-related peptide (SRP). Phylogenetic analysis suggested that the origin of each of these peptides predated the separation of tetrapods and teleosts. It is likely that CRF family genes in modern vertebrates evolved from an ancestor gene that gave rise to the CRF/Urocortin 1 and Urocortin 3(SCP)/Urocortin 2(SRP) branches through a gene duplication event. These two ancestor genes then gave rise to additional paralogs through a second round of gene duplication. Each of these four genes is tightly conserved ranging from the > 96% identity found for CRF to the > 55% for Urocortin 3/SCP, thus suggesting that these peptides played essential signaling roles over the 550 million years of vertebrate evolution. The finding of type 2 CRF receptor-selective Urocortin 3/SCP and Urocortin 2/SRP not only provided an opportunity to understand the physiology regulated by these novel ligands but also that of related peptides. The present review focuses on the recent findings of selective CRF receptor ligands, the evolution of signaling molecules associated with the CRF pathway as well as the implications of a complete inventory of CRF family ligands, receptors, and binding proteins in genomes of different organisms. In addition, new findings on CRF receptor subtype-dependent functions derived from studies using Urocortin 3/SCP and Urocortin 2/SRP are discussed. Instead of the traditional analysis of single-gene function in endocrine research, the complete assembly of CRF-associated signaling molecules throughout evolution can provide an integrated view for understanding the physiology and pathophysiology of all CRF family peptides and their receptors, thereby providing new therapeutic approaches for the pathology associated with stress.
Introduction
eukaryotic model organisms include that of baker's yeast, Saccharomyces cerevisiae, the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, two species of puffer fish (Fugu rubripes and Tetraodon nigroviridis), mouse, and human. In addition, genome sequencing for dozens of other species are ongoing. The availability of these genome sequences provides an unprecedented opportunity to discover novel signaling molecules that have
Over the past decade, whole-genome sequencing projects for numerous organisms have been completed. Thus far, the completed genomes of *E-mail:
[email protected] tparalogous: genes evolved from gene duplication in a species; orthologous: genes evolved from speciation.
115
116 coevolved with diverse polypeptide hormones that were previously discovered using traditional approaches. During the 1950s, Guillemin and Rosenberg, and Saffran and Schally observed independently the presence of a factor in hypothalamic extracts (termed corticotropin-releasing factor (CRF) that could stimulate the release of adrenocorticotropic hormone (ACTH) from anterior pituitary cells in vitro (Guillemin, 1967; Peterson and Guillemin, 1974; Saffran and Schally, 1977; Furutani et al., 1983; Shibahara et al., 1983). Later, purification of this factor using ion exchange and high-performance liquid chromatography led to the isolation, synthesis, and characterization of a 41-amino acid hypothalamic ovine CRF (Spiess et al., 1981; Vale et al., 1981). This original work paved the way for numerous studies into the role of CRF as the predominant hypothalamic neuropeptide regulating adrenal glucocorticoid release via pituitary ACTH release, thus establishing the importance of the hypothalamuspituitary-adrenal (HPA) axis (Contarino et al., 1999; Dunn and Swiergiel, 1999; Swiergiel and Dunn, 1999; Turnbull et al., 1999; Muglia et al., 2000; Muller et al., 2000). The characterization of CRF was followed in the 1980s by the identification of the related peptide ligands urotensin I from teleost and sauvagine from amphibian, and in 1995, Urocortin 1 from mammals (Vaughan et al., 1995). Urotensin-I, originally characterized by Lederis and his associates in 1982 (Ichikawa et al., 1982; Lederis et al., 1982, 1983), is a structural homolog of CRF and shares similar biological properties. However, piscine urotensin-I, produced in the caudal neurosecretory system (urophysis), stimulates the release of glucocorticoids directly from the interrenal gland, apparently bypassing the pituitary. Another related peptide, sauvagine, isolated from the serous glands of frog, Phyllomedusa sauvagei, is 50% identical to CRF/ urotensin I and could be important for defense in some anurans. Because Urocortin 1 possesses approximately 50-60% sequence identity with urotensin-I, it has been proposed that Urocortin 1 and its anuran homolog, sauvagine, represent an orthologous peptide to the piscine urotensin-I in tetrapod lineage (Vaughan et al., 1995). The structural organization of all these CRF family genes is similar and all
prepropeptides could be divided into five functional segments: a signal peptide for secretion, an N-terminal prepropeptide, conserved proteolytic sites, an alpha-helical-forming bioactive peptide, and a C-terminal amidation donor residue. Studies on the solution structure of CRF using proton nuclear magnetic resonance spectroscopy suggest that CRF and related peptides comprise an N-terminal coil connected to an extended helix (Lau et al., 1983). In addition to similar secondary structures, all family proteins appear to share similar posttranslational modification features required for the generation of functional mature peptides; all mature peptides have a free N-terminus and are amidated at the C-terminus. The biological actions of CRF and Urocortin 1 are mediated via binding to two G protein-coupled receptors (GPCRs), type 1 and type 2 CRF receptors (CRF1 and CRF2) found throughout the CNS, and periphery (Potter et al., 1991, 1994; Chen et al., 1993; Vita et al., 1993; Suman-Chauhan et al., 1999). The two receptors are closely related and are members of the family of "brain-gut" neuropeptide receptors that include receptors for calcitonin, CGRP, vasoactive intestinal peptide, parathyroid hormone, pituitary adenylate cyclase-activating peptide, growth hormone-releasing factor, CRF, and secretin. These receptors belong to a subset of GPCRs, referred to as the class B family that also includes the receptors for other brain-gut neuropeptides found in vertebrates and invertebrates. Each of the two CRF receptor genes give rise to multiple-splicing variants (type I: cz, 13, Y; type II: a, 13) with distinct expression profiles and possibly alternative physiological function. Similar to a number of other type B GPCRs, ligand binding led to the activation of adenyl cyclase and cAMP production by CRF receptors. However, coupling of the CRF receptors to other G proteins has been reported (Dautzenberg and Hauger, 2002). In addition to CRF and Urocortin 1, recent studies based on structural and posttranslational characteristics as well as comparative genomic approaches have led to the identification of two mammalian CRF/ Urocortin l-like peptides, Urocortin 3/stresscopin (SCP) (Hsu and Hsueh, 2001; Reyes et al., 2001) and Urocortin 2/stresscopin-related peptide (SRP) (Hsu and Hsueh, 2001; Lewis et al., 2001), with limited sequence relatedness to other family peptides. Phylogenetic analysis and functional
117 characterization studies showed that these two novel peptides represent a distant evolutionary branch in the evolution of CRF family peptides and they emerged as early as CRF and Urocortin 1 during vertebrate evolution. Unlike known CRF family peptides, which activate both CRF1 and CRF2, Urocortin 3/SCP and Urocortin 2/SRP are selective type 2 CRF receptor agonists. In addition to having an important role in the regulation of the HPA axis, CRF receptors are directly involved in the regulation of diverse functions in the cardiovascular, endocrine, and immune systems. Due to the receptor selectivity of Urocortin 3/SCP and Urocortin 2/SRP, these peptides could be important in CRF2-mediated stress-coping responses for homeostasis maintenance in different tissues, thus giving the CRF receptor system a high flexibility and dynamic role in the adaptation to environmental challenges (Bale et al., 2000; Kishimoto et al., 2000; Hsu and Hsueh, 2001; Aggelidou et al., 2002). Because abnormal signaling by CRF receptors may contribute to the pathophysiology of stress-related disorders, abnormal regulation of Urocortin 3/SCP and Urocortin 2/SRP could be involved in the regulation of anxiety, depression, and cardiac and inflammatory disorders. Before genome sequencing, it was usually difficult to understand complex hormonal systems in an integrated manner, and scientists frequently resorted to the speculation that unexplained experimental results are due to the actions of unknown genes. With a complete inventory of CRF family peptides and their receptors, scientists will be able to move from a search approach in their investigations to a reconstructionistic mode.
Evolution of the CRF-associated signaling molecules
Evolution of the CRF family peptides- t w o independent evolutionary branches in vertebrates Four core genes in mammals and other vertebrates Recent analysis of genome sequences from both vertebrates and invertebrates provided a complete
repertoire of CRF family peptides. Studies based on both pairwise sequence comparison and phylogenetic profiling showed that there are a total of four unique CRF-related genes, CRF, Urocortin 1, Urocortin 3/ SCP, and Urocortin 2/SRP, in the genomes of different mammals (Fig. 1). Although it is impossible to exclude the possibility that there is an additional paralog that evolved at an accelerated pace in the mammalian genomes, thus avoiding the detection by the computational method, integrated analysis on the evolution of CRF-related peptides and their receptors indicated that such possibility is negligible. As will be discussed later, the sequences of both the CRF family ligands and CRF receptors are highly conserved and can be traced to invertebrates. For genes with such high conservation, the existence of additional paralogs with a similar function is unlikely. The four CRF family genes exhibited conservation in both sequence and function throughout the vertebrate lineage (Hsu and Hsueh, 2001; Lewis et al., 2001; Reyes et al., 2001). Among these family members, CRF has been isolated from different mammals, anurans, and fish (Lovejoy and Balment, 1999). The highly conserved CRF appeared to play a central role in the regulation of the HPA axis and to function as an important neuropeptide in the brains of all species studied. Studies on mice deficient for CRF showed that the stress-related activation of the HPA axis was absent in the mutant mice and these mice displayed increased locomotor activity (Dunn and Swiergiel, 1999; Swiergiel and Dunn, 1999) (Table 1). Urocortin 1 has also been cloned from multiple vertebrates including mouse, human, and sheep (Vaughan et al., 1995; Lovejoy and Balment, 1999). Earlier studies on the Urocortin 1 expression pattern in the brain suggested that the Urocortin 1 could be involved in the regulation of feeding, anxiety, and auditory processing. Indeed, studies on mice deficient for the Urocortin 1 gene showed that mutant mice exhibit heightened anxietylike behaviors in the elevated plus maze and openfield tests (Vetter et al., 2002; Wang et al., 2002). In addition, these mutant mice display an impaired acoustic startle response at the level of the inner ear, suggesting that Urocortin 1 is involved in the normal development of cochlear sensory-cell function and hearing (Vetter et al., 2002). The effect of Urocortin 1
118
hCRH mCRH sCRH1 sCRH2 pCRH hUrocortin mUrocortin pUrotensin I
gUrotensin I fsauvagine
SEEP',..ISLBFHLLP~VLEMARAEQLAQQAI'~NRKLME .. II SEEPI ISL~FHLL~EVLEMARAEQLAQQAHSNRKLME I I SEEP ISLiFHLLR~VLEMARAEQLAQQAHSNRKMMEIF SEEP ISL~FHLL~EVLEMARAEQLVQQAHSNRKMMEIF SEDP ISL~FKLLR~MMEMSKAEQLAQQAQNNRIMMELV
D P LSlmF"LL rLLE L A RTQS QRE RAEQNR I IFD SV
DDP~LS I ~ F H L L P rLLELARTQSQRERAEQNRI IFDSV S E E P ~ L S I i F H L L P KMIKE AKMENQKE QE L INRKLL D E F NDDP~ IS I ~ F H L L R ~MIE MARNENQRE QAGLNRKYL D EV QGPP~I'SI~LELL~ KMIE IEKQEKEKQQAANNRLLLDTI
thDH
RMP S~S I DLPnS~ L RQKLSLE KERKVHALRA~NRI~FL ND I
acDH alDH
TGS GPSL-~;rV'~NPL~ ~LRQRLLLE IARRRI~RQSQDQ IQANRE ILQT I
hUROI I/SRP mURO I I/ SRP pUROI I/SRP hUROI I I/SCP mUROIII/SCP pUROIII/SCP
!
MGMGP SL S IVNPMDVLRQRLLLE IARRRLRDAE EQ IKANKDFLQQI
V~LSL~IGILLRILLEQARYKAA~QAXTNAQILAHV S R F ~ S L . TI IL SVL IDLAKNQDMRS KAAANAELMAR I
TKF _"x_-S,I-~ IMNILFN IDKA~LRAKAAANAQLMAQI S RLTLS L ~ ~ I ~ L F DV AKAKNL RAKA AENARLL AH I
Fig. 1. Sequence alignment of vertebrate and invertebrate CRF/diuretic hormone family peptides. There are a total of four unique CRF-related genes, CRF, Urocortin 1, Urocortin 3/SCP, and Urocortin 2/SRP, in the genomes of different mammals. The insect diuretic hormone showed similarity to CRF family peptides in both primary and secondary structures. The structural determining motif between the activation and binding domain of each subgroup of peptides are enclosed by lines, h: human; m: mouse; p: puffer fish (Fugu rubripes); s: sucker (Catostomus commersoni); g: goldfish (Carassius auratus); f: frog (P. sauvagei); th: tobacco hornworm (Manduca sexta); ac: american cockroach (Periplaneta Americana); al: African migratory locust (Locusta migratoria).
on the acoustic startle response could be mediated through the Urocortin 1-expressing neuron projections from the region of the Edinger-Westphal nucleus (Vetter et al., 2002; Wang et al., 2002). However, in comparison with CRF1- and CRF2deficient mice, Urocortin 1-deficient mice appeared to exhibit minimal alteration in anxiety-like behavior as well as autonomic regulation in response to stress. In contrast, sauvagine and urotensin I are found only in anuran and teleosts, respectively. Sauvagine and urotensin I are clearly related in structure to the C R F / U r o c o r t i n 1, but the peptides are found exclusively in specific tissues. The former was from the serosal gland of the skin of P. sauvagei, a frog native to Central and South America (Montecucchi
and Henschen, 1981; Negri et al., 1983), whereas the latter was purified from the urophysis of two teleost fish, the white sucker C. commersoni (Lederis et al., 1982) and the carp Cyprinus carpio (Ichikawa et al., 1982). Because these two peptides exhibit pharmacological and sequence characteristics similar to C R F , they likely derived from additional lineage-specific gene duplication (Fig. 2), one duplication in anuran to produce sauvagine and an independent duplication in teleost to generate urotensin I. Subsequent divergence of the promoter region of these newly duplicated genes then gave rise to the distinct expression profile of these paralogous genes. Unlike the better characterized family members that clustered in a branch of the evolutionary
119 hUROIII/SCP
E mUROIII/SCP pUROIII/SCP hUROII/SRP mUROII/SRP gUrotensin
I
pURO hURO mURO hCRH mCRH sCRHI sCRH2 pCRH
Fig. 2. Evolution of the CRF/diuretic hormone family peptides. The four vertebrate family peptides likely derived from a single ancestor gene that also gave rise to diuretic hormones in insects through gene duplication. A second round of gene duplication of the CRF/Urocortin 1and Urocortin 3(SCP)/ Urocortin 2(SRP) ancestors generated the four core ligand genes in vertebrates, h: human; m: mouse; p: puffer fish (F. rubripes); s: sucker (C. commersoni); g: goldfish (C. auratus). dendrogram, the newly identified Urocortin 3/SCP and Urocortin 2/SRP form a separate branch, suggesting that these genes emerged as early as the CRF/ Urocortin 1 branch. Similar to CRF and Urocortin 1, studies on Urocortin 3/SCP have shown that this gene is expressed in specific areas of the brain (Li et al., 2002). Urocortin 3/SCP-positive neurons were found predominately within the hypothalamus and medial amygdala. In the hypothalamus, Urocortin 3/ SCP neurons were observed in the median preoptic nucleus and in the rostral perifornical area lateral to the paraventricular nucleus (Li et al., 2002). In the periphery, it can be detected in multiple tissues including the cardiovascular system, the gastrointestinal tract, and the skin (Hsu and Hsueh, 2001; Lewis et al., 2001). As many of these tissues are known to express high levels of CRF2, Urocortin 3/SCP is likely to be an endogenous ligand for CRF2 in these areas and play a role in mediating physiological functions, including food intake, gastrointestinal mobility, and neuroendocrine regulation (Li et al., 2002). In contrast, Urocortin 2/SRP is expressed in the rodent central nervous system, including stress-related cell groups in the hypothalamus
and brainstem, and peripheral tissues (Lewis et al., 2001; Reyes et al., 2001). Behaviorally, central Urocortin 2/SRP attenuates night time feeding, but with a time course distinct from that seen in response to CRF. In contrast to CRF, central Urocortin 2/ SRP failed to increase gross motor activity suggesting that Urocortin 2/SRP is involved in central autonomic and appetitive control, but not in generalized behavioral activation (Hsu and Hsueh, 2001; Reyes et al., 2001; Valdez et al., 2002). In addition, studies on the pufferfish Urocortin 3/SCP peptide suggested that the selective activation of CRF2 by Urocortin 3/ SCP and Urocortin 3/SRP is evolutionarily conserved and that physiology processes regulated by these two peptides are essential for stress regulation and adaptation in all vertebrates (Hsu and Hsueh, 2001).
Tracing of the CRF family peptides to the common ancestors of Insects and vertebrates In addition to vertebrate CRF family peptides, studies on insect diuretic hormones have shown that this group of highly conserved hormones exhibit sequence and structural similarity to vertebrate CRF family peptides. Diuretic hormones are essential for fluid secretion regulation by Malpighian tubules in insects and have been isolated from insects, including the tobacco hornworm, moth, and cockroach. The diuretic hormone peptides range from 40 to 47 amino acids and exhibit >18% sequence identity to vertebrate CRF family peptides. Based on GenBank sequence search, diuretic hormone homologs from the Drosophila and the Anopheles mosquito have also been deduced (Adams et al., 2000; Riehle et al., 2002). Although there is a possibility that the relationship between vertebrate CRF family peptides and insect diuretic hormones is a result of convergent sequence evolution, comparative analysis has shown that the ancestors of diuretic hormone and the diuretic hormone receptor likely coevolved to give rise to diuretic hormone/diuretic hormone receptor signaling in insects and the CRF receptor signaling in vertebrates. Assuming the diuretic hormone and CRF family peptides followed the same rate of evolution, all vertebrate CRF family peptides will have a similarity greater than that between the insect
120 diuretic hormone and the mammalian CRF family peptides.
CRF/Urocortin 1 and Urocortin 3(SCP)/ Urocortin 2(SRP) as two separate evolutionary branches with unique functional characteristics Although CRF/Urocortin 1 and Urocortin 3(SCP)/ Urocortin 2(SRP) have been characterized as family peptides for CRF receptors, the presence of multiple ligands for the two CRF receptor subtypes in a given species raises the question as to how the corresponding genes coevolved. Because we can trace the origin of both the ligand and the receptor to invertebrates, the common theme of the coevolution of ligands and receptors suggests that CRF family peptides evolved from a single ancestral gene and all family peptides in vertebrates could be identified without ambiguity using the current analytical tools (Darlison and Richter, 1999). When all four groups of orthologous peptides from different vertebrates are compared with each other, it is clear that CRF/Urocortin 1 and Urocortin 3(SCP)/ Urocortin 2(SRP) have unique group-specific primary sequences and structural characteristics (Figs. 1 and 2). Because homologous genes for each of the four unique mammalian peptides, CRF, Urocortin 1, Urocortin 3/SCP, and Urocortin 2/SRP, can be found in species from human to teleost, the orthologous relationship of each gene among different vertebrates can be established unambiguously. As mentioned earlier, functional characterization indicates that the evolution of Urocortin 3(SCP)/Urocortin 2(SRP) is as ancient as CRF and Urocortin 1 and that the four core CRF family genes in modern vertebrates evolved from two sequential gene duplications of an ancient CRF-like gene (Fig. 2). The first duplication resulted in a CRF-like gene and a Urocortin 3/SCP-like gene, which subsequently evolved into four distinct genes and each of these paralogous genes persisted during evolution.
Evolution of the CRF receptor family Although less information is available regarding the sequences of CRF receptors in different vertebrates,
two subtypes of CRF receptors, CRF1 and CRF2, have been identified in human, rat, mouse, Xenopus, and multiple species of fish (Lovenberg et al., 1995; Liaw et al., 1996; Pohl et al., 2001). Sequence analysis showed that CRF receptors are highly conserved and the mammalian and fish CRF receptors exhibited a >50% sequence identity. Earlier studies have shown that although CRF and Urocortin 1 are capable of interacting with both Type I and Type II receptors from different species, CRF appears to have a greater potency on CRF1 whereas Urocortin 1 exhibited equivalent potency on both types of receptors. Based on anatomical distribution and the characteristics of ligand-receptor interactions, Urocortin 1 has been postulated to be an endogenous ligand for CRF2 and CRF mainly activates CRF1 to stimulate ACTH release and, consequently, glucocorticoid production from the adrenal cortex (Contarino et al., 1999; Turnbull et al., 1999; Bale et al., 2000; Coste et al., 2000; Kishimoto et al., 2000). Expression analyses in different vertebrates have shown that CRF1 is the major form of the CRF receptor expressed in the pituitary gland, whereas CRF2 is expressed in diverse tissues. In addition to differences in distribution between CRF1 and CRF2, there exists a distinct pattern of distribution between the receptor splicing variants (Valdenaire et al., 1997; Palchaudhuri et al., 1999; Suman-Chauhan et al., 1999; Pisarchik and Slominski, 2001, 2002). The CRF2~ isoforms primarily expressed within the CNS, where CRF2[3 isoforms are found both centrally and peripherally. Within the brain, CRF2~ is the predominant isoform, whereas the CRF2[3 is localized primarily to nonneuronal structures. Peripherally, the highest levels of CRF2[3 mRNA were found in heart and skeletal muscle, with lower levels detected in lung and intestine. The data suggest that different receptor variants could subserve specific physiological roles both centrally and peripherally (Palchaudhuri et al., 1999; Pisarchik and Slominski, 2001, 2002). The phenotypes of mice deficient in either CRF1 or CRF2 demonstrate the critical role these receptors play (Table 1). CRFl-mutant mice have an impaired stress response and display decreased anxiety-like behavior (Smith et al., 1998; Timpl et al., 1998), whereas CRF2-mutant mice are hypersensitive to
121 Table 1. Mice deficient for CRF, Urocortin 1, CRFI, CRF2, or CRF-BP show different phenotypes in stress-related responses Phenotypes
Gene CRF
Anxiety and hypothalamic-pituitaryadrenal (HPA) axis
Absence of stress-related activation of the HPA axis Urocortin 1 Normal stress-related activation of the HPA axis CRF1 Impaired HPA axis CRF2 Heightened stress-related activation of the HPA axis CRF-BP Normal HPA axis function CRF1 + CRF2 Greater impairment of the HPA axis response as compared to the single receptor knockout
Stress-related behavioral responses
Unique phenotype
Increased locomotor activity Heightened anxiety-like behaviors
Impaired acoustic startle response
Decreased anxiety-like behavior Increased anxiety-like behavior; hypersensitive to stress Increased anxiogenic-like behavior Similar to CRFl-deficient mice but shows sexual dichotomy
stress and display increased anxiety-like behavior under select conditions (Bale et al., 2000; Coste et al., 2000; Kishimoto et al., 2000). Therefore, it appears that C R F 2 mediates a central anxiolytic response, opposing the anxiogenic effect of C R F mediated by CRF1. However, double-mutant mice deficient in both CRF1 and C R F 2 displayed an even greater impairment of their HPA axis response to stress than that of the C R F l - m u t a n t mice, suggesting that both CRF1 and C R F 2 have critical roles in the regulation of the H P A axis and the maintenance of homeostasis in response to stress (Smith et al., 1998; Timpl et al., 1998; Contarino et al., 1999; Turnbull et al., 1999; Bale et al., 2000; Coste et al., 2000; Kishimoto et al., 2000). In addition, studies on mice deficient in both CRF1 and C R F 2 showed that the role of these receptors is gender specific. Although the female double-mutant mice displayed anxiolytic-like behavior, the male double-mutant mice showed significantly more anxiety-like behavior. C R F receptors from different species share a characteristics long N-terminal extracellular domain containing a number of cysteines that are important in ligand binding. Studies on nonmammalian species have identified two C R F receptor homologs closely related to the mammalian CRF1 and CRF2, respectively, from Xenopus laevis (Dautzenberg et al., 1997) and chun salmon, Oncorhynchus keta (Pohl et al., 2001). Like the mammalian receptors, both types of receptors from nonmammalian vertebrates
do not differentiate the C R F branch of ligands; all CRF-like peptides including CRF, urotensin I, sauvagine, and Urocortin 1 can activate both nonmammalian receptors (Dautzenberg et al., 1997; Pohl et al., 2001). Similar to X. laevis and chun salmon, studies of the puffer fish genome showed that F. rubripes encodes two C R F receptor homologs (type 1 C R F receptor: SINFRUP00000079341; type 2 C R F receptor: CAAB01000850.1)with greater than 81% and 80% similarity to human CRF1 and CRF2, respectively (Fig. 3). In addition, studies on C R F receptors in a diploid catfish, Ameiurus nebulosus, have also been reported (Arai et al., 2001). Unlike all other species studied, the Ameiurus catfish encodes two distinct mammalian CRF1 homologs and a single C R F 2 ortholog (Arai et al., 2001). The first catfish CRF1 is highly expressed in the brain and its distribution pattern correlates with that of mammalian CRF1, whereas the second CRF1 homolog (CRF3) is mainly expressed in the pituitary gland, urophysis, and brain. In contrast, the catfish C R F 2 is most abundantly expressed in the atrium of the heart, reminiscent to that of mammalian CRF2. Therefore, there has been extensive conservation in the expression of the two types of C R F receptors in different organs during evolution. Because the two CRF1 homologs (CRF1 and CRF3) found in catfish showed greater similarity to each other than other teleos C R F receptor homologs, the additional CRFl-like gene (CRF3) found in the catfish
122 hCRFI
mCRFI cCRFI
pCRFI hCRF2
cCRF2 pCRF2
Fig. 3. Two distinct CRF receptors are conserved from pufferfish F. rubripes to human. Similar to other vertebrates, pufferfish encode two CRF receptors (type 1: SINFRUP00000079341; type 2: CAAB01000850.1) homologous to the mammalian CRF1 and CRF2, respectively. Human and fish CRF1 are clustered in one branch separate from the human and fish CRF2 homologs, h: human; m: mouse; p: puffer fish (F. rubripes); c: catfish (A. nebulosus). likely derived from an additional gene duplication of the ancestral CRF1 gene. Future studies on the functional characteristics of teleost Urocortin 3/SCP and Urocortin 2/SRP peptides and their native receptors should reveal whether the nonmammalian CRF receptors could differentiate Urocortin 3/SCP and Urocortin 2/SRP as found in mammalian receptors. As described earlier, the presence of close sequence homologs in insects provides support for understanding the evolution of the CRF family peptides and the coevolved CRF receptor signaling system. Similar to the vertebrate CRF family peptides, insect diuretic hormones mediate the action through a type B GPCR homologous to the mammalian CRF receptors (Reagan, 1994, 1995). The orthologous relationship between the vertebrate CRF receptors and the insect diuretic hormone receptors is obvious because the insect diuretic hormone receptors have the closest sequence homology to CRF receptors as compared to all known GPCRs. In insects, the diuretic hormone receptors activated adenylyl cyclase and the protein kinase A-dependent pathway in target tissues, the Malpighian tubules (Reagan, 1994, 1995, 1996; Wiehart et al., 2002). Of interest, functional studies using the Malpighian tubules
have actually shown that vertebrate peptides including sauvagine and CRF have a significant effect on cAMP production in insect cells (Audsley et al., 1995, 1997). Analysis of recently completed insect genomes showed that the Anopheles mosquito and Drosophila encode orthologs of the diuretic hormone and the diuretic hormone receptors that have been identified in other insects. The Anopheles mosquito and Drosophila encoded one (agCP6328) and two (CG8422-PA and CGG12370-PA) diuretic hormone receptor homologs, respectively, sharing a >60% amino acid identity with known cockroach and house cricket diuretic hormone receptors. Important, the two Drosophila diuretic hormone receptors showed distinct similarity to mammalian CRF1 (51% similarity) and CRF2 (57% similarity), respectively. These data provide a clear evolutionary trail for the origin of the CRF receptor signaling system from invertebrates to vertebrates. They also indicate that the divergence of the two CRF receptor subtypes may have taken place prior to the emergence of vertebrates. Because the diuretic hormone receptor represents one of the few type B GPCRs found in insects, CRF receptors, and diuretic hormone receptor likely derived from a common ancestral gene in invertebrates and represents one of the earliest forms of type B GPCR signaling.
Evolution of the CRF-binding protein
(CRF-BP) The biological activity of CRF and Urocortin 1 has been shown to be modulated by a secreted glycoprotein, the CRF-BP (Potter et al., 1991; Behan et al., 1995; Seasholtz et al., 2002). CRF-BP binding to CRF and Urocortin 1 leads to the sequestration of bioactive peptides and the inhibition of CRF-induced ACTH release by pituitary cells in vitro (Behan et al., 1995, 1996b; Cortright et al., 1995). Studies on mice deficient in CRF-BP showed that mutant mice exhibit a normal increase in ACTH and corticosterone after restraint stress; however, mutant mice exhibit a significant increase in anxiogenic-like behavior and a significant reduction of body weight in males (Karolyi et al., 1999). The increased anorectic and anxiogenic-like behavior likely is a result of increased "free" CRF and/or Urocortin 1 levels in the brain
123 (Seasholtz et al., 2002). Thus, CRF-BP plays an important role in maintaining appropriate levels of CRF and Urocortin 1 in the central nervous system (Karolyi et al., 1999; Seasholtz et al., 2002). Of interest, studies have shown that CRF-BP does not show appreciable interaction with Urocortin 3/SCP and Urocortin 2/SRP (Lewis et al., 2001), suggesting that the interaction between CRF-BP and the ligands diverged for peptides in the CRF/Urocortin 1 and Urocortin 3(SCP)/Urocortin 2(SRP) branches. The lack of constraint from CRF-BP could be important for the full action of Urocortin 3(SCP)/Urocortin 2 (SRP) during stress regulation. CRF-BP has been characterized from diverse vertebrates including human, mouse, sheep, rat, and Xenopus (Behan et al., 1993, 1995, 1996a; Valverde et al., 2001). Like CRF receptors, CRF-BPs from different vertebrates are highly conserved. For example, Xenopus and human CRF-BPs share >78% identity. A search of GenBank sequences showed that CRF-BP is a single copy gene in mammals and is unique among known genes. Analyses of GenBank databases also indicated that, unlike mammalian genomes, the pufferfish encodes two copies of the CRF-BP homologs (SINFRUP000000086841 and SINFRUP000000069650) showing a >70% similarity to mammalian CRF-BP. Because the two pufferfish CRF-BPs showed a divergence similar to that between mammalian and piscine CRF-BPs, they likely derived from a gene duplication event early in teleost evolution. The pufferfish CRF-BP homologs share the ten cysteine residues that have been shown to be essential for forming five disulfide bridges and maintaining the binding activity of mammalian CRF-BP (Fischer et al., 1994). Great conservation of sequence and structural characteristics in the piscine CRF-BP homologs suggest that these polypeptides could have a functional characteristic similar to the CRF-BP in mammals wherein they function as a binding protein for urotensin I and CRF, but not the piscine Urocortin 3/SCP and Urocortin 2/SRP.
Functional evolution of the CRF receptor signaling pathway The broad and overlapping distribution of CRF family peptides and their receptors have confounded
our view of CRF signaling in different tissues. Thus, it seems appropriate to investigate the origin of CRF function with a view toward identifying the divergent nature of this signaling pathway. In view of the fact that CRF receptors and their ligands show higher conservation when compared to most other peptide hormone/GPCR signaling systems during the last one billion years of evolution, this system likely maintained many similar functional features in different lineages. As illustrated by the study of the insect diuretic hormone system, the CRF receptor originated as a paracrine signaling system important for osmoregulation. In lower vertebrates, CRF receptor signaling pathway assumed additional functions. The hypothalamic CRF control of the pituitary gland in teleosts has been well established. In addition to the hypothalamic-hypophyseal system, fish possess a lineage-specific neurosecretory organ, urophysis, which secretes urotensin I. In fish, both the pituitary CRF and the urophysis urotensin I have been implicated in the regulation of osmoregulation, ionic balance, and vasodilation (Mainoya and Bern, 1982; Bern et al., 1985; Lenz et al., 1985). Although data is not available on hormones and receptors in primitive chordates, the presence of multiple CRF family ligands and receptors in modern vertebrates suggests that gene duplications and the subsequent divergence of the regulatory mechanism of these paralogous genes provide an advantage as vertebrates' niches evolved during evolution. Other than having a role in osmoregulation and vascular homeostasis, CRF family peptides play major roles in the stress responses of teleosts. Similar to that observed in the mammalian system, handling and confinement stress increased the plasma cortisol and POMC peptides within minutes. CRF and urotensin I have been reported to coexist in the fish hypothalamus and both exert ACTH-stimulating action on the pituitary, reminiscent to that found in mammals. Interestingly, CRF has been shown to regulate metamorphosis in response to pond drying in some amphibian species (Denver, 1997; Boorse and Denver, 2002), whereas CRF-BP is closely regulated by the thyroid hormone during tail resorption in the frog (Brown et al., 1996). Though the tripeptide thyrotropin-releasing hormone is active on TSH secretion in adults, CRF seems to assume the thyrotropin-releasing hormone activity in amphibian
124 larvae, suggesting that the CRF function could include the thyroid axes for coordinating the metamorphosis program during the transition from an aquatic environment to land. Although this role may seem to be unrelated, it is consistent with the role of CRF family peptides as osmoregulators and as a stress transducer between the environment and the physiological responses of an organism (Denver, 1997; Boorse and Denver, 2002). Thus, CRF family peptides are phylogenetically ancient developmental signaling molecule that allows developing organisms to coordinate physiological responses in a changing environment (Denver, 1997, 1999; Boorse and Denver, 2002). In addition, the presence of sauvagine and urotensin I in specific secretory tissues of lower vertebrates as paralogs of CRF illustrates the advantage of divergent evolution even though the number of signaling molecular modules remains similar. Recent comparative genomic analyses of gene families across different species have provided insight into the evolution and associated adaptation of different hormonal regulatory circuits (Sherwood et al., 2000; Leo et al., 2002; Riehle et al., 2002). It has been hypothesized that diverse hormones and receptors coevolved during evolution and the vertebrate endocrine mechanism rooted in invertebrate paracrine signaling system. Although experimental evidence is required for the functional assignment of genes isolated from different species, the conservation of gene function in different phylogenies and networks has provided a solid foundation for translating gene function from one species to another. The identification of the two evolutionarily ancient CRF family peptides, Urocortin 3/SCP and Urocortin 2/SRP, in different vertebrates provides critical elements for the future characterization of the physiology associated with CRF receptors and the better characterized CRF and Urocortin 1.
New findings derived from studies of the CRF2-selective ligands, Urocortin 3/SCP and Urocortin 2[SRP As CRF and Urocortin 1 possess high affinity for CRF1 and CRF2 as well as the CRF-BP at physiological concentrations, in vivo physiological
studies with these peptides produce many confounding observations. As mentioned earlier, recent characterization of CRF1 and CRF2 in mutant mice have revealed a far-reaching complexity and physiological importance for the CRF family peptides (Dautzenberg and Hauger, 2002). It has become clear that adaptive responses induced by stressors are mediated by the autonomic nervous system and two interrelated and somewhat antagonistic CRF receptor pathways (Steckler, 2004). CRF 1 is important for the initial flight and fight response whereas CRF2 could be essential for the delayed stress-coping responses prompted by the initial stress inducer. The cloning of Urocortin 3/SCP and Urocortin 2/SRP genes clarified many misconceptions and provided a unique opportunity in the study of stress regulation by CRF receptors. The ancient origin of these CRF2-selective ligands indicates that the prevalent view of stress regulation by the CRF unintentionally tilted to the side of stress induction and much less on the equally important stress-coping responses (Steckler, 2004). Although it is expected that future studies on Urocortin 3/SCP and Urocortin 2/SRP may reveal an undescribed stresscoping network in humans and improve the understanding of stress regulation and environmental adaptation in general, recent studies using these two peptides have unambiguously dissected the critical CRF receptor subtypes involved in a variety of CRFdependent functions (Steckler, 2004). These studied are summarized in the following section (Table 2).
CRFl-dependent functions A CTH release from the pituitary Earlier studies on CRF receptor expression and pharmacological analogs suggested that CRF is the main mediator for pituitary ACTH release through the CRF1 and that CRF/CRF1 signaling is important for proper function of the HPA. Consistent with this hypothesis, deletion of CRF or CRF1 led to altered HPA axis responses in mutant mice (Swiergiel and Dunn, 1999; Turnbull et al., 1999; Muglia et al., 2000) (Table 1). Studies on the effect of Urocortin 3/SCP and Urocortin 2/SRP on pituitary cells indicated that these CRF2-selective
125 Table 2. CRF receptor subtype-dependent functions CRFl-dependent functions
CRF2-dependentfunctions
1. ACTH release from pituitary 2. Protection of hippocampal neurons from excitotoxic insults 3. Regulation of locomotor activation 4. Regulation of colon mobility 5. Regulation of myometrial relaxation/contractility
1. Regulation of gastric mobility and food intake 2. Regulation of vasculature homeostasis
agonists have negligible stimulation on the secretion of ACTH from anterior pituitary both in vivo and in vitro (Hsu and Hsueh, 2001; Lewis et al., 2001; Reyes et al., 2001), confirming the selective property of these ligands in vivo and that CRF1 is the main mediator of pituitary ACTH release.
Regulation of locomotor activation It was thought that CRF1 and CRF2 mediate CRF actions in the central nervous system and that both could be involved in locomotor behaviors. Studies on CRF1 null mice showed that the CRF treatment resulted in increased levels of locomotion in wild type mice, whereas no change was observed in CRF1 null mice (Contarino et al., 2000). Likewise, mice deficient for CRF exhibited altered locomotor activity (Dunn and Swiergiel, 1999; Swiergiel and Dunn, 1999). This hypothesis was supported by studies showing that, in contrast to CRF, central administration of Urocortin 2/SRP failed to increase gross motor activity (Reyes et al., 2001). In fact, Urocortin 2/SRP actually mildly suppressed locomotor activity during the inactive phase (Reyes et al., 2001; Valdez et al., 2002). Therefore, the activational effects of CRF on locomotor activity observed in many earlier studies are mediated by the CRF1.
Protection of hippocampal neurons from excitotoxic insults Hippocampus neurons are vulnerable to damage during disease and stress conditions, including
cerebral ischemia and anxiety disorders. Although both CRF1 and CRF2 are present in the hippocampal regions of mammalian brain, CRF and Urocortin 1 protect hippocampal neurons from insults whereas Urocortin 2/SRP is ineffective (Pedersen et al., 2002). These studies clearly demonstrated that the neuroprotective effect of CRF and urocortin on the hippocampal neurons to oxidative and excitotoxic insults is mediated by CRF1 (Pedersen et al., 2002).
Regulation of colon mobilio, Peripheral CRF has been shown to stimulate colonic motor function and inhibit gastric emptying in different animals. Earlier studies have shown that CRF-related peptides exert similar activity on gastrointestinal function (Miampamba et al., 2002; Million et al., 2002; Saunders et al., 2002); however, it was not clear which type(s) of CRF receptor is important for colonic response. Studies using ovine CRF, a preferential CRF1 agonist, suggested that CRF1 could be important for the regulation of colon transit. In support of this hypothesis, colonic response was shown to be dose dependently blocked by the selective CRF1 receptor antagonists, NBI-27914 and CP154,526, but not the CRF2-selective peptide antisauvagine-30 (Martinez et al., 2002). To identify the exact receptor subtypes responsible for the regulation of colonic motor function, the effects of Urocortin 2/SRP on colon transit have been investigated in conscious rats. It was shown that Urocortin 2/SRP did not influence colonic transit whereas CRF stimulated distal colonic transit motor activity. Thus, the stimulation of colonic propulsive activity involves CRF1 and the nonselective ligands, CRF and Urocortin 1 (Miampamba et al., 2002; Saunders et al., 2002).
Regulation of myometrial relaxation~contractility It has been well established that CRF modulates myometrial relaxation/contractility and stimulates the nitric oxide system (Rivier, 2001). CRF caused increased basal or atrial natriuretic peptide-stimulated cGMP production and this mechanism may be operational during human pregnancy. Studies using receptor-selective antagonists have shown that
126 treatment of the nonselective antagonists, astressin and antalarmin, but not the CRF2-selective antagonist antisauvagine 30, blocks the CRF-stimulated cGMP production (Aggelidou et al., 2002). Consistent with this observation, incubation of myometrial cells with CRF, but not Urocortin 3/ SCP or Urocortin 2/SRP, induced mRNA and protein expression of different nitric oxide synthase isoforms (Aggelidou et al., 2002), indicating that during pregnancy signaling of CRF1, but not CRF2, contributes to the maintenance of myometrial quiescence (Aggelidou et al., 2002).
CRF2-dependentfunctions Regulation of gastric mobility and food intake Peripheral treatment with CRF/Urocortin 1 peptides inhibits gastric emptying and food intake in different mammals. Unlike colon motor activity that is mainly mediated by CRF1, studies have shown that the nonselective antagonist astressin B and the selective CRF2 antagonist antisauvagine-30 dose dependently antagonized CRF- or Urocortin 1-induced delayed gastric emptying action, whereas the selective CRF1 antagonists, NBI-27914 and CP-154,526, had no effect (Wang et al., 2001; Chen et al., 2002). Likewise, Urocortin 1-induced hypophagia was partially antagonized by antisauvagine-30 whereas the CRF1 antagonists, CP-154,526 and DMP904, had no effect (Wang et al., 2001). These data suggest that peripheral CRF/Urocortin 1-induced suppression of gastric emptying involves primarily CRF2 whereas the action on feeding is only partly mediated by CRF2 (Wang et al., 2001). Consistent with these data, studies on CRFl-deficient mice show that CRF decreased food intake equally in both wild type and CRFl-deficient mice (Contarino et al., 2000). Studies using Urocortin 3/SCP and Urocortin 2/SRP have shown that both peptides dose dependently inhibited gastric emptying and food intake (Hsu and Hsueh, 2001; Lewis et al., 2001; Reyes et al., 2001; Miampamba et al., 2002; Million et al., 2002; Saunders et al., 2002), reinforcing the hypothesis that peripheral effects of CRF family peptides on gastric mobility and appetite suppression are mediated through CRF2. Therefore, peripheral CRF
family peptides induce opposite actions on upper and lower gut transit through different CRF receptor subtypes: the activation of CRF1 stimulates colonic propulsive activity whereas the activation of CRF2 inhibits gastric emptying (Martinez et al., 2002).
Regulation of vasculature homeostasis Since its discovery two decades ago, potent CRF/ Urocortin 1 effects on the vascular system have been consistently observed. The diverse cardiovascular effects included coronary vasodilation, enhanced cardiac contractility and heart rate, and the protection of cardiomyocytes from ischemia simulated by glucose deprivation, acidosis, or hypoxia reoxygenation injury (Terui et al., 2001; Aggelidou et al., 2002; Brar et al., 2002; Gordon et al., 2002; Schulman et al., 2002). In addition, it has been suggested that CRF1 mediates CRF-induced blood pressure elevation, whereas peripheral CRF2 mediates the hypotensive effect of systemically administered CRF and Urocortin 1. Because CRF2 has been shown to be the main receptor expressed in different chambers of the heart and vascular system, the CRF family peptides likely exert their effects through CRF2 in an endocrine or paracrine manner (Kimura et al., 2002). Furthermore, it has been shown that Urocortin 1 effects on ventricular performance, bioenergetics, and cell survival are not secondary to the inotropic effects of Urocortin 1, and the survival effect of CRF family peptides is likely direct (Scarabelli et al., 2002). Because Urocortin 1 expression has also been detected in the heart, it has been suggested that Urocortin 1 and CRF2 could form a peripheral cardiac CRF system important in mediating the adaptive responses of the heart to stress (Coste et al., 2002). Studies using Urocortin 3/SCP and Urocortin 2/SRP demonstrated that these peptides also have potent suppressive effects on paw edema formation (Hsu and Hsueh, 2001), suggesting a role in vascular homeostasis. Recent studied have also demonstrated that Urocortin 3/SCP and Urocortin 2/SRP could protect cardiomyocytes from injuries (Chanalaris et al., 2003; Brar et al., 2004), but would not provoke the unwanted fight or flight response mediated by the pituitary CRF1. Therefore, Urocortin 3/SCP and Urocortin 2/SRP represent novel cardioprotective
127 agents of therapeutic use in treating the damaging effects of cardiac ischaemia and associated diseases (Latchman, 2002).
Conclusions and future direction Traditional methods in genetics and molecular biology focused on the characterization of individual genes and their products, whereas the new genomic approach takes advantage of our knowledge concerning the totality of genes in diverse organisms. With the completion of a number of genomes of different species, it becomes feasible to identify orthologous genes belonging to the CRF-signaling pathway throughout eukaryotic evolution. One approach to understanding the role of different family peptides is to consider the basic functions of individual genes in the context of evolution. Current evidence suggests that the origin of CRF-related peptides and their receptors predates insects and vertebrates, and CRF/Urocortin 1 and Urocortin 3 (SCP)/Urocortin 2(SRP) represent the two parallel branches of functional paralogs (Steckler, 2004). Therefore, future integration of the current knowledge on the CRF physiology with that of Urocortin 3/ SCP and Urocortin 2/SRP is essential for the full understanding of stress regulation and the environmental adaptation in vertebrates. The array of effects triggered by CRF/Urocortin 1 span almost all systems in the body, including nervous, endocrine, vasculature, muscular, bone, and immune. The complexity resembles programs that coordinate different tissues during development and these interacting ligands and receptors could play roles in a circuit that ultimately coordinates stress adaptation and homeostasis in different tissues in response to environmental changes. In the short time since their discovery, Urocortin 3/SCP and Urocortin 2/SRP have facilitated the experimental differentiation of a number of CRF receptor subtype-dependent functions (Steckler, 2004). This information serves as a first step for understanding the physiological pathways and functional properties of Urocortin 3 (SCP)/Urocortin 2(SRP)/CRF2 signaling. However, the exact CRF receptor(s) involved in a variety of CRF/urocortin responses remains to be studied (Bamberger and Bamberger, 2000; Baigent, 2001;
Cullen et al., 2001; Schulman et al., 2002). Future studies using Urocortin 3/SCP and Urocortin 2/SRP will reveal the exact receptor type(s) important for the majority of CRF receptor-mediated responses and allow improved pharmacological treatments for diseases associated with abnormalities in CRF receptor action (Steckler, 2004). In addition, future generations of knockout mice in which Urocortin 3/ SCP or Urocortin 2/SRP is disrupted will add valuable insight to this research. Likewise, studies on the integrated expression of these ligands and receptors in a tissue-specific and time-coordinated manner are important for the full understanding of this GPCR-signaling pathway. Only then can we gain a complete understanding of the functional overlap in these closely related ligands and receptors.
Acknowledgments We thank Caren Spencer for editorial assistance and Dr. Aaron Hsueh for comments on the manuscript.
References Adams, M.D., Celniker, S.E., Holt, R.A., Evans, C.A., Gocayne, J.D., Amanatides, P.G., Scherer, S.E., Li, P.W., Hoskins, R.A., Galle, R.F., George, R.A., Lewis, S.E., Richards, S., Ashburner, M., Henderson, S.N., Sutton, G.G., Wortman, J.R., Yandell, M.D., Zhang, Q., Chen, L.X., Brandon, R.C., Rogers, Y.H., Blazej, R.G., Champe, M., Pfeiffer, B.D., Wan, K.H., Doyle, C., Baxter, E.G., Helt, G., Nelson, C.R., Gabor, G.L., Abril, J.F., Agbayani, A., An, H.J., Andrews-Pfannkoch, C., Baldwin, D., Ballew, R.M., Basu, A., Baxendale, J., Bayraktaroglu, L., Beasley, E.M., Beeson, K.Y., Benos, P.V., Berman, B.P., Bhandari, D., Bolshakov, S., Borkova, D., Botchan, M.R., Bouck, J., Brokstein, P., Brottier, P., Burtis, K.C., Busam, D.A., Butler, H., Cadieu, E., Center, A., Chandra, I., Cherry, J.M., Cawley, S., Dahlke, C., Davenport, L.B., Davies, P., de Pablos, B., Delcher, A., Deng, Z., Mays, A.D., Dew, I., Dietz, S.M., Dodson, K., Doup, L.E., Downes, M., Dugan-Rocha, S., Dunkov, B.C., Dunn, P., Durbin, K.J., Evangelista, C.C., Ferraz, C., Ferriera, S., Fleischmann, W., Fosler, C., Gabrielian, A.E., Garg, N.S., Gelbart, W.M., Glasser, K., Glodek, A., Gong, F., Gorrell, J.H., Gu, Z., Guan, P., Harris, M., Harris, N.L., Harvey, D., Heiman, T.J., Hernandez, J.R., Houck, J., Hostin, D., Houston, K.A., Howland, T.J., Wei, M.H., Ibegwam, C., et al. (2000) The genome sequence of Drosophila melanogaster. Science, 287: 2185-2195.
128 Aggelidou, E., Hillhouse, E.W. and Grammatopoulos, D.K. (2002) Up-regulation of nitric oxide synthase and modulation of the guanylate cyclase activity by corticotropin-releasing hormone but not urocortin II or urocortin III in cultured human pregnant myometrial cells. Proc. Natl. Acad. Sci. USA, 99: 3300-3305. Arai, M., Assil, I.Q. and Abou-Samra, A.B. (2001) Characterization of three corticotropin-releasing factor receptors in catfish: a novel third receptor is predominantly expressed in pituitary and urophysis. Endocrinology, 142: 446-454. Audsley, N., Goldsworthy, G.J. and Coast, G.M. (1997) Circulating levels of Locusta diuretic hormone: the effects of feeding. Peptides, 18: 59-65. Audsley, N., Kay, I., Hayes, T.K. and Coast, G.M. (1995) Cross reactivity studies of CRF-related peptides on insect malpighian tubules. Comp. Biochem. Physiol. A Physiol., 110: 87-93. Baigent, S.M. (2001) Peripheral Corticotropin-releasing hormone and urocortin in the control of the immune response. Peptides, 22: 809-820. Bale, T.L., Contarino, A., Smith, G.W., Chan, R., Gold, L.H., Sawchenko, P.E., Koob, G.F., Vale, W.W. and Lee, K.F. (2000) mice deficient for corticotropin-releasing hormone receptor-2 display anxiety-like behaviour and are hypersensitive to stress. Nat. Genet., 24: 410-414. Bamberger, C.M. and Bamberger, A.M. (2000) The peripheral CRH/urocortin system. Ann. N. Y. Acad. Sci., 917: 290-296. Behan, D.P., Cepoi, D., Fischer, W.H., Park, M., Sutton, S., Lowry, P.J. and Vale, W.W. (1996a) Characterization of a sheep brain corticotropin releasing factor binding protein (Brain Research 709 (1996) 265-274) (BRES 11959). Brain Res., 732: 267. Behan, D.P., De Souza, E.B., Lowry, P.J., Potter, E., Sawchenko, P. and Vale, W.W. (1995) Corticotropin releasing factor (CRF) binding protein: a novel regulator of CRF and related peptides. Front. Neuroendocrinol., 16: 362-382. Behan, D.P., De Souza, E.B., Potter, E., Sawchenko, P., Lowry, P.J. and Vale, W.W. (1996b) Modulatory actions of corticotropin-releasing factor-binding protein. Ann. N. Y. Acad. Sci., 780: 81-95. Behan, D.P., Potter, E., Lewis, K.A., Jenkins, N.A., Copeland, N., Lowry, P.J. and Vale, W.W. (1993) Cloning and structure of the human corticotrophin releasing factor-binding protein gene (CRHBP). Genomics, 16: 63-68. Bern, H.A., Pearson, D., Larson, B.A. and Nishioka, R.S. (1985) Neurohormones from fish tails: the caudal neurosecretory system. I. "Urophysiology" and the caudal neurosecretory system of fishes. Recent Prog. Horm. Res., 41: 533-552. Boorse, G.C. and Denver, R.J. (2002) Acceleration of ambystoma tigrinum metamorphosis by corticotropin-releasing hormone. J. Exp. Zool., 293: 94-98.
Brar, B.K., Jonassen, A.K., Egorina, E.M., Chen, A., Negro, A., Perrin, M.H., Mjos, O.D., Latchman, D.S., Lee, K.F. and Vale, W. (2004) Urocortin-II and urocortin-III are cardioprotective against ischemia reperfusion injury: an essential endogenous cardioprotective role for corticotropin releasing factor receptor type 2 in the murine heart. Endocrinology, 145: 24-35; discussion 21-3. Brar, B.K., Stephanou, A., Knight, R. and Latchman, D.S. (2002) Activation of protein kinase B/Akt by urocortin is essential for its ability to protect cardiac cells against hypoxia/reoxygenation-induced cell death. J. Mol. Cell Cardiol., 34: 483-492. Brown, D.D., Wang, Z., Furlow, J.D., Kanamori, A., Schwartzman, R.A., Remo, B.F. and Pinder, A. (1996) The thyroid hormone-induced tail resorption program during Xenopus laevis metamorphosis. Proc. Natl. Acad. Sci. USA, 93: 1924-1929. Chanalaris, A., Lawrence, K.M., Stephanou, A., Knight, R.D., Hsu, S.Y., Hsueh, A.J. and Latchman, D.S. (2003) Protective effects of the urocortin homologues stresscopin (SCP) and stresscopin-related peptide (SRP) against hypoxia/reoxygenation injury in rat neonatal cardiomyocytes. J. Mol. Cell Cardiol., 35: 1295-1305. Chen, C.Y., Million, M., Adelson, D.W., Martinez, V., Rivier, J. and Tache, Y. (2002) Intracisternal urocortin inhibits vagally stimulated gastric motility in rats: role of CRF(2). Br. J. Pharmacol., 136: 237-247. Chen, R., Lewis, K.A., Perrin, M.H. and Vale, W.W. (1993) Expression cloning of a human corticotropin-releasing-factor receptor. Proc. Natl. Acad. Sci. USA, 90: 8967-8971. Contarino, A., Dellu, F., Koob, G.F., Smith, G.W., Lee, K.F., Vale, W. and Gold, L.H. (1999) Reduced anxiety-like and cognitive performance in mice lacking the corticotropinreleasing factor receptor 1. Brain Res., 835: 1-9. Contarino, A., Dellu, F., Koob, G.F., Smith, G.W., Lee, K.F., Vale, W.W. and Gold, L.H. (2000) Dissociation of locomotor activation and suppression of food intake induced by CRF in CRFRl-deficient mice. Endocrinology, 141: 2698-2702. Cortright, D.N., Nicoletti, A. and Seasholtz, A.F. (1995) Molecular and biochemical characterization of the mouse brain corticotropin-releasing hormone-binding protein. Mol. Cell Endocrinol., 111: 147-157. Coste, S.C., Kesterson, R.A., Heldwein, K.A., Stevens, S.L., Heard, A.D., Hollis, J.H., Murray, S.E., Hill, J.K., Pantely, G.A., Hohimer, A.R., Hatton, D.C., Phillips, T.J., Finn, D.A., Low, M.J., Rittenberg, M.B., Stenzel, P. and Stenzel-Poore, M.P. (2000) Abnormal adaptations to stress and impaired cardiovascular function in mice lacking corticotropin-releasing hormone receptor-2. Nat. Genet., 24: 403-409. Coste, S.C., Quintos, R.F. and Stenzel-Poore, M.P. (2002) Corticotropin-releasing hormone-related peptides and receptors: emergent regulators of cardiovascular adaptations to stress. Trends Cardiovasc. Med., 12: 176-182.
129 Cullen, M.J., Ling, N., Foster, A.C. and Pelleymounter, M.A. (2001) Urocortin, corticotropin releasing factor-2 receptors and energy balance. Endocrinology, 142: 992-999. Darlison, M.G. and Richter, D. (1999) Multiple genes for neuropeptides and their receptors: co-evolution and physiology. Trends Neurosci., 22: 81-88. Dautzenberg, F.M., Dietrich, K., Palchaudhuri, M.R. and Spiess, J. (1997) Identification of two corticotropin-releasing factor receptors from Xenopus laevis with high ligand selectivity: unusual pharmacology of the type 1 receptor. J. Neurochem., 69: 1640-1649. Dautzenberg, F.M. and Hauger, R.L. (2002) The CRF peptide family and their receptors: yet more partners discovered. Trends Pharmacol. Sci., 23: 71-77. Denver, R.J. (1997) Environmental stress as a developmental cue: corticotropin-releasing hormone is a proximate mediator of adaptive phenotypic plasticity in amphibian metamorphosis. Horm. Behav., 31: 169-179. Denver, R.J. (1999) Evolution of the corticotropin-releasing hormone signaling system and its role in stress-induced phenotypic plasticity. Ann. N. Y. Acad. Sci., 897: 46-53. Dunn, A.J. and Swiergiel, A.H. (1999) Behavioral responses to stress are intact in CRF-deficient mice. Brain Res., 845: 14-20. Fischer, W.H., Behan, D.P., Park, M., Potter, E., Lowry, P.J. and Vale, W. (1994) Assignment of disulfide bonds in corticotropin-releasing factor-binding protein. J. Biol. Chem., 269: 4313-4316. Furutani, Y., Morimoto, Y., Shibahara, S., Noda, M., Takahashi, H., Hirose, T., Asai, M., Inayama, S., Hayashida, H., Miyata, T. and Numa, S. (1983) Cloning and sequence analysis of cDNA for ovine corticotropinreleasing factor precursor. Nature, 301: 537-540. Gordon, J.M., Dusting, G.J., Woodman, O.L. and Ritchie, R.H. (2002) Cardioprotective action of the CRF peptide urocortin against simulated ischemia in adult rat cardiomyocytes. Am. J. Physiol. Heart Circ. Physiol. Guillemin, R. (1967) The Adenohypophysis and its hypothalamic control. Annu. Rev. Physiol., 29: 313-348. Hsu, S.Y. and Hsueh, A.J. (2001) Human stresscopin and stresscopin-related peptide are selective ligands for the type 2 corticotropin-releasing hormone receptor. Nat. Med., 7: 605-611. Ichikawa, T., McMaster, D., Lederis, K. and Kobayashi, H. (1982) Isolation and amino acid sequence of urotensin I, a vasoactive and ACTH-releasing neuropeptide, from the carp (Cyprinus carpio) urophysis. Peptides, 3: 859-867. Karolyi, I.J., Burrows, H.L., Ramesh, T.M., Nakajima, M., Lesh, J.S., Seong, E., Camper, S.A. and Seasholtz, A.F. (1999) Altered anxiety and weight gain in corticotropinreleasing hormone-binding protein-deficient mice. Proc. Natl. Acad. Sci. USA, 96:11595-11600. Kimura, Y., Takahashi, K., Totsune, K., Muramatsu, Y., Kaneko, C., Darnel, A.D., Suzuki, T., Ebina, M., Nukiwa, T.
and Sasano, H. (2002) Expression of urocortin and corticotropin-releasing factor receptor subtypes in the human heart. J. Clin. Endocrinol. Metab., 87: 340-346. Kishimoto, T., Radulovic, J., Radulovic, M., Lin, C.R., Schrick, C., Hooshmand, F., Hermanson, O., Rosenfeld, M.G. and Spiess, J. (2000) Deletion of crhr2 reveals an anxiolytic role for corticotropin-releasing hormone receptor-2. Nat. Genet., 24: 415-419. Latchman, D.S. (2002) Urocortin. Int. J. Biochem. Cell Biol., 34:907-910. Lau, S.H., Rivier, J., Vale, W., Kaiser, E.T. and Kezdy, F.J. (1983) Surface properties of an amphiphilic peptide hormone and of its analog: corticotropin-releasing factor and sauvagine. Proc. Natl. Acad. Sci. USA, 80: 7070-7074. Lederis, K., Letter, A., McMaster, D., Ichikawa, T., MacCannell, K.L., Kobayashi, Y., Rivier, J., Rivier, C., Vale, W. and Fryer, J. (1983) Isolation, analysis of structure, synthesis, and biological actions of urotensin I neuropeptides. Can. J. Biochem. Cell Biol., 61: 602-614. Lederis, K., Vale, W., Rivier, J., MacCannell, K.L., McMaster, D., Kobayashi, Y., Suess, U. and Lawrence, J. (1982) Urotensin I - a novel CRF-like peptide in catostomus commersoni urophysis. Proc. West Pharmacol. Soc., 25: 223-227. Lenz, H.J., Fisher, L.A., Vale, W.W. and Brown, M.R. (1985) Corticotropin-releasing factor, sauvagine, and urotensin I: effects on blood flow. Am. J. Physiol., 249: R85-90. Leo, C.P., Hsu, S.Y. and Hsueh, A.J. (2002) Hormonal genomics. Endocr. Rev., 23: 369-381. Lewis, K., Li, C., Perrin, M.H., Blount, A., Kunitake, K., Donaldson, C., Vaughan, J., Reyes, T.M., Gulyas, J., Fischer, W., Bilezikjian, L., Rivier, J., Sawchenko, P.E. and Vale, W.W. (2001) Identification of urocortin III, an additional member of the corticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor. Proc. Natl. Acad. Sci. USA, 98: 7570-7575. Li, C., Vaughan, J., Sawchenko, P.E. and Vale, W.W. (2002) Urocortin III-immunoreactive projections in rat brain: partial overlap with sites of type 2 corticotrophin-releasing factor receptor expression. J. Neurosci., 22: 991-1001. Liaw, C.W., Lovenberg, T.W., Barry, G., Oltersdorf, T., Grigoriadis, D.E. and de Souza, E.B. (1996) Cloning and characterization of the human corticotropin-releasing factor-2 receptor complementary deoxyribonucleic acid. Endocrinology, 137: 72-77. Lovejoy, D.A. and Balment, R.J. (1999) Evolution and physiology of the corticotropin-releasing factor (CRF) family of neuropeptides in vertebrates. Gen. Comp. Endocrinol., 115: 1-22. Lovenberg, T.W., Liaw, C.W., Grigoriadis, D.E., Clevenger, W., Chalmers, D.T., De Souza, E.B. and Oltersdorf, T. (1995) Cloning and characterization of a functionally distinct corticotropin-releasing factor receptor subtype from rat brain. Proc. Natl. Acad. Sci. USA, 92: 836-840.
130 Mainoya, J.R. and Bern, H.A. (1982) Effects of teleost urotensins on intestinal absorption of water and nacl in tilapia, sarotherodon mossambicus, adapted to fresh water or seawater. Gen. Comp. Endocrinol., 47: 54-58. Martinez, V., Wang, L., Rivier, J.E., Vale, W. and Tache, Y. (2002) Differential actions of peripheral corticotropin-releasing factor (CRF), Urocortin II, and Urocortin III on gastric emptying and colonic transit in mice: role of CRF receptor subtypes 1 and 2. J. Pharmacol. Exp. Ther., 301: 611-617. Miampamba, M., Maillot, C., Million, M. and Tache, Y. (2002) Peripheral CRF activates myenteric neurons in the proximal colon through CRF(1) receptor in conscious rats. Am. J. Physiol. Gastrointest. Liver Physiol., 282: G857-865. Million, M., Maillot, C., Saunders, P., Rivier, J., Vale, W. and Tache, Y. (2002) Human Urocortin II, a new CRF-related peptide, displays selective CRF(2)-mediated action on gastric transit in rats. Am. J. Physiol. Gastrointest. Liver Physiol., 282: G34-40. Montecucchi, P.C. and Henschen, A. (1981) Amino acid composition and sequence analysis of sauvagine, a new active peptide from the skin of Phyllomedusa sauvagei. Int. J. Pept. Protein Res., 18: 113-120. Muglia, L.J., Bethin, K.E., Jacobson, L., Vogt, S.K. and Majzoub, J.A. (2000) Pituitary-adrenal axis regulation in CRH-deficient mice. Endocr. Res., 26: 1057-1066. Muller, M.B., Keck, M.E., Zimmermann, S., Holsboer, F. and Wurst, W. (2000) Disruption of feeding behavior in CRH receptor 1-deficient mice is dependent on glucocorticoids. Neuroreport, 11: 1963-1966. Negri, L., Melchiorri, P., Montecucchi, P.C., Henschen, A., D'Urso, R., Iellamo, R. and Falaschi, P. (1983) Sauvagine induces release of adrenocorticotropin, beta-endorphin and corticosterone in rats. Pharmacol. Res. Commun., 15: 427-438. Palchaudhuri, M.R., Hauger, R.L., Wille, S., Fuchs, E. and Dautzenberg, F.M. (1999) Isolation and pharmacological characterization of two functional splice variants of corticotropin-releasing factor type 2 receptor from tupaia belangeri. J. Neuroendocrinol., 11: 419-428. Pedersen, W.A., Wan, R., Zhang, P. and Mattson, M.P. (2002) Urocortin, but not urocortin II, protects cultured hippocampal neurons from oxidative and excitotoxic cell death via corticotropin-releasing hormone receptor type I. J. Neurosci., 22: 404-412. Peterson, R.E. and Guillemin, R. (1974) The hormones of the hypothalamus. Am. J. Med., 57: 591-600. Pisarchik, A. and Slominski, A. (2002) Corticotropin releasing factor receptor type 1: molecular cloning and investigation of alternative splicing in the hamster skin. J. Invest. Dermatol., 118:1065-1072. Pisarchik, A. and Slominski, A.T. (2001) Alternative splicing of CRH-R1 receptors in human and mouse skin: identification of new variants and their differential expression. Faseb. J., 15: 2754-2756.
Pohl, S., Darlison, M.G., Clarke, W.C., Lederis, K. and Richter, D. (2001) Cloning and functional pharmacology of two corticotropin-releasing factor receptors from a teleost fish. Eur. J. Pharmacol., 430: 193-202. Potter, E., Behan, D.P., Fischer, W.H., Linton, E.A., Lowry, P.J. and Vale, W.W. (1991) Cloning and characterization of the cDNAs for human and rat corticotropin releasing factorbinding proteins. Nature, 349: 423-426. Potter, E., Sutton, S., Donaldson, C., Chen, R., Perrin, M., Lewis, K., Sawchenko, P.E. and Vale, W. (1994) Distribution of corticotropin-releasing factor receptor mRNA expression in the rat brain and pituitary. Proc. Natl. Acad. Sci. USA, 91:8777-8781. Reagan, J.D. (1994) Expression cloning of an insect diuretic hormone receptor. A member of the calcitonin/secretin receptor family. J. Biol. Chem., 269: 9-12. Reagan, J.D. (1995) Functional expression of a diuretic hormone receptor in baculovirus-infected insect cells: evidence suggesting that the N-terminal region of diuretic hormone is associated with receptor activation. Insect Biochem. Mol. Biol., 25: 535-539. Reagan, J.D. (1996) Molecular cloning and function expression of a diuretic hormone receptor from the house cricket, Acheta domesticus. Insect. Biochem. Mol. Biol., 26: 1-6. Reyes, T.M., Lewis, K., Perrin, M.H., Kunitake, K.S., Vaughan, J., Arias, C.A., Hogenesch, J.B., Gulyas, J., Rivier, J., Vale, W.W. and Sawchenko, P.E. (2001) Urocortin II: a member of the corticotropin-releasing factor (CRF) neuropeptide family that is selectively bound by type 2 CRF receptors. Proc. Natl. Acad. Sci. USA, 98: 2843-2848. Riehle, M.A., Garczynski, S.F., Crim, J.W., Hill, C.A. and Brown, M.R. (2002) Neuropeptides and peptide hormones in Anopheles gambiae. Science, 298: 172-175. Rivier, C. (2001) Role of gaseous neurotransmitters in the hypothalamic-pituitary-adrenal axis. Ann. N. Y. Acad. Sci., 933: 254-264. Saffran, M. and Schally, A.V. (1977) The status of the corticotropin releasing factor (CRF). Neuroendocrinology, 24: 359-375. Saunders, P.R., Maillot, C., Million, M. and Tache, Y. (2002) Peripheral corticotropin-releasing factor induces diarrhea in rats: role of CRF1 receptor in fecal watery excretion. Eur. J. Pharmacol., 435: 231-235. Scarabelli, T.M., Pasini, E., Stephanou, A., Comini, L., Curello, S., Raddino, R., Ferrari, R., Knight, R. and Latchman, D.S. (2002) Urocortin promotes hemodynamic and bioenergetic recovery and improves cell survival in the isolated rat heart exposed to ischemia/reperfusion. J. Am. Coll. Cardiol., 40: 155-161. Schulman, D., Latchman, D.S. and Yellon, D.M. (2002) Urocortin protects the heart from reperfusion injury via upregulation of p42/p44 MAPK signaling pathway. Am. J. Physiol. Heart Circ. Physiol., 283: H1481-1488.
131 Seasholtz, A.F., Valverde, R.A. and Denver, R.J. (2002) Corticotropin-releasing hormone-binding protein: biochemistry and function from fishes to mammals. J. Endocrinol., 175: 89-97. Sherwood, N.M., Krueckl, S.L. and McRory, J.E. (2000) The origin and function of the pituitary adenylate cyclaseactivating polypeptide (PACAP)/glucagon superfamily. Endocr. Rev., 21: 619-670. Shibahara, S., Morimoto, Y., Furutani, Y., Notake, M., Takahashi, H., Shimizu, S., Horikawa, S. and Numa, S. (1983) Isolation and sequence analysis of the human corticotropin-releasing factor precursor gene. Embo. J., 2: 775-779. Smith, G.W., Aubry, J.M., Dellu, F., Contarino, A., Bilezikjian, L.M., Gold, L.H., Chen, R., Marchuk, Y., Hauser, C., Bentley, C.A., Sawchenko, P.E., Koob, G.F., Vale, W. and Lee, K.F. (1998) Corticotropin releasing factor receptor 1-deficient mice display decreased anxiety, impaired stress response, and aberrant neuroendocrine development. Neuron, 20:1093-1102. Spiess, J., Rivier, J., Rivier, C. and Vale, W. (1981) Primary structure of corticotropin-releasing factor from ovine hypothalamus. Proc. Natl. Acad. Sci. USA, 78: 6517-6521. Steckler, T. (2005). CRF antagonists as novel treatment strategies for stress-related disorders. In: Steckler, T., Kalin, N. and Reul, J.M.H.M. (Eds.), Handbook of Stress and the Brain, Part 1, Elsevier, Amsterdam, pp. 371-400. Suman-Chauhan, N., Carnell, P., Franks, R., Webdale, L., Gee, N.S., McNulty, S., Rossant, C.J., Van Leeuwen, D., MacKenzie, R. and Hall, M.D. (1999) Expression and characterisation of human and rat CRF2alpha receptors. Eur. J. Pharmacol., 379: 219-227. Swiergiel, A.H. and Dunn, A.J. (1999) CRF-deficient mice respond like wild-type mice to hypophagic stimuli. Pharmacol. Biochem. Behav., 64: 59-64. Terui, K., Higashiyama, A., Horiba, N., Furukawa, K.I., Motomura, S. and Suda, T. (2001) Coronary vasodilation and positive inotropism by urocortin in the isolated rat heart. J. Endocrinol., 169: 177-183. Timpl, P., Spanagel, R., Sillaber, I., Kresse, A., Reul, J.M., Stalla, G.K., Blanquet, V., Steckler, T., Holsboer, F. and Wurst, W. (1998) Impaired stress response and reduced anxiety in mice lacking a functional corticotropin-releasing hormone receptor 1. Nat. Genet., 19: 162-166. Turnbull, A.V., Smith, G.W., Lee, S., Vale, W.W., Lee, K.F. and Rivier, C. (1999) CRF type I receptor-deficient mice exhibit a pronounced pituitary-adrenal response to local inflammation. Endocrinology, 140:1013-1017.
Valdenaire, O., Giller, T., Breu, V., Gottowik, J. and Kilpatrick, G. (1997) A new functional isoform of the human CRF2 receptor for corticotropin-releasing factor. Biochim. Biophys. Acta, 1352: 129-132. Valdez, G.R., Inoue, K., Koob, G.F., Rivier, J., Vale, W. and Zorrilla, E.P. (2002) Human urocortin II: mild locomotor suppressive and delayed anxiolytic-like effects of a novel corticotropin-releasing factor related peptide. Brain Res., 943: 142-150. Vale, W., Spiess, J., Rivier, C. and Rivier, J. (1981) Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and beta-endorphin. Science, 213: 1394-1397. Valverde, R.A., Seasholtz, A.F., Cortright, D.N. and Denver, R.J. (2001) Biochemical characterization and expression analysis of the Xenopus laevis corticotropin-releasing hormone binding protein. Mol. Cell Endocrinol., 173: 29-40. Vaughan, J., Donaldson, C., Bittencourt, J., Perrin, M.H., Lewis, K., Sutton, S., Chan, R., Turnbull, A.V., Lovejoy, D., Rivier, C., Rivier, J., Sawenchko, P.E. and Vale, W. (1995) Urocortin, a mammalian neuropeptide related to fish urotensin I and to corticotropin-releasing factor. Nature, 378: 287-292. Vetter, D.E., Li, C., Zhao, L., Contarino, A., Liberman, M.C., Smith, G.W., Marchuk, Y., Koob, G.F., Heinemann, S.F., Vale, W. and Lee, K.F. (2002) Urocortin-deficient mice show hearing impairment and increased anxiety-like behavior. Nat. Genet, 31: 363-369. Vita, N., Laurent, P., Lefort, S., Chalon, P., Dumont, X., Kaghad, M., Gully, D., Le Fur, G., Ferrara, P. and Caput, D. (1993) Cloning and expression of a complementary DNA encoding a high affinity human neurotensin receptor. FEBS Lett., 317: 139-142. Wang, L., Martinez, V., Rivier, J.E. and Tache, Y. (2001) Peripheral urocortin inhibits gastric emptying and food intake in mice: differential role of CRF receptor 2. Am. J. Physiol. Regul. Integr. Comp. Physiol., 281: R1401-1410. Wang, X., Su, H., Copenhagen, L.D., Vaishnav, S., Pieri, F., Shope, C.D., Brownell, W.E., De Biasi, M., Paylor, R. and Bradley, A. (2002) Urocortin-deficient mice display normal stress-induced anxiety behavior and autonomic control but an impaired acoustic startle response. Mol. Cell Biol., 22: 6605-6610. Wiehart, U.I., Nicolson, S.W., Eigenheer, R.A. and Schooley, D.A. (2002) Antagonistic control of fluid secretion by the Malpighian tubules of Tenebrio molitor: effects of diuretic and antidiuretic peptides and their second messengers. J. Exp. Biol., 205: 493-501.
This Page Intentionally Left Blank
T. Steckler, N.H. Kalin and J.M.H.M. Reul (Eds.) Handbook of Stress and the Brain, Vol. 15
ISBN 0-444-51173-3 Copyright 2005 Elsevier B.V. All rights reserved CHAPTER 2.2
Molecular regulation of the CRF system P.H. Roseboom 1'3'*, N.H. Kalin ~'2, T. Steckler 4 and F.M. Dautzenberg 4'* 1Department of Psychiatry, University of Wisconsin, 6001 Research Park Blvd, Madison, WI 53719, USA 2Department of Psychology, University of Wisconsin, 1202 West Johnson Street, Madison, WI 53706, USA 3Department of Pharmacology, University of Wisconsin, 1300 University Avenue, Madison, W153706, USA 4johnson and Johnson Pharmaceutical Research & Development, Janssen Pharmaceutica N.V., Turnhoutseweg 30, 2340 Beerse, Belgium
Abstract: The corticotropin-releasing factor (CRF) system, including the urocortin peptides, is a key regulator of how the body responds to stress. A large amount of preclinical data in rodents and nonhuman primates implicates the CRF system in mediating the various physiological and psychological responses to stress. Importantly, alterations in the CRF system are associated with depression and anxiety disorders in humans. The goal of this chapter is to review what is known about the molecular mechanisms that regulate the activity of the CRF system. The role of the CRF system in stress-induced psychopathology is initially reviewed. Stress-induced molecular changes that are associated with activation of the CRF system are then described, along with the effects of manipulations that mimic or block the effects of stress. CRF receptor regulation is outlined in detail including an overview of recent data implicating the role of G-protein receptor kinase 3 in the phosphorylation and desensitization of the CRF1 receptor. The limited data on the regulation of the CRF2 receptors is also described. Finally, preliminary data from the use of microarrays and gene chips aimed at identifying stress-induced changes in gene expression that are CRF receptor dependent or independent will be described. A detailed understanding of the molecular mechanisms that mediate the stress-induced changes in the CRF system will enable identification of novel targets for the treatment and prevention of stress-related disorders.
The CRF system and stress-induced psychopathology
1990; De Souza, 1995; Kalin, 1997; Koob and Heinrichs, 1999). Numerous clinical and preclinical reports indicate that the CRF system mediates behavioral, autonomic, neuroendocrine, and immune responses to stress. Furthermore, alterations in the CRF system are often associated with stress-related psychopathology, such as depression and anxiety disorders (Mitchell, 1998; Arborelius et al., 1999). More recently, additional components of the CRF system have been identified. CRF and the related endogenous peptide agonists urocortin 1 (Vaughan et al., 1995), urocortin 2 (Reyes et al., 2001), and urocortin 3 (Lewis et al., 2001) bind to the two cloned CRF receptors, designated CRF1 and CRF2 (Chen et al., 1993; Perrin et al., 1995). A CRF-binding protein (CRF-BP) that putatively buffers the effects
The corticotropin-releasing factor (CRF) system is one of the major peptide systems implicated in regulating the stress response. CRF is a 41-amino acid peptide that was originally discovered as a novel hypothalamic factor controlling the release of pituitary proopiomelanocortin peptides (Guillemin and Rosenberg, 1955; Saffran and Schally, 1956; Vale et al., 1981). It has since been found to play an important role in coordinating various components of the stress response (Dunn and Berridge, *Corresponding author. E-mail:
[email protected] rE-mail:
[email protected] 133
134 of endogenous CRF system ligands has also been identified (Potter et al., 1991). Several isoforms of the receptors have been cloned including CRFz(a), CRFz(b), and CRF2(c) receptors (Lovenberg et al., 1995a; Kostich et al., 1998; Hauger et al., 2003b). CRF2(a) and CRF2(c) are the subtypes that predominate in the brain (Lovenberg et al., 1995b; Kostich et al., 1998). Urocortin 1, urocortin 2, and urocortin 3 are likely important in mediating stress-induced behavior because studies have noted that CRF knockout mice exhibit normal stress-induced behavioral responses that can be blocked by CRF receptor antagonism (Weninger et al., 1999). There is a great deal of clinical and preclinical evidence implicating the CRF system in stressinduced psychopathology, such as depression and anxiety disorders (for review Mitchell, 1998; Arborelius et al., 1999). Elevated levels of CRF in the cerebrospinal fluid of depressed patients have been reported (Nemeroff et al., 1984), and increased numbers of CRF-expressing neurons in the paraventricular nucleus of the hypothalamus (PVN) of depressed patients have been observed (Raadsheer et al., 1994). In addition, altered levels of CRF binding sites in the brains of suicide victims have been reported (Nemeroff, 1988). Recent efforts are directed toward developing CRF1 antagonists for treating stress-related psychiatric disorders (McCarthy et al., 1999; Steckler, 2005). The first open label trial of such a compound in depressed patients revealed significant reductions in depression and anxiety after CRF1 antagonist treatment (Zobel et al., 2000). In addition to clinical data, there are numerous preclinical studies implicating the CRF system in stress-induced psychopathology. Early studies administering CRF intraventricularly to rodents and primates reported that this peptide caused marked behavioral effects that mimicked fear-related and depressive responses (Kalin et al., 1983; Sherman and Kalin, 1986; Dunn and Berridge, 1990; Koob et al., 1993). Infusion of nonselective CRF antagonists such as alpha-helical CRF9_41 blocked the effects of stress on behavior (Kalin, 1985; Koob and Heinrichs, 1999). CRF-overexpressing mice demonstrate behavioral phenotypes that are consistent with increased levels of stress (Stenzel-Poore et al., 1992, 1994; van der Meer et al., 2001; van Gaalen et al., 2002), while CRF~ receptor knockout mice display responses indicative
of decreased anxiety-like behavior (Smith et al., 1998; Timpl et al., 1998; Contarino et al., 1999). Data from recent site-specific antagonist administration and antisense oligonucleotide studies suggest that the CRF2(a) receptor has a role similar to that of the CRF1 receptor in mediating fearful behaviors (Ho et al., 2001; Bakshi et al., 2002). It should also be noted that the CRF and urocortin peptides mimic the anorectic effects of stress most likely through activation of the CRF2 receptor (Krahn et al., 1986; Arase et al., 1988; Spina, 1996). Results from three reports of CRF2 receptor knockout mice (Bale et al., 2000; Coste et al., 2000; Kishimoto et al., 2000) are inconsistent. Two of these studies suggest that the behavioral profile of CRF2 receptor knockouts increases stress-like responding as evidenced by a decrease in open arm entries in the elevated plus maze (Bale et al., 2000; Kishimoto et al., 2000). However, other aspects of the behavioral profile indicate either no alteration of stress-related responding (Bale et al., 2000; Coste et al., 2000), or a decrease in anxiety-like behaviors (Kishimoto et al., 2000). There has been one study characterizing mice deficient in both CRF1 and CRF2 receptors (Bale et al., 2002). These mice display a greater impairment of the hypothalamic-pituitary-adrenal (HPA) axis response to stress than CRF1 knockout mice. The behavioral effects described are complex and sexually dichotomous, with female double-mutant mice displaying anxiolytic-like behavior and male doublemutant mice demonstrating significantly greater anxiety-like behaviors compared to females. In addition, this study hypothesized that the CRF2 receptor genotype of the mothers had a significant effect on the anxiety-like behaviors of the male pups regardless of the pup's genotype. Male pups born from dams that have one (heterozygous) or both copies (homozygous) of the CRF2 receptor gene knocked out display significantly more anxiety-like behavior (Bale et al., 2002). Nevertheless in the light of the rather inconsistent results obtained with the CRF2 receptor knockout mice, data obtained from transgenic animals should be treated with caution and might not reflect the situation in animals that carried the gene through their entire development. Small molecule CRF1 and CRF2 receptor-specific antagonists might better address questions concerning the involvement of both receptors in the development of
135 stress-related and depressive disorders. In addition, studies have shown that the central distribution of CRF2 receptors in humans and primates is much broader than in rodents (Kostich et al., 1998; Palchaudhuri et al., 1999; Sanchez et al., 1999) suggesting a more prominent role for CRF2 receptors in mediating behavior in primates and humans. In summary, there is a great deal of preclinical data indicating the importance of the various components of the CRF system in mediating the various physiological and behavioral responses to stress. A detailed understanding of the roles played by each of the two known CRF receptors is emerging, and evidence is accumulating that implicates the CRF system in stress-induced psychopathologies, such as depression and anxiety disorders.
can produce an increase in CRF mRNA levels in the PVN (for review Bakshi and Kalin, 2000). Outside the hypothalamus, the components of the CRF system are widely expressed throughout the brain. In some instances, the extrahypothalamic CRF system is regulated differently from the hypothalamic CRF system. For example, stress-induced release of CRF from the amygdala appears to occur under a more limited set of circumstances compared to stressinduced release of CRF from the PVN (Bakshi and Kalin, 2000). Extrahypothalamic CRF is thought to play an important role in a variety of psychological and physiological responses to stress. The main question is how exposure to acute and repeated stress affects the expression of CRF and other components of the CRF system. Acute stress
Stress-mediated activation of the CRF system The data above indicate the necessity for understanding how stress exerts its effects on the CRF system. There is considerable evidence that stress activates the hypothalamic CRF system, resulting in regulation of the HPA axis. In response to an acute stress, CRF that is produced in the PVN is secreted into the median eminence, enters the portal vessels, and is transported to CRF1 receptor binding sites in the anterior pituitary. Binding of CRF to the CRF1 receptor and subsequent receptor activation leads to increased intracellular cAMP levels within pituitary corticotrophs. This results in immediate release of adrenocorticotropic hormone (ACTH) into the general circulation, and also a long-term increase in ACTH synthesis. The circulating ACTH then binds to its specific receptor, the melanocortin 2 receptor, in the adrenal cortex and thereby stimulates the release of glucocorticoids. Glucocorticoids (i.e. cortisol) distribute throughout the body and produce physiological changes to enable the body to acutely respond to the stressful situation. In addition, via a negative receptor feedback loop to the brain, glucocorticoids inhibit further activation of the HPA system (Kaplan, 1992). Activation of the hypothalamic CRF system is evidenced by an increase in CRF gene expression in the PVN. A wide variety of acute psychological, physical, physiological, or immunological stressors
In rats, restraint stress for 1 h or less or ethanol withdrawal have been shown to increase the release of C R F peptide and the expression of CRF mRNA in the amygdala (Kalin et al., 1994; Pich et al., 1995; Hsu et al., 1998; Merali et al., 1998) and in various thalamic nuclei (Hsu et al., 2001). This stress-induced increase in CRF peptide release occurs immediately with onset of restraint (Pich et al., 1995; Merali et al., 1998) and mRNA changes are seen as early as 1 h after cessation of stress (Hsu et al., 1998). Three hours of restraint stress also increases rat urocortin 1 mRNA in the Edinger-Westphal nucleus (Weninger et al., 2000). CRF1 receptor mRNA is increased in the PVN following a variety of acute stressors (Makino et al., 1995; Aguilera et al., 1997; Bonaz and Rivest, 1998). Restraint stress increases mRNA expression for CRF-BP in the pituitary and amygdala (Lombardo et al., 2001), and food deprivation increases CRF-BP mRNA in the amygdala and hypothalamic regions (Timofeeva et al., 1999). Repeated stress
The effects of repeated stress on expression of CRF mRNA have been most extensively studied. Repeated stress (restraint or immobilization) produces elevation in basal levels of CRF mRNA levels in the PVN (Mamalaki et al., 1992; Bartanusz et al., 1993). Although acute stress elevates CRF mRNA in the
136 amygdala, repeated stress has not been found to alter basal levels of amygdalar CRF mRNA (Mamalaki et al., 1992). However, chronic social stress produces an elevation of CRF mRNA in this brain area, but decreases CRF mRNA levels in the PVN (Albeck et al., 1997). In the pituitary, two weeks of restraint stress decreased CRF1 receptor mRNA (Makino et al., 1995). However, other groups were unable to replicate this effect (Aguilera et al., 1997). In certain strains of mice, cortical levels of CRF1 receptor mRNA increase in response to repeated restraint stress (Giardino et al., 1996). In rats, however, CRF1 receptor mRNA levels in several extrahypothalamic regions including the cortex, amygdala, and hippocampus appear not to be affected by repeated restraint stress (Makino et al., 1995; Iredale et al., 1996). However, when the repeated stress paradigm consists of a variable, unpredictable, multimodal regimen, a significant reduction in CRF~ receptor gene expression occurs in the cortex, and a significant increase of transcript is seen in the hippocampus (Iredale et al., 1996). Only one study reported the effects of repeated immobilization stress on CRFz(a) receptor gene expression and showed a small (12%) decrease in CRFz(a) receptor mRNA in the ventromedial hypothalamus with stress (Makino et al., 1999).
Effects o f repeated stress on response to acute stress
Repeated exposure of rats to stress prevents the acute stress-induced increase of CRF mRNA in the amygdala when the acute stressor is identical to the repeated stressor (Mamalaki et al., 1992). Similarly, the acute stress-induced increase in CRF-BP mRNA in the amygdala is blocked by prior exposure to repeated restraint stress (Lombardo et al., 2001). The acute stress-induced increase in CRF mRNA in the PVN is significantly dampened by prior exposure to repeated restraint (Ma et al., 1997, 1999). However, the acute response is still present if the challenge is with a heterotypic stressor (Ma et al., 1999). There is also evidence for some involvement of the arginine vasopressin system in regulating the activity of the HPA axis in response to repeated stress. This system controls how the HPA axis responds to a homotypic
stressor; however, the CRF system controls the HPA axis response to a heterotypic stressor (Bakshi and Kalin, 2000).
Developmental stress
A large body of literature exists indicating that separating both animals and humans from their mothers is a significant stressor that can negatively impact the emotional development of the infant (Bowlby, 1973; Carlson and Earls, 1997). In rat pups, during the stress hyporesponsive period (postnatal days 3-14), an intense stressor, such as 24h of maternal separation, is necessary to activate the HPA axis. This results in decreased expression of CRF mRNA in the PVN (Smith et al., 1997; van Oers et al., 1998; Dent et al., 2000). In contrast, restraint stress of 30 min or less given to postnatal day 12 pups immediately following 24 h of maternal deprivation results in an increase in CRF mRNA in the PVN. This increase was seen 15 rain after the cessation of the stress compared to nonrestrained controls (Dent et al., 2000). CRF mRNA levels are also increased in the amygdala of pups that have been exposed to a maternal deprivation/cold stress exposure (Hatalski et al., 1998). The stress of maternal deprivation results in heightened responsiveness of the CRF system to stressors that occur after the stress hyporesponsive period. For example, rat pups that are subjected to maternal separation on postnatal day 3 demonstrate an increase in CRF mRNA expression in the PVN relative to nondeprived rats that are stressed two weeks later (van Oers et al., 1998). Alterations in the responsiveness of the CRF system following maternal deprivation of rat pups is maintained into adulthood (Anisman et al., 1998; Francis et al., 1999b; Heim and Nemeroff, 1999). Interestingly, the direction of the change in responsiveness is dependent upon the length of maternal separation. Short bouts of separation from the mother (3-15 rain per bout once a day for 2 weeks) results in a profile in adulthood that is indicative of lower levels of anxiety. These rats also have decreased basal levels of hypothalamic CRF mRNA and median eminence CRF peptide as adults compared to control rats (Plotsky and Meaney, 1993). In contrast, when separation from the mother is
137 extended to 3 h or more, the pups develop into adults displaying increased stress-like responding. This change is associated with increased CRF gene expression (Plotsky and Meaney, 1993; Rots et al., 1996; Wigger and Neumann, 1999). The intense stress of an endotoxin insult to rat pups elevates basal levels of CRF gene expression and enhances stress-induced HPA axis response in adulthood (Shanks et al., 1995). Similar findings have been reported in nonhuman primates. CSF levels of CRF are basally and persistently elevated in adult macaques whose mothers have been exposed to 3 months of variable foraging demand compared to macaques whose mothers faced a predictable foraging demand (Coplan et al., 1996, 2000). This condition results in greater uncertainty in food availability for mother and infant and likely alters mother-infant interactions. In addition, macaques raised by variable foraging demand-exposed mothers develop impaired affiliative social behaviors in adulthood (Andrews and Rosenblum, 1994). In rodents it has been shown that mothering styles can be passed on to offspring by nongenomic modes of transmission. For example, it has been shown that pups will develop the mothering style of the mother who raises them regardless of whether she is their biological mother (Francis et al., 1999a). A similar pattern of behavioral transmission may occur in primates. In rhesus monkeys there is a relationship between birth order and cortisol concentration with an early position in the birth order associated with higher basal cortisol concentrations (Kalin et al., 1998). In summary, it has been proposed that the perinatal environment and mothering style can shape the offspring's stress-coping system that persists into adulthood and in part this is related to alterations in the CRF system. It is likely that, at some level, the setting of the stress-coping system involves the CRF system. An intriguing suggestion from this theory is that stress in the early days of life has a profound impact on how an individual responds to stress for the remainder of its life (Anisman et al., 1998; Francis et al., 1999b; Heim and Nemeroff, 1999). It is possible that stressors occurring early in life may have a more profound impact on the stress-response profile than any effects of acute or prolonged stress occurring in adulthood. Therefore, it is important to continue to identify how, on a molecular level, developmental
stress affects the C R F family of genes (Bakshi and Kalin, 2000). This may ultimately lead to the identification of novel drug targets for treatment in adulthood of developmental stress-induced pathologies such as depression and anxiety disorders.
Effects on the CRF system of hormonal and pharmacological manipulations that either mimic or block the effects of stress
Mimicking the effects of stress It is known that stress results in release of CRF and activation of the HPA axis, ultimately leading to the release of cortisol or corticosterone into the circulation. Therefore, it is possible to pharmacologically mimic effects of stress by administering either C R F or glucocorticoids. Glucocorticoids affect various components of the CRF system. For example, corticosterone administration decreases CRF m R N A levels in the PVN (Makino et al., 1995). Extensive studies that have been performed on this topic are covered in greater detail in Kino and Chrousos, 2005. Though expression of brain CRF receptors does not readily change in response to various manipulations, administration of high intracerebral doses of CRF can downregulate brain CRF receptor expression. Four daily intracisternal injections of CRF (5 nmol/day) decreased CRF receptor binding in the amygdala (Hauger et al., 1993). Downregulation of CRF1 receptors was also reported in rat pups 4h after administration of 0.75 nmol CRF into the left lateral cerebral ventricle, with a decrease of 21% in the frontal cortex (Brunson et al., 2002). In addition, this 0.75 nmol CRF treatment also caused persistent increases in CRF1 m R N A levels in the frontal cortex and hippocampal CA3, but not in the CA1 or amygdala (Brunson et al., 2002). A CRF-induced change in the level of CRF2 receptor m R N A or CRF2 receptor binding was not detected.
Blocking the effects of stress Treatment with various psychopharmacologic agents alters the dysregulation of the HPA axis that is
138 observed in some depressed patients. It is possible that some antidepressant and anxiolytic agents produce their effects through actions on the CRF system. In rodents, acute and chronic treatment with these agents has been shown to affect the CRF system. For example, both acute and chronic alprazolam administration decreased CRF concentrations in the locus coeruleus (Skelton et al., 2000). In the same study, alprazolam also decreased CRF mRNA expression in the central nucleus of the amygdala, and decreased expression of CRF1 receptors and mRNA levels in the basolateral amygdala. In contrast, alprazolam resulted in increased CRFz(a) receptor binding in the lateral septum and ventromedial hypothalamus. Urocortin 1 mRNA expression was also increased in the Edinger-Westphal nucleus that supplies preganglionic parasympathetic nerve fibers to the eye (Skelton et al., 2000). The authors of this study postulated that two separate CRF systems exist that could coordinately and inversely be regulated by stress and other manipulations. There have been several studies reporting on the effects of antidepressant treatment on the CRF system. In depressed humans successful antidepressant therapy is associated with decreased cortisol concentrations as well as reductions in CSF-CRF concentrations (Heuser et al., 1998). Some preclinical studies show that long-term antidepressant treatment (4-8 weeks) decreases CRF mRNA levels in the PVN (Brady et al., 1991, 1992; Fadda et al., 1995; Aubry et al., 1999). However, other studies have failed to demonstrate a decrease in CRF mRNA in the PVN following similar antidepressant treatments (Jensen et al., 1999; Stout et al., 2002). Finally, four weeks of treatment with amitriptyline has been shown to decrease the levels of CRF1 receptor mRNA in the rat amygdala (Aubry et al., 1999). At least one study has described how the effects of long-term antidepressant treatment influence the effects of stress on the CRF system. Four weeks of treatment with either the dual serotonin/noradrenaline reuptake inhibitor venlafaxine or the monoamine oxidase inhibitor tranylcypromine inhibited both acute and chronic stress-induced increases in CRF mRNA levels in the rat PVN (Stout et al., 2002). This study suggests that antidepressants may decrease the sensitivity of CRF-expressing neurons in the PVN to the effects of stress.
Molecular mechanisms underlying effects of stress on expression of the CRF family of genes The above discussions summarized the effects of stress on the CRF system. To understand at a molecular level how stress alters CRF gene expression, we must first understand the promoter regions that control expression from each of the genes in the CRF family.
CRF Stress-related increases in CRF gene transcription are thought to be mediated via activation of a variety of different kinases resulting in phosphorylation of cAMP-response element binding protein (CREB) (Chen et al., 2001). The promoter for the CRF genehas been extensively characterized. The cAMPresponse element (CRE) that is present in the promoter region of the CRF gene at -224 nucleotides affects basal promoter activity as well as cAMP-dependent, 12-O-tetradecanoylphorbol- 13acetate-dependent, and depolarization-dependent transcriptional activation of the CRF promoter (Spengler et al., 1992; Mugele et al., 1993; GuardiolaDiaz et al., 1994). The CRE binds the transcription factor CREB that is activated via phosphorylation. Glucocorticoids appear to regulate CRF expression through direct inhibition of gene transcription (Herman et al., 1992). Following binding of ligand, glucocorticoid receptors translocate to the nucleus and bind to a negative glucocorticoid response element (nGRE) at bases -279 to -249 that inhibits CRF gene transcription (Malkoski et al., 1997; Malkoski and Dorin, 1999). Interestingly, mutating or eliminating this nGRE from the promoter sequence diminishes cAMP-dependent induction of gene transcription, indicating that there is recruitment of proteins capable of binding to elements within the nGRE that contribute to cAMP-inducible expression. Interestingly, the nGRE site contains an AP-1 site that binds c-fos and c-jun transcription factors, both of which are known to be stimulated by activation of the cAMP pathway. Mutations within this nGRE that disrupted either GR or AP-1 binding activity abolished glucocorticoid-dependent repression of gene expression. In summary, there are
139 elements within the nGRE that contribute to the glucocorticoid-dependent repression of gene expression and cAMP-dependent stimulation of gene expression (Malkoski and Dorin, 1999). This complex activity of the nGRE within the CRF gene is not novel, and several examples of coregulation between GR and AP-1 proteins have been reported (Diamond et al., 1990; Zhang et al., 1991). Finally the human cell line BE(2)-M17 expresses CRF protein following differentiation with retinoic acid. In this cell line, the POU transcription factor Brn-2 is required for retinoic acid-induced expression of the CRF gene. However, overexpression of Brn-2 in the absence of retinoic acid is not sufficient to induce CRF m R N A expression (Ramkumar and Adler, 1999). This study suggests that Brn-2 is important in mediating retinoic acid-induced CRF gene expression. Brn-2 is expressed in the parvocellular neurons of the PVN that synthesize CRF, and the CRF promoter contains multiple binding sites for Brn-2 (Li et al., 1993). However, the involvement of this transcription factor in vivo in the regulation of CRF expression remains to be demonstrated. When Brn-2 was overexpressed in vivo in the mouse PVN using an adenoviral vector delivery strategy, no effect on CRF gene expression was demonstrated (Wong and Murphy, 2003).
Urocortin 1 The urocortin 1 gene, like the CRF1 gene, contains two exons, with exon 2 containing the entire coding region of the urocortin 1 m R N A (Park et al., 2000). Similar to the CRF gene, the urocortin 1 promoter and Y-upstream region contains CRE elements and a Brn-2 binding site (Zhao et al., 1998). This CRE element seems to be important for basal transcriptional activity and protein kinase A (PKA)-mediated responses (Zhao et al., 1998).
14 GRE (Chen et al., 2003). Transient transfection experiments have shown that the urocortin 2 promoter is driven by glucocorticoid activation (Nanda et al., 2002; Chen et al., 2003). Additionally results from transfection experiments show that steroid hormone antagonists block the actions of glucocorticoids, and prevent dexamethasone induction of urocortin 2 gene expression, suggesting a direct glucocorticoid receptor-mediated effect (Chen et al., 2003). These results are consistent with in vivo mouse studies demonstrating dexamethasoneinduced increases in urocortin 2 gene expression in the hypothalamus and brain stem (Chen et al., 2003). In contrast to the urocortin 2 gene, characterization of the promoter region of the urocortin 3 gene has not been reported.
CRF-binding protein The CRF-binding protein (CRF-BP) is thought to buffer the actions of CRF at the synapse (Kemp et al., 1998). This protein preferentially binds CRF and urocortin 1 but displays no affinity for urocortin 2 or 3 (Dautzenberg and Hauger, 2002). As also observed for its natural ligands CRF and urocortin 1, CRF-BP gene expression may be regulated by nuclear factors binding to a CRE element in the Y-upstream promoter region, as elevation of intracellular cAMP concentrations increase CRF-BP gene transcription (Kemp et al., 1998; McClennen and Seasholtz, 1999). Furthermore, glucocorticoids repress the transcription of the CRF-BP gene (McClennen and Seasholtz, 1999). Besides CRE and GRE elements, acute phase response elements have been located in the promoter region of the CRF-BP gene, one of which has been shown to promote binding of NF-KB (Kemp et al., 1998).
CRF1 and CRF2 receptors Urocortins 2 and 3 Recently the urocortin 2 promoter region was identified (Chen et al., 2003). In contrast to CRF and urocortin 1, glucocorticoids appear to play a dominant role in the regulation of urocortin 2 gene expression as its Y-flanking region is clustered with
CRF1 and CRF2 receptors are members of the class B subfamily of G protein-coupled receptors (GPCRs) (Dautzenberg and Hauger, 2002), which are characterized by genes with several introns, in contrast to the majority of members of the GPCR superfamily (Gentles and Karlin, 1999).
140 The genomic organization of the rat and human CRF~ gene has been reported (Tsai-Morris et al., 1996; Sakai et al., 1998). Both genes span more than 36 (rat) and 20 kilobases (human), respectively, with similar organization and location of exons and exon/ intron boundaries (Sakai et al., 1998). The rat gene contains 13 exons and 12 introns (Tsai-Morris et al., 1996), whereas the human gene contains 14 exons and 13 introns (Sakai et al., 1998). The additional 87 bp exon (number 5 in the human gene) gives rise to a splice variant with a 29 amino acid insertion in the first intracellular domain of the receptor (Chen et al., 1993). It is important to note that in humans a large variety of splice variants have been observed (Dautzenberg et al., 2001b), which arise from exonskipping and encode truncated versions of this receptor. Most of these variants have not been found in other species. None of these splice variants have been shown to be functional, or to act as dominantnegative receptors, reducing the action of the normal CRF~ receptor in vitro or in vivo. Thus, the International Union of Pharmacology subcommittee on the nomenclature of C R F receptors has recently discarded and refused to number these variants, as has been done with the functional CRF2 receptor variants CRF2(a), CRFz(b), and CRF2(c) (Hauger et al., 2003b). A short segment of the rat CRF1 gene promoter has been obtained with the cloning of the gene and several consensus binding sites for transcriptional factors have been determined (Tsai-Morris et al., 1996). However, it is not known
bl
b2 d
c ~.
which nuclear factors bind to the promoter region of the CRF~ gene and regulate its transcription. The gene encoding the CRF2 receptor is more complex than that of the CRF~ receptor. The human CRF2 gene is more than 50 kb in size and spans 15 exons and 14 introns (Dautzenberg et al., 2000a). The first four exons encode the N-terminal segments of the three human CRF2 receptor splice variants, with exons 1 and 2 encoding the CRFz(b) variant, followed by exon 3 encoding the CRF2(c) variant, and finally exon 4, which gives rise to the CRFz(a) variant (Dautzenberg et al., 2000a; Fig. 1). The two CRFz(b)specific exons are separated from each other by a 10.5 kb intron (Fig. 1). It was recently speculated that the three CRF2 receptor splice variants may not be generated by excision of exons from the p r e - m R N A but by alternate usage of multiple promoter sites (Nanda et al., 2001; Catalano et al., 2003). Indeed, as exons 1, 3, and 4 encode the start methionine triplet A T G for each of the three splice variants, it is likely that these variants arise from alternate promoter usage (Catalano et al., 2003). First crude mapping of the putative promoter regions revealed various consensus sites for tissue-specific nuclear binding factors (Catalano et al., 2003). For the CRFz(b) promoter region, consensus sequences for vascular factors have been found, whereas the CRF2(c) promoter contains pituitary specific nuclear factor consensus sequences (Catalano et al., 2003). However, it is important to note that in contrast to rodents that show tissue-specific expression of the CRFz(a) and
a "
m
e~ 9
t. ~ t~
tt~ ~
~ ~-
~ t",l
~ tt~
~"
Fig. 1. Schematic representation of the gene encoding the human CRF2 receptor. The exons (El-15) are represented as grey boxes and their sizes are indicated above. The introns (I1-14) are drawn as lines. Exons 1 and 2 encode the N-terminal residues of the human CRFz(b) splice variants and are separated by a large 10.5 kb intron. Exon 3, which is specific for the CRF2(c)variant is downstream of the CRFz(b)-specificexons, whereas exon 4, which encodes the CRFz(a)variant, is separated from exon 3 by a 4.1 kb intron. The first exon common for all CRF2 receptor variants is only separated from the CRF2(a)-specific exon by a very small intron of 136 bp in size.
141
CRF2(b) receptors, in humans all three variants are coexpressed in several tissues. Additionally, the CRF2(a) variant dominates in all human tissues that have been analyzed, including the heart and pituitary (Valdenaire et al., 1997; Kostich et al., 1998). Thus, the factors regulating the generation of either splice variants are by far more complex than described by Catalano and colleagues. Nevertheless, it is clear that the expression of the CRF2 gene is very tightly regulated. To date only a few cell lines have been reported to express very low levels of CRF2 receptors, including the A-431 epidermoid (Kiang et al., 1998), the A7R5 aortic (Kageyama et al., 2000), and the AR-5 hippocampal cells (Sheriff et al., 2001). Interestingly, primary hippocampal neurons, when brought into tissue culture, seem to downregulate their CRF2 receptors (Pedersen et al., 2002). It is tempting to speculate that either a silencer region within the CRF2 gene may be activated when these cells are taken out of their natural context, or that in vivo neighboring neurons may secrete factors that are required for active transcription of the CRF2 gene and that these factors are missing in the primary cultures. Silencer regions have recently been identified in the CRF2 gene (Nanda et al., 2001).
CRF receptor signaling CRF receptors belong to the superfamily of G-proteincoupled receptors that are characterized by the presence of seven transmembrane domains. The CRF receptors belong to the class B subfamily, which includes receptors with small peptide ligands like growth hormone-releasing factor, calcitonin, vasoactive intestinal peptide, parathyroid hormone, and others (Segre and Goldring, 1993). The two CRF receptor subtypes share 70% identity in their amino acid sequence (Spiess et al., 1998). Agonist binding to either CRF receptor causes activation of adenylate cyclase (AC) through the stimulatory G-protein (Gs) and thereby results in an increase in the production of cAMP, which can then bind to the regulatory subunit of PKA. The cAMP-bound regulatory subunit dissociates from the catalytic subunit, thereby activating it and resulting in phosphorylation of a wide variety of proteins (McKnight et al., 1988). One of these proteins is the transcription factor CREB;
phosphorylation of CREB converts it into a powerful activator of gene transcription (Gonzalez and Montminy, 1989). In vitro studies demonstrate that CRF receptors are positively coupled to AC. CRF stimulation of AC activity has been demonstrated in pituitary cell culture (Labrie et al., 1982; Litvin et al., 1984) and CRF treatment of homogenates from a wide variety of rat brain regions increases AC activity (Wynn et al., 1984; Chen et al., 1986; Battaglia et al., 1987). In addition, CRF stimulation of a variety of CRF1 receptor-expressing neuronal-like cell lines elevates AC activity (Dieterich and DeSouza, 1996; Hogg et al., 1996; Iredale et al., 1996; Hauger et al., 1997; Schoeffter et al., 1999; Dautzenberg et al., 2000b; Roseboom et al., 2001b). Lastly, CRF treatment of a variety of cell lines transfected with vectors that express CRF1 receptors under control of a constitutively active promoter elevates intracellular cAMP levels (Chen et al., 1993; Xiong et al., 1995; Dieterich et al., 1996; Suman-Chauhan et al., 1999). Similar results have also been reported for the CRF2(a) receptor (Lovenberg et al., 1995a; Suman-Chauhan et al., 1999). In contrast to the extensive data on CRFstimulated AC, relatively less has been done to characterize the effects of CRF on PKA activity. CRF receptor activation increases PKA activity in a pituitary cell culture (Aguilera et al., 1983; Litvin et al., 1984). In addition, many of the effects of CRF on cell physiology are blocked by inhibitors of PKA activity, providing evidence that CRF receptor activation increases PKA activity in vitro. For example, activation of both CRF~ receptor and CRF2(a) receptor in transfected cells results in CREB phosphorylation that is blocked by the PKA inhibitor H89 (Rossant et al., 1999). In addition to the cAMP pathway, other second messenger pathways including MAP kinase, calcium, and phospholipase C have been implicated in the actions of CRF (Rossant et al., 1999). A recent study identified a variety of G-proteins that couple CRF receptors to various intracellular signaling pathways in the mouse hippocampus (Blank et al., 2003). The G-proteins and intracellular pathways that were activated depended on the strain of mouse used. In BALB/c mice, CRF stimulation coupled CRF receptors to Gq/ll and was associated
142 with protein kinase C (PKC) activation. In C57BL/ 6N mice, CRF stimulation coupled CRF receptors to Gs, Gq/11, and Gi and was associated with PKA activation. Application of CRF to mouse hippocampal slices from both strains of mice increased neuronal activity based on intracellular recording data. As expected, this CRF-dependent increase in BALB/c mice was blocked by an inhibitor of PKC, bisindoylmaleimide, and in C57BL/6N mice by an inhibitor of PKA, H-89. This study indicates that CRF receptors can couple to multiple G-proteins and intracellular pathways and varies with the strain of mouse that is studied.
G protein receptor kinases (GRKs) and CRF1 receptor desensitization Sustained exposure of the anterior pituitary and the brain to stress or high levels of administered CRF decreased CRF binding sites (Hauger et al., 1993; Fuchs and Flugge, 1995; Brunson et al., 2002), desensitized CRF-stimulated cAMP accumulation, and decreased ACTH release in corticotrophs (Hauger and Dautzenberg, 1999; Aguilera et al., 2001). Since alterations of signaling properties in vivo are impossible to measure, cellular systems are required for measuring long-term effects of CRF treatment. Several pituitary- and brain-derived cell lines expressing CRF1 receptors endogenously have been utilized to mimic the in vivo situation and have been compared to standard cell culture lines that recombinantly express CRF1 receptors, such as HEK293, COS, and Ltk cells. Likewise, CRF treatment of anterior pituitary cells or AtT20 cells causes large reductions in CRFstimulated ACTH release resulting from downregulation of CRF~ receptors and desensitization of CRF1 receptor-stimulated cAMP accumulation (Reisine and Hoffman, 1983; Hoffman et al., 1985). In principle, CRF1 receptor desensitization can be subdivided into two groups: homologous and heterologous (see following section). During homologous desensitization, only agonist-activated GPCRs desensitize, mainly due to phosphorylation through specific GRKs, followed by binding of cytosolic proteins called arrestins. Arrestins then facilitate sequestration of the respective GPCRs and result in receptor
internalization (Ferguson, 2001). In an environment favoring GRK action (i.e. preincubation with physiological or pharmacological levels of CRF), a subsequent challenge with CRF revealed a marked decrease in CRF-stimulated cAMP accumulation in human retinoblastoma Y79 and human neuroblastoma IMR-32 cells (Hauger et al., 1997; Aguilera et al., 2001; Dautzenberg et al., 2001a; Roseboom et al., 2001b). Homologous CRF1 receptor desensitization occurs in neuron-like cells exposed to physiological concentrations of CRF without any changes in CRF1 mRNA levels, even after 24 h of continuous agonist exposure (Hauger et al., 1997; Aguilera et al., 2001; Roseboom et al., 2001b). However, CRF1 receptor mRNA expression decreases in anterior pituitary cells exposed to CRF for several hours (Pozzoli et al., 1996). Homologous CRF1 receptor desensitization can be markedly inhibited in Y79 cells after a large reduction in GRK3 expression is induced by uptake of a GRK3 antisense oligonucleotide or transfection of a GRK3 antisense cDNA construct (Dautzenberg et al., 2001a). These findings suggest that GRK3mediated phosphorylation contributes to the homologous desensitization of brain CRF1 receptors. However, a high degree of CRF1 receptor phosphorylation was detected in COS cells expressing an epitope-tagged CRF1 receptor after exposure to CRF (Hauger et al., 2000). Because COS cells express GRK2, but not GRK3 (Menard et al., 1997), GRK2 appears to be capable of desensitizing CRF1 receptors in this cell system. Homologous desensitization of CRF1 receptors was significantly less in transfected HEK293 and Ltk cells when compared to native cells endogenously expressing CRF1 receptors (Dieterich et al., 1996; Hauger et al., 1997). It is possible that receptor reserve present in the transfected HEK293 and Ltk cells may compensate for agonist-mediated receptor desensitization (Fig. 2). Indeed, preincubation of transfected HEK293 cells with pharmacological levels of CRF strongly elevates basal cAMP levels but only minimally reduces CRFstimulated cAMP levels (Fig. 2). In contrast, CRF challenge of Y79 retinoblastoma cells that endogenously express CRF1 receptors only minimally elevates basal cAMP levels, but CRF-stimulated cAMP production is strongly reduced (Fig. 2). This is similar to results reported for the IMR-32 cell
143 HEK-hCRF1
Y79-hCRF1 45
400 350
I 40 i I 35 i
!
300
30
250 ~: o
~
1"
251
2oo N [] basal
15o !
i~~....
[ ] 100 nM CRF
lOO
:iii:/i~'/) i
20
***
~" 15 i
[]basal
--- i
[] 100 nM CRF
10i
0~ control
10 n M C R F
100 n M C R F
x 60 m i n
x 60 m i n
'---control
10 n M C R F
100 n M C R F
x 60 m i n
x 60 m i n
Fig. 2. Desensitization of cAMP responses in HEK293 cells recombinantly expressing human CRFI receptors (HEK-hCRFI) vs. Y79 retinoblastoma cells endogenously expressing the human CRF~ receptor (Y79-hCRF1). Both cell lines were exposed to a physiological CRF concentration (10 nM), extensively washed with > 1000-fold cellular volume of physiological buffers and then restimulated with a maximally active CRF concentration (100nM). In HEK-hCRF~ cells, despite a 10-fold increase in basal cAMP levels, only small reductions (up to 17%) of CRF-mediated cAMP production were observed, likely due to a large reserve of spare receptors. In contrast, strong reductions of CRF-mediated cAMP accumulation (up to 60% loss of second messenger responsiveness), accompanied by only minimal alterations of basal cAMP levels, were observed in Y79-hCRF~ cells after CRF preincubation. Statistical significance: *p < 0.05 vs. control; **p < 0.01 vs. control; ***p < 0.001 vs. control.
line, a line that also endogenously expresses CRFI receptors (Roseboom et al., 2001b). Studies aimed at understanding the physiological regulation of receptors should consider using endogenously expressing cell lines versus experiments utilizing transfected cells that express super-physiological levels of receptor.
Heterologous regulation of CRFt receptors by PKC In contrast to homologous desensitization, heterologous mechanisms can attenuate receptor responsiveness independent of agonist binding. Heterologous desensitization normally occurs when a receptor unbound by agonist is phosphorylated via a specific second messenger kinase that has been activated via another G P C R (Penn and Benovic, 1998). Since CRF1 and CRF2 receptors mainly couple to the stimulatory Gs protein (Hauger et al., 2003b), it was originally expected that P K A may also be involved in the heterologous desensitization of C R F receptors as demonstrated for other Gs-coupled G P C R s (Penn and Benovic, 1998; K o h o u t and Lefkowitz, 2003). However, both CRF~ and CRF2 receptors lack the classical P K A
consensus sequence Arg-Arg-X-Ser (where X can represent any amino acid). Indeed, our laboratories have not found an involvement of P K A in either homologous or heterologous C R F receptor desensitization (Hauger et al., 1997, 2000; Dautzenberg et al., 2001 a; Roseboom et al., 2001 b). Several potential phosphorylation sites Arg/ Lys-X-Ser-X-Arg for P K C have been identified in intracellular domains, especially the C-terminus of all vertebrate CRF1 and CRF2 receptors (Dautzenberg and Hauger, 2002). It has recently been shown that P K C activation potently desensitizes the CRF1 receptor, even in the absence of its ligand, whereas P K C inhibitors reduce the magnitude of C R F receptor desensitization (Hauger et al., 2003a). Furthermore, in transiently transfected COS cells, P K C activation results in CRF1 receptor phosphorylation (Hauger et al., 2003a), suggesting that PKC-mediated CRF1 receptor phosphorylation may be involved in physiological regulation of CRF1 receptors. Interestingly, in the recombinant setting, a high degree of basal CRF1 receptor phosphorylation was observed that could be decreased with specific P K C inhibitors (Hauger et al., 2003a; Fig. 3). It remains to be determined if this high degree of basal CRF1 receptor phosphorylation is restricted to the artificial overexpression system.
144 + 0
=9 -
0
0
.~ N
0
N
~~~ r,.)
~)
c,.)
r,.)
~i. ~:~:~~i~~%%i , i~i~~:~' .i~:~:i~% i.: .........,~i',:!~!~.~%%':~.;ii::"W; .......... ~':':~'~::ii ~"~.........~ ' :.~,~'~!~
75 kDa
70 kDa - -
i~..
?.
Fig. 4. Western blot probed with a mouse GRK3-specific monoclonal antibody. Y79 cells were exposed to physiological C R F concentrations (10nM) for up to 24h. G R K 3 protein levels increased more than 3-fold ('-~350%) compared to control cells not exposed to C R F .
~--- CRF1
Fig. 3. PKC- and CRF-mediated phosphorylation of the human CRF1 receptor. The 32p-labeled human epitope-tagged CRF~ receptor was exposed to a P K C activator or to 1 gM C R F in the presence or absence of P K C inhibitors for 5 min, followed by an immunoprecipitation step and SDS-PAGE. The 66 k D a band representing the phosphorylated CRFl-receptor was ~ 2.5-fold greater in C R F - and PKC-activated cells. In contrast P K C inhibitors blocked the basal phosphorylation > 2-fold.
Ligand-dependent CRF receptor adaptations CRF and urocortin 1 bind to the CRF1 receptor with similar affinity, and both peptides stimulate cAMP production with a similar potency in cells expressing endogenous or recombinant CRF1 receptors (Hauger et al., 2003b). In addition, both ligands desensitize second messenger responses of the CRF1 receptor in Y79 cells with a similar potency and time course (Dautzenberg et al., 2002). However, while CRF promotes CRF1 receptor phosphorylation (Hauger et al., 2000), this has yet to be examined for urocortin 1. Interestingly, CRF rapidly upregulates GRK3 mRNA, the main GRK capable of phosphorylating and desensitizing the CRFI receptor (Dautzenberg et al., 2001a). Urocortin 1 exposure has no effect on
GRK3 mRNA levels (Dautzenberg et al., 2002). In agreement with its potency to upregulate GRK3 mRNA levels, CRF exposure to Y79 cells also increases GRK3 protein levels (Fig. 4), albeit only during long-term exposure (Dautzenberg et al., 2002). It can be speculated that elevated GRK3 levels promote a faster desensitization and internalization of CRF1 receptors. Based on the data above, it appears that long-term elevated levels of CRF or urocortin 1 produce differential CRF receptor adaptation.
Regulation of CRF2 receptors Stress in the form of an inflammatory response can downregulate CRF2 receptor mRNA expression in the mouse heart (Heldwein et al., 1997), and left ventricular hypertrophy can increase urocortin 1 mRNA expression and downregulate C R F z ( b ) receptor mRNA expression in the human heart (Nishikimi et al., 2000). When aortic smooth muscle A7R5 cells or ventricular myocytes are exposed to urocortin 1 or CRF, a dose-dependent reduction in CRF2 receptor mRNA expression occurs (Kageyama et al., 1999; Coste et al., 2001). However, the homologous desensitization of CRF2 receptors has not been investigated to date, mainly due to the lack of cell lines expressing significant amounts of endogenous CRF2 receptors. A few cell lines have been reported to endogenously express CRF2 receptor mRNA, among those are the above mentioned A7R5 rat aortic
145 (Kageyama et al., 2000), the A-431 human epidermoid (Kiang et al., 1998), and the AR-5 rat amygdalar (Kasckow et al., 1999) cell lines. The A7R5 cell line expresses the CRF2(b) receptors that increase cAMP following CRF treatment. This cAMP increase is blocked by the CRF receptor antagonist Astressin (Kageyama et al., 2002). In the A-431 cell line, CRF receptor activation alters intracellular Ca 2+ levels (Kiang, 1994), activates PKC (Kiang et al., 1994), and phosphorylates phospholipase C-gamma (Kiang et al., 1998). The AR-5 amygdalar cells express the CRF2(a) receptor and CRF treatment increases cAMP in these cells. The cells also express neuropeptide Y (NPY) receptors and NPY treatment can decrease CRF-induced increases in cAMP (Sheriff et al., 2001). This latter finding is of interest because CRF and NPY can produce opposite effects on anxiety-like behaviors when injected into the amygdala, with CRF producing anxiogenic responses (Liang and Lee, 1988) and NPY producing anxiolytic responses (Heilig et al., 1993). The results from the AR-5 cells led the authors to suggest that the opposite effects of CRF and NPY on anxiety-like behaviors may be mediated in part by the opposing actions of these two peptides on cAMP accumulation in the amygdala (Sheriff et al., 2001). Additionally, nicotinic agonists can increase CRF mRNA levels in AR-5 cells (Kasckow et al., 1999), which is interesting because acetylcholine induces CRF release from the rat amygdala and hypothalamus, possibly through nicotinic receptors (Raber et al., 1995). Overall, however, the usefulness of these cell lines is limited by the relatively low second messenger responses seen upon receptor stimulation. Furthermore, CRF2 receptor mRNA and protein levels seem to decrease rapidly when primary cells are established from tissues shown to express CRF2 mRNA in situ (Pedersen et al., 2002).
Microarray studies to identify genes affected by the CRF system Great advances have been made in the areas of gene chip and laser capture technology, where the expression patterns of thousands of genes can be investigated within single cells. However, these relatively new techniques also present challenges in data analysis and interpretation.
DNA microarray chips are templates of genes of interest made of cDNA clones or oligonucleotides. Using this technology, small amounts of tissue are required, for example, a few cells which can be microdissected out of a brain area of interest using laser capture methodology (Simone et al., 1998). The importance of this technology in understanding basic mechanisms is illustrated by the estimation that 50-60% of known genes are expressed in the brain (Sandberg et al., 2000), an organ that is believed to exhibit the greatest complexity of gene expression throughout the body (Colantuoni et al., 2000). Studies using microarray technology to understand how stress affects gene expression through CRF system activation and how these responses may be altered in pathological conditions are scarce. Recently, changes in the expression of genes that are involved in intracellular calcium signaling, neurogenesis, and myelination have been reported in several brain regions in transgenic mice overexpressing CRF (Peeters et al., 2002), suggesting that adaptive mechanisms exist to compensate for life-long exposure to elevated levels of CRF. Both downregulation of enhancers of glucocorticoid receptor signaling (11B-HSD 1) and upregulation of repressors of this signaling pathway (FK506 binding protein 5) were observed in transgenic mice, suggesting adaptation to the six-fold increase in plasma glucocorticoid levels seen in these CRF overexpressing mice. At a cellular level, stress and increased HPA axis activity have been shown to decrease neurogenesis (Gould et al., 2000). This is consistent with the reduction in neurogenesis in CRF overexpressing mice. In addition to an overactive HPA axis, inhibited neurogenesis may be relevant to the development of mood disorders (Yuan et al., 2003). In summary, these microarray experiments in transgenic mice identify the compensatory changes that take place in the brain in response to elevation of CRF and corticosterone from an early age, and may identify those changes that underlie stress-induced psychopathology. A recent study compared the mRNA expression patterns from the whole brains of mice lacking a functional CRF1 receptor to that of mice that had received 40 mg/kg of the CRF1 antagonist R121919 administered orally for 1 or 7 days. Importantly, the alterations in gene expression seen in the knockout mice were mimicked by 7-day treatment with the
146 CRF1 antagonist (Landgrebe et al., 2002). There was a strain difference, with the antagonist having a bigger effect in wildtype mice of a 129SvJ background than in wildtype mice of a 129Ola/CD 1 background. Therefore, for certain genetic backgrounds, administration of CRF1 receptor antagonists can mimic the changes seen in CRF1 receptor knockout mice. In studying the effects of acute CRF exposure and of blockade of the CRF1 receptor with R121919 in AtT-20 cell cultures, microarray analysis of 7256 genes revealed altered gene expression in about 90 genes that was attenuated by the antagonist (Peeters et al., 2002). Known targets of CRF1 signaling that were altered included immediate early genes such as Jun/B, Nurrl, and Nurr77. For Nurrl, it has been shown that CRF signaling leads to induction of mRNA through PKA- and calcium calmodulin kinase II-dependent mechanisms, while Nurr77 transactivation is regulated through a MAPK-dependent pathway (Kovalovsky et al., 2002). Moreover, several previously unknown targets involved in this signaling cascade were identified and subsequently confirmed by quantitative real timepolymerase chain reaction (qRT-PCR), demonstrating the usefulness of microarrays to accelerate the discovery of the function of unknown genes. Another microarray study using Clontech nylon filter arrays focusing on the rat septal brain region revealed that 19 genes with altered expression in response to restraint stress (Roseboom et al., 2001a). Each of these genes could potentially mediate adaptive or maladaptive responses to stress and could serve as novel targets for therapeutic intervention. In this study Pat Roseboom and Ned Kalin also identified 16 genes with altered expression in response to treatment with the non-selective CRF receptor antagonist [D-Phe 12, Nle 21'38, C~-MeLeu37]CRF12_41 (D-Phe). These changes occurred in the absence of stress, demonstrating intrinsic effects of D-Phe. Some of these changes may be the result of antagonism of baseline CRF neurotransmission, whereas others may be the result of D-Phe acting through as yet unidentified pathways. Most importantly, we determined that D-Phe blocked the effects of stress on 13 of 19 genes that were altered in response to restraint (Roseboom et al., 2001a). Ned Kalin and Pat Roseboom are in the process of confirming these changes with qRT-PCR, and so far have confirmed
the D-Phe blockade of the stress-induced increase in somatostatin mRNA expression. This study demonstrates that stress-induced activation of CRF receptors results in downstream effects in a variety of biochemical pathways. Future studies will determine the extent to which these effects are physiologically relevant. We also established that stress induces changes in 6 genes through mechanisms other than CRF receptor activation, for example an increase in ~-calcium/calmodulin-dependent protein kinase II. Although gene expression changes need to be confirmed by qRT-PCR and in situ hybridization, the present results illustrate novel pathways that may be involved in mediating stress effects. In addition, these newly implicated genes could lead to novel targets for the development of therapeutic agents for the treatment of stress-related disorders.
Future directions
There is a great deal of preclinical and clinical data associating alterations in the CRF system with stress-induced psychopathologies, such as depression and anxiety disorders. Ultimately, it is important to identify the molecular mechanisms that account for these alterations in the CRF system. Several questions remain to be addressed. As with many other genes, polymorphisms in the genes of the CRF system may alter the body's response to stressful stimuli and have a great impact on the susceptibility to stress-induced pathology. So far, there is no report on polymorphisms within the coding regions of the various genes of the CRF system. In addition, polymorphisms may exist in the promoter and 5'-upstream regions, as well as in the large introns that may up- or downregulate gene transcription and/ or mRNA translation. It is important to note that the promoter and 5'-upstream regions, especially in the CRF receptor genes and the genes encoding urocortin 2 and urocortin 3, are either poorly characterized or not identified yet. Thus, extensive studies unraveling their structure and identifying transcription factor interactions may facilitate understanding the complex nature of CRF system regulation. Drugs that influence these promoter regions either directly, or indirectly by altering the actions of promoterspecific transcription factors, represent a novel
147 approach to correcting or preventing stress-induced psychopathology. Current studies utilizing gene-chip technology may identify novel biochemical pathways mediating the C R F receptor-dependent effects of stress on the brain. Identifying alterations in these pathways in the brains of animals that show a pathological response to stress may lead to novel drug targets for the development of "anti-stress" medications that could significantly improve quality of life and productivity for a large fraction of the human population.
Abbreviations AC ACTH AP-1 CRF CRE CREB CRF-BP CRF~ CRF2 D-Phe GR GRE GRK GPCRs Gs HPA qRT-PCR nGRE NPY PKA PKC PVN
adenylate cyclase adrenocorticotropic hormone activator protein- 1 corticotropin-releasing factor cAMP-response element cAMP-response element binding protein corticotropin-releasing factor binding protein CRF~ receptor CRF2 receptor [D-Phe 12, Nle 21'38, C~-MeLeu 37] CRF12_41 glucocorticoid receptor glucocorticoid response element G protein-coupled receptor kinase G-protein-coupled receptors stimulatory G-protein hypothalamic-pituitary-adrenal quantitative real time-polymerase chain reaction negative glucocorticoid response element nuropeptide Y cAMP-dependent protein kinase protein kinase C paraventricular nucleus of the hypothalamus
Acknowledgments This work was supported by N I H grant MH40855 and the U W HealthEmotions Research Institute
(Madison, WI). The authors would like to thank Stephany G. Jones and Kim A. Jochman for advice and encouragement during the preparation of this review. Ned H. Kalin and Patrick H. Roseboom have equity positions in Promoter Neurosciences, LLC (Madison, WI), and have applied for patents through the Wisconsin Alumni Research Foundation (WARF, Madison, WI) related to promoter-based treatments to alter C R F regulation. In addition to other pharmaceutical companies, Ned H. Kalin is also a scientific consultant to Neurocrine Biosciences (San Diego, CA). Ned H. Kalin and Patrick H. Roseboom had a contractual arrangement with Johnson and Johnson to investigate the effects of C R F receptor antagonists in cell culture.
References Aguilera, G., Harwood, J.P., Wilson, J.X., Morell, J., Brown, J.H. and Catt, K.J. (1983) Mechanisms of action of corticotropin-releasing factor and other regulators of corticotropin release in rat pituitary cells. J. Biol. Chem., 258: 8039-8045. Aguilera, G., Jessop, D.S., Harbuz, M.S., Kiss, A. and Lightman, S.L. (1997) Differential regulation of hypothalamic pituitary corticotropin releasing hormone receptors during development of adjuvant-induced arthritis in the rat. J. Endocrinol., 153: 185-191. Aguilera, G., Rabadan-Diehl, C. and Nikodemova, M. (2001) regulation of pituitary corticotropin releasing hormone receptors. Peptides, 22: 769-774. Albeck, D.S., McKittrick, C.R., Blanchard, D.C., Blanchard, R.J., Nikulina, J., McEwen, B.S. and Sakai, R.R. (1997) Chronic social stress alters levels of corticotropin-releasing factor and arginine vasopressin mRNA in rat brain. J. Neurosci., 17: 4895-4903. Andrews, M.W. and Rosenblum, L.A. (1994) The development of affiliative and agonistic social patterns in differentially reared monkeys. Child Dev., 65: 1398-1404. Anisman, H., Zaharia, M.D., Meaney, M.J. and Merali, Z. (1998) Do early-life events permanently alter behavioral and hormonal responses to stressors? Int. J. Dev. Neurosci., 16: 149-164. Arase, K., York, D.A., Shimizu, H., Shargill, N. and Bray, G.A. (1988) Effects of corticotropin-releasing factor on food intake and brown adipose tissue thermogenesis in rats. Am. J. Physiol., 255: E255-259. Arborelius, L., Owens, M.J., Plotsky, P.M. and Nemeroff, C.B. (1999) The role of corticotropin-releasing factor in depression and anxiety disorders. J. Endocrinol., 160: 1-12. Aubry, J.M., Pozzoli, G. and Vale, W.W. (1999) Chronic treatment with the antidepressant amitriptyline decreases
148 CRF-R1 receptor mRNA levels in the rat amygdala. Neurosci. Lett., 266: 197-200. Bakshi, V.P. and Kalin, N.H. (2000) Corticotropin-releasing hormone and animal models of anxiety: gene-environment interactions. Biol. Psychiatry, 48:1175-1198. Bakshi, V.P., Smith-Roe, S., Newman, S.M., Grigoriadis, D.E. and Kalin, N.H. (2002) Reduction of stress-induced behavior by antagonism of corticotropin-releasing hormone 2 (CRHz) receptors in lateral septum or CRH1 receptors in amygdala. J. Neurosci., 22: 2926-2935. Bale, T.L., Contarino, A., Smith, G.W., Chan, R., Gold, L.H., Sawchenko, P.E., Koob, G.F., Vale, W.W. and Lee, K.F. (2000) Mice deficient for corticotropin-releasing hormone receptor-2 display anxiety-like behaviour and are hypersensitive to stress. Nat. Genet., 24: 410-414. Bale, T.L., Picetti, R., Contarino, A., Koob, G.F., Vale, W.W. and Lee, K.F. (2002) Mice deficient for both corticotropinreleasing factor receptor 1 (CRFR1) and CRFR2 have an impaired stress response and display sexually dichotomous anxiety-like behavior. J. Neurosci., 22: 193-199. Bartanusz, V., Jezova, D., Bertini, L.T., Tilders, F.J., Aubry, J.M. and Kiss, J.Z. (1993) Stress-induced increase in vasopressin and corticotropin-releasing factor expression in hypophysiotrophic paraventricular neurons. Endocrinology, 132: 895-902. Battaglia, G., Webster, E.L. and DeSouza, E.B. (1987) Characterization of corticotropin-releasing factor receptormediated adenylate cyclase activity in the rat central nervous system. Synapse, 1: SYN1. Blank, T., Nijholt, I., Grammatopoulos, D.K., Randeva, H.S., Hillhouse, E.W. and Spiess, J. (2003) Corticotropin-releasing factor receptors couple to multiple G-proteins to activate diverse intracellular signaling pathways in mouse hippocampus: role in neuronal excitability and associative learning. J. Neurosci., 23: 700-707. Bonaz, B. and Rivest, S. (1998) Effect of a chronic stress on CRF neuronal activity and expression of its type 1 receptor in the rat brain. Am. J. Physiol., 275: R1438-R1449. Bowlby, J. (1973) Attachment and Loss, Vol. II: Separation, Basic Books, New York. Brady, L.S., Gold, P.W., Herkenham, M., Lynn, A.B. and Whitfield, H.J., Jr. (1992) The antidepressants fluoxetine, idazoxan and phenelzine alter corticotropin-releasing hormone and tyrosine hydroxylase mRNA levels in rat brain: therapeutic implications. Brain Res., 572: 117-125. Brady, L.S., Whitfield, H.J., Jr., Fox, R.J., Gold, P.W. and Herkenham, M. (1991) Long-term antidepressant administration alters corticotropin-releasing hormone, tyrosine hydroxylase, and mineralocorticoid receptor gene expression in rat brain. Therapeutic implications. J. Clin. Invest., 87: 831-837. Brunson, K.L., Grigoriadis, D.E., Lorang, M.T. and Baram, T.Z. (2002) Corticotropin-Releasing hormone (CRH) downregulates the function of its receptor (CRF1)
and induces CRF1 expression in hippocampal and cortical regions of the immature rat brain. Exp. Neurol., 176: 75-86. Carlson, M. and Earls, F. (1997) Psychological and neuroendocrinological sequelae of early social deprivation in institutionalized children in Romania. Ann. N. Y. Acad. Sci., 807:419-428. Catalano, R.D., Kyriakou, T., Chen, J., Easton, A. and Hillhouse, E.W. (2003) Regulation of corticotropin-releasing hormone type 2 receptors by multiple promoters and alternative splicing: identification of multiple splice variants. Mol. Endocrinol., 17: 395-410. Chen, A., Vaughan, J. and Vale, W.W. (2003) Glucocorticoids regulate the expression of the mouse urocortin II gene: a putative connection between the corticotropin releasing factor receptor pathways. Mol. Endocrinol., 17: 1622-1639. Chen, F.M., Bilezikjian, L.M., Perrin, M.H., Rivier, J. and Vale, W. (1986) Corticotropin releasing factor receptormediated stimulation of adenylate cyclase activity in the rat brain. Brain Res., 381: 49-57. Chen, R., Lewis, K.A., Perrin, M.H. and Vale, W.W. (1993) Expression cloning of a human corticotropin-releasing-factor receptor. Proc. Natl. Acad. Sci. USA, 90: 8967-8971. Chen, Y., Hatalski, C.G., Brunson, K.L. and Baram, T.Z. (2001) Rapid phosphorylation of the CRE binding protein precedes stress-induced activation of the corticotropin releasing hormone gene in medial parvoceUular hypothalamic neurons of the immature rat. Brain Res. Mol. Brain Res., 96: 39-49. Colantuoni, C., Purcell, A.E., Bouton, C.M. and Pevsner, J. (2000) high throughput analysis of gene expression in the human brain. J. Neurosci. Res., 59: 1-10. Contarino, A., Dellu, F., Koob, G.F., Smith, G.W., Lee, K.F., Vale, W. and Gold, L.H. (1999) Reduced anxiety-like and cognitive performance in mice lacking the corticotropinreleasing factor receptor 1. Brain Res., 835: 1-9. Coplan, J.D., Andrews, M.W., Rosenblum, L.A., Owens, M.J., Friedman, S., Gorman, J.M. and Nemeroff, C.B. (1996) Persistent elevations of cerebrospinal fluid concentrations of corticotropin-releasing factor in adult nonhuman primates exposed to early-life stressors: implications for the pathophysiology of mood and anxiety disorders. Proc. Natl. Acad. Sci. USA, 93: 1619-1623. Coplan, J.D., Smith, E.L., Trost, R.C., Scharf, B.A., Altemus, M., Bjornson, L., Owens, M.J., Gorman, J.M., Nemeroff, C.B. and Rosenblum, L.A. (2000) Growth hormone response to clonidine in adversely reared young adult primates: relationship to serial cerebrospinal fluid corticotropin-releasing factor concentrations. Psychiatry Res., 95: 93-102. Coste, S.C., Heldwein, K.A., Stevens, S.L., Tobar-Dupres, E. and Stenzel-Poore, M.P. (2001) IL-lalpha and TNFalpha down-regulate CRH receptor-2 mRNA expression in the mouse heart. Endocrinology, 142: 3537-3545. Coste, S.C., Kesterson, R.A., Heldwein, K.A., Stevens, S.L., Heard, A.D., Hollis, J.H., Murray, S.E., Hill, J.K., Pantely,
149 G.A., Hohimer, A.R., Hatton, D.C., Phillips, T.J., Finn, D.A., Low, M.J., Rittenberg, M.B., Stenzel, P. and Stenzel-Poore, M.P. (2000) Abnormal adaptations to stress and impaired cardiovascular function in mice lacking corticotropin-releasing hormone receptor-2. Nat. Genet., 24: 403-409. Dautzenberg, F.M. and Hauger, R.L. (2002) The CRF peptide family and their receptors: yet more partners discovered. Trends Pharmacol. Sci., 23: 71-77. Dautzenberg, F.M., Braun, S. and Hauger, R.L. (2001a) GRK3 mediates homologous desensitization of CRF1 receptors: a potential mechanism regulating stress adaptation. Am. J. Physiol. Regul. Integr. Comp. Physiol., 280: R935-946. Dautzenberg, F.M., Higelin, J. and Teichert, U. (2000b) Functional characterization of corticotropin-releasing factor type 1 receptor endogenously expressed in human embryonic kidney 293 cells. Eur. J. Pharmacol., 390: 51-59. Dautzenberg, F.M., Huber, G., Higelin, J., Py-Lang, G. and Kilpatrick, G.J. (2000a) Evidence for the abundant expression of arginine 185 containing human CRF(2alpha) receptors and the role of position 185 for receptor-ligand selectivity. Neuropharmacology, 39:1368-1376. Dautzenberg, F.M., Kilpatrick, G.J., Hauger, R.L. and Moreau, J. (2001b) Molecular biology of the CRH receptors - in the mood. Peptides, 22: 753-760. Dautzenberg, F.M., Wille, S., Braun, S. and Hauger, R.L. (2002) GRK3 regulation during CRF- and urocortin-induced CRF~ receptor desensitization. Biochem. Biophys. Res. Commun., 298: 303-308. De Souza, E.B. (1995) Corticotropin-releasing factor receptors: physiology, pharmacology, biochemistry and role in central nervous system and immune disorders. Psychoneuroendocrinology, 20:789-819. Dent, G.W., Smith, M.A. and Levine, S. (2000) Rapid induction of corticotropin-releasing hormone gene transcription in the paraventricular nucleus of the developing rat. Endocrinology, 141: 1593-1598. Diamond, M.I., Miner, J.N., Yoshinaga, S.K. and Yamamoto, K.R. (1990) Transcription factor interactions: selectors of positive or negative regulation from a single DNA element. Science, 249: 1266-1272. Dieterich, K.D. and DeSouza, E.B. (1996) Functional corticotropin-releasing factor receptors in human neuroblastoma cells. Brain Res., 733:113-118. Dieterich, K.D., Grigoriadis, D.E. and De Souza, E.B. (1996) Homologous desensitization of human corticotropin-releasing factor~ receptor in stable transfected mouse fibroblast cells. Brain Res., 710: 287-292. Dunn, A.J. and Berridge, C.W. (1990) Physiological and behavioral responses to corticotropin-releasing factor administration: is CRF a mediator of anxiety or stress responses? Brain Res. Brain Res. Rev., 15: 71-100. Fadda, P., Pani, L., Porcella, A. and Fratta, W. (1995) Chronic imipramine, L-sulpiride and mianserin decrease corticotropin
releasing factor levels in the rat brain. Neurosci. Lett., 192: 121-123. Ferguson, S.S. (2001) Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacol. Rev., 53: 1-24. Francis, D., Diorio, J., Liu, D. and Meaney, M.J. (199%) nongenomic transmission across generations of maternal behavior and stress responses in the rat. Science, 286: 1155-1158. Francis, D.D., Caldji, C., Champagne, F., Plotsky, P.M. and Meaney, M.J. (1999b) The role of corticotropin-releasing factor - norepinephrine systems in mediating the effects of early experience on the development of behavioral and endocrine responses to stress. Biol. Psychiatry, 46: 1153-1166. Fuchs, E. and Flugge, G. (1995) Modulation of binding sites for corticotropin-releasing hormone by chronic psychosocial stress. Psychoneuroendocrinology, 20: 33-51. Gentles, A.J. and Karlin, S. (1999) Why are human G-proteincoupled receptors predominantly intronless? Trends Genet., 15: 47-49. Giardino, L., Puglisi-Allegra, S. and Ceccatelli, S. (1996) CRHR1 mRNA expression in two strains of inbred mice and its regulation after repeated restraint stress. Brain Res. Mol. Brain Res., 40:310-314. Gonzalez, G.A. and Montminy, M.R. (1989) Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell, 59: 675-680. Gould, E., Tanapat, P., Rydel, T. and Hastings, N. (2000) Regulation of hippocampal neurogenesis in adulthood. Biol. Psychiatry, 48: 715-720. Guardiola-Diaz, H.M., Boswell, (7. and Seasholtz, A.F. (1994) The cAMP-responsive element in the corticotropin-releasing hormone gene mediates transcriptional regulation by depolarization. J. Biol. Chem., 269: 14784-14791. Guillemin, R. and Rosenberg, B. (1955) Humoral hypothalamic control of anterior pituitary: a study with combined tissue cultures. Endocrinology, 57: 599-607. Hatalski, C.G., Guirguis, C. and Baram, T.Z. (1998) Corticotropin releasing factor mRNA expression in the hypothalamic paraventricular nucleus and the central nucleus of the amygdala is modulated by repeated acute stress in the immature rat. J. Neuroendocrinol., 10: 663-669. Hauger, R.L. and Dautzenberg, F.M. (1999) Regulation of the stress response by corticotropin-releasing factor receptors. In: Conn, P.M. and Freeman, M.E. (Eds.), Neuroendocrinology in Physiology and Medicine, Humana Press, Totowa, NJ, pp. 261-286. Hauger, R.L., Dautzenberg, F.M., Flaccus, A., Liepold, T. and Spiess, J. (1997) Regulation of corticotropin-releasing factor receptor function in human Y-79 retinoblastoma cells: rapid and reversible homologous desensitization but prolonged recovery. J. Neurochem., 68: 2308-2316.
150 Hauger, R.L., Grigoriadis, D.E., Dallman, M.F., Plotsky, P.M., Vale, W.W. and Dautzenberg, F.M. (2003b) International union of pharmacology. XXXVI. Current status of the nomenclature for receptors for corticotropin-releasing factor and their ligands. Pharmacol. Rev., 55: 21-26. Hauger, R.L., Irwin, M.R., Lorang, M., Aguilera, G. and Brown, M.R. (1993) High intracerebral levels of CRH result in CRH receptor downregulation in the amygdala and neuroimmune desensitization. Brain Res., 616: 283-292. Hauger, R.L., Olivares-Reyes, J.A., Braun, S.D., Catt, K.J. and Dautzenberg, F.M. (2003a) Mediation of CRF1 receptor phosphorylation and desensitization by protein kinase C: a possible role in stress adaptation. J. Pharmacol. Exp. Ther., 306: 794-803. Hauger, R.L., Smith, R.D., Braun, S., Dautzenberg, F.M. and Catt, K.J. (2000) Rapid agonist-induced phosphorylation of the human CRF receptor, type 1: a potential mechanism for homologous desensitization. Biochem. Biophys. Res. Commun., 268: 572-576. Heilig, M., McLeod, S., Brot, M., Heinrichs, S.C., Menzaghi, F., Koob, G.F. and Britton, K.T. (1993) Anxiolytic-like action of neuropeptide Y: mediation by Y1 receptors in amygdala, and dissociation from food intake effects. Neuropsychopharmacology, 8: 357-363. Heim, C. and Nemeroff, C.B. (1999) The impact of early adverse experiences on brain systems involved in the pathophysiology of anxiety and affective disorders. Biol. Psychiatry, 46:1509-1522. Heldwein, K.A., Duncan, J.E., Stenzel, P., Rittenberg, M.B. and Stenzel-Poore, M.P. (1997) Endotoxin regulates corticotropin-releasing hormone receptor 2 in heart and skeletal muscle. Mol. Cell Endocrinol., 131: 167-172. Herman, J.P., Schafer, M.K., Thompson, R.C. and Watson, S.J. (1992) Rapid regulation of corticotropin-releasing hormone gene transcription in vivo. Mol. Endocrinol., 6: 1061-1069. Heuser, I., Bissette, G., Dettling, M., Schweiger, U., Gotthardt, U., Schmider, J., Lammers, C.H., Nemeroff, C.B. and Holsboer, F. (1998) Cerebrospinal fluid concentrations of corticotropin-releasing hormone, vasopressin, and somatostatin in depressed patients and healthy controls: response to amitriptyline treatment. Depress Anxiety, 8: 71-79. Ho, S.P., Takahashi, L.K., Livanov, V., Spencer, K., Lesher, T., Maciag, C., Smith, M.A., Rohrbach, K.W., Hartig, P.R. and Arneric, S.P. (2001) Attenuation of fear conditioning by antisense inhibition of brain corticotropin releasing factor-2 receptor. Brain Res. M ol. Brain Res., 89: 29-40. Hoffman, A.R., Ceda, G. and Reisine, T.D. (1985) Corticotropin-releasing factor desensitization of adrenocorticotropic hormone release is augmented by arginine vasopressin. J. Neurosci., 5: 234-242. Hogg, J.E., Myers, J. and Hutson, P.H. (1996) The human neuroblastoma cell line, IMR-32, expresses functional corticotropin-releasing factor receptors. Eur. J. Pharmacol., 312:257-261.
Hsu, D.T., Chen, F.L., Takahashi, L.K. and Kalin, N.H. (1998) Rapid stress-induced elevations in corticotropin-releasing hormone mRNA in rat central amygdala nucleus and hypothalamic paraventricular nucleus: an in situ hybridization Analysis. Brain Res., 788: 305-310. Hsu, D.T., Lombardo, K.A., Herringa, R.J., Bakshi, V.P., Roseboom, P.H. and Kalin, N.H. (2001) Corticotropinreleasing hormone messenger RNA distribution and stressinduced activation in the thalamus. Neuroscience, 105:911-921. Iredale, P.A., Terwilliger, R., Widnell, K.L., Nestler, E.J. and Duman, R.S. (1996) Differential regulation of corticotropinreleasing factor (1) receptor expression by stress and agonist treatments in brain and cultured cells. Mol. Pharmacol., 50: 1103-1110. Jensen, J.B., Jessop, D.S., Harbuz, M.S., Mork, A., Sanchez, C. and Mikkelsen, J.D. (1999) Acute and long-term treatments with the selective serotonin reuptake inhibitor citalopram modulate the HPA axis activity at different levels in male rats. J. Neuroendocrinol., 11: 465-471. Kageyama, K., Bradbury, M.J., Zhao, L., Blount, A.L. and Vale, W.W. (1999) Urocortin messenger ribonucleic acid: tissue distribution in the rat and regulation in thymus by lipopolysaccharide and glucocorticoids. Endocrinology, 140:5651-5658. Kageyama, K., Gaudriault, G.E., Bradbury, M.J. and Vale, W.W. (2000) Regulation of corticotropin-releasing factor receptor type 2 beta messenger ribonucleic acid in the rat cardiovascular system by urocortin, glucocorticoids, and cytokines. Endocrinology, 141: 2285-2293. Kageyama, K., Gaudriault, G.E., Suda, T. and Vale, W.W. (2002) Regulation of corticotropin-releasing factor receptor type 2beta mRNA via cyclic AMP pathway in A7r5 aortic smooth muscle cells. Cell Signal, 15: 17-25. Kalin, N.H. (1985) Behavioral effects of ovine corticotropinreleasing factor administered to rhesus monkeys. Fed. Proc., 44: 249-253. Kalin, N.H. (1997) The neurobiology of fear. Sci. Am, Mysteries of the Mind: 76-83. Kalin, N.H., Shelton, S.E., Kraemer, G.W. and McKinney, W.T. (1983) Corticotropin-Releasing factor administered intraventricularly to rhesus monkeys. Peptides, 4: 217-220. Kalin, N.H., Shelton, S.E., Rickman, M. and Davidson, R.J. (1998) Individual differences in freezing and cortisol in infant and mother rhesus monkeys. Behav. Neurosci., 112:251-254. Kalin, N.H., Takahashi, L.K. and Chen, F.L. (1994) Restraint stress increases corticotropin-releasing hormone mRNA content in the amygdala and paraventricular nucleus. Brain Res., 656: 182-186. Kaplan, N.M. (1992) The adrenal glands. In: Griffin, J.E. and Ojeda, S.R. (Eds.), Textbook of Endocrine Physiology, Oxford University Press, New York, pp. 247-275. Kasckow, J.W., Regmi, A., Sheriff, S., Mulchahey, J. and Geracioti, T.D., Jr. (1999) Regulation of corticotropinreleasing factor messenger RNA by nicotine in an immortalized amygdalar cell line. Life. Sci., 65: 2709-2714.
151 Kemp, C.F., Woods, R.J. and Lowry, P.J. (1998) The corticotrophin-releasing factor-binding protein: an act of several parts. Peptides, 19:1119-1128. Kiang, J.G. (1994) Corticotropin-releasing factor increases [Ca2+]i via receptor-mediated Ca2+ channels in human epidermoid A-431 cells. Eur. J. Pharmacol., 267: 135-142. Kiang, J.G., Ding, X.Z., Gist, I.D., Jones, R.R. and Tsokos, G.C. (1998) Corticotropin-releasing factor induces phosphorylation of phospholipase C-gamma at tyrosine residues via its receptor 2beta in human epidermoid A-431 cells. Eur. J. Pharmacol., 363: 203-210. Kiang, J.G., Wang, X. and McClain, D.E. (1994) Corticotropin-releasing factor increases protein kinase C activity by elevating membrane-bound alpha and beta isoforms. Chin J Physiol., 37: 105-110. Kino, T. and Chrousos, G.P. (2005) Glucocorticoid effects on gene expression. In: Steckler, T., Kalin, N. and Reul, J.M.H.M. (Eds.), Handbook of Stress and the Brain, Part 1, Elsevier, Amsterdam, pp. 295-312. Kishimoto, T., Radulovic, J., Radulovic, M., Lin, C.R., Schrick, C., Hooshmand, F. Hermanson, O., Rosenfeld, M.G. and Spiess, J. (2000) Deletion of crhr2 reveals an anxiolytic role for corticotropin-releasing hormone receptor2. Nat. Genet, 24: 415-419. Kohout, T.A. and Lefkowitz, R.J. (2003) Regulation of G protein-coupled receptor kinases and arrestins during receptor desensitization. Mol. Pharmacol., 63: 9-18. Koob, G.F. and Heinrichs, S.C. (1999) A role for corticotropin releasing factor and urocortin in behavioral responses to stressors. Brain Res, 848: 141-152. Koob, G.F., Heinrichs, S.C., Pich, E.M., Menzaghi, F., Baldwin, H., Miczek, K. and Britton, K.T. (1993) The role of corticotropin-releasing factor in behavioural responses to stress. Ciba. Found Symp., 172: 277-289. Kostich, W.A., Chen, A., Sperle, K. and Largent, B.L. (1998) Molecular identification and analysis of a novel human corticotropin-releasing factor (CRF) receptor: the CRF2g . . . . receptor. Mol. Endocrinol, 12: 1077-1085. Kovalovsky, D., Refojo, D., Liberman, A.C., Hochbaum, D., Pereda, M.P., Coso, O.A., Stalla, G.K., Holsboer, F. and Arzt, E. (2002) Activation and induction of NUR77/NURR1 in corticotrophs by CRH/cAMP: involvement of calcium, protein kinase A, and MAPK pathways. Mol. Endocrinol, 16: 1638-1651. Krahn, D.D., Gosnell, B.A., Grace, M. and Levine, A.S. (1986) CRF antagonist partially reverses CRF- and stress-induced effects on feeding. Brain Res. Bull., 17: 285-289. Labrie, F., Veilleux, R. and LeFevre, G. (1982) Corticotropinreleasing factor stimulates accumulation of adenosine 3' • 5'monophasphate in rat pituitary corticotrophs. Science, 216: 1007-1008. Landgrebe, J., Wurst, W. and Welzl, G. (2002) Permutationvalidated principal components analysis of microarray data. Genome. Biol., 3:research0019.0011-0019.0011.
Lewis, K., Li, C., Perrin, M.H., Blount, A., Kunitake, K., Donaldson, C., Vaughan, J., Reyes, T.M., Gulyas, J., Fischer, W., Bilezikjian, L., Rivier, J., Sawchenko, P.E. and Vale, W.W. (2001) Identification of urocortin III, an additional member of the corticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor. Proc. Natl. Acad. Sci. USA, 98: 7570-7575. Li, P., He, X., Gerrero, M.R., Mok, M., Aggarwal, A. and Rosenfeld, M.G. (1993) Spacing and orientation of bipartite DNA-binding motifs as potential functional determinants for POU domain factors. Genes Dev., 7: 2483-2496. Liang, K.C. and Lee, E.H.Y. (1988) Intra-amygdala injections of corticotropin releasing factor facilitate inhibitory avoidance learning and reduce exploratory behavior in rats. Psychopharmacology (Berl). 96: 232-236. Litvin, Y., PasMantier, R., Fleischer, N. and Erlichman, J. (1984) Hormonal activation of the cAMP-dependent protein kinases in AtT20 cells. J. Biol. Chem., 259: 10296-10302. Lombardo, K.A., Herringa, R.J., Balachandran, J.S., Hsu, D.T., Bakshi, V.P., Roseboom, P.H. and Kalin, N.H. (2001) Effects of acute and repeated restraint stress on corticotropinreleasing hormone binding protein mRNA in rat amygdala and dorsal hippocampus. Neurosci. Lett., 302: 81-84. Lovenberg, T.W., Chalmers, D.T., Liu, C. and De Souza, E.B. (1995b) CRFzalpha and CRF2beta receptor mRNAs are differentially distributed between the rat central nervous system and peripheral tissues. Endocrinology, 136:4139-4142. Lovenberg, T.W., Liaw, C.W., Grigoriadis, D.E., Clevenger, W., Chalmers, D.T., DeSouza, E.B. and Oltersdorf, T. (1995a) Cloning and characterization of a functionally distinct corticotropin-releasing factor receptor subtype from rat brain. Proc. Natl. Acad. Sci. USA, 92: 836-840. Ma, X., Levy, A. and Lightman, S. (1997) Emergence of an isolated arginine vasopressin (AVP) response to stress after repeated restraint: a study of both AVP and corticotropinreleasing hormone messenger ribonucleic acid (RNA) and heteronuclear RNA. Endocrinology, 138: 4351-4357. Ma, X.M., Lightman, S.L. and Aguilera, G. (1999) Vasopressin and corticotropin-releasing hormone gene responses to novel stress in rats adapted to repeated restraint. Endocrinology, 140: 3623-3632. Makino, S., Asaba, K., Nishiyama, M. and Hashimoto, K. (1999) Decreased type 2 corticotropin-releasing hormone receptor mRNA expression in the ventromedial hypothalamus during repeated immobilization stress. Neuroendocrinology, 70: 160-167. Makino, S., Schulkin, J., Smith, M., Pacak, K., Palkovits, M. and Gold, P. (1995) Regulation of corticotropin-releasing hormone receptor messenger ribonucleic acid in the rat brain and pituitary by glucocorticoids and stress. Endocrinology, 136: 4517-4525. Malkoski, S.P. and Dorin, R.I. (1999) Composite glucocorticoid regulation at a functionally defined negative glucocorticoid response element of the human
152 corticotropin-releasing hormone gene. Mol. Endocrinol., 13: 1629-1644. Malkoski, S.P., Handanos, C.M. and Dorin, R.I. (1997) Localization of a negative glucocorticoid response element of the human corticotropin releasing hormone gene. Mol. Cell Endocrinol., 127: 189-199. Mamalaki, E., Kvetnansky, R., Brady, L.S., Gold, P.W. and Herkenham, M. (1992) Repeated immobilization stress alters tyrosine hydroxylase, corticotropin-releasing hormone and corticosteroid receptor messenger ribonucleic acid levels in rat brain. J. Neuroendocrinol., 4: 689-699. McCarthy, J.R., Heinrichs, S.C. and Grigoriadis, D.E. (1999) Recent advances with the CRF~ receptor: design of small molecule inhibitors, receptor subtypes and clinical indications. Curr. Pharm. Des., 5: 289-315. McClennen, S.J. and Seasholtz, A.F. (1999) Transcriptional regulation of corticotropin-releasing hormone-binding protein gene expression in astrocyte cultures. Endocrinology, 140:4095-4 103. McKnight, G.S., Clegg, C.H., Uhler, M.D., Chrivia, J.C., Cadd, G.G., Correll, L.A. and Otten, A.D. (1988) Analysis of the cAMP-Dependent protein kinase system using molecular genetic approaches. Recent Prog. Horm. Res., 44: 307-335. Menard, L., Ferguson, S.S., Zhang, J., Lin, F.T., Lefkowitz, R.J., Caron, M.G. and Barak, L.S. (1997) Synergistic regulation of beta2-adrenergic receptor sequestration: intracellular complement of beta-adrenergic receptor kinase and beta-arrestin determine kinetics of internalization. Mol. Pharmacol., 51: 800-808. Merali, Z., McIntosh, J., Kent, P., Michaud, D. and Anisman, H. (1998) Aversive and appetitive events evoke the release of corticotropin-releasing hormone and bombesinlike peptides at the central nucleus of the amygdala. J. Neurosci., 18: 4758-4766. Mitchell, A.J. (1998) The role of corticotropin releasing factor in depressive illness: a critical review. Neurosci. Biobehav Rev., 22: 635-651. Mugele, K., Kulger, H.I. and Spiess, J. (1993) Immortalization of a fetal rat brain cell line that expresses corticotropinreleasing factor mRNA. DNA Cell Biol., 12:119-126. Nanda, S.A., Jones, S.G., Kalin, N.H. and Roseboom, P.H. (2002) Regulation of human urocortin II promoter activity. Soc. Neurosci. Abstract, 28: 769.762. Nanda, S.A., Roseboom, P.H., Nash, G.A., Digre, J.R. and Kalin, N.H. (2001) Regulation of the human corticotropinreleasing hormone2 receptor promoter activity. Soc. Neurosci. Abstract, 27: 914.913. Nemeroff, C.B. (1988) The role of corticotropin-releasing factor in the pathogenesis of major depression. Pharmacopsychiatry, 21: 76-82. Nemeroff, C.B., Widerlov, E., Bissette, G., Walleus, H., Karlsson, I., Eklund, K., Kilts, C.D., Loosen, P.T. and Vale, W. (1984) Elevated concentrations of CSF corticotro-
pin-releasing factor-like immunoreactivity in depressed patients. Science, 226:1342-1344. Nishikimi, T., Miyata, A., Horio, T., Yoshihara, F., Nagaya, N., Takishita, S., Yutani, C., Matsuo, H., Matsuoka, H. and Kangawa, K. (2000) Urocortin, a member of the corticotropin-releasing factor family, in normal and diseased heart. Am. J. Physiol. Heart Circ. Physiol., 279:H3031-H3039. Palchaudhuri, M.R., Hauger, R.L., Wille, S., Fuchs, E. and Dautzenberg, F.M. (1999) Isolation and pharmacological characterization of two functional splice variants of corticotropin-releasing factor type 2 receptor from tupaia belangeri. J. Neuroendocrinol., 11: 419-428. Park, J.H., Lee, Y.J., Na, S.Y. and Kim, K.L. (2000) Genomic organization and tissue-specific expression of rat urocortin. Neurosci. Lett., 292: 45-48. Pedersen, W.A., Wan, R., Zhang, P. and Mattson, M.P. (2002) Urocortin, but not urocortin II, protects cultured hippocampal neurons from oxidative and excitotoxic cell death via corticotropin-releasing hormone receptor type I. J. Neurosci., 22: 404-412. Peeters, P., Moechars, D., Grohlmann, H., Swagemakers, S., Kass, S., Langlois, X., Stenzel-Poore, M., Bakker, M. and Steckler, T. (2002) Gene expression profiling of CRF signaling. Soc. Neurosci. Abstract, 28: 223.222. Penn, R.B. and Benovic, J.L. (1998) Mechanisms of receptor regulation. In: Conn, P.M. (Ed.), Handbook of physiology, section 7: Endocrinology, American Physiological Society, Bethesda, MD, pp. 125-164. Perrin, M., Donaldson, C., Chen, R., Blount, A., Berggren, T., Bilezikjian, L., Sawchenko, P. and Vale, W. (1995) Identification of a second corticotropin-releasing factor receptor gene and characterization of a cDNA expressed in heart. Proc. Natl. Acad. Sci. USA, 92: 2969-2973. Pich, E.M., Lorang, M., Yeganeh, M., Rodriguez de Fonseca, F., Raber, J., Koob, G.F. and Weiss, F. (1995) Increase of extracellular corticotropin-releasing factor-like immunoreactivity levels in the amygdala of awake rats during restraint stress and ethanol withdrawal as measured by microdialysis. J. Neurosci., 15: 5439-5447. Plotsky, P.M. and Meaney, M.J. (1993) Early, postnatal experience alters hypothalamic corticotropin-releasing factor (CRF) mRNA, median eminence CRF content and stressinduced release in adult rats. Brain Res. Mol. Brain Res., 18: 195-200. Potter, E., Behan, D.P., Fischer, W.H., Linton, E.A., Lowry, P.J. and Vale, W.W. (1991) Cloning and characterization of the cDNAs for human and rat corticotropin releasing factor-binding proteins. Nature, 349: 423-425. Pozzoli, G., Bilezikjian, L.M., Perrin, M.H., Blount, A.L. and Vale, W.W. (1996) Corticotropin-Releasing factor (CRF) and glucocorticoids modulate the expression of type 1 CRF receptor messenger ribonucleic acid in rat anterior pituitary cell cultures. Endocrinology, 137: 65-71.
153 Raadsheer, F., Hoogendijk, W., Stare, F., Tilders, F. and Swaab, D. (1994) Increased numbers of corticotropinreleasing hormone expressing neurons in the hypothalamic paraventricular nucleus of depressed patients. Neuroendocirnology, 60: 436-444. Raber, J., Koob, G.F. and Bloom, F.E. (1995) Interleukin-2 (IL-2) induces corticotropin-releasing factor (CRF) release from the amygdala and involves a nitric oxide-mediated signaling; comparison with the hypothalamic response. J. Pharmacol. Exp. Ther., 272: 815-824. Ramkumar, T. and Adler, S. (1999) A Requirement for the POU transcription factor, Brn-2, in corticotropin-releasing hormone expression in a neuronal Cell Line. Mol. Endocrinol, 13: 1237-1248. Reisine, T. and Hoffman, A. (1983) Desensitization of corticotropin-releasing factor receptors. Biochem. Biophys. Res. Commun., 111:919-925. Reyes, T.M., Lewis, K., Perrin, M.H., Kunitake, K.S., Vaughan, J., Arias, C.A., Hogenesch, J.B., Gulyas, J., Rivier, J., Vale, W.W. and Sawchenko, P.E. (2001) Urocortin II: a member of the corticotropin-releasing factor (CRF) neuropeptide family that is selectively bound by type 2 CRF receptors. Proc. Natl. Acad. Sci. USA, 98: 2843-2848. Roseboom, P.H., Bakshi, V.P., Herringa, R.J. and Kalin, N.H. (2001a) Microarray analysis of restraint stress-induced gene expression changes in the rat septum. Soc. Neurosci. Abstract, 27: 739.733. Roseboom, P.H., Urben, C.M. and Kalin, N.H. (2001b) Persistent corticotropin-releasing factor l receptor desensitization and downregulation in the human neuroblastoma cell line IMR-32. Brain Res. Mol. Brain Res., 92: 115-127. Rossant, C.J., Pinnock, R.D., Hughes, J., Hall, M.D. and McNulty, S. (1999) Corticotropin-releasing factor type 1 and type 2alpha receptors regulate phosphorylation of calcium/ cyclic adenosine 3',5'- monophosphate response elementbinding protein and activation of p42/p44 mitogen-activated protein kinase. Endocrinology, 140: 1525-1536. Rots, N.Y., deJong, J., Workel, J.O., Levine, S., Cools, A.R. and DeKloet, E.R. (1996) Neonatal maternally deprived rats have as adults elevated basal pituitary-adrenal activity and enhanced susceptibility to apomorphine. J. Neuroendocrinol., 8:501-506. Saffran, M. and Schally, A.V. (1956) The release of corticotrophin by anterior pituitary tissue in vitro. Can. J. Biochem. Physiol., 33:408-415. Sakai, K., Yamada, M., Horiba, N., Wakui, M., Demura, H. and Suda, T. (1998) The genomic organization of the human corticotropin-releasing factor type-1 receptor. Gene, 219: 125-130. Sanchez, M.M., Young, L.J., Plotsky, P.M. and Insel, T.R. (1999) Autoradiographic and in situ hybridization localization of corticotropin-releasing factor 1 and 2 receptors in nonhuman primate brain. J. Comp. Neurol., 408: 365-377.
Sandberg, J.A., Parker, V.P., Blanchard, K.S., Sweedler, D., Powell, J.A., Kachensky, A., Bellon, L., Usman, N., Rossing, T., Borden, E. and Blatt, L.M. (2000) Pharmacokinetics and tolerability of an antiangiogenic ribozyme (ANGIOZYME) in healthy volunteers. J. Clin. Pharmacol., 40: 1462-1469. Schoeffter, P., Feuerbach, D., Bobirnac, I., Gazi, L. and Longato, R. (1999) Functional, endogenously expressed corticotropin-releasing factor receptor type 1 (CRF~) and CRF1 receptor mRNA expression in human neuroblastoma SH-SY5Y cells. Fundam. Clin. Pharmacol., 13: 484-489. Segre, G.V. and Goldring, S.R. (1993) Trends Endocrin. Metab., 4: 309-314. Shanks, N., Larocque, S. and Meaney, M.J. (1995) Neonatal endotoxin exposure alters the development of the hypothalamic-pituitary-adrenal axis: early illness and later responsivity to stress. J. Neurosci., 15: 376-384. Sheriff, S., Dautzenberg, F.M., Mulchahey, J.J., Pisarska, M., Hauger, R.L., Chance, W.T., Balasubramaniam, A. and Kasckow, J.W. (2001) Interaction of neuropeptide Y and corticotropin-releasing factor signaling pathways in AR-5 amygdalar cells. Peptides, 22: 2083-2089. Sherman, J.E. and Kalin, N.H. (1986) Corticotropin releasing hormone: effects on stress-related behavior in rats. In: Moody, T. (Ed.) Neural. and Endocrine Peptides and Receptors, Plenum Publ Co., New York, pp. 195-204. Simone, N.L., Bonner, R.F., Gillespie, J.W., Emmert-Buck, M.R. and Liotta, L.A. (1998) Laser-capture microdissection: opening the microscopic frontier to molecular analysis. Trends Genet, 14: 272-276. Skelton, K.H., Nemeroff, C.B., Knight, D.L. and Owens, M.J. (2000) Chronic administration of the triazolobenzodiazepine alprazolam produces opposite effects on corticotropinreleasing factor and urocortin neuronal systems. J. Neurosci., 20: 1240-1248. Smith, G.W., Aubry, J.M., Dellu, F., Contarino, A., Bilezikjian, L.M., Gold, L.H., Chen, R., Marchuk, Y., Hauser, C., Bentley, C.A., Sawchenko, P.E., Koob, G.F., Vale, W. and Lee, K.F. (1998) Corticotropin releasing factor receptor 1-deficient mice display decreased anxiety, impaired stress response, and aberrant neuroendocrine development. Neuron, 20:1093-1102. Smith, M.A., Kim, S.Y., VanOers, H.J.J. and Levine, S. (1997) Maternal deprivation and stress induce immediate early genes in the infant rat brain. Endocrinology, 138: 4622-4628. Spengler, D., Rupprecht, R., Van, L.P. and Holsboer, F. (1992) Identification and characterization of a 3', 5'-cyclic adenosine monophosphate-responsive element in the human corticotropin-releasing gene promoter. Mol. Endocrinol., 6: 1931-1941. Spiess, J., Dautzenberg, F.M., Sydow, S., Hauger, R.L., Ruhmann, A. and Radulowic, J. (1998) Molecular properties of the CRF receptor. Trends Endocrin Metab., 9: 140-145. Spina, M. (1996) Appetite-suppressing effects of urocortin, a CRF-related neuropeptide. Science, 273:1561-1564.
154 Steckler, T. (2005) CRF antagonists as novel treatment strategies for stress-related disorders. In: Steckler, T., Kalin, N. and Reul, J.M.H.M. (Eds.), Handbook of Stress and the Brain, Part 1, Elsevier, Amsterdam, pp. 371-400. Stenzel-Poore, M.P., Cameron, V.A., Vaughan, J., Sawchenko, P.E. and Vale, W. (1992) Development of cushing's syndrome in corticotropin-releasing factor transgenic mice. Endocrinology, 130: 3378-3386. Stenzel-Poore, M.P., Heinrichs, S.C., Rivest, S., Koob, G.F. and Vale, W.W. (1994) Overproduction of corticotropinreleasing factor in transgenic mice: a genetic model of anxiogenic behavior. J. Neurosci., 14: 2579-2584. Stout, S.C., Owens, M.J. and Nemeroff, C.B. (2002) Regulation of corticotropin-releasing factor neuronal systems and hypothalamic-pituitary-adrenaI axis activity by stress and chronic antidepressant treatment. J. Pharmacol. Exp. Ther., 300: 1085-1092. Suman-Chauhan, N., Carnell, P., Franks, R., Webdale, L., Gee, N.S., McNulty, S., Rossant, C.J., Van Leeuwen, D., MacKenzie, R. and Hall, M.D. (1999) Expression and characterisation of human and rat CRF2alpha receptors. Eur. J. Pharmacol., 379: 219-227. Timofeeva, E., Deshaies, Y., Picard, F. and Richard, D. (1999) Corticotropin-releasing hormone-binding protein in brain and pituitary of food-deprived obese (fa/fa) zucker rats. Am. J. Physiol., 277: R1749-R1759. Timpl, P., Spanagel, R., Sillaber, I., Kresse, A., Reul, J.M., Stalla, G.K., Blanquet, V., Steckler, T., Holsboer, F. and Wurst, W. (1998) Impaired stress response and reduced anxiety in mice lacking a functional corticotropin-releasing hormone receptor 1. Nat. Genet., 19: 162-166. Tsai-Morris, C.H., Buczko, E., Geng, Y., Gamoa-Pinto, A. and Dufau, M.L. (1996) The genomic structure of the rat corticotropin releasing factor receptor. J. Biol. Chem., 271: 14519-14525. Valdenaire, O., Giller, T., Breu, V., Gottowik, J. and Kilpatrick, G. (1997) A new functional isoform of the human CRF2 receptor for corticotropin-releasing factor. Biochim. Biophys. Acta., 1352: 129-132. Vale, W., Spiess, J., Rivier, C. and Rivier, J. (1981) Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and betaendorphin. Science, 213: 1394-1397. van der Meer, M., Baumans, V., Olivier, B. and van Zutphen, B.L. (2001) Impact of transgenic procedures on behavioral and physiological responses in postweaning mice. Physiol. Behav., 73: 133-143. van Gaalen, M.M., Stenzel-Poore, M.P., Holsboer, F. and Steckler, T. (2002) Effects of transgenic overproduction of CRH on anxiety-like behaviour. Eur. J. Neurosci., 15: 2007-2015. van Oers, H.J., de Kloet, E.R. and Levine, S. (1998) Early vs. late maternal deprivation differentially alters the
endocrine and hypothalamic responses to stress. Brain Res. Dev. Brain Res., 111: 245-252. Vaughan, J., Donaldson, C., Bittencourt, J., Perrin, M.H., Lewis, K., Sutton, S., Chan, R., Turnbull, A.V., Lovejoy, D., Rivier, C. et al. (1995) Urocortin, a mammalian neuropeptide related to fish urotensin I and to corticotropin-releasing factor. Nature, 378: 287-292. Weninger, S.C., Dunn, A.J., Muglia, L.J., Dikkes, P., Miczek, K.A., Swiergiel, A.H., Berridge, C.W. and Majzoub, J.A. (1999) Stress-induced behaviors require the corticotropin-releasing hormone (CRH) receptor, but not CRH. Proc. Natl. Acad. Sci. USA, 96: 8283-8288. Weninger, S.C., Peters, L.L. and Majzoub, J.A. (2000) Urocortin expression in the edinger-westphal nucleus is up-regulated by stress and corticotropin-releasing hormone deficiency. Endocrinology, 141: 256-263. Wigger, A. and Neumann, I.D. (1999) Periodic maternal deprivation induces gender-dependent alterations in behavioral and neuroendocrine responses to emotional stress in adult rats. Physiol. Behav., 66: 293-302. Wong, L.F. and Murphy, D. (2003) Adenoviral-mediated overexpression of Brn2 in the rat paraventricular nucleus: no effect on vasopressin or corticotrophin releasing factor RNA levels. Mol. Cell Endocrinol., 200: 165-175. Wynn, P.C., Hauger, R.L., Holmes, M.C., Millan, M.A., Catt, K.J. and Aguilera, G. (1984) Brain and pituitary receptors for corticotropin releasing factor: localization and differential regulation after adrenalectomy. Peptides, 5: 1077-1084. Xiong, Y., Xie, L.Y. and Abou-Samra, A.B. (1995) Signaling properties of mouse and human corticotropin-releasing factor (CRF) receptors: decreased coupling efficiency of human type II CRF receptor. Endocrinology, 136: 1828-1834. Yuan, P.X., Zhou, R., Farzad, N., Gray, N.A., Du, J. and Manji, H.K. (2005) Enhancing resilience to stress: the role of signaling cascades. In: Steckler, T., Kalin, N. and Reul, J.M.H.M. (Eds.), Handbook of Stress and the Brain, Part 1, Elsevier, Amsterdam, pp. 751-772. Zhang, X.K., Dong, J.M. and Chiu, J.F. (1991) Regulation of alpha-fetoprotein gene expression by antagonism between AP-1 and the glucocorticoid receptor at their overlapping binding site. J. Biol. Chem., 266: 8248-8254. Zhao, L., Donaldson, C.J., Smith, G.W. and Vale, W.W. (1998) The structures of the mouse and human urocortin genes (Ucn and UCN). Genomics, 50: 23-33. Zobel, A.W., Nickel, T., Kunzel, H.E., Ackl, N., Sonntag, A., Ising, M. and Holsboer, F. (2000) Effects of the highaffinity corticotropin-releasing hormone receptor 1 antagonist R121919 in major depression: the first 20 patients treated. J. Psychiatr. Res., 34: 171-181.
T. Steckler, N.H. Kalin and J.M.H.M. Reul (Eds.) Handbook of Stress and the Brain, Vol. 15
ISBN 0-444-51173-3 Copyright 2005 Elsevier B.V. All rights reserved CHAPTER 2.3
Behavioral consequences of altered corticotropin-releasing factor activation in brain" a functionalist view of affective neuroscience Stephen C. Heinrichs* Boston College, Department of Psychology, McGuinn Hall, 140 Commonwealth Avenue, Chestnut Hill, MA 02467, USA
Abstract: Organisms exposed to challenging stimuli that alter the status quo inside or outside of the body are required for survival purposes to generate appropriate coping responses which counteract the departure from homeostasis. Identification of an executive control mechanism within the brain capable of coordinating the multitude of endocrine, physiological and functional coping responses has high utility for discovery of efficacious pharmacological tools in stressor-exposed organisms under normal or pathological conditions. One such mechanism is the corticotropinreleasing factor (CRF)/urocortin family of neuropeptides and receptors which constitutes an affective regulatory system, so called due to the integral role this neuropeptide system plays in controlling core components of affect such as arousal, emotionality, and aversive processes. In particular, available evidence from pharmacological intervention in multiple species and phenotyping of mutant mice is that CRF/urocortin systems mediate negatively valenced activating, avoidant, and distressing components of affective responses. It is suggested that affective regulation is exerted by CRF/urocortin systems within the brain based upon the sensitivity of local brain sites to CRF/urocortin ligand administration and the necessary but insufficient nature of the hypothalamo-pituitary-adrenocortical activation following stressor exposure. Moreover, these same stress neuropeptides may constitute a mechanism for learning to avoid noxious stimuli by forming so-called emotional memories. In additional to presentation of experimental evidence relevant for understanding the role of brain CRF/urocortin from a functional perspective, a conceptual framework is provided for extrapolation of animal model findings to humans and for viewing CRF/urocortin activation as a continuum measure which links normal and pathological states.
Introduction
citation of the mass of descriptive and experimental studies performed in animals that support the role of C R F as a mediator of interoceptive coping reactions to environmental or physiological challenges. However, the advent of small molecule ligands for C R F receptors suitable for administration in human beings begs the question of what utility, if any, antistress pharmacology via C R F manipulations in animals provides in the context of human biology and psychopathology. The present chapter tackles the pressing task of predicting the consequences of brain C R F system activation or deactivation in man based upon the adaptive utility of C R F systems and
The close of two complete decades of research into the biology of the corticotropin-releasing factor (CRF) family of neuropeptides seems an opportune moment to ask a simple but beguiling question: what is the ultimate function of these brain signalling systems? This question is typically addressed by
*Tel.: 617-552-0852; Fax: 617-552-0523; E-mail:
[email protected] 155
156 the homologous and analogous characteristics of CRF system activation in multiple species. The functionalist thesis most strongly supported by the available experimental evidence is that activating, avoidant and learning/memory functions of CRF systems have evolved in order to serve affective regulatory functions in man and animals. One means of assessing the ultimate functions of CRF systems is to identify cross-species homologies in the comparative biology of this peptide, that is, the extent to which characteristics of a contemporary organism have been inherited from ancestors. The term "homology" is used here to denote evidence of a shared underlying mechanism for a particular behavioral function that is expressed in a variety of species but derived from a single evolutionary point of origin. Comparative studies of CRF system biology in animals employing genetic and neurobiological techniques are thus suited to address the issue of homology. For example, the earliest known functions of CRF family peptides are related to osmoregulation and diuresis which are exerted by all variants of the urotensin-I family of CRF-like peptides (Lovejoy and Balment, 1999). Recently evolved vertebrate species exhibit physiological effects of CRF activation related to sympathetic activation and reproduction (Lovejoy and Balment, 1999). In particular, CRF expression in amniotes is associated with descending autonomic nerves that innervate smooth and cardiac muscle and various visceral glands and organs (Lovejoy and Balment, 1999). This cluster of consequences of brain CRF activation can be conceptualized as an action set designed to accomplish important biological functions such as avoiding and escaping life-threatening events. From the perspective of ultimate functionality, such affect programs can be viewed as evolutionarily derived mechanisms for successfully passing genes onto coming generations (Lohman et al., 2000). Brain CRF systems can be hypothesized to modulate three specific aspects of affective programming: (1) the activation of negatively valenced action sets related to avoidance and escape tendencies, (2) the amplitude of activation as measured by motor output or subjective arousal, and (3) the acquisition of new information necessary for optimal performance in learning and memory tasks. Affective valence can be defined as a bipolar scale defining
a continuum of subjective feeling states from the positive extreme of pleasantness to the negative extreme of unpleasantness (Bradley and Lang, 2000). Energized brain CRF systems would be expected to excite unpleasant states associated with the emotional labels anger, sadness, or fear (Panksepp, 1998). It should be noted that the neural substrates of reward which presumably mediate the pleasantly valenced affect programs are well characterized (Rolls, 1999) but beyond the scope of this chapter. Activation is similarly construed as a measure of the intensity of affect program expression ranging from an unaroused state to high arousal (Bradley and Lang, 2000). Energized brain CRF systems would be expected to induce features of high arousal such as excitement and motor activity. These orthogonal valence/arousal scales of affect programming have been proposed as primary motivational systems of the brain in multiple species (Rolls, 1999; Russell, 2003). Finally, brain plasticity required for learning and memory functions necessary to accomplish classical conditioning and working memory are hardwired aspects of brain affect programming (LeDoux, 2000). In particular, fear conditioning in response to noxious stimulus exposure is reported to promote emotional memories which are particularly vivid and enduring (LeDoux, 2000). The remaining sections of this review are organized to present the thesis that CRF systems mediate the valence, intensity, and learned flexibility of affect programs.
Relevance of CRF systems for affective neuroscience Environmental change invokes an integrated state of endocrine, autonomic, and behavioral activation that is critically dependent on CRF substrates within the central nervous system. A contemporary understanding of the role of classic corticotropin secretagogues has emerged in which functional targets for peptide hormones such as CRF exist in brain sites unrelated to neuroendocrine hypophysiotropic circuits and even entirely outside of the brain. Thus, direct neurobiological actions of CRF acting as a synaptic transmitter at specialized extra-hypothalamic receptors must be coordinated with the capacity of CRF to stimulate secretion of pituitary hormones.
157 Factors that mobilize brain CRF systems appear to have one feature in common, the ability to disturb homeostasis. For example, demands on the organism may be induced either internally or externally by exposure to physical trauma, infection, or social conflict. Coping responses to such deflections in steady state that include sympathetic nervous system activation, promotion of negative energy balance and augmentation of vigilance and emotionality appear to be CRF dependent. Comparative pharmacology of CRF receptor agonists suggests that CRF, urocortin 1, sauvagine, and urotensin consistently mimic, and peptide CRF receptor antagonists consistently lessen, functional consequences of stressor exposure. Together with the development of novel nonpeptide CRF receptor antagonists, a growing number of CRF receptor selective ligands are available to elucidate the neurobiology and physiological role of CRF systems. The classification of CRF as a peptide with activating, arousing, and anxiogenic properties is based on a broad and comprehensive in vivo testing battery whose dependent measures reflect changes in locomotion, choice accuracy, energy balance, speciestypical social interactions, and many other constructs (Koob, 1999; Steckler and Holsboer, 1999). CRF administrated intracerebroventricularly results in behavioral, physiological, and autonomic responses that are similar to those observed when animals are exposed to a stressor. The striking behavioral effects include decreased food consumption, altered locomotor activity, diminished sexual behavior, sleep disruption, and anxiogenic-like increases in emotionality (see also chapter by Zorrilla and Koob, this volume). These functional responses to pharmacological doses of exogenous CRF are analogous to those produced in animals by exposure to a species-typical, naturalistic stressor such as social conflict (Meerlo et al., 1996; Buwalda et al., 1997). This evidence implicates CRF as a physiological mediator of stress responses or stress-induced psychopathology. Since the frank and nonspecific behavioral actions of exogenous CRF receptor agonists are sufficiently robust to mask more subtle functional effects that would otherwise emerge (see section of Energy Balance and Reward), a more meaningful classification of animal models that reveal the true physiological significance of CRF neurobiology may be
reflected in behavioral actions of CRF receptor antagonists. Indeed, complete reversal of the actions of exogenous CRF is accomplished by coadministration of a competitive receptor antagonist such as s-helical CRF (9-41) in pharmacological competition studies irrespective of the nature of the testing protocol (Dunn and Berridge, 1990). Accordingly, dependent measure selection ought not be based on efficacy of CRF itself or the results of CRF receptor antagonist competition studies but rather on testing contexts that reveal intrinsic actions of the CRF receptor antagonist alone. This strategy has been applied extensively in studies employing numerous dependent measures used to reveal antistress actions of CRF receptor antagonists following stressor pretreatment. Thus, central administration of a peptide CRF receptor antagonist, or alternatively CRF/urocortin immunoneutralization, blocks or attenuates the behavioral responses to centrally administered CRF and also reverses the effects of a variety of stressors including restraint, foot shock, hemorrhage, insulin-induced hypoglycemia, cholecystokinin administration, and ether exposure (Ida et al., 2000; Ohata et al., 2000; Heinrichs and De Souza, 2001). CRF receptor antagonists reverse not only decreases but also increases in behavior associated with stress, suggesting that the effects of CRF receptor antagonists are stress dependent and not due to nonspecific changes in behavior. The bulk of these studies suggest that efficacy of CRF receptor antagonists can be generalized to many stress situations and is reproducible (Table 1). Current studies in the field are now largely focused on the real world relevance and exact nature of stressors employed in experiments designed to reveal the true physiological role of CRF in biological systems.
Neuroendocrine and neurotransmitter interactions
Hypophysiotropic CRF neurons which mediate ACTH release (see chapter by Fulford and Harbuz, this volume) do not act autonomously but rather are reciprocally regulated by a wide variety of neurochemically and functionally distinct brain peptides and neurotransmitters (Grossman et al., 1993; Menzaghi et al., 1993). CRF neurons within hypothalamus
158 Table 1. Testing contexts sensitive to the behavioral actions of CRF system activation by receptor agonist administration or CRF system inactivation by competitive receptor antagonist administration Test
Ligand and behavioral action
References
Evidence for mediation of negatively valenced affective events by CRF Elevated Plus-Maze CRF suppresses exploration and CRF receptor antagonist reverses stress, drug and genotypically induced suppression of exploration Startle reactivity CRF facilitates startle amplitude and CRF receptor antagonist blocks fear-potentiated startle Defensive burying CRF enhances defensive burying and CRF receptor antagonist reduces defensive burying Defensive withdrawal CRF enhances and CRF receptor antagonist attenuates defensive withdrawal Evidence for mediation of activation by CRF Locomotor activity CRF enhances locomotion and CRF receptor antagonist reduces stress and drug-induced locomotion Sleep latency and architecture CRF promotes wakefullness and CRF receptor antagonists delay spontaneous awakening Food intake CRF decreases food intake and CRF receptor antagonist reverses stress and drug-induced anorexia Evidence for mediation of learning and memory performance by CRF Taste/place conditioning CRF produces aversion and CRF receptor antagonist weakens drug-induced place aversion Amphetamine-induced stereotypy CRF enhances sensitization and CRF receptor antagonist attenuates stress-induced sensitization Conditioned emotional response CRF induces conditioned fear and CRF receptor antagonist blocks acquisition of conditioned emotional response Conditioned freezing CRF facilitates acquisition of immobility and CRF receptor antagonist blocks stress-induced facilitation of immobility
apparently interact with peptides such as vasopressin, endorphins, and neuropeptide Y as well as gammaamino-butyric acid (GABA), catecholamine, noradrenergic, and indolamine systems to modulate adrenocorticotropin secretion (Plotsky et al., 1989). W h e n relevant tissues are isolated and examined using in vitro techniques, m a n y of these interactions persist suggesting neuronal codistribution or mediation by short synaptic circuits. This multiplicity of afferent input attests to the need for precise homeostatic control of the H P A axis and suggests a
Griebel et al., 1998; Heinrichs et al., 2002
Swerdlow et al., 1989; Arborelius et al., 2000
Korte et al., 1994; Basso et al., 1999
Arborelius et al., 2000; Heinrichs and Joppa, 2001
Sarnyai et al., 1992; Menzaghi et al., 1994
Ehlers et al., 1986a; Chang and Opp, 1999; Lancel et al., 2002 Bell et al., 1998; Jones et al., 1999
Heinrichs et al., 1991; Benoit et al., 2000a
Cador et al., 1989; Cole et al., 1990b
Cole et al., 1987
Heinrichs et al., 1997b; Bakshi et al., 1999
role for C R F in particular as an integrative neuropeptide which consolidates relevant input from dispersed sources. The hypothesis that C R F systems constitute the final c o m m o n pathway for mediation of h y p o t h a l m o pituitary-adrenal (HPA) tone has been tested experimentally (Cole et al., 1990a). Using the technique of immunoneutralization in which the pituitary actions of C R F in portal blood are limited by prior introduction of a C R F antibody that sequesters and inactivates the peptide, n u m e r o u s studies have shown
159 that HPA-activating properties of footshock, social defeat, and pharmacological stressors, such as cocaine and cholecystokinin, are CRF dependent. Moreover, while other ACTH secretagogues, such as vasopressin exert synergistic activation of HPA tone in the presence of CRF by virtue of clearly defined codistribution and copackaging of these hypophysiotropic factors in the paraventricular nucleus and median eminence, these actions appear to be mediated predominantly or exclusively via increased release of endogenous CRF (Brunani et al., 1995). Several extrapituitary effects of other neurotransmitters and nonCRF neuropeptide systems including feeding suppression, increased emotionality, and fever induction also appear to the CRF dependent (Gray, 1993). For instance, the anxiogenic-like and anorexic actions of serotonin agonists, such as fenfluramine as well as cholecystokinin, caffeine, and estradiol, are blunted or reversed by reduction in CRF tone accomplished by CRF immunoneutralization or central administration of a CRF receptor antagonist. Similarly, anxiogenic-like behavior produced by central administration of cholecystokinin octapeptide is reversed by concurrent administration of a CRF receptor antagonist or CRF antiserum in a dose-dependent manner. In addition, behavioral despair, anorexic actions, and antinociceptive effects of cytokines such as interleukin-1 appear to be CRF dependent. These results suggest that a possible countermeasure for departure from behavioral homeostasis is to normalize CRF tone regardless of whether CRF is directly or indirectly involved in the behavioral dysregulation.
CRF-binding protein The majority of late gestational maternal plasma CRF is bound to a high affinity CRF-binding protein (CRF-BP) which neutralizes the receptor agonist's ACTH-releasing properties (Lowry et al., 1996). Thus, maternal plasma CRF-BP levels determines the amount of "free" CRF that will bind to pituitary CRF receptors and thereby modulate the activity of the pituitary-adrenocortical axis during late human pregnancy. Many workers have now demonstrated that CRF is substantially elevated during the third trimester of human pregnancy and
that this process is likely to participate in a cascade of events which eventually leads to parturition (Behan et al., 1993). This beneficial biological action of CRF is presumably exerted without undesirable Cushingoid-like symptoms of pituitary-adrenal overactivation due to the simultaneous, buffering presence of CRF-BP. In contrast to humans, all other species examined to date do not express CRF-BP in the liver and plasma. The predominant tissues expressing CRF-BP in all species are the brain and pituitary gland. With respect to the central nervous system and the role of CRF-BP, it has been demonstrated, by immunohistochemistry and in situ hybridization techniques, that CRF-BP is expressed in various areas of rat brain including the cerebral cortex, amygdala, hippocampus as well as sensory relay nuclei associated with the auditory, olfactory, vestibular, and trigeminal systems (Potter et al., 1992). Of note, there are brain areas that are enriched with CRF and CRF-BP but contain low densities of receptors and conversely, other brain areas that are enriched with receptors and devoid of CRF-BP. Thus, the differential distribution of brain CRF-BP and CRF receptors presents multiple distinct sites of interaction with CRF (Behan et al., 1993). A number of observations suggest that there is a membrane associated form of the CRF-BP within the brain (Behan et al., 1995a). Using a ligand immunoradiometric assay to detect the CRF-BP, a specific CRF-BP-like activity could be solubilized from sheep, rat, and human brain membranes. Further purification of crude rat and sheep brain membranes by sucrose density gradient centrifugation and detergent solubilization localized CRF-BP activity to the plasma membrane fraction. The detergent solubilized rat and human brain membrane preparations have similar pharmacological profiles to the recombinant protein present in human plasma. A large proportion of CRF in normal human brain may be complexed to the CRF-BP and thus unavailable for actions at the receptor. It is likely that the interaction between CRF and membrane-associated CRF-BP in brain is important in maintaining synaptic CRF concentrations either by presynaptic uptake or by modulating the quantity of neuropeptide that activates CRF receptors at the membrane interface (Turnbull and Rivier, 1997).
160 The identification of a membrane-associated form of the CRF-BP in brain with kinetic and pharmacological characteristics comparable to those previously observed for human plasma CRF-BP provides a novel target to modulate endogenous CRF levels in select brain areas enriched in CRF-BP. Selective peptide ligands that dissociate CRF from the CRF-BP, termed CRF-BP ligand inhibitors, mimic a number of behavioral effects of CRF including food intake suppression (Heinrichs et al., 1999; Bjenning and Rimvall, 2000) and locomotor activation (Heinrichs and Joppa, 2001). CRF-BP ligand inhibitors also normalize the reduced levels of unbound
CRF in postmortem cerebral cortex of Alzheimer's patients to those seen in age-matched controls (Behan et al., 1995b) and exert significant cognitive-enhancing properties in animal models of learning and memory such as the Morris water maze and visual discrimination tests without producing any overt anxiogenic actions characteristic of CRF receptor agonists (Behan et al., 1995a; Radulovic et al., 2000; Zorrilla et al., 2001). Thus, CRF-BP represents a novel target for the symptomatic treatment of cognitive deficits associated with neurodegenerative dementia (see section on Learning and Memory Modulation) (Fig. 1).
-'~'600
Vehicle
r/h CRF(1-41)
r/h CRF (6-33)
o 500-
.~_
l= 400.
c)
~o 300. t--
o 200. O .
> 100. ,.,.,.
o
20 t Wildtype o co15. cr (D i,,.. u-10.
CRF Transgenic
tl-~ 0
5.
Dose R121919 (mg/kg p.o.) Fig. 3. CRF Type 1 receptor antagonist attenuated the anxiogenic-like phenotype of CRF-transgenic mice and diminished sexual receptivity of female CRF-transgenic mice. Motor activity (mean-F SEM) measured over 10min in wildtype control or CRF-transgenic mice administered subchronic 0 or 10mg/kg p.o. x 3 doses of R121919 in the Two-Compartment model of anxiety (top panel). Frequency of mounting (mean + SEM) of experimental female wildtype and CRF-transgenic mice treated with a 0 or 20mg/kg p.o. doses of R121919 by male conspecifics during a 20min sexual receptivity test (bottom panel). *p 70%) of noradrenaline in the brain, following treatment with the neurotoxin, DSP-4, there was a twofold increase in the concentration of extracellular noradrenaline in the frontal cortex (Hughes and
Stanford, 1998). Another limitation of both these approaches, as experience with humans confirms, is that they ignore the factors that determine vulnerability to stress, and its long-term impact, which leads to physical and mental illnesses in some individuals but not others. In order to avoid these problems, and yet investigate how any difference in neurochemical responses might parallel behaviour (or vice versa) we have studied two inbred strains of rats, which express innate differences in their behavioural responses to stress: the Maudsley Reactive (MR) and Nonreactive (Wistar, MNRA) rats. These animals are ideal for experiments investigating the relationship between noradrenergic transmission and the behavioural response to aversive environmental stimuli because the criterion for their inbreeding is their behavioural response to a novel environment (the 'open field'). The behaviour of these rats has been studied a good deal in the past (see Broadhurst, 1975) and, albeit controversially, was deemed to hold the key to innate differences in 'emotionality'. We carried out behavioural and microdialysis studies in these two strains of rat, using the light/dark exploration box, as described above. Only in the M R strain were time spent/visit and activity/visit in the light arena less than the dark one (McQuade and Stanford, 2001). This is consistent with evidence that M R and M N R A rats differ in their exploratory behaviour (Broadhurst, 1975) and that these differences are seen most clearly under high-stress conditions (Sara et al., 1994). In parallel microdialysis experiments, there was no difference in the concentration of extracellular noradrenaline in M R and M N R A rats while they were in the neutral zone. As in outbred SpragueDawley rats, noradrenaline efflux in the frontal cortex was transiently increased in both M R and M N R A rats when they were confined within the brightly lit but not the dark, test arena of the light/dark exploration box. Noradrenaline efflux was also increased in the brightly lit arena in the hypothalamus of both strains of Maudsley rats. However, in M N R A but not M R rats, the noradrenaline response was maintained for at least 2 h (i.e. until the animals were returned to the neutral zone of the light/ dark exploration box) (McQuade and Stanford, 2001).
497 It is striking that it is the M N R A rats, which are regarded as being the more resistant to stress, that showed a prolonged hypothalamic noradrenaline response. Whether this means that the increased efltux had a stress-protective effect is as yet unknown. However, it is notable that a prolongation of the noradrenaline response was also evident in outbred Sprague-Dawley rats that had been repeatedly exposed to, and showed some behavioural habituation to, the light zone of the light/dark exploration box (see above; McQuade and Stanford, 2000). Further evidence to support the possibility that augmentation or prolongation of a noradrenaline response increases behavioural resistance to an aversive environment comes from reports that basal firing rate of neurones in the locus coeruleus and incidence of burst firing was greater in the MNRA than the MR strain (Verbanac et al., 1994), albeit in anaesthetised rats. Consistent with this, there was a greater increase in tyrosine hydroxylase activity in the locus coeruleus of MNRA rats after a bout of footshock (Blizard et al., 1983). These changes are possibly explained by a lower sensitivity of ~2-autoreceptors to activation by agonists (Verbanac et al., 1994). Finally, a recent comparative study of outbred Sprague-Dawleys and the Lewis (low stress responsivity) and Wistar Kyoto (high stress responsivity) strains of rats showed that stress-induced increases in mRNA for tyrosine hydroxylase and noradrenaline etttux were greatest in the Lewis rats. Such findings have led to the conclusion that increased noradrenergic transmission is an essential component of coping with stress (Pardon et al., 2002). Despite these promising leads, the relationship between noradrenergic transmission and the stress response in the Maudsley inbred strains is not straightforward (reviewed by Blizard and Adams, 2002). There are reports of a greater increase in the concentration of noradrenaline in the plasma of MR rats after immobilisation (Blizard et al., 1983). Also, the increase in DOPAC concentration (used as an index of noradrenaline synthesis) in the locus coeruleus and ventrolateral medulla (which contains the A1/C1 nucleus) after a bout of immobilisation was greater in MR than in MNRA rats (Buda et al., 1994). Clearly, more research is needed to explain these apparently disparate findings. Also, it is
important to establish whether a difference in the cortical noradrenaline response is apparent when MR and M N R A rats are exposed to conditioned environmental stimuli, as described above.
Noradrenergic coding of coping with stress It is now clear that there is no stereotypical noradrenaline response to stress, but that the role and consequences of central noradrenergic transmission depend on the type or severity of the stimulus, the brain region to which these neurones project and individual differences in the neurobiological coding of behaviour. On this basis, it is likely that the optimal behavioural response to a given environmental stimulus requires a specific noradrenergic response (or combination of responses). Evidence for such a proposal first emerged from a series of studies of peripheral catecholamine responses to psychological stress, carried out by Frankenhaeuser's group (e.g. Frankenhaeuser, 1968; reviewed by Stanford, 1993). In these experiments, subjects with low basal urinary noradrenaline excretion perceived themselves to cope better with the stress of an audiovisual conflict test, the Stroop test, and actual task control was better, in those subjects that rallied the greatest noradrenaline response. Conversely, high baseline noradrenaline secretors performed best in an unstressed condition and it was the subjects who showed the smallest increase in plasma noradrenaline who actually coped better with the test stress. A caveat of this proposal is that it appears that changes in plasma catecholamines during psychological stress are evident only under carefully controlled conditions. In a study of three different groups of cardiac patients, plasma catecholamines exceeded those commonly reported in studies of relaxed, young, healthy individuals, and in none of these patients was it possible to demonstrate an increase in plasma catecholamines during a psychological stress (the Stroop test: Stanford et al., 1997). Moreover, in heart transplant patients, haemodynamic responses to the Stroop test were clearly disrupted and there was a greater reliance on hormonal secretion of catecholamines. Yet, this seemed to have no effect on their emotional response to the stress (Salmon et al., 2001).
498 Nevertheless, based on Frankenhaeuser's findings, and those describing correlations between individual differences in neurochemical markers and rats' behavioural responses during stress (see above), it was suggested that the relationship between central noradrenergic transmission and coping with stress is described by a bell-shaped curve (Stanford, 1993). Accordingly, optimal coping with stress rests on rallying an appropriate noradrenergic response; this could be determined genetically and/or by the individuals' previous experience of that stimulus.
A
.c_ c~ 0
0
NA transmission -"--,
B
0
.... - Decrease
-
Increase
C
~
Noradrenergic transmission Fig. 5. Schematic diagram showing the hypothetical relationship between noradrenergic transmission and an individual's resistance to stress. A: Optimal coping is attained when the brain rallies a specific noradrenergic response (~ this could be determined genetically and/or by previous experience of the stress. Either a reduction or an increase in noradrenergic transmission diminishes coping. B: The shaded area depicts the relationship between noradrenergic transmission and stress resistance in normal individuals (as shown in 'A'). If there is a leftward shift of the curve then the (predetermined) noradrenergic response that would be optimal in normal individuals, now produces suboptimal stress resistance (~ One remedy for such a dysfunction is to reduce noradrenergic transmission so as to restore optimal coping. C: In the case of a rightward shift of the curve, a predetermined noradrenergic response to a specific stimulus, that would be optimal in normal individuals, will again produce suboptimal coping (~ This time, the remedy is to increase noradrenergic transmission. In both B and C, an alternative way to restore optimal coping would be to reverse the shift in the noradrenergic transmissioncoping curve. This could explain the changes in mood that occur after chronic administration of drugs or behavioural strategies that cause long-latency changes in neurochemical factors that influence noradrenergic transmission.
According to such a scheme, either a deficit or overactivity of noradrenergic transmission would impair the ability to cope with the stimulus (Fig. 5A). It is also possible that the neurochemical coding of coping with stress can be disrupted by a shift, to either the right or the left, of the curve defining the relationship between noradrenergic transmission and coping (Fig. 5B and C). U n d e r these circumstances, a given noradrenergic response, which would be optimal in normal subjects, would now produce a suboptimal coping response (see also Stanford, 1996). The remedy would be to adjust the extent to which noradrenergic neurones are activated by an acute stress, or to recruit long-term adaptive changes that restore the appropriate relationship between the neuronal response and coping. Obviously, noradrenergic transmission is not the only factor that determines the behavioural response to even simple environmental stimuli. Indeed, a bellshaped dose-response curve itself confirms the intervention of one or more factors that govern noradrenergic transmission. Ultimately, it is these interactions between noradrenergic neurones and other neurotransmitters that determine the role of noradrenergic transmission in the neurochemical coding of coping with stress.
References
Abercrombie, E.D. and Jacobs, B.L. (1987) Single-unit response of noradrenergic neurons in the locus coeruleus of freely moving cats. I. Acutely presented stressful and nonstressful stimuli. J. Neurosci., 7: 2837-2843. Abercrombie, E.D., Keller, R.W. and Zigmond, M.J. (1988) Characterization of hippocampal norepinephrine release as measured by microdialysis perfusion: pharmacological and behavioral studies. Neuroscience, 27: 897-904. Aston-Jones, G., Rajkowski, J. and Cohen, J. (2000) Locus coeruleus and regulation of behavioral flexibility and attention. Prog. Brain Res., 126: 165-182. Blizard, D.A. and Adams, N. (2002) The Maudsley reactive and nonreactive strains: a new perspective. Behav. Genet., 32: 277-299. Blizard, D.A., Freedman, L.S. and Liang, B. (1983) Genetic variation, chronic stress and the central and peripheral noradrenergic systems. Am. J. Physiol., 245: R600-R605. Broadhurst, P.I. (1975) The Maudsley reactive and nonreactive strains of rats: a survey. Behav. Genet., 5: 288-319.
499 Buda, M., Lachuer, J., Devauges, V., Barbagli, B., Blizard, D. and Sara, S.J. (1994) Central noradrenergic reactivity to stress in Maudsley rat strains. Neurosci. Lett., 167:33-36 Cadogan, A.K., Kendall, D.A., Fink, H. and Marsden, C.A. (1994) Social interaction increases 5-HT release and cAMP efflux in the rat ventral hippocampus in vivo. Behav. Pharmacol., 5: 299-305. Cenci, M.A., Kalen, P., Mandel, R.J. and Bjorkland, A. (1992) Regional differences in the regulation of dopamine and noradrenaline release in medial frontal cortex, nucleus accumbens and caudate-putamen: a microdialysis study in the rat. Brain Res., 581: 217-228. Crawley, J. and Goodwin, F.K. (1980) Preliminary report of a simple animal behaviour model for the anxiolytic effects of benzodiazepines. Pharmacol. Biochem. Behav., 13: 167-170. Dalley, J.W. and Stanford, S.C. (1994) Graded changes in noradrenaline (NA) efflux in dialysates of the rat cortex evoked by non-noxious stressors. J. Psychopharmacol., 8 (suppl.): A47. Dalley, J.W. and Stanford, S.C. (1995) Incremental changes in extracellular noradrenaline availability in the frontal cortex induced by naturalistic environmental stimuli: a microdialysis study in the freely moving rat. J. Neurochem., 65: 2644-2651. Dalley, J.W., Mason, K. and Stanford, S.C. (1996) Increased levels of extracellular noradrenaline in the frontal cortex of rats exposed to naturalistic environmental stimuli: modulation by acute systemic administration of diazepam or buspirone. Psychopharmacol., 127: 47-54. Dayas, C.V. and Day, T.A. (2001) Opposing roles for medial and central amygdala in the initiation of noradrenergic cell responses to a psychological stressor. Eur. J. Neuroscience, 15: 1712-1718. Drijfhout, W.J., Kemper, R.H., Meerlo, P., Koolhaas, J.M. and Westerink, B.H. (1995) A telemetry study of the chronic effects of microdialysis probe implantation on the activity pattern and temperature rhythm of the rat. J. Neurosci. Methods, 61: 191-196. Fallon, J.H., Koziell, D.A. and Moore, R.Y. (1978) Catecholamine innervation of the basal forebrain. J. Comp. Neurol., 180: 509-532. Feenstra, M.G. (2000) Dopamine and noradrenaline release in the prefrontal cortex in relation to unconditioned and conditioned stress and reward. Prog. Brain Res. 126: 133-163. Feenstra, M.G.P., Botterblom, M.H.A. and Uum, J.F.M. (1998) Local activation of metabotropic glutamate receptors inhibits the handling-induced increased release of dopamine in the nuclues accumbens but not that of dopamine or noradrenaline in the prefrontal cortex: comparison with inhibition of ionotropic receptors. J. Neurochem., 70: 1104-1113.
Feenstra, M.G.P., Teske, G., Botterblom, M.H.A., and De Bruin, J.P.C. (1999) Dopamine and noradrenaline release
in the prefrontal cortex of rats during classical aversive and appetitive conditioning to a contextual stimulus: interference by novelty effects. Neurosci. Lett., 272:179-182. Feenstra, M.G.P., Botterblom, M.H.A. and Masterbroek, S. (2000) Dopamine and noradrenaline efflux in the prefrontal cortex in the light and dark period: effects of novelty and handling and comparison to the nucleus accumbens. Neuroscience, 100: 741-748. Frankenhaeuser, M. (1968) Catecholamine excretion as related to cognitive and emotion reaction patterns. Psychosomatic Med., 30: 109-120. Frankenhaeuser, M. (1971) Behavior and circulating catecholamines. Brain Res., 31: 241-262. Fritschy, J.-M. and Grzanna, R. (1989) Immunohistochemical analysis of the neurotoxic effects of DSP-4 identifies two populations of noradrenergic axon terminals. Neuroscience, 30: 181-197. Fulford, A.J. and Marsden, C.A. (1997a) Social isolation in the rat enhances cz2-autoreceptor function in the hippocampus in vivo. Neuroscience, 77: 57-64. Fulford, A.J. and Marsden, C.A. (1997b) Conditioned release of 5-hyroxytryptamine in vivo in the nucleus accumbens following isolation-rearing in the rat. Neuroscience, 83: 481-487. Fulford, A.J. and Marsden, C.A. (1997c) Effect of isolationrearing on noradrenaline release in rat hypothalamus and hippocampus in vitro. Brain Res., 748: 93-99. Fulford, A.J. and Marsden, C.A. (1998) Effect of isolationrearing on conditioned dopamine release in vivo in the nucleus accumbens of the rat. J. Neurochem., 70: 384-390. Graeff, F.G., Guimaraes, F.S., de Andrade, T.G. and Deakin, W.F. (1996) Role of 5-HT in stress, anxiety and depression. Pharmacol. Biochem. Behav., 54: 129-141. Grant, S.J., Aston-Jones, G. and Redmond, D.E. (1988) Responses of primate locus coeruleus neurons to simple and complex sensory stimuli. Brain Res. Bull., 21: 401-410. Hall, F.S., Humby, T., Wilkinson, L.S. and Robbins, T.W. (1997) The effects of isolation-rearing of rats on the behavioural response to food and environmental novelty. Physiol. Behav., 62: 281-290. Herman, J.P. and Cullinan, W.E. (1997) Neurocircuitry of stress: central control of the hypothalmo-pituitary-adrenocortical axis. Trends Neurosci., 20: 74-84. Hirata, H. and Aston-Jones, G. (1994) A novel long-latency response of locus coeruleus neurons to noxious stimuli: mediation by peripheral C-fibres. J. Neurophysiol., 71: 1752-1761. Hughes, Z.A. and Stanford, S.C. (1998) A partial noradrenergic lesion induced by DSP-4 increases extracellular noradrenaline concentration in rat frontal cortex: a microdialysis~study in vivo. Psychopharmacol., 136: 299-303. Jones, G.H., Hernandez, T.D., Kendall, D.A., Marsden, C.A. and Robbins, T.W. (1992) Dopaminergic and serotonergic function following isolation rearing in rats, a study of
500 behavioural responses and post-mortem and in vivo neurochemistry. Pharmacol. Biochem. Behav., 43: 17-35. Kawahara, H., Kawahara, Y. and Westerink, B.H.C. (2000) The role of afferents to the locus coeruleus in the handling stress-induced increase in the release of noradrenaline in the medial prefrontal cortex: a dual-probe microdialysis study in the rat brain. Eur. J. Pharmacol., 387: 279-286. Lapiz, M.D.S., Mateo, Y., Durkin, S., Parker, T. and Marsden, C.A. (2001) Effects of central noradrenaline depletion by the selective neurotoxin DSP-4 on the behaviour of the isolated rat in the elevated plus maze and water maze. Psychopharmacology, 155: 251-259. McQuade, R. and Stanford, S.C. (2000) A microdialysis study of the noradrenergic response in rat frontal cortex and hypothalamus to a conditioned cue for aversive, naturalistic environmental stimuli. Psychopharmacol., 148: 201-208. McQuade, R. and Stanford, S.C. (2001) Differences in central noradrenergic and behavioural responses of Maudsley nonreactive and Maudsley reactive inbred rats on exposure to an aversive novel environment. J. Neurochem., 76: 21-28. McQuade, R., Creton, D. and Stanford, S.C. (1999) Effect of novel environmental stimuli on rat behaviour and central noradrenaline function measured by in vivo microdialysis. Psychopharmacol., 145: 393-400. Muchimapura, S., Mason, R. and Marsden, C.A. (2002) Isolation rearing in the rat disrupts the hippocampal response to stress. Neuroscience, 112: 697-706. Muchimapura, S., Mason, R. and Marsden, C.A. (2003) The effect of isolation rearing on pre- and post-synaptic serotonergic function in the rat dorsal hippocampus. Synapse, 47:209-217. Neophytou, S.I., Aspley, S., Butler, S., Beckett, S. and Marsden, C.A. (2001) Effects of lesioning noradrenergic neurones in the locus coeruleus on conditioned and unconditioned aversive behaviour in the rat. Prog. Neuropsychopharmacol. Biol. Psychiatry, 25: 1307-1321. Pacak, K., Armando, I., Fukuhara, K., Kvetnansky, R., Palkovits, M., Kopin, I.J. and Goldstein, D.S. (1992) Noradrenergic activation in the paraventricular nucleus during acute and chronic immobilization stress in rats: an in vivo microdialysis study. Brain Res., 589:91-96 Pardon, M.-C., Gould, G.G., Garcia, A., Phillips, L., Cook, M.C., Miller, S.A., Mason, P.A. and Morilak, D.A. (2002) Stress reactivity of the brain noradrenergic system in three rat strains differing in their neuroendocrine and behavioural responses to stress: implications for susceptibility to stress-related neuropsychiatric disorders. Neuroscience. 115: 229-242. Pezzone, M.A., Lee, W.-S., Hoffman, G.E., Pezzone, K.M. and Rabin, B.S. (1993) Activation of brainstem catecholaminergic neurons by conditioned and unconditioned aversive stimuli as revealed by c-Fos immunoreactivity. Brain Res., 608: 310-318.
Phillips, G.D., Howes, S.R., Whitelaw, R.B., Wilkinson, L.S. and Robbins, T.W. (1994) Isolation rearing enhances the locomotor response to cocaine and a novel environment, but impairs the intravenous administration of cocaine. Psychopharmacology, 115:407-418. Rasmussen, K. and Jacobs, B.L. (1986) Single unit activity of locus coeruleus neurons in the freely moving cat. II. Conditioning and pharmacologic studies. Brain Res., 371: 335-344 Rosario, L.A. and Abercrombie, E.D. (1999) Individual differences in behavioural reactivity: correlation with stressinduced norepinephrine efflux in the hippocampus of Sprague-Dawley rats. Brain Res. Bull., 48: 595-602. Salmon, P. and Stanford, S.C. (1989) Beta-adrenoceptor density correlates with behaviour of rats in the open field. Psychopharmacology, 98:412-416. Salmon, P. and Stanford, S.C. (1992) Research strategies for decoding the neurochemical basis of resistance to stress. J. Psychopharmacol., 6: 1-7. Salmon, P., Stanford, S.C., Mikhail, G., Zielinski, S. and Pepper, J.R. (2001) Hemodynamic and emotional responses to a psychological stressor after cardiac transplantation. Psychosomatic. Med., 63: 289-299. Sara, S.J. and Segal, M. (1991) Plasticity of sensory responses of locus coeruleus neurons in the behaving rat: implications for cognition. Prog. Brain. Res., 88: 571-585. Sara, S.J., Devauges, V., Biegon, A. and Blizard, D.A. (1994) The Maudsley rat strains as a probe to investigate noradrenaline-cholinergic interaction in cognitive function. J. Physiol., 88: 337-345. Shanks, N., Griffiths, J. and Anisman, H. (1994) Norepinephrine and serotonin alterations following stressor exposure: mouse strain differences. Pharmacol. Biochem. Behav., 49: 57-65. Stanford, S.C. (1993) Monoamines in response and adaptation to stress. In: Stanford, S.C and Salmon, P (Eds.), Stress: From Synapse to Syndrome. Academic Press, London, pp. 281-331. Stanford, S.C. (1995) Central noradrenergic neurones and stress. Pharmac. Ther., 68: 297-343. Stanford, S.C. (1996) Stress: a major variable in the psychopharmacological response. Pharmacol. Biochem. Behav., 54: 211-217. Stanford, S.C. and Salmon, P. (1989) Neurochemical correlates of behavioural responses to frustrative nonreward in the rat: implications for the role of central noradrenergic neurones in behavioural adaptation to stress. Exp. Brain. Res., 75: 133-138. Stanford, S.C., Fillenz, M. and Ryan, E. (1984) The effect of repeated mild stress on cerebral cortical adrenoceptors and noradrenaline synthesis in the rat. Neurosci. Lett., 45: 163-167. Stanford, S.C., Parker, V. and Morinan, A. (1988) Deficits in exploratory behaviour in socially-isolated rats are not
501 accompanied by changes in cerebral cortical adrenoceptor binding. J. Affect. Disord., 15: 175-180. Stanford, S.C., Mikhail, G., Salmon, P., Gettins, D., Zielinski, S. and Pepper, J.R. (1997) Psychological stress does not affect plasma catecholamines in subjects with cardiovascular disorder. Pharmacol. Biochem. Behav., 58:1167-1174. Tanaka, M., Tsuda, A., Yokoo, H., Yoshida, M., Mizoguchi, K. and Shimizu, T. (1991) Psychological stress-induced increase in noradrenaline release in rat brain regions are attenuated by diazepam but not by morphine. Pharmacol. Biochem. Behav., 39: 191-195. Varty, G.B., Marsden, C.A. and Higgins, G.A. (1999) Reduced synaptophysin immunoreactivity in the dentate gyrus of prepulse inhibition-impaired isolation-reared rats. Brain Res., 824: 197-203. Verbanac, J.S., Commissaris, R.L., Altman, H.J. and Pitts, D.K. (1994) Electrophysiological characteristics of
locus coeruleus neurons in the Maudsley reactive (MR) and non-reactive (MNRA) rat strains, Neurosci. Lett., 179: 137-140. Wongwitdecha, N. and Marsden, C.A. (1996) Effect of social isolation on the reinforcing properties of morphine in the conditioned place preference test. Pharmacol. Biochem. Behav., 53: 531-534. Wright, I.K., Ismail, H., Upton, N. and Marsden, C.A. (1991) Resocialisation of isolation-reared rats does not alter their anxiogenic profile in the elevated X-maze model of anxiety. Physiol. Behav., 50:1129-1132. Yokoo, H., Tanaka, M., Yoshida, M., Tsuda A., Takahiki, T. and Mizoguchi, K. (1990) Direct evidence of conditioned fear-elicited enhancement of noradrenaline release in the rat hypothalamus assessed by intracranial microdialysis. Brain Res., 536: 305-308.
This Page Intentionally Left Blank
T. Steckler, N.H. Kalin and J.M.H.M. Reul (Eds.) Handbook of Stress and the Brain, Vol. 15
ISBN 0-444-51173-3 Copyright 2005 Elsevier B.V. All rights reserved CHAPTER 4.6
Stress, corticotropin-releasing factor and serotonergic neurotransmission Astrid C.E. Linthorst* The Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, Dorothy Hodgkin Building, University of Bristol, Whitson Street, Bristol BS1 3NY, UK
Abstract: An intricate interplay between different brain neurotransmitter and neuropeptide systems coordinates the neuroendocrine, autonomic and behavioural responses to stress. Under normal conditions, the body has appropriate mechanisms to respond to acute stressful challenges, but chronic stress may evolve in maladaptive coping processes, resulting in an enhanced risk for illness. Disturbances at the level of both corticotropin-releasing factor (CRF) and serotonin (5-HT) have been implicated in the etiology of stress-related psychiatric disorders, such as major depression and anxiety. Therefore, this chapter is dedicated to the effects of stress on serotonergic neurotransmission and the role of CRF herein. The first part of this chapter covers the neuroanatomical evidence for interactions between CRF and 5-HT at the level of the raphe nuclei. Further, the effects of stress and CRF on different aspects of serotonergic neurotransmission will be addressed, including expression of c-fos in the raphe nuclei, serotonin synthesis and firing rate of serotonergic neurons. Moreover, the effects of five selected stressors (immune stress, forced swimming, tail pinch, electric shock/fear conditioned stress and predator stress), differing in their physical and psychological impact, on the levels of 5-HT and its metabolite 5-hydroxyindoleacetic acid in forebrain regions will be discussed. The data presented here underscore the concept that stress, possibly via activation of the CRF system, affects serotonergic neurotransmission. Stress seems to exert predominantly stimulatory effects on the 5-HT system, at least in higher brain structures such as the hippocampus. Long-term changes in the CRF system, e.g. chronic elevation of brain levels of CRF or dysfunctioning of CRF receptor type 1, have profound consequences for the stress responsiveness of the hippocampal 5-HT system. Notwithstanding the vast amount of data on the effects of stress on serotonergic neurotransmission, the wide variety of experimental protocols used until now has uncovered the clear need for more systematic and neuroanatomically detailed approaches to further elucidate the interactions between stress, CRF and 5-HT. Such strategies will increase our knowledge on 'healthy' stress processing and may consequently lead to a better understanding of the putative maladaptive processes involved in the etiology of stress-related psychiatric and other disorders.
Introduction
stressful situations. Chronic stress may, however, lead to maladaptive stress coping processes and, consequently, to an enhanced risk for illness. In this respect, it is of interest that the prevalence of stressrelated psychiatric disorders, such as major depression and anxiety, seems to increase considerably. It is, therefore, of utmost importance to develop and propagate programmes to improve stress coping strategies in (young) individuals. However, to tackle stress-related health problems and to improve coping
The increasing load of psychological and physical stress weighs heavily on a vast number of people in our society. Under normal circumstances the body will exert appropriate responses to overcome acute
*Tel.: +44 117 33 13140; E-mail:
[email protected] 503
504 strategies, it will be essential to increase our understanding of the processing of stress at the level of the central nervous system. Interactions between different brain neurotransmitter and neuropeptide systems coordinate the neuroendocrine, autonomic and behavioural responses to stress. In this chapter, I will discuss the present knowledge on the effects of stress on the serotonin (5-HT) system in animals, because this neurotransmitter is known to regulate hypothalamic-pituitaryadrenocortical (HPA) axis activity (Dinan, 1996; Lowry, 2002; Carrasco and Van de Kar, 2003) and behaviour (Lucki, 1998) during stressful challenges. Moreover, disturbed functioning of serotonergic neurotransmission seems to play a key role in the etiology of depression, as evidenced by the decreased levels of 5-hydroxyindoleacetic acid (5-HIAA, the metabolite of 5-HT) in the cerebrospinal fluid, the specific changes in the expression of 5-HT receptors and the therapeutical efficacy of selective serotonin reuptake inhibitors (SSRIs) observed in depressed patients (for review see Mann, 1998; Maes and Meltzer, 1995; Ressler and Nemeroff, 2000). As has been excellently described in other chapters in this Handbook (see Section 2. Hypothalamic Hormones involved in stress responsivity), corticotropin-releasing factor (CRF) is a key mediator of the stress-induced activation of the HPA axis and the autonomic nervous system, and of behavioural responses to stress. During the past two decades evidence has been accumulating for a role of a hyperactive CRF system in major depression. In depressed patients, enhanced levels of CRF in the cerebrospinal fluid (Nemeroff et al., 1984), decreased numbers of CRF binding sites in the frontal cortex (Nemeroff et al., 1988), an elevated level of CRF mRNA expression in the hypothalamic paraventricular nucleus (Raadsheer et al., 1995) and an increased number of CRF-expressing neurons in this nucleus (Raadsheer et al., 1994) have been found. Hence, because both CRF and 5-HT are involved in the regulation of the stress response and both systems seem to be malfunctioning in mood disorders, this chapter will focus on the present evidence for a role of CRF in the regulation of serotonergic neurotransmission under basal and stress conditions. Furthermore, observations showing that chronic changes in the CRF system have profound
consequences for the responsiveness of serotonergic neurotransmission to stress will also be discussed. The information presented in this chapter underscores the concept that stress, via activation of the CRF system, affects serotonergic neurotransmission. Long-term changes in the status of the CRF system, for instance due to the experience of chronic stress, may lead to changes in the responsiveness of 5-HT to stressful challenges. Whether such changes in serotonergic neurotransmission eventually precipitate in psychiatric illness or mental imbalance should be subject of further investigations.
Relationship between serotonin and corticotropin-releasing factor at the neuroanatomical level Serotonergic neurons, located in the brainstem, are mainly confined to the raphe nuclei (Dahlstr6m and Fuxe, 1964; Steinbusch, 1981). A rostral and a caudal 5-HT system can be distinguished (T6rk, 1990; Jacobs and Azmitia, 1992). The rostral 5-HT system consists of neurons located in the caudal linear nucleus, the dorsal (DRN) and median raphe nucleus (MRN) and the supralemniscal region (B9 group). This system provides a dense serotonergic innervation of the whole forebrain. On the other hand, the caudal 5-HT system, encompassing the raphe pallidus nucleus, the raphe obscurus nucleus, the raphe magnus nucleus and serotonergic cell bodies in the ventral lateral medulla, projects to different targets in the spinal cord. Connections between the raphe nuclei and with other brainstem structures have also been described. A neuroanatomical basis for a relationship between CRF and 5-HT is now emerging. CRFimmunoreactive cell bodies and fibres have been demonstrated in the rostral and caudal raphe nuclei (Cummings et al., 1983; Swanson et al., 1983; Sakanaka et al., 1987; Ruggiero et al., 1999; Kirby et al., 2000; Valentino et al., 2001). Cell bodies immunoreactive for the CRF-like neuropeptide urocortin 1 (Vaughan et al., 1995) have also been detected in the DRN and MRN, and in the raphe magnus nucleus after colchicine treatment (Kozicz et al., 1998; Bittencourt et al., 1999). Moreover, a
505 dense distribution of urocortin 1-immunoreactive fibres has been observed in the D R N together with a low to moderate innervation of the M R N and the caudal raphe nuclei (Bittencourt et al., 1999). In contrast to the clear presence of CRF and urocortin 1 fibres in the DRN, no (or very few) immunoreactive fibres (or m R N A levels) for the recently discovered CRF-related neuropeptide urocortin 3 (Lewis et al., 2001) have been found in this nucleus and in the other serotonergic cell body regions (Li et al., 2002). Unfortunately, no detailed information on the localization of urocortin 2, another member of the CRF family (Reyes et al., 2001), in the raphe nuclei is available yet. Some recent studies have begun to focus on the topographical distribution of CRF-immunoreactive fibres in the DRN. Although CRF-immunoreactive fibres are present throughout the DRN, a distinct rostral to caudal innervation pattern has been described. Dense CRF innervation has been found in the interfascicular and ventromedial regions at rostral to medial levels, which shifts to dorsal and dorsolateral regions in the caudal D R N (Kirby et al., 2000; Lowry et al., 2000; Valentino et al., 2001). CRF-immunoreactive fibres are found in close association with neurons immunopositive for tryptophan hydroxylase (TPH; the rate-limiting enzyme for the synthesis of serotonin) (Lowry et al., 2000) or 5-HT (Kirby et al., 2000), but also with nonserotonergic neurons. A detailed electron microscopy study revealed that CRF axons in the D R N make preferentially synaptic contacts with dendrites and (non-CRF) axon terminals (Valentino et al., 2001). In the dorsal and dorsolateral D R N the dendritic contacts are in majority asymmetric (excitatory) and in the ventromedial/interfascicular regions largely symmetric (inhibitory). Moreover, the percentage of contacts of CRF fibres with axon terminals is higher in the rostral ventromedial/interfascicular D R N as compared to the caudal dorsolateral D R N (Valentino et al., 2001). Of interest to note is that the distribution of CRF fibres and 5-HT neurons in the D RN is not identical. For instance, a high number of 5-HT neurons is found in the ventromedial/interfascicular region of the caudal DRN, whereas the CRF innervation is relatively low in this region as compared to the dorsolateral aspects at the same rostralcaudal level. This observation suggests that CRF
fibres may form synaptic contacts also with nonserotonergic neurons. Indeed, a recent study has demonstrated immunoreactivity for CRF receptors associated with GABA-immunopositive neurons (Roche et al., 2003). CRF and the urocortins show characteristic binding affinities for the two types of CRF receptors presently known (for review see Reul and Holsboer, 2002). Whereas CRF binds relatively selectively to CRF receptor type 1 (CRF~) over CRF receptor type 2 (CRF2), urocortin 1 shows high-binding affinity for both the receptor types (Chen et al., 1993; Vaughan et al., 1995; Lovenberg et al., 1995). In contrast, urocortin 2 and urocortin 3 seem to represent selective ligands for CRF2 (Lewis et al., 2001; Reyes et al., 2001). A low to moderate CRF~ m R N A expression has been found in both the rostral and caudal raphe nuclei (Chalmers et al., 1995; Bittencourt and Sawchenko, 2000; Van Pett et al., 2000). The M R N contains moderate and the D R N and the caudal linear nucleus higher levels of CRF2 m R N A (Chalmers et al., 1995; Bittencourt and Sawchenko, 2000; Van Pett et al., 2000), but expression of CRF2 m R N A seems to be absent in the caudal serotonergic cell groups. However, CRF2-immunoreactive neuronal profiles have been demonstrated recently in the rostral as well as in the caudal raphe nuclei (Lowry et al., 2002). Immunocytochemistry studies applying a double-labeling approach have shown the presence of CRF~ in TPH-positive neurons in the raphe nuclei (Lowry et al., 2002). Of interest is also the observation that CRF-R immunoreactivity can be found in GABAergic neurons in the dorsolateral region of the D R N (Roche et al., 2003). According to the manufacturer's specifications, the antibody for CRF1 used in the latter study may, however, show cross reactivity with CRF2. Based on the above-described findings, it is evident that the CRF system is in a unique position to influence serotonergic neurotransmission by direct and indirect (GABAergic interneurons) mechanisms at the level of the raphe nuclei. However, the exact interplay between the different members of the CRF family, the role of the two types of CRF-R, and their functional implications is subject of intensive research. It should be emphasized that further detailed studies addressing the topographical specificity of the relationship between the CRF and 5-HT
506 systems are needed. Such studies should also include more elaborated investigations in the MRN, because this raphe nucleus plays an important role in the regulation of the output of the hippocampus and the cortex, brain regions fundamentally involved in stress coping strategies.
Raphe nuclei: stress- and corticotropin-releasing factor-induced alterations in immediate early gene expression and firing rate of serotonergic neurons
c-los Several studies, implementing the immediate early gene product c-fos as a functional marker for neuronal activation (for review see Kovacs, 1998), have shown that stress may result in activation of raphe neurons. Different psychological stress models such as the exposure to an elevated plus maze (Silveira et al., 1993) or a conditioned fear stress paradigm (Pezzone et al., 1993; Beck and Fibiger, 1995; Ishida et al., 2002), social defeat stress (Martinez et al., 1998), restraint (Watanabe et al., 1994; Cullinan et al., 1995) and inescapable tailshock (as compared to escapable tailshock; Grahn et al., 1999) increase c-fos expression within the DRN and/or the MRN. The mixed psychological and physical stressor forced swimming also results in elevated levels of c-fos mRNA in the DRN and MRN (Cullinan et al., 1995). In contrast, immune stress (for instance induced by intraperitoneal injection of the bacterial endotoxin lipopolysaccharide (LPS) or intravenous administration of interleukin-1) seems to have no effect on c-fos expression in the DRN or MRN (Ericsson et al., 1994; Elmquist et al., 1996; Laflamme et al., 1999). Until now little attention has been paid to the detailed neuroanatomical topography of stressinduced expression of c-fos in the different raphe nuclei. A recent study by the group of Rita Valentino, however, shows that 15min of forced swimming in water of 25~ increases c-fos immunoreactivity especially in the dorsolateral DRN (Roche et al., 2003). A high percentage of the c-fos-positive neurons were doubly labeled for GABA and enveloped by CRF fibres. Interestingly, these GABA neurons also contained CRF-R (Roche et al., 2003). Hence, these
authors have speculated that, at least in this subregion of the DRN, stress results (via CRF-R) in stimulation of inhibitory interneurons leading to inhibition of 5-HT neurons and consequently to lower levels of 5-HT in specific terminal regions. This possibility is underscored by the observation that conditioned fear stress results in c-fos immunostaining of not only 5-HT, but also of GABAimmunopositive neurons in the DRN (Ishida et al., 2002). The effect of stress on c-fos expression in the raphe nuclei seems to be mimicked by i.c.v, administration of CRF and urocortin 1. Both the neuropeptides increase c-fos immunoreactivity profoundly in the DRN but only moderately in the MRN (Bittencourt and Sawchenko, 2000). Moderate effects of these neuropeptides were also demonstrated in the caudal raphe nuclei, i.e. raphe magnus, pallidus and obscurus nuclei (Bittencourt and Sawchenko, 2000). In contrast, urocortin 2 has no effects on c-fos expression in the raphe nuclei (Reyes et al., 2001).
Firing rate of serotonergic neurons Different types of serotonergic neurons have been described in the rostral raphe nuclei. The majority of 5-HT neurons fire solitary spikes in a slow (0.3-5 Hz) and regular discharge pattern (Aghajanian et al., 1968; Aghajanian and Vandermaelen, 1982), which is closely related to the sleep-wake cycle. The highest levels of discharge are observed during active waking. The discharge rate, however, decreases during quiet waking and even further during non-rapid eye movement sleep. Finally, these 5-HT cell bodies are almost salient during rapid eye movement sleep (see Jacobs and Azmitia, 1992). A substantial number of 5-HT neurons (about 25%) in the cat DRN and MRN has been found to be tonically activated specifically during oral-buccal movements, such as chewing, licking and grooming. These neurons are not activated during other behaviours and even decrease their firing rate during locomotion and orienting behaviours (Fornal et al., 1996). In contrast, a small subpopulation of serotonergic neurons has been described in a region between the medial longitudinal fasciculi at the caudal interface of the DRN and the MRN. The firing frequency of these 5-HT neurons is not clearly related to the vigilance
507 state of the animal (Rasmussen et al., 1984). Finally, recent studies report on 5-HT neurons in the D R N (and to a lesser extent in the MRN) that fire pairs of spikes or bursts of spikes in a very short (< 20ms) time interval but with a highly regular pattern (Haj6s et al., 1995; Morzorati and Johnson, 1999). Mimicking the burst-firing pattern of 5-HT neurons by electrical stimulation of the D RN produces a greater increase in the extracellular levels of 5-HT in the medial prefrontal cortex than observed after single pulse stimulation (Gartside et al., 2000). Barry Jacobs and colleagues have carefully analysed the effects of stress on the firing patterns of 5-HT neurons in the raphe nuclei of the cat. During different types of stress, including thermoregulatory and glucoregulatory challenges as well as noise stress and painful stimuli, the firing rate of 5-HT neurons in the D R N and M R N is not different from discharge rates found during a normal phase of active waking (for review see Jacobs and Azmitia, 1992). These observations have led to the proposition that 5-HT plays no specific role during stress, but that this neurotransmitter merely facilitates motor output and coordinates the concurrent neuroendocrine and autonomic responses, together with the suppression of sensory processing (Jacobs and Fornal, 1999). More recent studies on the effects of CRF on discharge rate suggest, however, that there are, at least in the DRN, distinct subsets of 5-HT neurons. Intracerebroventricular (i.c.v.) and intraraphe administration of low doses of CRF decrease the in vivo firing rate of 5-HT neurons in rats under halothane anaesthesia in the rostral and medial aspects of the D R N without an apparent dorsoventral or mediolateral organization (Price et al., 1998; Kirby et al., 2000). In contrast, an in vitro study by Lowry and colleagues showed a CRF-induced rise in the firing rate of a specific set of neurons located in the ventral and interfascicular region of the caudal D R N (Lowry et al., 2000). No effect was found on 5-HT neurons in the dorsomedial region of the D R N at the same rostral-caudal level in the latter study. Although the respective influence of anaesthesia and of the absence of afferent input in the in vivo and in vitro studies cannot be excluded at this moment, the data suggest the presence of discrete subsets of CRF-sensitive 5-HT neurons in the DRN. Together with the distinct efferent innervation pattern of the
forebrain by the different subregions of the D R N and the M R N (there is evidence for a rostrocaudal, mediolateral and dorsoventral encephalotopy within the DRN; see discussion in Valentino et al., 2001), these data put forward that 5-HT neurons may display a topographically specific response to stress. The effects of stress and CRF on the subpopulation of 5-HT neurons that fire in a burst-like pattern are not known yet. Studies on stress-induced changes in the firing rate of these neurons will be of high relevance, given the observation that electrical stimulation of the D R N in a burst-like pattern causes a greater rise in extracellular 5-HT in the prefrontal cortex than single pulse stimulation (Gartside et al., 2000).
Effects ofstress and corticotropin-releasing factor on the synthesis of serotonin Serotonin is synthesized from the essential amino acid L-tryptophan, which is transported from the blood into the brain via an L-type neutral amino acid transporter in the blood-brain barrier. Proteins in the diet are the principal source of L-tryptophan. In serotonergic neurons L-tryptophan is hydroxylated into 5-hydroxytryptophan (5-HTP) by the enzyme TPH, forming the rate-limiting step in the synthesis of 5-HT. Next, 5-HTP is decarboxylated into 5-HT by the enzyme aromatic L-amino acid decarboxylase (AADC). A recent report indicates that two isoforms of TPH may exist, TPH1 and TPH2, responsible for the synthesis of 5-HT in the periphery and the brain respectively (Walther et al., 2003). TPH becomes activated during situations where a replenishment of the stores of 5-HT is needed. For instance, during electrical stimulation of serotonergic cell bodies a (frequency-dependent) rise in the synthesis of 5-HT from L-tryptophan can be observed (Shields and Eccleston, 1972; Herr et al., 1975; Boadle-Biber et al., 1986). The activation of TPH involves protein phosphorylation by calcium/ calmodulin-dependent protein kinase II (Lysz and Sze, 1978; Hamon et al., 1981; Kuhn and Lovenberg, 1982; Ehret et al., 1989) and/or cAMP-dependent protein kinase A (Johansen et al., 1995; see also Stenfors and Ross, 2002; see also Mockus and Vrana, 1998). There is also evidence that the availability of
508 (free) L-tryptophan to the brain can affect the level of synthesis of 5-HT under certain circumstances (Fernstrom and Wurtman, 1971; Knott and Curzon, 1972; for further discussion see Boadle-Biber, 1993). As described above, distinct effects of stress on neuronal activation and on the firing rate of 5-HT neurons have been found. Therefore, the question arises whether stress also affects the synthesis of 5-HT and whether it does so in a stressor and neuroanatomically specific manner. Initial work by Azmitia and McEwen has shown that prolonged cold and electric footshock stress increase TPH activity in the midbrain and forebrain of rats; an effect which is prevented by adrenalectomy (Azmitia and McEwen, 1974). The group of Margaret Boadle-Biber has collected interesting and comprehensive data on the effects of loud sound stress on 5-HT synthesis, using an ex vivo TPH activity assay and an in vivo 5-HTP accumulation paradigm, with emphasis on dissecting the different effects of acute versus chronic stress. One session of randomly presented sound stress results in a short-lasting increase in TPH activity in supernatants of the cortex and midbrain of Fischer 344 rats (Boadle-Biber et al., 1989). Repeated exposure of the animals to the sound stress paradigm induces a persistent elevation of TPH activity which is still present 24 h after the last stress session (Boadle-Biber et al., 1989). The effects of sound stress on TPH activity are related to increases in firing rate, because no effects of sound stress were found after pretreatment of the animals with the 5-HT1A receptor agonist gepirone, which inhibits the discharge of 5-HT neurons (Corley et al., 1992). Interestingly, the effects of sound stress on TPH activity are blocked by adrenalectomy (Singh et al., 1990) and are mimicked by i.c.v, administration of CRF (Singh et al., 1992). Moreover, glucocorticoid receptors (GRs) seem to play a permissive role in the CRF- and sound stressinduced activation of TPH (Singh et al., 1990, 1992). Studies, in which the accumulation of 5-HTP after inhibition of AADC by m-hydroxylbenzylamine (NSD 1015) was assessed, revealed that sound stress increases 5-HT synthesis in the M R N but not in the D R N (Dilts and Boadle-Biber, 1995; Daugherty et al., 2001). A similar specific activation only in the M R N is observed after exposure to forced swim stress or tailshock (Corley et al., 2002). The notion that stress may cause a neuroanatomically specific effect
on 5-HT synthesis is further underscored by the observation that i.c.v, administration of CRF has no effect on 5-HTP accumulation after AADC inhibition in the mediobasal hypothalamus (Van Loon et al., 1982). Moreover, also repeated immobilization stress seems to exert neuroanatomically distinct effects on TPH activity. Whereas Miklos Palkovits and colleagues found no changes in TPH activity in various brain regions, such as the hypothalamus, amygdala, hippocampus and DRN, after repeated (5 times) immobilization stress (Palkovits et al., 1976), Culman et al. observed increased TPH activity in the locus coeruleus, decreased activity in the suprachiasmatic nucleus together with no effects in the D R N after seven immobilization sessions (Culman et al., 1984). In contrast, this stress paradigm has been found to increase TPH m R N A levels 10-fold in the D R N and 6-fold in the MRN, without affecting TPH m R N A levels in the pineal gland (Chamas et al., 1999). Interestingly, measurement of 5-HTP accumulation by microdialysis revealed that l h of immobilization increases 5-HT synthesis in the medial prefrontal cortex and the nucleus accumbens (Nakahara and Nakamura, 1999). The effects of stress, and especially chronic stress, on 5-HT synthesis are highly relevant also with respect to the pathophysiology of affective and anxiety disorders, in which a deficit in 5-HT neurotransmission has been suggested. Indeed depressive symptoms may exacerbate following a low-tryptophan diet (Delgado et al., 1990). Clearly more detailed studies will be needed, also implementing the effects of stress on the phosphorylation of TPH. From the presently available data, however, the picture is emerging that stress can influence the synthesis of 5-HT, in a neuroanatomical and stress duration specific manner, by changing the activity of the rate-limiting enzyme TPH.
Effects of stress and corticotropin-releasing factor on the levels of serotonin and 5-hydroxyindoleacetic acid in forebrain terminal regions The previous sections of this chapter have delineated specialized interactions between the CRF and 5-HT systems at the neuroanatomical level, together with
509 stressor- and site-specific effects of stress on the firing rate of raphe neurons and the synthesis of 5-HT. The impact of stress-induced alterations in serotonergic neurotransmission for behaviour and neuroendocrine regulation will, however, highly depend on the ultimate changes in the extracellular levels of 5-HT in the terminal regions. The present section will, therefore, be devoted to the question whether stress in a stressor- and brain structure-specific manner influences terminal levels of 5-HT, with an emphasis on its extracellular level as assessed by in vivo microdialysis. The effects of five stressors, varying in intensity and their physical versus psychological character, and of CRF and urocortin 1 will be discussed. A summary of the effects of these stressors on the levels of 5-HT and 5-HIAA in terminal regions is presented in Table 1.
Immune stress
Immune stress is often considered to represent a so-called systemic stressor (Li et al., 1996; Herman and Cullinan, 1997), indicating its immediate physiologic threat. During an infectious challenge the organism responds with a variety of defence mechanisms, among which are the activation of the HPA axis and the development of fever. The behavioral changes frequently observed during infection, i.e. fatigue, loss of appetite, immobility and social disinterest, are collectively termed as 'sickness behaviour' (Hart, 1988; Dantzer et al., 1991), and indicate that immune stress may also possess a psychological element, involving higher brain structures. We have provided extensive evidence showing that an infectious challenge results in a highly specific answer of the brain 5-HT system. Intraperitoneal (i.p.) injection of LPS results in a prolonged rise in extracellular levels of 5-HT (Fig. 1A) and 5-HIAA in the hippocampus of rats (Linthorst et al., 1995b). This effect can be mimicked by i.p. (Merali et al., 1997) and i.c.v, injection of interleukin113 (Linthorst et al., 1995b) and interleukin-2, but not tumor necrosis factor-~ (Pauli et al., 1998). The stimulatory effects of immune stress on hippocampal serotonergic neurotransmission are underscored by studies on the turnover of 5-HT, as indicated by an increase in the ratio between the post-mortem levels
of 5-HIAA and 5-HT (Kabiersch et al., 1988; Zalcman et al., 1994; Lacosta et al., 1999). Moreover, studies on tissue levels of 5-HT and on tissue and dialysate levels of 5-HIAA have shown that the stimulatory effects of immune activation on serotonergic neurotransmission are not confined to the hippocampus, but can also be found in other higher brain structures such as the frontal cortex (Dunn and Welch, 1991; Dunn, 1992; Zalcman et al., 1994; Lavicky and Dunn, 1995). In contrast, we observed no effect of i.p. injection of LPS on extracellular levels of 5-HT (Fig. 1B), and only a minute rise in 5-HIAA, in the preoptic area of the rat (Linthorst et al., 1995a). A similar observation in this brain region has been made after administration of the pyrogen muramyl dipeptide in cats (Wilkinson et al., 1991). At the level of the hypothalamus predominantly stimulatory effects of systemic immune stimulation and administration of interleukin-1 on 5-HT turnover and dialysate levels of 5-HIAA have been described (Mefford and Heyes, 1990; Dunn, 1992; Mohankumar et al., 1993; Shintani et al., 1993; Lavicky and Dunn, 1995; Lacosta et al., 1999), although also a decrease in tissue levels of 5-HIAA after i.c.v, injection of interleukin-1 and tumornecrosis factor-~ has been observed (Connor et al., 1998). The effects of immune stress on serotonergic neurotransmission are also interesting from a neuroanatomical point of view. As described above, administration of LPS and interleukin-1 do not induce c-fos activation in the raphe nuclei (Ericsson et al., 1994; Elmquist et al., 1996; Laflamme et al., 1999). Hence, it has been suggested that the immune stress-induced alterations in 5-HT levels are generated locally in the terminal regions. This possibility is underlined by our observation that local infusion of interleukin-lB into the hippocampus by reversed dialysis indeed results in a rise in extracellular 5-HT in this brain region (Linthorst et al., 1994).
Forced swim stress
Forced swim stress is a widely used stress paradigm in rats and mice, mainly applied to assess putative antidepressant properties of drugs in the so-called Porsolt forced swim test paradigm (Porsolt et al., 1977;
510 Table 1. Summary of the effects of stress on 5-HT and 5-HIAA levels in terminal regions as reported in the literature Stressor
Brain structure
Observations
References
Immune stress (lipopolysaccharide; cytokines)
Hippocampus
t 5-HT and 5-HIAA (m)
Linthorst et al., 1994, 1995b; Merali et al., 1997; Pauli et al., 1998 Kabiersch et al., 1988; Zalcman et al., 1994 Connor et al., 1998; Lacosta et al., 1999 Dunn and Welch, 1991; Dunn, 1992; Zalcman et al., 1994 Lavicky and Dunn, 1995 Wilkinson et al., 1991; Linthorst et al., 1995a Dunn and Welch, 1991 Mefford and Heyes, 1990; Dunn, 1992 Mohankumar et al., 1993; Shintani et al., 1993; Lavicky and Dunn, 1995 Connor et al., 1998 Lacosta et al., 1999 Rueter and Jacobs, 1996; Pefialva et al., 2002; Linthorst et al., 2002; Fujino et al., 2002 Adell et al., 1997 Kirby et al., 1995 Rueter and Jacobs, 1996; Fujino et al., 2002 Adell et al., 1997 Kirby et al., 1995 Connor et al., 2000 Rueter and Jacobs, 1996; Adell et al., 1997 Kirby et al., 1995 Connor et al., 2000 Rueter and Jacobs, 1996 Kirby et al., 1995 Kirby et al., 1995 Kal6n et al., 1989; Pei et al., 1990; Vahabzadeh and Fillenz, 1994; Rueter and Jacobs, 1996; Fujino et al., 2002 Pei et al., 1990 Rueter and Jacobs, 1996; Fujino et al., 2002 Pei et al., 1990 Rueter and Jacobs, 1996 Rueter and Jacobs, 1996 Pei et al., 1990 Pei et al., 1990
t turnover (p) 1" 5-HT and 5-HIAA (p) Frontal cortex
t turnover (p)
Preoptic area
t 5-HIAA (m) 5-HT (m), minute rise in 5-HIAA
Hypothalamus
,+ turnover (p) 1" turnover (p) 1" 5-HT and 5-HIAA (m)
Forced swim stress
Hippocampus
$ 5-HIAA (p) i" 5-HIAA (p) 1" 5-HT, 5-HIAA biphasic (m)
Frontal cortex
4 5-HT (m) +. 5-HT, $ 5-HIAA (m) 1" 5-HT (m)
Amygdala
$ 5-HT (m) -. 220
;~ 22o
180
180
180
140
140
140
~. 100
100
~. 100
U.J
6o
......................... 5 10 15
20
25
0
Day 4 and 5
;~ 220
~_, . . . . . . . . . . . . . . . . . . . . . . . . 5 10 15
o~100
O 100
~ 6o
~
25
6o
20
25
Frequency (Hz)
Withdrawal Day 2 S
180
20
0
.~ 220
140
15
~
Z 180
10
~ 8o
.~ 220
140
Frequency (Hz)
25
Withdrawal Day 1
S
5
20
Frequency (Hz)
Frequency (Hz)
0
......................... 5 10 15
180
~ 140 o 100 ......................... 5 10 15
Frequency (Hz)
20
25
~ ~o
0
5
10
15
20
25
Frequency (Hz)
Fig. 4. Effets of 3a, 50~-THP(I 5 mg/kg body weight) and placebo on EEG power densitity within non-REM sleep in male Wistar rats. Data represent mean 4- SEM (n = 8 per group) and are shown as percentage of the average power density during the baseline condition. The lines in the panels day 1, day 2 and 3 and day 4 and 5 indicate the frequency bands with a significant difference between both treatment groups. Modified according to Damianisch et al. (2001).
sedation, alteration of sleep architecture and development of tolerance have to be taken into account, especially when considering long-term treatment with this new class of drugs.
this compound (Zorumski et al., 2000). It remains to be determined whether 3a-reduced neuroactive steroids will receive further consideration as putative anesthetics in humans.
Anesthesia
Nootropic properties, cognition and dementia disorders
3a-reduced neuroactive steroids may also exert antinociceptive (Frye and Duncan, 1994; Nadeson and Goodchild, 2001) and anesthetic (Korneyev and Costa, 1996) effects in various animal models. 3a-reduced neuroactive steroids such as 3a, 5[3-THP (pregnanolone) may exert strong sedative (Schulz et al., 1996) or even anesthetic (Carl et al., 1990) effects in humans. A mixture of alphaxolone and alphadolone has also been developed (Althesin | as an anesthetic in humans (Gyermek and Soyka, 1975). However, solubility problems and a hypersensitivity to the respective solvent have led to the withdrawal of
Pregnenolone sulfate (PS) and D H E A sulfate (DHEA-S) display GABA antagonistic properties and exert complex effects at N M D A receptors (Zorumski et al., 2000). Moreover, sulfate derivatives of pregnanolone have been shown to exert neuroprotective effects via inhibition of N M D A receptor function (Weaver et al., 1997b). In addition to the effects of D H E A via the cell membrane, potential antiglucocorticoid effects of D H E A have been reported in vivo (Browne et al., 1993; Araneo and Daynes, 1995). Therefore, such steroids might possess nootropic properties. Indeed, early studies have
552 suggested that intracerebroventricular administration of pregnenolone and pregnenolone sulfate leads to an amelioration in various memory tasks in rodents (Flood et al., 1992). Moreover, also DHEA has been suggested to enhance memory retention in mice (Flood et al., 1988). In aged rats, low PS levels have been found in the hippocampus and were correlated with memory deficits that could be transiently corrected by PS injection (Vallee et al., 1997). Also prolonged intracerebroventricular infusion of PS enhanced cognitive performance in mice (Ladurelle et al., 2000). However, valid clinical data concerning the memory-enhancing properties of pregnenolone in dementia disorders are lacking to date. There is evidence that DHEA levels decrease with age (Thomas et al., 1994) and decreased concentrations of DHEA have been reported in patients suffering from Alzheimer's disease and multi-infarct dementia (Sunderland et al., 1989; NS.sman et al., 1991). Decreased DHEA-S concentrations may constitute an enhanced risk for the development of Alzheimer's disease (Hillen et al., 2000). Thus, an interplay between neuroactive steroids and the HPA system may be of importance for the pathophysiology of dementia disorders. Meanwhile, DHEA is sold as an antiaging drug especially in the USA. However, systematic research as to whether DHEA supplementation may enhance cognitive performance in normal aging people or in dementia disorders is scarcely available. One open study suggested beneficial effects of DHEA-S on daily living in patients with multiinfarct dementia (Azuma et al., 1999). However, controlled studies with DHEA in Alzheimer's diesase or multi-infarct dementia are not available to date.
Antipsychotic properties Epidemiological studies suggest that the onset of psychiatric symptoms may be related to changes in the secretion of gonadal hormones (Hallonquist et al., 1993; Hfifner et al., 1993). Moreover, there is a difference between pre- and post-menopausal women with an increased vulnerability for the onset of schizophrenic episodes after the menopause (Hfifner et al., 1993). Thus, it may be hypothesized that a sudden drop of steroid concentrations may
contribute to the development of such disorders and a steroid replacement might be of therapeutic value. In contrast to haloperidol, progesterone neither produced catalepsy nor antagonized amphetamineinduced stereotypy. However, both progesterone and haloperidol but not 3~,5~-THP (Rupprecht et al., 1999) effectively restored the disruption of the prepulse inhibition (PPI) of the acoustic startle response that was evoked by apomorphine. This behavioral profile of progesterone is compatible with the possibility that progesterone itself shares some properties with atypical antipsychotics, which may be relevant for the development and treatment of psychotic disturbances, e.g. postpartum psychosis. It has recently been demonstrated that the atypical neuroleptic agent olanzapine may increase the concentrations of 3~, 5~-THP in rat brain (Marx et al., 2000). Also clozapine, in contrast to haloperidol, may enhance the concentrations of both 3~, 5~-THP and of progesterone in rat brain in a time and dose dependent fashion (Barbaccia et al., 2001). Thus, neuroactive steroids might also contribute to the pharmacological profile of atypical antipsychotic drugs.
Premenstrual dysphoric disorder, pregnancy and postpartum period Concentrations of neuroactive steroids vary throughout the menstrual cycle and throughout pregnancy, which is accompanied by changes in GABAA receptor plasticity (Concas et al., 1998). In patients with premenstrual dysphoric disorder (PMDD), decreased levels of 3cz, 5~-THP have been reported during the luteal phase (Rapkin et al., 1997; Bicikova et al., 1998; Monteleone et al., 2000) which might contribute to the development of mood symptoms and irritability. Both at baseline and after mental stress, an enhanced ratio 3~,5~-THP/cortisol has been observed (Girdler et al., 2001). Interestingly, PMDD patients with a high symptom score had lower levels of 3cz,5~-THP when compared with less symptomatic patients (Girdler et al., 2001). Treatment with citalopram may enhance the sensitivity of GABAA receptors to modulation by 3~, 5[3THP in women suffering from PMDD (Sundstr6m and B/ickstr6m, 1998) and the importance of
553 neuroactive steroid-serotonergic interactions is further underlined by an increased response of 3~,5cz-THP to challenge with L-tryptophan in patients with P M D D (Rasgon et al., 2001). Thus, the interplay between the serotonergic system and neuroactive steroids may contribute to the efficacy of selective serotonin reuptake inhibitors in the treatment of P M D D (Steiner et al., 1995). During pregnancy, there is a rise in the concentrations of progesterone and of an array of neuroactive steroids (Pearson Murphy et al., 2001). While progesterone concentrations decline rapidly after delivery, neuroactive steroids are still elevated several weeks postpartum (Pearson Murphy et al., 2001). There was a tendency for increased concentrations of neuroactive steroids in depressed women during the latter half of pregnancy when compared with nondepressed women (Pearson Murphy et al., 2001). Thus, neuroactive steroids might also contribute to psychiatric complaints during pregnancy and the postpartum period.
Antidepressant properties and major depression and stress Stress is a key factor of major depression which is accompanied by an overdrive of the HPA-system. 3~-reduced neuroactive steroids may suppress the expression of vasopressin and CRF in rats (Patchev et al., 1994; Patchev et al., 1997). As endogenous 3cz-reduced neuroactive steroids rise during stress, e.g. during a forced swimming procedure (Paul and Purdy, 1992), such an increase of endogenous neuroactive steroids might contribute to the termination of a stress period as a counterregulatory mechanism. The selective serotonin reuptake inhibitor (SSRI) fluoxetine may enhance the concentrations of 3~, 5~-THP in different areas of the rat brain (Uzunov et al., 1996; Serra et al., 2001). At the molecular level, it has recently been demonstrated that SSRIs may shift in the activity of the 3~-hydroxysteroid oxidreductase, which catalyzes the conversion of 5czDHP into 3~, 5~-THP, towards the reductive direction thereby enhancing the formation of 3cz, 5~-THP (Griffin and Mellon, 1999). In addition, 3~, 5~-THP has been suggested to possess antidepressant-like
effects in mice using the Porsolt swim test (Khisti et al., 2000). These preclinical findings suggest that 3~-reduced neuroactive steroids such as 3~, 5~-THP may play a role for the treatment of depression with antidepressant drugs. Indeed, the concentrations of the GABA agonistic neuroactive steroids 3~, 5~-THP and pregnanolone were reduced in the plasma of depressed patients, while there was an increase in 313, 5cz-THP, an antagonistic isomer for 3cz,5~-THP (Romeo et al., 1998). This disequilibrium of neuroactive steroids could be corrected by treatment with fluoxetine throughout several weeks (Romeo et al., 1998; Uzunova et al., 1998) (Fig. 5). In contrast to the preclinical data, also tri- and tetracyclic antidepressants interfered with the composition of neuroactive steroids in a similar way as did SSRIs (Romeo et al., 1998). However, treatment of depressed patients with repetitive transcranial magnetic stimulation did not affect the concentrations of neuroactive steroids neither in responders nor in nonresponders to this nonpharmacological treatment (Padberg et al., 2002). Studies investigating DHEA or DHEA-S concentrations in depression have yielded divergent results. While one study noted a decrease in DHEA-S concentrations associated with depression (BarrettConnor et al., 1999), other studies reported an increase during major depression (Heuser et al., 1998; Fabian et al., 2001). Elevated baseline DHEA-S concentrations have even been suggested to predict non-response to electroconvulsive therapy (ECT) (Maayan et al., 2000). Nevertheless, treatment with DHEA either as the only medication or as an adjunct to stable antidepressant medication may exert beneficial effects on depressed mood (Wolkowitz et al., 1997; Wolkowitz et al., 1999). Therefore, the role of DHEA in depression and antidepressant therapy should receive further consideration in the future.
Ethanol tolerance and withdrawal Animal studies have shown that systemic ethanol administration may elevate the concentrations of 3cz, 5cz-THP in rat brain (Janis et al., 1998) and that 3~, 5~-THP might contribute to the pharmacological actions of ethanol (van Doren et al., 2000). On the other hand, 3cz,5~-THP protects against
554
12
o
8
E c9progesterone
4
0
93a, 5a-THP 0
10
20
30
40
50
controls
93a, 51~-THP
Days of fluoxetine treatment in patients with depression
931~, 5a-THP 5 4
o E
3 2 1
0 0
10
20
30
40
50
controls
Days of fluoxetine treatment in patients with depression
Fig. 5. Plasma concentrations of neuroactive steroids in depressed patients during treatment with 20mg fluoxetine. The asterisks indicate significant differences from baseline values. Modified according Romeo et al. (1998).
bicuculline-induced seizures during ethanol withdrawal (Devaud et al., 1995). Interestingly, an abstinence syndrome with increased seizure liability may also occur after discontinuation of GABAergic steroids (Janis et al., 1998; Smith et al., 1998b) which may be related to changes in the kinetics of GABAA receptor channels. Moreover, rats selectively bred for high sensitivity to ethanol exhibit also an enhanced sensitivity to 3~-reduced neuroactive steroids (Korpi et al., 2001). However, also pregnenolone sulfate (PS) and DHEA-S have been suggested to be involved in the development of tolerance to ethanol in mice (Barbosa and Morato, 2001). In patients suffering from ethanol abuse, the concentrations of 3~, 5~-THP are markedly reduced during ethanol withdrawal (Romeo et al., 1996; Romeo et al., 2000) and normalized within four weeks (Romeo et al., 2000). The reduced concentrations of 3~, 5~-THP might contribute to the enhanced seizure liability of such patients during ethanol withdrawal. Treatment with fluoxetine results in an
earlier rise in 3~,5~-THP concentrations and is accompanied by a decrease of depression and anxiety during ethanol withdrawal.
Anxiolytic properties and anxiety disorders Positive allosteric modulators of GABAA receptors, e.g. benzodiazepines, are effective anxiolytic substances. Thus, also 3~-reduced neuroactive steroids should exert anxiolytic effects. Indeed, such steroids were effective anxiolytics in different animal models, e.g. the elevated plus maze test (Crawley et al., 1986; Bitran et al., 1991; Wieland et al., 1991). Also progesterone as a precursor molecule for 3~-reduced neuroactive steroids may act as an anxiolytic via GABAA receptors (Bitran et al., 1995). Meanwhile, anxiolytic properties have also been demonstrated for synthetic analogues of 3~-reduced neuroactive steroids (Vanover et al., 2000). 3~-reduced neuroactive steroids may further counteract the anxiogenic effects of CRF and reduce the expression of the
555 CRF gene (Patchev et al., 1994). Although the anxiolytic effects of 3~-reduced neuroactive steroids in animal models are promising, putative side effects such as toxicity, sedation and withdrawal effects have to be taken into consideration and no firm conclusion can be drawn at the moment whether such steroids are superior to benzodiazepines as anxiolytics. First studies in patients with panic disorder from our research group suggest that 3~-reduced neuroactive steroids may play a pivotal role in human anxiety in that they may serve as a counterregulatory mechanism against the occurrence of spontaneous panic attacks (Str6hle et al., 2002). Studies of neuroactive steroids during experimentally induced panic attacks in patients with panic disorder and healthy controls in our research group showed that there is a pronounced decrease in 3~, 5~-THP and 3~, 513-THP together with an increase 313, 5~z-THP following both cholecystokinin tetrapeptide (CCK4) and sodium lactate administration in patients with panic disorder (Str6hle et al., in press). However, such changes do not occur in healthy controls
Conclusions
Neuroactive steroids may modulate neuronal function through their concurrent influence on neuronal excitability and gene expression (Fig. 6). This intracellular cross-talk between genomic and nongenomic steroid effects provides the molecular basis for steroid action in the brain and the future development of such compounds in neuropsychopharmacology, both with regard to putative clinical Steroid hormones:
Neuroactive steroids: 1713-estradiol progesterone 3~, 5~-THP 3~,ps5~-THDOC
Cl-
DHEA-S
(Str6hle et al., in press). These changes in neuroactive steroid composition might result in a decreased GABAergic tone that may be related to the pathophysiology of panic attacks in patients with panic disorder. Although treatment with paroxetine did not further increase the concentrations of GABAergic neuroactive steroids, antidepressants such as SSRIs might be effective as antipanic agents also through stabilizing the equilibrium of endogenous neuroactive steroids (Str6hle et al., 2002).
steroid
~..
1713-estrad io I dihydrotestosterone progesterone aldosterone corticosterone cortisol /
norepinephrine
C a ++
/
dooamine
membrat~e
GABAA receptor 5-HT 3 receptor nicotinic acetylcholine receptor NMDA receptor kainate receptor AMPA receptor glycine receptor sigma receptor oxytocin receptor
oxidationllREDUCTiON
(NAPSE
nucleu~
MINUTES
HOURS NONGENOMIC
HSP90
DAYS
MONTHS
GENOMIC
Fig. 6. Nongenomic and genomic effects of neuroactive steroids. Abbreviations: BDZ, benzodiazepines; R, receptor; G, G-protein; PKA, protein kinase A; HSP 90, heat shock protein 90; GR, glucocorticoid receptor; MR, mineralocorticoid receptor; PR, progesterone receptor; ER, estrogen receptor. Reproduced with permission from Rupprecht and Holsboer (1999).
556 effects and side effects. One i m p o r t a n t issue is specificity. As yet, no naturally occurring steroid with a really specific and selective action at a distinct steroid receptor or n e u r o t r a n s m i t t e r receptor has been identified. A n o t h e r issue that deserves further consideration is t r e a t m e n t duration. While behavioural properties of neuroactive steroids are quite well characterized in n u m e r o u s paradigms after acute administration, studies on the consequences of longterm a d m i n i s t r a t i o n of such c o m p o u n d s are widely lacking to date. As a prerequisite for further clinical exploitation of such steroids in n e u r o p s y c h o p h a r m a cology especially these types of studies are particularly needed in the future. In conclusion, e n d o g e n o u s or exogenous neuroactive steroids offer a considerable potential in the t r e a t m e n t of neuropsychiatric disorders. F u t u r e studies addressing the effects of neuroactive steroids on multiple n e u r o t r a n s m i t t e r receptors and the behavioural consequences of longterm administration will be crucial to explore the n e u r o p s y c h o p h a r m a c o l o g i c a l potential of this yet unexploited class of drugs.
Acknowledgements The studies on neuroactive steroids at the MaxPlanck-Institute of Psychiatry and the D e p a r t m e n t of Psychiatry, Ludwig Maximilian University, M u n i c h are s u p p o r t e d by the G e r h a r d Hel3 P r o g r a m m of the Deutsche F o r s c h u n g s g e m e i n s c h a f t and the G e r m a n F e d e r a l Research Ministry within the p r o m o t i o n a l emphasis " C o m p e t e n c e Nets in Medicine".
References Akwa, Y., Morfin, R.F., Robel, P. and Baulieu, E.E. (1992) Neurosteroid metabolism. 7 alpha-hydroxylation of dehydroepiandrosterone and pregnenolone by rat brain microsomes. Biochem. J., 288: 959-964. Araneo, B. and Daynes, R. (1995) Dehydroepiandrosterone functions as more than.an antiglucocorticoid in preserving immunocompetence after thermal injury. Endocrinology, 136: 393-401. Azuma, T., Nagai, Y., Saito, T., Funauchi, M., Matsubara, T. and Sakoda, S. (1999) The effect of dehydroepiandrosterone sulfate administration to patients with multi-infarct dementia. J. Neurol. Sci., 162: 69-73.
Barbaccia, M.L., Affricano, D., Purdy, R.H., Maciocco, E., Spiga, F. and Biggio, G. (2001) Clozapine, but not haloperidol, increases brain concentrations of neuroactive steroids in the rat. Neuropsychopharmacology, 25: 489-497. Barbosa, A.D. and Morato, G.S. (2001) Influence of neurosteroids on the development of rapid tolerance to ethanol in mice. Eur. J. Pharmacol., 431: 179-188. Barrantes, F.J., Antollini, S.S., Bouzat, C.B., Garbus, I. and Massol, R.H. (2000) Nongenomic effects of steroids on the nicotinic acetylcholine receptor. Kidney Int., 57: 1382-1389. Barrett-Connor, E., von Muhlen, D., Laughlin, G.A. and Kripke, A. (1999) Endogenous levels of dehydroepiandrosterone sulfate, but not other sex hormones, are associated with depressed mood in older women: the Rancho Bernardo Study. J. Am. Geriatr. Soc., 47: 685-691. Baulieu, E.E. (1991) Neurosteroids: A new function in the brain. Biol. Cell., 71: 3-10. Baulieu, E.E. (1998) Neurosteroids: a novel function of the brain. Psychoneuroendocrinology, 23: 963-987. B/ickstr6m, T., Zetterlund, B., Blom, S. and Romano, M. (1984) Effects of intravenous progesterone infusions on the epileptic discharge frequency in women with partial epilepsy. Acta Neurol. Scand., 69: 240-248. Beekman, M., Ungard, J.T., Gasior, M., Carter, R.B., Dijkstra, D., Goldberg, S.R. and Witkin, J.M. (1998) Reversal of behavioral effects of pentylenetetrazol by the neuroactive steroid ganaxolone. J. Pharmacol. Exp. Ther., 284: 868-877. Belelli, D., Lan, N.C. and Gee, K.W. (1990) Anticonvulsant steroids and the GABA/benzodiazepine receptor-chloride ionophore complex. Neurosci. Biobehav. Rev., 14: 315-322. Bicikova, M., Dibbelt, L., Hill, M., Hampl, R. and Starka, L. (1998) Allopregnanolone in women with premenstrual syndrome. Hormon. Metab. Res., 30: 227-230. Bicikova, M., Tallov/t, J., Hill, M., Krausova, Z. and Hampl, R. (2000) Serum concentrations of some neuroactive steroids in women suffering from mixed anxiety-depressive disorder. Neurochem. Res., 25: 1623-1627. Bitran, D., Hilvers, R.J. and Kellogg, C. K. (1991) Anxiolytic effects of 3~-hydroxy-5~ [13]-pregnan-20-one: endogenous metabolites of progesterone that are active at the GABAA receptor. Brain Res., 561:157-161. Bitran, D., Shiekh, M. and McLeod, M. (1995) Anxiolytic effect of progesterone is mediated by the neurosteroid allopregnanolone at brain GABAA receptors. Journal of Neuroendocrinology, 7: 171-177. Browne, E.S., Porter, J.R., Correa, G., Abadie, J. and Svec, F. (1993) Dehydroepiandrosterone regulation of the hepatic glucocorticoid receptor in the Zucker rat. The obesity research program. J. Steroid Biochem. Molec. Biol., 45: 517-524. Bullock, A.E., Clark, A.L., Grady, S.R., Robinson, S.F., Slobe, B.S., Marks, M.J. and Collins, A.C. (1997) Neurosteroids modulate nicotinic receptor function in mouse
557 striatal and thalamic synaptosomes. J. Neurochem., 68: 2412-2423. Carl, P., H6gskilde, S., Nielsen, J.W., S6rensen, M.B., Lindholm, M., Karlen, B. and Bfickstr6m, T. (1990) Pregnanolone emulsion: a preliminary pharmacokinetic and pharmacodynamic study of a new intravenous agent. Anaesthesia, 45: 189-197. Carter, R.B., Wood, P.L., Wieland, S., Hawkinson, J.E., Belelli, D., Lambert, J.J., White, H.S., Wolf, H.H., Mirsadeghi, S., Tahir, S.H., Bolger, M.B., Lan, N.C. and Gee, K.W. (1997) Characterization of the anticonvulsant properties of ganaxolone (CCD 1042; 3~-hydroxy-3-methyl5~-pregnan-20-one), a selective high-affinity steroid modulator of the GABAA receptor. J. Pharmacol. Exp. Ther., 280:1284-1295. Celotti, F., Melcangi, R.C. and Martini, L. (1992) The 5~-reductase in the brain: molecular aspects and relation to brain function. Front. Neuroendocrinol., 13: 163-215. Compagnone, N.A. and Mellon, S.H. (2000) Neurosteroids: Biosynthesis and function of these novel neuromodulators. Front. Neuroendocrinol. 21, 1-56. Concas, A., Mostallino, M.C., Porcu, P., Follesa, P., Barbaccia, M.L., Trabucchi, M., Purdy, R.H., Grisenti, P. and Biggio, G. (1998) Role of brain allopregnanolone in the plasticity of 3,-aminobutyric acid type A receptor in rat brain during pregnancy and after delivery. Proc. Natl. Acad. Sci. USA, 95: 13284-13289. Crawley, J.N., Glowa, J.R., Majewska, M.D. and Paul, S.M. (1986) Anxiolytic activity of an endogenous adrenal steroid. Brain Res., 398: 382-385. Damianisch, K., Rupprecht, R. and Lancel, M. (2001) The influence of subchronic administration of the neurosteroid allopregnanolone on sleep in the rat. Neuropsychopharmacology, 25: 576-584. Devaud, L.L., Purdy, R.H. and Morrow, A.E. (1995) The neurosteroid 3~-hydroxy-5~-pregnan-20-one, protects against bicuculline-induced seizures during ethanol withdrawal in rats. Alcoholism: Clinical and Experimental Research, 19:350-355. Edgar, D.M., Seidel, W.F., Gee, K.W., Lan, N.C., Field, G., Xia, H., Hawkinson, J.E., Wieland, S., Carter, R.B. and Wood, P.L. (1997) CCD-3693: An orally bioavailable analog of the endogenous neuroactive steroid, pregnanolone, demonstrates potent sedative hypnotic actions in the rat. J. Pharmacol. Exp. Ther., 282: 420-429. Evans, R.M. (1988) The steroid and thyroid hormone receptor superfamily. Science, 240: 889-895. Fabian, T.J., Dew, M.A., Pollock, B.G., Reynolds, C.F., Mulsant, B.H., Butters, M.A., Zmuda, M.D., Linares, A.M., Trottini, M. and Kroboth, P.D. (2001) Endogenous concentrations of DHEA and DHEA-S decrease with remission of depression in older adults. Biol. Psychiatry, 50: 767-774.
Flood, J.F., Morley, J.E. and Roberts, E. (1992) Memoryenhancing effects in male mice of pregnenolone and of steroids metabolically derived from it. Proc. Natl. Acad. Sci. USA, 89: 1567-1571. Flood, J.F., Smith, G.E. and Roberts, E. (1988) Dehydroepiandrosterone and its sulfate enhance memory retention in mice. Brain Res., 447: 269-278. Friess, E., Tagaya, H., Trachsel, L., Holsboer, F. and Rupprecht, R. (1997) Progesterone-induced changes in sleep in male subjects. Am. Journal Physiol., 272: E885-E891. Frye, C.A. and Duncan, J.E. (1994) Progesterone metatolites effective at the GABAA receptor complex modulate pain sensitivity in rats. Brain Res., 643: 194-203. Frye, C.A. and Scalise, T.J. (2000) Anti-seizure effects of progesterone and 3~,5~-THP in kainic acid and perforant pathway models of epilepsy. Psychoneuroendocrinology, 25: 407-420. Gasior, M., Carter, R.B., Goldber, S.R. and Witkin, J.M. (1997) Anticonvulsant and behavioral effects of neuroactive steroids alone and in conjuction with diazepam. J. Pharmacol. Exp. Ther., 282: 543-553. Gasior, M., Carter, R.B. and Witkin, J.M. (1999) Neuroactive steroids: potential therapeutic use in neurological and psychiatric disorders. Trends Pharmacol. Sci., 20: 107-112. Gee, K.W., Bolger, M.B., Brinton, R.E., Coirini, H. and McEwen, B.S. (1988) Steroid modulation of the chloride ionophore in rat brain: Structure-activity requirements, regional dependance and mechanism of action. J. Pharmacol. Exp. Ther., 246: 803-812. Girdler, S.S., Straneva, P.A., Light, K.C., Pedersen, C.A. and Morrow, A.L. (2001) Allopregnanolone levels and reactivity to mental stress in premenstrual dysphoric disorder. Biol. Psychiatry., 49: 788-797. Grazzini, E., Guillon, G., Mouillac, B. and Zingg, H.H. (1998) Inhibition of oxytocin receptor function by direct binding of progesterone. Nature, 392: 509-512. Griffin, L.D. and Mellon, S.H. (1999) Selective serotonin reuptake inhibitors directly alter activity of neurosteroidogenic enzymes. Proc. Natl. Acad. Sci. USA, 96: 13512-13517. Grobin, A.C. and Morrow, A.L. (2000) 3~-hydroxy-5~pregnan-20-one exposure reduces GABA(A) receptor ~4 subunit mRNA levels. Eur. J. Pharmacol., 409: R1-R2. Gyermek, L. and Soyka, L.F. (1975) Steroid anesthetics. Anesthesiology, 42:116-121. Hallonquist, J.D., Seeman, M.V., Lang, M. and Rector, N.A. (1993) Variation in symptom severity over the menstrual cycle of schizophrenics. Biol. Psychiatry, 33: 207-209. Hfifner, H., Riecher-R6ssler, A., an der Heiden, W., Maurer, K., Ffitkenheuer, B. and L6ffler, W. (1993) Generating and testing a causal explanation of the gender difference in age at first onset of schizophrenia. Psychological Med., 23: 925-940.
558 Herzog, A.G. (1995) Progesterone therapy in women with complex partial and secondary generalized seizures. Neurology, 45: 1660-1662. Heuser, I., Deuschle, M., Luppa, P., Schweiger, U., Standhardt, H. and Weber, B. (1998) Increased diurnal plasma concentrations of dehydroepiandrosterone in depressed patients. J. Clin. Endocrinol. Metab., 83: 3130-3133. Hillen, T., Lun, A., Reischies, F.M., Borchelt, M., Steinhagen-Thiessen, E. and Schaub, R.T. (2000) DHEA-S plasma levels and incidence of Alzheimer's Disease. Biol. Psychiatry, 47: 161-163. Janis, G.C., Devaud, L.L., Mitsuyama, H. and Morrow, A.L. (1998) Effects of chronic ethanol consumption and withdrawal on the neuroactive steroid 3~-hydroxy-5~-pregnan20-one in male and female rats. Alcohol. Clin. Exp. Res., 22:2055-2061. Khisti, R.T., Chopde, C.T. and Jain, S.P. (2000) Antidepressantlike effect of the neurosteroid 3~-hydroxy-5~-pregnan-20-one in mice forced swim tegt. Pharmacol. Biochem. Behav., 67: 137-143. Korneyev, A. and Costa, E. (1996) Allopregnanolone (THP) mediates anesthetic effects of progesterone in rats. Horm. Behav., 30: 37-43. Korpi, E.R., Makela, R., Romeo, E., Guidotti, A., Uusi-Oukari, M., Furnari, C., di Michele, F., Sarviharju, M., Xu, M. and Rosenberg, P.H. (2001) Increased behavioral neurosteroid sensitivity in a rat line selectively bred for high alcohol sensitivity. Eur. J. Pharmacol., 421: 31-38. Ladurelle, N., Eychenne, B., Denton, D., Blair-West, J., Schumacher, M., Robel, P. and Baulieu, E. (2000) Prolonged intracerebroventricular infusion of neurosteroids affects cognitive performances in the mouse. Brain Res., 858: 371-379. Lambert, J.J., Belelli, D., Hill-Venning, C. and Peters, J.A. (1995) Neurosteroids and GABAA receptor function. Trends Pharmacol. Sci., 16: 295-303. Lancel, M., Faulhaber, J., Holsboer, F. and Rupprecht, R. (1996) Progesterone induces changes in sleep EEG comparable to those of agonistic GABAA receptor modulators. Am. J. Physiol., 271: E763-E772. Lancel, M., Faulhaber, J., Schiffelholz, T., Romeo, E., di Michele, F., Holsboer, F. and Rupprecht, R. (1997) Allopregnanolone affects sleep in a benzodiazepine-like fashion. J. Pharmacol. Exp. Ther., 282: 1213-1218. Maayan, R., Yagorowski, Y., Grupper, D., Weiss, M., Shtaif, B., Kaoud, M.A. and Weizman, A. (2000) Basal plasma dehydroepiandrosterone sulfate level: a possible predictor for response to electroconvulsive therapy in depressed psychotic inpatients. Biol. Psychiatry, 48: 693-701. Maitra, R. and Reynolds, J.N. (1998) Modulation of GABAA receptor function by neuroactive steroids: evidence for heterogeneity of steroid sensitivity of recombinant GABAA receptor isoforms. Can. J. Physiol. Pharmacol., 76: 909-920.
Majewska, M.D., Harrison, N.L., Schwartz, R.D., Barker, J.L. and Paul, S.M. (1986) Steroid hormone metabolites are barbiturate-like modulators of the GABA receptor. Science, 232: 1004-1007. Maricq, A.V., Peterson, A.S., Brake, A.J., Myers, R.M. and Julius, D. (1991) Primary structure and functional expression of the 5HT3 receptor, a serotonin-gated ion channel. Science, 254: 432-437. Marx, C.E., Duncan, G.E., Gilmore, J.H., Lieberman, J.A. and Morrow, A.L. (2000) Olanzapine increases allopregnanolone in the rat cerebral cortex. Biol. Psychiatry, 47: 1000-1004. McEwen, B.S. (1991) Non-genomic and genomic effects of steroids on neural activity. Trends Pharmacol. Sci., 12: 141-147. Mellon, S.H. (1994) Neurosteroids: Biochemistry, modes of action and clinical relevance. J. Clin. Endocrinol. Metab., 78: 1003-1008. Mendelson, W.B., Martin, J.V., Perlis, M., Wagner, R., Majewska, M.D and Paul, S.M. (1987) Sleep induction by an adrenal steroid in the rat. Psychopharmacology, 93: 226-229. Monaghan, E.P., McAuley, J.W. and Data, J.L. (1999) Ganaxolone: a novel positive allosteric modulator of the GABA(A) receptor complex for the treatment of epilepsy. Expert Opin. Investig. Drugs, 8: 1663-1671. Monaghan, E.P., Navalta, L.A., Shum, L., Ashbrock, D.W. and Lee, D.A. (1997) Initial human experience with ganaxolone, a neuroactive steroid with antiepileptic activity. Epilepsia, 38: 1026-1031. Monnet, F.P., Mah~, V., Robel, P. and Baulieu, E.E. (1995) Neurosteroids, via sigma receptors, modulate the [3H]norepinephrine release evoked by N-methyl-D-aspartate in the rat hippocampus. Proc. Natl. Acad. Sci. USA, 92: 3774-3778. Nadeson, R. and Goodchild, C.S. (2001) Antinociceptive properties of neurosteroids III: experiments with alphadolone given intravenously, intraperitoneally, and intragastrically. Br. J. Anaesth., 86: 704-708. Nfisman, B., Olsson, B., Bfickstr6m, T., Eriksson, S., Grankvist, K., Viitanen, M. and Bucht, G. (1991) Serum dehydroepiandrosterone sulfate in Alzheimer's disease and in multiinfarct dementia. Biol. Psychiatry, 30: 684-690. Orchinik, M., Murray, T.F. and Moore, F.L. (1991) A corticosteroid receptor in neuronal membranes. Science, 252: 1848-1851. Padberg, F., di Michele, F., Zwanzger, P., Romeo, E., Bernardi, G., Schfile, C., Baghai, T.C., Ella, R., Pasini, A. and Rupprecht, R. (2002) Plasma concentrations of neuroactive steroids before and after repetitive transcranial magnetic stimulation (rTMS) in major depression. Neuropsychopharmacology, 27: 874-878. Pappas, T.D., Gametchu, B. and Watson, C.S. (1995) Membrane estrogen receptors identified by multiple antibody labelling and impeded-ligand binding. FASEB J., 9:404-410.
559 Park-Chung, M., Wu, F.S. and Farb, D.H. (1994) 3cz-hydroxy5beta-pregnan-20-one sulfate: a negative modulator of the NMDA-induced current in cultured neurons. Mol. Pharmacol., 46: 146-150. Patchev, V.K., Montkowski, A., Rouskova, D., Koranyi, L., Holsboer, F. and Almeida, O.F.X. (1997) Neonatal treatment of rats with the neuroactive steroid tetrahydrodeoxycorticosterone (THDOC) abolishes the behavioral and neurondocrine consequences of adverse early life events. J. Clin. Invest., 99: 962-966. Patchev, V.K., Shoaib, M., Holsboer, F. and Almeida, O.F.X. (1994) The neurosteroid tetrahydroprogesterone counteracts corticotropin-releasing hormone-induced anxiety and alters the release and gene expression of corticotropin-releasing hormone in the rat hypothalamus. Neurosci., 62: 265-271. Paul, S.M. and Purdy, R.H. (1992) Neuroactive steroids. FASEB J., 6:2311-2322. Pearson Murphy, B.E., Steinberg, S.I., Hu, F.-Y. and Allison, C.M. (2001) Neuroactive ring A-reduced metabolites of progesterone in human plasma during pregnancy: elevated levels of 5alpha-dihydroprogesterone in depressed patients during the latter half of pregnancy. J. Clin. Endocrinol. Metab., 86: 5981-5987. Prince, R.J. and Simmonds, M.A. (1992) 5[3-pregnan-3[3-ol-20one, a specific antagonist at the neurosteroid site of the GABAA receptor-complex. Neurosci. Lett.,135: 273-275. Ramirez, V.D. and Zheng, J. (1996) Membrane sex-steroid receptors in the brain. Front. Neuroendocrinol., 17: 402-439. Rapkin, A.J., Morgan, M., Goldman, L., Brann, D.W., Simone, D. and Mahesh, V.B. (1997) Progesterone metabolite allopregnanolone in women with premenstrual syndrome. Obstet. Gynecol., 90: 709-714. Rasgon, N., Serra, M., Biggio, G., Pisu, M. G., Fairbanks, L., Tanavoli, S. and Rapkin, A. (2001) Neuroactive steroidserotonergic interaction: responses to an intravenous L-tryptophan challenge in women with premenstrual syndrome. Eur. J. Endocrinol., 145: 25-33. Reddy, D.S. and Rogawski, M.A. (2000) Chronic treatment with the neuroactive steroid ganaxolone in the rat induces anticonvulsant tolerance to diazepam but not to itself. J. Pharmacol. Exp. Ther., 295: 1241-1248. Romeo, E., Brancati, A., De Lorenzo, A., Fucci, P., Furnari, C., Pompili, E., Sasso, G. F., Spalletta, G., Troisi, A. and Pasini, A. (1996) Marked decrease of plasma neuroactive steroids during alcohol withdrawal. Clin. Neuropharmacol., 19: 366-369. Romeo, E., Pompili, E., di Michele, F., Pace, M., Rupprecht, R., Bernardi, G. and Pasini, A. (2000) Effects of fluoxetine, indomethacine and placebo on 3~, 5cz tetrahydroprogesterone (THP) plasma levels in uncomplicated alcohol withdrawal. World J. Biol. Psychiatry, 1: 101-104. Romeo, E., Str6hle, A., Spalletta, G., di Michele, F., Hermann, B., Holsboer, F., Pasini, A. and Rupprecht, R. (1998) Effects of antidepressant treatment on neuroactive
steroids in major depression. Am. J. Psychiatry, 155: 910-913. Rupprecht, R., Berning, B., Hauser, C.A.E., Holsboer, F. and Reul, J.M.H.M. (1996) Steroid receptor mediated effects of neuroactive steroids: characterization of structure-activity relationship. Eur. J. Pharmacol., 303: 227-234. Rupprecht, R. and Holsboer, F. (1999) Neuroactive steroids: mechanisms of action and neuropsychopharmacological perspectives. Trends Neurosci., 22:410-4 16. Rupprecht, R., Koch, M., Montkowski, A., Lancel, M., Faulhaber, J., Harting, J. and Spanagel, R. (1999) Assessment of neuroleptic-like properties of progesterone. Psychopharmacology, 143: 29-38. Rupprecht, R., Reul, J.M.H.M., Trapp, T., van Steensel, B., Wetzel, C., Damm, K., Zieglgfinsberger, W. and Holsboer, F. (1993) Progesterone receptor-mediated effects of neuroactive steroids. Neuron, 11: 523-530. Schulz, H., Jobert, M., Gee, K.W. and Ashbrook, D.W. (1996) Soporific effect of the neurosteroid pregnanolone in relation to the substance's plasma level: a pilot study. Neuropsychobiology, 34:106-112. Serra, M., Pisu, M.G., Muggironi, M., Parodo, V., Papi, G., Sari, R., Dazzi, L., Spiga, F., Purdy, R.H. and Biggio, G. (2001) Opposite effects of short- versus long-term administration of fluoxetine on the concentrations of neuroactive steroids in rat plasma and brain. Psychopharmacology, 158: 48-54. Smith, S.S., Gong, Q.H., Hsu, F.-C., Markowitz, R.S., ffrenchMullen, J.M.H. and Li, X. (1998a) GABAA receptor cz4 subunit suppression prevents withdrawal properties of an endogenous steroid. Nature, 392: 926-930. Smith, S.S., Gong, Q.H., Li, X., Moran, M.H., Bitran, D., Frye, C.A. and Hsu, F.C. (1998b) Withdrawal from 3~-OH5~-pregnan-20-one using a pseudopregnancy model alters the kinetics of hippocampal GABAA-gated current and increases the GABAA receptor alpha4 subunit in association with increased anxiety. J. Neurosci., 18: 5275-5284. Steiner, M., Steinberg, S., Stewart, D., Carter, D., Berger, C., Reid, R., Grover, D. and Streiner, D. (1995) Fluoxetine in the treatment of premenstrual dysphoria. N. Engl. J. Med., 332, 1529-1534. Str6hle, A., Romeo, E., di Michele, F., Pasini, A., Yassouridis, A., Holsboer, F. and Rupprecht, R. (2002) GABAA receptor modulatory neuroactive steroid composition in panic disoder and during paroxetine treatment. Am. J. Psychiatry, 159: 145-147. Str6hle, A., Romeo, E., di Michele, F., Pasini, A., Hermann, B., Gajewski, G., Holsboer, F. and Rupprecht, R. (2003) Induced panic attacks shift GABAA receptor modulatory steroid composition in patients with panic disorder: preliminary results. Archives of General Psychiatry, 60:161-168. Su, T.-P., London, E.D. and Jaffe, J.H. (1988) Steroid binding at cy receptors suggests a link between endocrine, nervous, and immune systems. Science, 240: 219-221.
560 Sunderland, T., Merril, C.R., Harrington, M.G., Lawlor, B. A., Molchan, S.E., Martinez, R. and Murphy, D.L. (1989) Reduced plasma dehydroepiandrosterone concentrations in Alzheimer's disesase. Lancet, II: 570-572. Sundstr6m, I. and Bfickstr6m, T. (1998) Citalopram increases pregnanolone sensitivity in patients with premenstrual syndrome: an open trial. Psychoneuroendocrinology, 23: 73-88. Thomas, G., Frenoy, N., Legrain, S., Sebag-Lanoe, R., Baulieu, E.E. and Debuire, B. (1994) Serum dehydroepiandrosterone sulfate levels as an individual marker. J. Clin. Endocrinol. Metab., 79: 1273-1276. Truss, M. and Beato, M. (1993) Steroid hormone receptors: interaction with deoxyribonucleic acid and transcription factors. Endocrine Rev., 14: 459-479. Uzunov, D.P., Cooper, T.B., Costa, E. and Guidotti, A. (1996) Fluoxetine-elicited changes in brain neurosteroid content measured by negative ion mass fragmentography. Proc. Natl. Acad. Sci., USA 93: 12599-12604. Uzunova, V., Sheline, Y., Davis, J.M., Rasmusson, A., Uzunov, D.P., Costa, E. and Guidotti, A. (1998) Increase in the cerebrospinal fluid content of neurosteroids in patients with unipolar major depression who are receiving fluoxetine or fluvoxamine. Proc. Natl. Acad. Sci.USA, 95: 3239-3244. Valera, S., Ballivet, M. and Bertrand, D. (1992) Progesterone modulates a neuronal nicotinic acetylcholine receptor. Proc. Natl. Acad. Sci.USA, 89: 9949-9953. Vallee, M., Mayo, W., Darnaudery, M., Corpechot, C., Young, J., Koehl, M., Le Moal, M., Baulieu, E.E., Robel, P. and Simon, H. (1997) Neurosteroids: deficient cognitive performance in aged rats depends on low pregnenolone sulfate levels in the hippocampus. Proc. Natl. Acad. Sci. USA, 94: 14865-14870. VanDoren, M.J., Matthews, D.B., Janis, G.C., Grobin, A.C., Devaud, L.L. and Morrow, A.L. (2000) Neuroactive steroid 3alpha-hydroxy-5alpha-pregnan-20-one modulates electrophysiological and behavioral actions of ethanol. J. Neuroscience, 20: 1982-1989. Vanover, K.E., Rosenzweig-Lipson, S., Hawkinson, J.E., Lan, N.C., Belluzzi, J.D., Stein, L., Barrett, J.E., Wood, P.L. and Carter, R.B. (2000) Characterization of the anxiolytic properties of a novel neuroactive steroid, Co 2-
6749 (GMA-839; WAY-141839; 3~, 21-Dihydroxy-313-trifluoromethyl-19-nor-513-pregnan-20-one), a selective modulator of 3,-aminobutyric acidA receptors. J. Pharmacol. Exp. Ther., 295: 337-345. Weaver, C.E. Jr., Marek, P., Park-Chung, M., Tam, S.W. and Farb, D.H. (1997b). Neuroprotective activity of a new class of steroidal inhibitors of the N-methyl-D-aspartate receptor. Proc. Natl. Acad. Sci. USA, 94: 10450-10454. Weaver, C.E. Jr., Park-Chung, M., Gibbs, T.T. and Farb, D.H. (1997a) 17]3-estradiol protects against NMDA-induced excitotoxicity by direct inhibition of NMDA receptors. Brain Res., 761: 338-341. Wehling, M. (1997) Specific, nongenomic actions of steroid hormones. Annu. Rev. Physiol., 59: 365-393. Wetzel, C.H.R., Hermann, B., Behl, C., Pestel, E., Rammes, G., Zieglgfinsberger, W., Holsboer, F. and Rupprecht, R. (1998) Functional antagonism of gonadal steroids at the 5-HT3 receptor. Mol. Endocrinol., 12: 1441-1451. Wieland, S., Lan, N.C., Mirasedeghi, S., Gee, K.W. (1991) Anxiolytic activity of the progesterone metabolite 50~-pregnan-3~-ol-20-one. Brain Res., 565: 263-268. Wolkowitz, O.M., Reus, V.I., Keebler, A., Nelson, N., Friedland, M., Brizendine, L. and Roberts, E. (1999) Double-blind treatment of major depression with dehydroepiandrosterone. Am. J. Psychiatry, 156: 646-649. Wolkowitz, O.M., Reus, V.I., Roberts, E., Manfredi, F., Chan, T., Raum, W.J., Ormiston, S., Johnson, R., Canick, J., Brizendine, L. and Weingartner, H. (1997) Dehydroepiandrosterone (DHEA) treatment of depression. Biol. Psychiatry, 41: 311-318. Wu, F.-S., Gibbs, T.T. and Farb, D.H. (1990) Inverse modulation of 3,-aminobutyric acid- and glycine-induced currents by progesterone. Mol. Pharmacol., 37: 597-602. Wu, F.-S., Gibbs, T.T. and Farb, D.H. (1991) Pregnenolone sulfate: A positive allosteric modulator at the N-Methyl-Daspartate receptor. Mol. Pharmacol., 40: 333-336. Zakon, H.H. (1998) The effects of steroid hormones on electrical activity of excitable cells. Trends Neurosci., 21: 202-207. Zorumski, C.F., Mennerick, S., Isenberg, K.E. and Covey, D.F. (2000) Potential clinical uses of neuroactive steroids. Curr. Opin. Investig. Drugs, 1: 360-369.
T. Steckler, N.H. Kalin and J.M.H.M. Reul (Eds.) Handbook of Stress and the Brain, Vol. 15
ISBN 0-444-51173-3 Copyright 2005 Elsevier B.V. All rights reserved CHAPTER 4.9
Endogenous opioids, stress, and psychopathology Andrea L.O. Hebb, Sylvie Laforest and Guy Drolet* Centre de Recherche en Neurosciences, CHUL, UniversitO Laval, QC, Canada
Abstract: The maintenance of homeostasis during stressful conditions is mediated through a complex, highly interactive organization of neuroanatomical pathways in the central nervous system (CNS). Specific neurotransmitter systems initiate immune, endocrine, metabolic, cardiovascular, respiratory, and behavioral changes in response to stress. Among these neurotransmitter systems, endogenous opioids (enkephalin, [3-endorphin, and dynorphin) represent major modulatory systems in responding and adapting to stress. The endogenous opioids are important neuromodulators, acting at multiple levels of the CNS, participating in the mediation, modulation, and regulation of the stress response, including neuroendocrine (hypothalamo-pituitary-adrenal, HPA axis), autonomic (sympathoadrenal axis), and behavioral (fear, anxiety, memory, locomotor activity, mood, perception of reward) responses. Endogenous opioids also play a fundamental role in stressor-induced analgesia and the modulation of organismic defensive repertoires. Although being part of the same peptide family, the endogenous opioids do not act as a single functional unit in stress regulation. Indeed, there is growing evidence demonstrating the existence of functionally opposed opioid systems affecting emotional and perceptual experiences. Therefore, conflicting reports on the functional roles of endogenous opioids in stress regulation are likely to reflect the fact that different opioid systems may have opposite actions depending on the site of action, the subtype ofopioid receptor involved, or the stressor conditions. The present review is certainly not an exhaustive evaluation of the past years on endogenous opioids and stress. Although in this review we have focused our attention mainly on the traditional opioid peptides (enkephalin, dynorphin, 13-endorphin) and receptors (mu, ~t-OP; delta, 8-OP; kappa, K-OR) and their roles in stress adaptation, we will also introduce new endogenous opioid members (endomorphin and nociceptin/orphanin FQ). We will also review how psychological stressors induced alterations in endogenous opioid peptides and how this could have important implications for anxiety, depression, and panic disorder.
Opioid and stress: endogenous opioids
review, see Bolles and Fanselow, 1982; Holaday, 1983; Akil et al., 1984; Howlett and Rees, 1986; Przewlocki et al., 1991; Hayden-Hixson and Nemeroff, 1993; Yamada and Nabeshima, 1995; Akil et al., 1998; Russell and Douglas, 2000). In fact, these anatomical and functional opioid characteristics argue against the conceptualization of the endogenous opioid peptides as a single, functional unit. Three distinct (classical) families of endogenous opioid peptides have been identified to date; the enkephalins, dynorphins, and [3-endorphin. Each family is derived from different multifunctional precursor polypeptides: proenkephalin (proenkephalin A), prodynorphin (proenkephalin B), and proopioimelanocortin (POMC), respectively. The
The endogenous opioids and their receptor systems are ubiquitous, located with varying densities throughout the central, peripheral, and autonomic nervous systems as well as in several endocrine tissues and target organs. This widespread distribution is consistent with the involvement of endogenous opioids in a broad range of functions and behavior, including regulation of pain, reinforcement, and reward, release of neurotransmitters, as well as autonomic and neuroendocrine modulation (for *Corresponding author. Tel.: + 1(418) 654 2152; Fax: + 1(418) 654 2753; E-mail:
[email protected] 561
562 endogenous opioid peptides produce their biological effects through three main types of receptors (all being members of the family of seven transmembrane G-protein-coupled receptors) referred to as mu (~t-OP), delta (~5-OP), and kappa (~-OP). The different types of opioid receptors have been defined based on pharmacological- and radioligand-binding experiments (Mansour et al., 1987), and more recently by their recent cloning (Mansour et al., 1994; Uhl et al., 1994). Although in vitro studies indicated that each opioid peptide has affinity for more than one type of opioid receptor (enkephalins and ~5-OP, dynorphins and ~:-OP, endorphin and la-OP), proenkephalin and prodynorphin cleavage products are able to bind g-OP, ~5-OP and ~c-OP receptors (Quirion and Pert, 1981; Wuster et al., 1981). More recently, additional endogenous opioid peptides have been characterized; endomorphin-1 and endomorphin-2 appear to have properties consistent with neurotransmitter/neuromodulator actions in mammals. Endomorphin-1 (Tyr-Pro-Trp-PheNH2, EM-1) and endomorphin-2 (Tyr-Pro-Phe-PheNH2, EM-2) are peptides recently isolated from brain that show the highest affinity and selectivity for the g-OR of all the known endogenous opioids (Zadina et al., 1997). The distribution of endomorphins is consistent with a role for the peptides in the modulation of diverse functions, including perception of pain, autonomic, neuroendocrine, and some homeostatic functions as well as modulation of responses to stress (Martin-Schild et al., 1999; Horvath, 2000). However, the physiological significance of endomorphins in the stress response still has to be determined. A novel group of a related but nonopioid system, the nociceptin/orphanin FQ (N/OFQ) and its receptor, has been characterized in the brain (Meunier et al., 1995; Reinscheid et al., 1995). The endogenous ligand of the N/OFQ system is a heptadecapeptide that binds with high affinity to the nociceptin-orphanin peptide (NOP) receptor (formerly know as ORL 1). The N/OFQ has a high degree of sequence identity to the other endogenous opioids (especially with dynorphin A), but does not bind to traditional opioid receptors. In the same vein, the NOP receptor, which is also G-protein-coupled receptor, shows low binding affinities for selective opioid agonists and antagonists. The functions and
distribution of N/OFQ and NOP receptors have been described and reviewed in detail elsewhere (Mogil and Pasternak, 2001; Reinscheid and Civelli, 2002). There is a very important bibliographic source of information on endogenous opioids published each year since 1979. This interesting series of 24 papers constitute an annual source on research concerning the opioid system, including stress and behavior (Olson et al., 1979, 1980, 1981, 1982; Olson et al., 1983, 1984, 1985, 1986, 1987, 1989a,b, 1990, 1991, 1992, 1993, 1994, 1995, 1996, 1997, 1998; Vaccarino et al., 1999; Vaccarino and Kastin, 2000, 2001; Bodnar and Hadjimarkou, 2002).
Opioid and stress: anatomical distribution All traditional endogenous opioid systems are widely represented in regions which are heavily involved in the stress response, including the hypothalamus, pituitary, and adrenal gland. Similarly opioid neurons are found in central autonomic centers which receive important opioidergic innervation. More specifically, central endorphinergic neurons originate from two nuclei, the arcuate nucleus in the posterior hypothalamus and the nucleus tractus solitarius in the brainstem. Both endorphin groups have extensive projections to other areas providing a rich network of POMC fibers throughout the brain (Palkovits et al., 1987; Schafer et al., 1991). POMC fibers originating from the arcuate nucleus innervate hypothalamic nuclei, including the paraventricular nucleus of the hypothalamus (PVH), the median eminence, and limbic structures including the septum, bed nucleus of the stria terminalis, amygdala, and the preoptic area (Palkovits et al., 1987; Schafer et al., 1991). In the brainstem, endorphinergic fibers are observed in the parabrachial nucleus, the ventrolateral medulla, the nucleus tractus solitarius, the dorsal motor nucleus of the vagus, and the nucleus ambiguus, regions involved in autonomic regulation. The neuronal distribution of the enkephalinderived and dynorphin-derived peptides is far more complex than the neuronal distribution of the POMC family. Therefore, the description of the structures containing enkephalin or dynorphin perikarya and
563 fibers has been restricted to the nuclei that may be involved in the neurobiology of stress. The complete distribution of enkephalin and dynorphin neurons (as seen by immunohistochemistry and in situ hybridization histochemistry) and fibers throughout the brain have been extensively described by previous studies (Watson et al., 1982; Khachaturian et al., 1983; Guthrie and Basbaum, 1984; Petrusz et al., 1985; Fallon and Leslie, 1986; Harlan et al., 1987; Menetrey and Basbaum, 1987; Hurd, 1996). In brief, neurons containing enkephalin- or dynorphinderived peptides can be found virtually at all levels of the brain, from the telencephalon to the spinal cord. Enkephalinergic neurons are present in most regions of the telencephalon, including the cerebral and piriform cortex, amygdala, septum, bed nucleus of the stria terminalis, and the preoptic area. In the hypothalamus, perikarya are seen in most nuclei (including the PVH). At the level of the brainstem, enkephalin neurons are identified in the parabrachial nucleus, ventrolateral medulla (nucleus paragigantocellularis, lateral reticular nucleus), and nucleus tractus solitarius. The distribution of enkephalin fibers and terminals is roughly similar to that of neuronal perikarya. Dense enkephalin innervation is found in the parabrachial nucleus, the nucleus tractus solitarius, and the lateral reticular nucleus which are nuclei involved in autonomic regulation. A moderate innervation is also found in PVH, median eminence, amygdala, LC, and ventrolateral medulla. Dynorphin-derived peptide neurons are present in the cerebral cortex, amygdala, the dentate gyrus of the hippocampal formation, PVH, supraoptic nucleus, parabrachial nucleus, nucleus tractus solitarius, and ventrolateral medulla. Areas containing a relatively high density of dynorphin fibers are the hippocampus, septum, median eminence, LC, dorsal motor nucleus of the vagus, and the lateral reticular nucleus. Each opioid receptor type demonstrates a distinct anatomical distribution which was previously determined by binding studies (Mansour et al., 1987; Blackburn et al., 1988) and, more recently, by in situ hybridization histochemistry (Mansour et al., 1994, 1995) to visualize the expression of their mRNAs. In relation with the structures that are involved in the neurobiology of stress, cells expressing ~t-OR are distributed in such regions as the septum, bed nucleus
of the stria terminalis, hippocampus (dentate gyrus), amygdala, medial preoptic area, locus coeruleus, parabrachial nucleus, nucleus tractus solitarius, and dorsal motor nucleus of the vagus. Neurons expressing 8-OR are located in hippocampus, amygdala, and lateral reticular nucleus. Finally, the neurons expressing K-OR are localized in regions such as piriform cortex, medial preoptic area, bed nucleus of the stria terminalis, amygdala, PVH, supraoptic nucleus, median eminence, and the nucleus tractus solitarius.
Opioid and stress: adaptation to stress The relevance of the role(s) played by endogenous opioids in adaptation to stress may be considered critical in the etiology and pathology of certain physiological disorders associated with repeated or prolonged stress, such as cardiovascular diseases, affective, and behavior disorders. Although, several attempts have been made to assess stress-induced alterations of opioid receptors, the results of the studies are varied, even contradictory (Akil et al., 1984; Grossman, 1986; Howlett and Rees, 1986; Szekely, 1990; Przewlocki et al., 1991). Many possibilities have been advanced to explain discrepancies in the involvement of endogenous opioids in stress processes. The obvious technical considerations, such as species, agonist, and antagonist specificity, dose selectivity, route of administration, time course of the response, state and nature of the anesthetic, and stress levels, have all been offered to account for these differences. Moreover, the existence of contradictory results may also reflect the fact that opioid pathways in the brain do not act as a single entity. Furthermore, endogenous opioid peptides are involved in many physiological processes which are not directly related to the stress response. There is solid evidence indicating major involvement for enkephalin, 13-endorphin, and dynorphin in both stress-induced physiological and behavioral responses (Howlett and Rees, 1986; Katoh et al., 1990; Szekely, 1990; Przewlocki et al., 1991; Katoh et al., 1992; Pechnick, 1993). Endogenous opioids may exert their action on the HPA axis and the autonomic nervous system which are major effector systems that
564 serve to maintain homeostasis during exposure to stressors. Indeed, considerable neuroanatomical and pharmacological data exist suggesting an involvement of opioid-derived peptides in the regulation of the sympathetic nervous system and cardiovascular system (Holaday, 1983; Morilak et al., 1990a,b; Drolet et al., 1991a,b; McCubbin, 1993) as well as on the role of opioids in neuroendocrine regulation, particularly at the level of the paraventricular nucleus of the hypothalamus. The importance of the PVH in the central regulation and coordination of the stress response is well recognized. Indeed, the PVH is one important coordinating center of the stress system having virtually all the corticotropin-releasing factor (CRF) neurons that control the release of adrenocorticotropic hormone (ACTH) at the level of the median eminence (Sawchenko, 1986; Swanson et al., 1986, 1987; Palkovits, 1987; Sawchenko et al., 1993). Furthermore, the PVH is one of the few structures that project directly onto preganglionic sympathetic neurons (Strack et al., 1989a,b), as well as onto the preganglionic neurons of the parasympathetic nervous system (Lawrence and Pittman, 1985). Many studies have examined the expression of enkephalin mRNA (and dynorphin mRNA) within the PVH. It was demonstrated that stress caused adaptive increases in Enkephalin gene expression in these neurons, enkephalin mRNA levels are increased in the PVH after acute or chronic stress including intraperitoneal injection of hypertonic saline (Lightman and Young, 1987a,b, 1988, 1989; Harbuz and Lightman, 1989; Watts, 1992; Young and Lightman, 1992), ether stress (Watts, 1991; Ceccatelli and Orazzo, 1993), restraint (Ceccatelli and Orazzo, 1993), morphine withdrawal (Lightman and Young, 1987a, 1988; Harbuz et al., 1991), or colchicine injection (Ceccatelli and Orazzo, 1993). These findings all suggest a role for opioids in the PVH with respect to some aspects of adaptation of the organism to stress. We have recently investigated the effects of acute and chronic exposure to psychological stress on enkephalin-neuron activation (enkephalin mRNA and Fos immunoreactivity) in the PVH (Dumont et al., 2000) and in the ventrolateral medulla (Mansi et al., 2000), which provides the highest density of enkephalinergic parvocellular PVH afferents (Beaulieu et al., 1996). Acute immobilization caused a marked increase in
both the number of Fos-ir and Fos-enkephalin double-labeled cells in all the parvocellular subdivisions of the paraventricular nucleus of the hypothalamus as well as in the caudal and rostral ventrolateral medulla. Chronic immobilization had no effect on basal Fos labeling of both regions, but had opposite effects on the basal number of enkephalin cells (PVH: 43% increase and ventrolateral medulla: 50% decrease). Moreover, chronically stressed rats displayed an attenuated Fos response (PVH: 67% decrease and ventrolateral medulla: 100% decrease) to subsequent immobilization exposure. Conversely, there was no significant attenuation of the activation of PVH-enkephalin neurons. By contrast, the stress-induced activation of enkephalin neurons in the ventrolateral medulla was completely abolished following chronic immobilization. These results indicate that chronic psychological stress induced a differential, apparently regionspecific adaptation response of the enkephalin system. The influence of enkephalin neurons in the PVH appears to be increased following chronic stress as suggested by the increased number of basal enkephalin neurons and their sustained activation (Dumont et al., 2000). Conversely, the enkephalinergic influence originating from the ventrolateral medulla is virtually removed following exposure to chronic psychological stress (Mansi et al., 2000). These observations therefore imply that during acute stress exposure, activation of enkephalin neurons from PVH and ventrolateral medulla regulates some aspects of the stress response. During chronic stress conditions, this decrease of activation of the enkephalinergic input from ventrolateral medulla may be translated into a decrease inhibition in the PVH, while the resistance to habituation of the activation of enkephalin neurons within the PVH could contribute to buffer the potentially detrimental effects of a physiological response (i.e., HPA and autonomic axis) to stress.
A role for opioids in the amygdaloid complex in adaptation to stress There is strong evidence indicating that endogenous opioids (and particularly enkephalin) are involved in attenuating or terminating stress responses
565 (i.e., defensive reactions of the organism) (Tanaka et al., 1988, 1989, 2000; McCubbin, 1993; Janssens et al., 1995; Mansi et al., 2000; Curtis et al., 2001). For instance, naloxone (a nonselective opioid receptor antagonist) induced a greater increment in the HPA responses (ACTH and cortisol) in chronically stressed animals as compared to unstressed control animals, suggesting that the impact of opioid systems had increased due to chronic stress (Janssens et al., 1995). In the same vein, Tanaka's group reported that central administration of opioid agonists (including enkephalin itself) can attenuate not only stressinduced increases in norepinephrine release in cortex and limbic structures, including the amygdala, but also emotional responses shown during stress exposure (i.e., 60 rain immobilization stress) (Tanaka et al., 2000). A substantial literature exists implicating opioids in the amygdala in modulation of the stress response, and specifically in attenuating the impact of psychological stressors. Direct injection of opioid agonists within the amygdala diminishes anxiety-like behavior (File and Rodgers, 1979; Rodgers and File, 1979; Good and Westbrook, 1995). Injection of an enkephalin analog into the central nucleus of the amygdala produced attenuation of cold restraintinduced gastric mucosal lesions in rats while intraamygdala (central nucleus) naloxone administration potentiated restraint-induced gastric pathology (Ray et al., 1988; Ray and Henke, 1990). An enkephalinergic pathway from the amygdala to the periacquaductal gray nucleus has also been shown to be involved in the suppression of anxiety-related behaviors (Shaikh et al., 1991a,b; Siegel et al., 1997). Further, in addition to modulating overt physiological and behavioral expressions of anxiety elicited in direct response to stressful stimuli, opioids in the amygdala have also been shown to impair fear conditioning, an aspect of the stress response that may be related to a more general role for opioids in processes such as learning and memory (Westbrook et al., 1997). Morphine injected within the amygdala (central nucleus) impaired the acquisition of fear in rats exposed to a hot-plate apparatus (Good and Westbrook, 1995). Activation of opioid receptors in the central nucleus of the amygdala during exposure of rats to the hot plate may have prevented the formation of the excitatory connections between
representations of the apparatus cues and the noxious thermal stimulation, thereby reducing the ability of these cues to provoke fear (Good and Westbrook, 1995). This is consistent with a previous study (Gallagher et al., 1982) that showed injection of an enkephalin analogue within the central nucleus of the amygdala attenuated the acquisition of classically conditioned heart-rate responding in rabbits. The advent of knockout mice for opioid genes brought further evidence for a major role of enkephalin and 8-OR in anxiety-like behavior. Enkephalin knockout mice exhibit an elevated level of anxiety-like behavior (Konig et al., 1996; Kieffer, 1999; Ragnauth et al., 2001). Consistent with that, the 8-OR knockout mice also showed higher anxiety in the elevated plus maze and the light-dark box, suggesting that the activity of 8-OR may diminish anxiety (Filliol et al., 2000). The amygdala could represent a major site for these effects since overexpression of proenkephalin in the amygdala potentiates the anxiolytic effects of benzodiazepines. Indeed, Kang et al. (2000) addressed the role of enkephalin in the control of anxiety-like behavior and anxiety-reducing actions of benzodiazepines, using a recombinant, replication-defective herpes virus carrying human preproenkephalin cDNA that was delivered to rat amygdala. While enkephalin gene infection alone did not reduce anxiety-like behavior, rats infected with enkephalin gene exhibited a greater response to the anxiolytic effect of diazepam when compared to rats infected with a control virus containing the lacZ gene. The enhancement of diazepam action by enkephalin transfection was naloxone-reversible, region-specific, and correlated with the time course of preproenkephalin expression. These findings implicate amygdala opioid peptides in regulating the anxiolytic effects of benzodiazepines (Kang et al., 2000). Considerably fewer studies have addressed the physiological functions of dynorphin (and ~:-OR) within the amygdaloid complex. The existence of functionally opposed opioid systems affecting emotional and perceptual experiences has been proposed on the basis that the g-OR and/or 8-OR mediate the euphorigenic properties (rewarding effect) of opioid agonists, while ~:-OR seem to mediate aversive effects (Pan, 1998; Matsuzawa et al., 1999; Nobre et al., 2000; Sante et al., 2000; Ge et al., 2002). However,
566 recent research, using local microinjections within the infralimbic cortex (Wall and Messier, 2000a,b) or systemic injections (Privette and Terrian, 1995; Agmo and Belzung, 1998) revealed an anxiolytic role for •-OP agonists as well. Therefore, amygdaloid dynorphin and enkephalin neurons could potentially exert either different or similar effects in response to stressful stimuli. Activation of the enkephalinergic system may attenuate, whereas activation of the dynorphinergic system may potentiate the response to stressors. Alternatively, it is also justified to postulate that dynorphin as well as enkephalin may contribute to attenuating the impact of psychological stressors in the amygdaloid complex. As a result, changes in the relative activity of dynorphinergic compared to enkephalinergic neuronal systems within the amygdala invoked by stressors may be important in behavioral and physiological changes induced by stress. More research is needed to elucidate and understand the role(s) played by each endogenous peptide and their receptors within the amygdaloid complex in the stress response.
Stressor-induced alterations in endogenous opioid peptides: implications for anxiety, depression, and panic The putative influence of aversive life events to the provocation, maintenance, and exacerbation of psychological disturbance is well documented (Breier, 1989; Anisman and Zacharko, 1990, 1992; Masure, 1994; Loas, 1996; Cui and Vaillant, 1997; Risch, 1997; Kessing et al., 1998; Kim and Yoon, 1998; Bremner, 1999; Weiss et al., 1999). The notion that stressful life events provoke or exacerbate psychopathology in humans is appealing. The severity of any stressor experience may be defined by the release kinetics of neurotransmitters associated with mood, affect, and motivation and putative neurotransmitters, which modulate individual experiences associated with anxiety and cognitive appraisal of environmental stimuli (see Zacharko et al., 1995). The following discussion outlines the mesolimbic opioid alterations, specifically enkephalin, associated with stressor imposition and the propensity of such neural variations to influence anxiety and motivation.
Human studies
Among clinicians it is well versed that uncontrollable aversive life events result in the formation of attributions concerning the stressor, leading to negative expectancies and alterations in mood which ultimately provoke feelings of anxiety as well as depression (Abramson et al., 1978; Maier, 1984; Breier et al., 1987; Brown and Siegel, 1988; Metalsky and Joiner, 1992; Porteous and Tyndall, 1994). Increased perceptions of stressful life events, a concomitant of anxiety disorders, impairs the anatomy, physiology, and behavioral functions of the prefrontal cortex (Diorio et al., 1993) and hippocampus (McEwen and Sapolsky, 1995; Sapolsky, 1996), damage thought to be related to an increase in cortisol levels and glucocorticoids associated with stress (Van Dijken et al., 1992; Bremner, 1999). In humans, the hippocampus, prefrontal cortex, and amygdala are believed to contribute to emotion, in particular fear-related negative affect and affective working memory (Morgan et al., 1993; Morgan and LeDoux, 1995; Jinks and McGregor, 1997; Davidson and Irwin, 1999). In a recent magnetic resonance imaging study of mood disorders, experimenters demonstrated a reduction in the mean gray matter volume of the prefrontal cortex (Drevets et al., 1998) and amygdala (Sheline et al., 1998, 1999) in subjects with recurrent episodes of major depressive disorder. Similarly, functional neuroimaging studies have identified abnormalities of resting blood flow and glucose metabolism in the medial prefrontal cortex and amygdala of depressed individuals which are correlated with depression severity (Drevets, 1999). Liberzon et al. (2002) employed positron-emission tomography to measure cerebral blood flow and ~t-OR binding in limbic and cortical structures among subjects during presentation of emotionally salient visual pictures. It was found that emotionally charged stimuli induced an increase in cerebral blood flow to the prefrontal cortex and lateral amygdala and was associated with reduced g-OP binding in these identical sites. Immunohistochemical and retrograde tracing has identified g-OR on parabrachial neurons projecting to the amygdala in the rat (Chamberlin et al., 1999). Furthermore, D-Ala 2, N-Me-Phe 4, Gly-O15-enkephalin (DAMGO)
567 induced la-OP activation in the parabrachial nucleus attenuated the impact of relatively severe psychological stressors as revealed by cardiovascular attenuation (Kiritsy-Roy et al., 1986; Marson et al., 1989; Sun et al., 1996; Wisniewska and Wisniewski, 1996). Moreover, the central and basolateral amygdaloid nuclei (Gelsema et al., 1987; Soltis et al., 1997) and the ventral tegmental area (VTA) (van den Buuse, 1998) provide prominent parabrachial innervation and accordingly may influence cardiovascular responsivity to environmental challenge. Enkephalinergic neurons having ascending projections into the A10 region (VTA) are located in the dorsal raphe, dorsal tegmental nucleus, and brainstem nuclei associated with respiration and cardiovascular control. These data suggest an anatomical basis for the abnormal hedonic, motivational, neuroendocrine, and autonomic manifestations evident in clinical cases as well as provide a neural model for the increased sensitivity of individuals with psychological dysfunction to aversive life events and to recurrent and severe episodes of illness. Exposure to life stressors provoke increased CSF [3-endorphin levels among individuals with mild anxiety disorders (Eriksson et al., 1989; Darko et al., 1992; Goodwin et al., 1993; Baker et al., 1997; Westrin et al., 1999), while panic patients and individuals with major depression display reduced CSF [3-endorphin levels (Zis et al., 1985). Severe anxiety syndromes, including panic and posttraumatic stress disorder/individuals with panic disorder or posttraumatic stress disorder, also display augmented levels of aggression (Korn et al., 1997; Southwick et al., 1999). For example, some panic patients report an increased incidence of panicassociated suicidal ideation, engage in property destruction, initiate physical assaults, and may exhibit homicidal tendencies (Korn et al., 1997). Moreover, the incidence of aggressive episodes among patients with posttraumatic stress disorder appeared to increase with attending symptom severity (McFall et al., 1999). Interestingly, mice lacking ~5-OP (Filliol et al., 2000) or preproenkephalin-derived peptides display increased fear and anxiety in novel environments and increased aggression toward conspecifics (Konig et al., 1996; McFall et al., 1999). In a similar vein, naltrexone has been reported to increase aggression and blood pressure in an
individual with posttraumatic stress disorder (Ibarra et al., 1994) and induce panic attacks in individuals with panic disorder (Maremmani et al., 1998). Aggression in animal subjects, individuals with panic disorder, and posttraumatic stress disorder has also been associated with increased sensitivity of 5-HT1A receptors in the raphe nuclei and hypothalamus (Korn et al., 1997; Southwick et al., 1999). Met- and leu-enkephalin and [3-endorphin are colocalized with 5-HT in the caudal raphe nuclei (Korn et al., 1997; Southwick et al., 1999). These data suggest that increased anxiety-like behavior may be associated with a dysinhibition of 5-HT in raphe nuclei (Albert and Walsh, 1982; Ferris et al., 1997, 1999). In conclusion, it would seem appropriate to target pharmacological manipulations which effect g-OP and 8-OP to the treatment of psychological disturbance in which enhanced vulnerability to stressors is a prominent feature of psychopathology (e.g., severe depression, anxiety).
Animal studies: psychological
stressors
Exposure of animals to species-specific predatory cues may provide a relevant simulation of clinical psychopathology (Ferris et al., 1999). For example, exposure of rats to predators and predator odor incites anxiety-like behaviour in the elevated plus maze (McGregor and Dielenberg, 1999), increased acoustic startle (Adamec et al., 1997; Plata-Salaman et al., 2000), increased freezing in novel environments (Hotsenpiller and Williams, 1997), and decreased sucrose consumption (Calvo-Torrent et al., 1999). Exposure of rats to a cat is associated with an enhanced acoustic startle response for up to seven days following predator exposure (Adamec et al., 1998). Drugs that decrease anxiety, including diazepam (Bitsios et al., 1999; Cannizzaro et al., 2001) and enkephalin ~-OR and ~5-OR agonists (Tilson et al., 1986; Vivian and Miczek, 1998), attenuate startle. Rats which are exposed to predator cues display enhanced risk-assessment activity, including defensive burying, stretch attend, and freezing behaviors (Kemble and Bolwahnn, 1997; Dielenberg et al., 2001) (for a review, see Blanchard et al., 1998) which are attenuated by diazepam and imipramine pretreatment (Blanchard et al., 1993;
568 Molewijk et al., 1995; Grewal et al., 1997). Exposure of rats or mice to fox odor (TMT), weasel odor (PT), or cat odor is associated with increased opioiddependent analgesia and freezing in animals relative to control odors (Lester and Fanselow, 1985; Kavaliers, 1988; Lichtman and Fanselow, 1990; Hotsenpiller and Williams, 1997; Kavaliers and Choleris, 1997; Kavaliers et al., 1997). Among nonhuman primates, naloxone increased and morphine decreased vocalizations among infants separated from their mother (Kalin et al., 1988). These findings were replicated in rats exposed to a predator. In particular, morphine decreased (Shepherd et al., 1992) while naloxone increased (Blanchard et al., 1991) ultrasonic emissions in rats exposed to a cat. It has recently been demonstrated in our laboratory (Hebb et al., 2002a,b) that a 10-min predator odor exposure (fox odor) increased anxietylike behavior in the light-dark box and enhanced acoustic startle in mice relative to mice exposed to saline or the pungent control odor butyric acid. Mice exposed to TMT displayed enhanced freezing relative to control mice which was associated with reduced time spent in the light of the test apparatus. Anxietylike behavior in the light-dark box following predator odor exposure was accompanied by decreased proenkephalin gene expression in the core of the nucleus accumbens and increased proenkephalin gene expression in the basolateral, central, and medial amygdala. In particular, among mice exposed to fox odor, enhanced levels of anxiety-like behavior in mice was associated with increased activation of enkephalin neurons in the basolateral, medial, and central amygdaloid nuclei gene expression while the overall level of enkephalin transcript was decreased in these animals. However, enhanced levels of freezing among mice not assessed for anxiety in the light-dark test was associated with decreased activation of enkephalin neurons in the central amygdala relative to mice which exhibited less freezing to predator odor. It is well established that the central amygdala plays a prominent role in the acquisition and mediation of fear responses (Roozendaal et al., 1991) and is the major output pathway to the basolateral amygdala and many subcortical nuclei that mediate fear-related behaviors, including freezing and startle (Rosen et al., 1998). The medial amygdala has been associated with a variety of neuroendocrine as well as behavioral
responses, including mating and aggression. The induction of enkephalin in the medial amygdala of mice is consistent with a role of this nucleus in behavioral arousal and social memory (Vochteloo and Koolhaas, 1987; Kollack-Walker and Newman, 1995). Interestingly, it has recently been reported that the male rats display an increase in delta-opioid receptor immunoreactivity in the medial nucleus of the amygdala compared to female rats (Wilson et al., 2002), which may underlie sex differences in response to stressor experiences (Heinsbroek et al., 1988). Klein et al. (1998) reported that in male rats opioid blockade by peripheral naloxone administration enhanced unconditioned freezing in the home cage following footshock imposition. However, conditioned freezing was associated with increased enkephalin mRNA from the central amygdala, suggesting that enkephalin within this nucleus is associated with learning and memory (Petrovich et al., 2000). It will be recalled that preproenkephalindeficient mice display increased anxiety in the open field and elevated plus-maze tests (Petrovich et al., 2000; Ragnauth et al., 2001). Psychological stressors increase amygdaloid ~5-OR receptor binding (Pohorecky et al., 1999) and la-OR in the ventral tegmental area (Nikulina et al., 1999), paralleling anxiety induction in rats and mice (MacNeil et al., 1997; Blanchard et al., 1998; Moynihan et al., 2000). In other investigations, employing various psychological stressors, evidence exists to support a role of enkephalin in the stress response of animals. For example, the social interaction and defeat paradigm indices correlate with extracellular met-enkephalin availability in the rostral nucleus accumbens (Bertrand et al., 1997) and ~t-OR mRNA density in the VTA (Nikulina et al., 1999), respectively. Indeed, naltrexone increased initial contact latency and decreased active interaction duration among rats in a social-interaction paradigm (Zhang et al., 1996). Pretreatment with naloxone (1 mg/kg s.c.) antagonized increased behavioral activity (e.g., ambulation, rearing, sniffing) of rats exposed to a psychological stressor (perception of another rat receiving footshocks) while having no effect on behavior of control animals or animals subjected to footshock (VandenBerg et al., 1998). In rats, social isolation (e.g., seven days) was associated with decreased proenkephalin mRNA in the nucleus
569 accumbens (Angulo et al., 199 l) and increased ~-OR binding in the prefrontal cortex, the amygdala, nucleus accumbens, and hypothalamus (Pohorecky et al., 1999) compared to group-housed rats. Interestingly, prior stressor history of mice influences locomotor activity to subsequent administration of enkephalin agonists (Calenco-Choukroun et al., 1991). For example, intra-VTA administration of the ~-OR agonists in the rat induced hyperactivity in a familiar (i.e., home cage), unfamiliar (four-hole box), and an open-field environment, while DAGO enhanced locomotion in a familiar but not in the unfamiliar environment or the open-field paradigm 24 h following a 3-day habituation baseline schedule. The environment-~t-OP and 6-OP associated increase in locomotor activity was blocked by systemic naloxone (0.3mg/kg) in mice (Calenco-Choukroun et al., 1991). Moreover, such stressor scenarios may define the profile of symptoms and determine vulnerability (e.g., latency to the emergence of psychological dysfunction) to anxiety disorders. Such predisposing neurochemical variations may (a) identify region- specific neural sequelae contributing to behavioral sensitization and conditioning among subjects; (b) provide a parallel for the development of psychological disturbance in human subjects; and (c) suggest therapeutic-intervention strategies.
Mesolimbic opioid availability underlying coping and therapeutic efficacy of antidepressant regimens It is well documented that endogenous opioids contribute to the expression of affect and motivation in human (Cohen et al., 1984; Zis et al., 1985; Castilla-Cortazar et al., 1998) and animal subjects (Hernandez et al., 1997). Indeed, major affective disorder has been associated with decreased cerebrospinal (CSF) endorphin concentrations (Djurovic et al., 1999). Endogenous opioids have been implicated in the mechanism of action of antidepressant therapies (de Felipe et al., 1985, 1989). In fact, the involvement of opioid systems in clinical depression is based on clinical and animal studies which report that inhibitors of enkephalin-degrading
enzymes (e.g., RB 101) have antidepressant properties in various paradigms (Tejedor-Real et al., 1998), chronic imipramine treatment promotes the expression of the ~t-OR in the hippocampus and frontal cortex of the rat (de Gandarias et al., 1998), chronic imipramine and desipramine, inhibit the enkephalindegrading aminopeptidase in a concentration-dependent manner in rat brain (Gallego et al., 1998; de Gandarias et al., 1999), and endogenous opioids have been implicated in the mechanism of action of antidepressant therapies (de Felipe et al., 1985, 1989). Inhibitors of enkephalin-degrading enzymes (de Felipe et al., 1985, 1989; Tejedor-Real et al., 1998) demonstrate antidepressant properties which are linked to specific opioid and nonopioid receptor subtypes (e.g., ~-OR; Tejedor-Real et al., 1998) in various animal paradigms. For example, in rodents the antidepressant effects of RB 101 in conditioned suppression of motility (CSM) test were reversed by the selective 6 opioid receptor antagonist, naltrindole (Baamonde et al., 1992). Met-enkephalin administration attenuated suppression of motility among mice placed in an environment previously paired with electric shock, an effect that was reversed by naloxone and 6-OHDA lesions of nucleus accumbens neurons (Katoh et al., 1991). Acute administration of imipramine (10 mg/kg, i.p.) decreased proenkephalin mRNA (20%) and prodynorphin mRNA (25%) in the nucleus accumbens and striatum, while chronic administration (10 mg/kg i.p. twice daily for 10 days) increased nucleus accumbens met-enkephalin and proenkephalin gene levels (Dziedzicka-Wasylewska and Rogoz, 1995; Dziedzicka-Wasylewska and Papp, 1996) as well as prodynorphin mRNA 24 h following cessation of treatment (Kurumaji et al., 1988; Przewlocki et al., 1997). Similar changes in the levels of met-enkephalin in the rat hippocampus, striatum, hypothalamus, and pituitary were reported following chronic administration of the antidepressant drugs, tianeptine and fluoxetine (Dziedzicka-Wasylewska et al., 2002). Treatment-resistant depression in clinical populations sometimes responds to electroconvulsive shock-therapy treatment which promotes increased enkephalin in brain (Hong et al., 1979; Holaday et al., 1986; Lason et al., 1987; He et al., 1989), including hippocampus (He et al., 1989). Typically, chronic antidepressant regimes (4-6 weeks) are necessary to restore mood in clinical depression (Stimmel, 1995).
570 It may be that chronic antidepressant therapy is necessary to promote stable levels of enkephalin in central sites before any noticeable change in mood and affect occurs. Taken together, coping and antidepressant agents favor mesolimbic enkephalin release among subjects which may blunt the impact of the stressor or stressor-associated cues. It has been reported that the release of enkephalin within mesolimbic sites reduces the effect of the stress response by decreasing numerous physiological responses, including emotional and affective states (Dziedzicka-Wasylewska and Papp, 1996). It should be considered that while mild stressors or the cues associated with aversive events may elicit cognitions which reenlist neurochemical variations associated with the original stressor experience (Ahmed and Koob, 1997; Erb et al., 1998), coping may prompt anxiolytic agent release. In humans, hyperactivity of endogenous opioids has been postulated to underlie defensive coping styles, characterized by orientation away from threatening stimuli thereby minimizing distress and negative emotions (Janssen and Arntz, 1996, 1997; Jamner and Leigh, 1999). The exact relationship between increased endogenous opioids and coping has not been defined (Goodwin and Barr, 1997; Jamner and Leigh, 1999). Cardiac patients demonstrated enhanced blood pressure and heart rate accompanied by increased plasma levels of metenkephalin, dynorphin, and 13-endorphin in response to a psychological stressor (mental arithmetic test) (Fontana et al., 1998). Systemic administration of the opioid antagonists, naloxone and naltrexone, enhanced cardiovascular indices induced by a cognitive stressor in normal and cardiac patients (Fontana et al., 1997, 1998; McCubbin et al., 1998). Interestingly, naloxone and naltrexone also decreased approach behaviour among phobic individuals (Arntz, 1993; Janssen and Arntz, 1996, 1997) and attenuated the anxiolytic influence of diazepam among patients awaiting surgery (Duka et al., 1982). Interestingly, naloxone can also reverse placebo analgesia (Benedetti and Amanzio, 1997), suggesting that there is a modulatory effect of opioid function on subject expectations which may be extended to anticipatory anxiety. In mice, naloxone attenuated the anxiolytic properties of diazepam and chlordiazepoxide in mice in the light-dark and elevated plus-maze paradigms (Agmo et al., 1995;
Belzung and Agmo, 1997). In contrast to benzodiazepine administration, chronic benzodiazepine withdrawal elicits significant anxiety (Fontaine et al., 1984; Otto et al., 1993). Such behavioural reactivity to anxiolytic withdrawal has been linked to decreased met-enkephalin immunoreactivity in the nucleus accumbens (Kurumaji et al., 1988; Przewlocki et al., 1997). It should be considered that acute and chronic administration of the antidepressant/anxiolytic agent imipramine effects neurochemically distinct profiles of met and leu enkephalin release within the ventral tegmental area and nucleus accumbens (Dziedzicka-Wasylewska and Rogoz, 1995). The variable sensitivity of central mesocorticolimbic sites to antidepressant regimens is consistent with data outlining differential responsivity of mesocorticolimbic sites to stressor imposition (Zacharko and Anisman, 1991). It should be emphasized that cognitive and performance-based therapeutic interventions among anxiety patients frequently introduce coping strategies to reduce anxiety during pharmacotherapy and eventual drug taper (Shear et al., 1991; Nagy et al., 1993; Spiegel et al., 1994). Such interventions attempt to reduce the saliency of associational cues which precipitate anxiety. In fact, the efficacy of performance-associated phobic treatment, for example, can be reliably traced to cognitive appraisal of performance self-adequacy and coping repertoires (Williams et al., 1989). Perceived control over footshock enhances cerebrospinal (CSF) release of benzodiazepine-like agent among rats (Piva et al., 1991; Drugan et al., 1994, 1997), which may detract from the severity of the experience. Yet, the source of such neurochemical activity may be associated with mesocorticolimbic sensitivity to stressor controllability. Indeed, the release of "anxiolytic" agents following stressor imposition has been detected in the amygdala (Kang et al., 1999), mesocortex (Cabib and Puglisi-Allegra, 1996), the ventral tegmental area, and the nucleus accumbens (DziedzickaWasylewska and Papp, 1996). Indeed, it has been suggested that endogenous enkephalin release among animal subjects may, in some instances, diminish the propensity of restraint to sustain protracted alterations (e.g., one week) of mesocortical dopamine release (Cuadra et al., 1999) and anxiety-like behavior in animals in the light-dark paradigm
571 (Cancela et al., 1995). Central administration of g-OR and 8-OR opioid receptor agonists, before or after footshock, reduced stressor-associated deficits in locomotor activity (Hebb et al., 1997) and VTA brain stimulation deficits (Zacharko et al., 1998) among mice. Systemic administration of met-enkephalin (10 mg/kg) 30 min before a 6-h restraint stress reduced the stressor-induced increase in plasma corticosterone among mice (Sverko et al., 1997), while daily naltrindole (lmg/kg) from birth to postnatal day 19 inhibited subsequent increased corticosterone release in 25-day-old female rats in response to a 3-min swim session (Fernandez et al., 1999). Moreover, systemic administration of RB-101 or naltrexone reduced and exaggerated freezing to shock-associated environmental cues, respectively, in mice (Baamonde et al., 1992; Calcagnetti and Schechter, 1994). The enkephalin-inhibitor RB- 101, met-enkephalin, leu-enkephalin as well as the specific 8-OP agonist, BUBU, also reversed escape deficits among rats previously subjected to uncontrollable footshock (Tejedor-Real et al., 1993, 1995, 1998). The effects of RB-101 on escape deficits were attenuated by prior administration of the DA receptor antagonist, SCH-23390, or the selective 8 opioid receptor antagonist, naltrindole (Tejedor-Real et al., 1998). In contrast, naloxone potentiated the effect of inescapable shock among rats (Tejedor-Real et al., 1998). Taken together, acute exposure of animal and human subjects to stressors promotes enkephalin release within the mesocortex, nucleus accumbens, and ventral tegmental area. Such a neurochemical signature within the mesolimbic system may be influenced by the severity of the environmental experience and determine behavioral responsivity to stressor applications (Siegel et al., 1984; Imperato et al., 1992; Izumi, 1998; Giardino et al., 1999). Mesolimbic opioid receptor density appears to coincide with somatodendritic dopamine, cholecystokinin, and GABA interneuron density (Mansour et al., 1988; Kalivas, 1993). Notably, central and basolateral nuclei amygdaloid neurochemical perturbations associated with benzodiazepine-GABA receptor variations including alterations in GABA and glutamate (Davis et al., 1994; Kunos and Varga, 1995; Soltis et al., 1997; Walker and Davis, 1997) most likely potentiate the efficacy of anxiolytic agents, including diazepam. It should be emphasized
that la-OP have been identified on both GABA- and DA-containing neurons and 8-OP have been detected on afferent inputs to GABA neurons in the VTA (Kalyuzhny and Wessendorf, 1997). Such anatomical data provide a putative framework for a la/8-OR interaction in the VTA that may impact on stressorassociated behavioral deficits. The effects of opioid activity on the anxiolytic properties of benzodiazepine-like substances have been attributed to specific opioid receptor subtypes (Tsuda et al., 1996; TejedorReal et al., 1998; Abbadie et al., 2000). In summary, increased enkephalin availability may represent a modulatory system in stress adaptation in both animal and human subjects and avert exaggerated behavioral and neurochemical cascades associated with reexposure to stressors or stressor-associated cues previously associated with initial stressor presentations. In effect, behavioral-coping strategies and antidepressant regimens favor mesolimbic enkephalin release which may blunt the impact of the stressor or stressor-associated cues. In animals, a variety of environmental stressors, both nocioceptive and nonpainful threat stimuli (Lightman and Young, 1987a; Rodgers and Randall, 1987; Sribanditmongkol et al., 1994; Yamada and Nabeshima, 1995; Beaulieu et al., 1996), elicit opioid release and changes in opioid receptor binding (Stuckey et al., 1989) which may mediate specific types of stress analgesia (Zadina et al., 1999) which is blocked by opioid antagonists and interacts with anxiolytic (Kelly et al., 1986; Watkins and Mayer, 1986) and antidepressant (Zurita et al., 1999) agents. For example, chronic footshock induced a 40-50% reduction of Met and Leu enkephalin levels in whole brain of rats (McGivern et al., 1983) and a time-dependent decrease in the level of immunoreactive metenkephalin in the medial A10 region (VTA), which may be indicative of a footshock-induced increase in release and metabolism of met-enkephalin (Kalivas et al., 1988). Exposure of young rats to a male intruder increased preproenkephalin mRNA in the central amygdaloid nucleus and enhanced heat analgesia (Wiedenmayer et al., 2002). Stressorinduced analgesia is thought to be an integral and adaptive component of the defensive repertoire (Amit and Galina, 1986; Fanselow, 1986; Rodgers and Randall, 1987; Vaccarino and Kastin, 2001).
572 It has also been proposed that endogenous opioid mechanisms, specifically leu-enkephalin availability and delta receptor activation, are involved in stimulus processing and early stages of learning in conditioning tasks among animals (Schulteis and Martinez, 1992; Hernandez and Watson, 1997; Martinez et al., 1998). Indeed, salient environmental experiences modulate peripheral leu-enkephalin availability and may increase the associated memory strength and preparedness of the animal to future challenges (Martinez and Weinberger, 1987, 1988; Derrick et al., 1991; Schulteis and Martinez, 1993). One of the mechanisms associated with the expression of anxiety incorporates an amygdaloid-opioid neurochemical mosaic which may encode the saliency of the environmental experience, consistent with an amygdaloid-dependent modulation of emotional memory (Ferry et al., 1999). Central administration of the 61-OP agonist D-Pen 2, L-Pen 5 (DDLPE) decreased passive-avoidance learning in rats trained to avoid footshock (Ukai et al., 1997). It has been demonstrated that central ~5-OP activation following uncontrollable footshock and novelty stress in CD-1 mice precipitated an increase in locomotor activity with reexposure to only the identical, milder form of the original stressor. The behavioral response of ~-OR activation following stressor imposition were also dose and time dependent (Hebb et al., 1997). Indeed, endogenous 5-OR activation may increase the saliency of particular attributes or characteristics of environmental stimuli (Hernandez and Watson, 1997). Clearly, the distribution of ~t-OP and 6-OR in mesolimbic sites contributes to coping (Goodwin and Barr, 1997). Increased preproenkephalin gene expression in central sites, including the amygdala, nucleus accumbens, locus coeruleus, among others, associated with fear and anxiety, presumably underlies compensatory physiological responses that attenuate the deleterious effects of uncontrollable stressor applications (Dumont et al., 2000; Curtis et al., 2001). Conceptually, it is conceivable that central la-OP and ~5-OR activation interferes with the encoding of stressor-associated events. Such processes may alter long-term responsivity of organisms to future stressor encounters. In human and animal subjects increased preproenkephalin availability is associated with mood elevation and coping ability which may detract from
the aversiveness of the stressor experience. In animal and human subjects, activation of ~t-OP and ~5-OP in the basolateral amygdala, ventral tegmental area, and mesocortex attenuates the aversiveness of psychological stressors. The temporal expression of enkephalin in mesolimbic sites associated with specific indices of anxiety and fear would delineate specific behavioral subsets associated with stressor exposure and predict vulnerability to future-like events. In particular, activation of ~t-OP and 6-OR within the ventral tegmental area, nucleus accumbens, and amygdala may diminish anxiety and motivational loss accompanying stressor exposure, permit expression of coping behavior, detract from the saliency of the stressor, and alter long-term responsivity to ensuing stressor encounters. The relative contributions of 5-OP and ~t-OP activation to motivated behavior is predicated on mesocorticolimbic site-specific differences in opioid receptor density as well as environmental stimuli which may modulate the release of variable neurotransmitter and neuropeptide systems with different affinities for the ~5-OP and ~t-OR sub-types.
Conclusion
Clearly, prominent behavioral correlates of central opioid activation are in evidence following stressor manipulations. In particular, opioid availability in diverse brain areas associated with anxiety-like behaviors (i.e., enkephalin within the amygdala) may contribute to affective aspects of an anxietyprovoking situation following stressor imposition. Opioid peptide availability is linked to colocalization of other neurotransmitters in distinct central sites which suggests that opioids may modulate (a) different aspects of anxiety, including anticipatory reactions to anxiogenic stimuli, (b) variations in cognitive arousal and vigilance, and (c) modulation of behavior and central neurochemical activity. This seemingly overlap of function may supply an organism with many stress responses, but more importantly the ability to manufacture assorted behavioral reactions in response to specific stressful situations. Likewise, multiple anxiogenic agents and putative neurotransmitters or neuromodulators in the mesolimbic system as well as prefrontal cortex and brainstem would appear to participate in the
573 p r o m o t i o n or alleviation of stress-induced s y m p t o m s a n d m o o d alterations. In any event, these d a t a suggest t h a t p h a r m a c o l o g i c a l m a n a g e m e n t of stressi n d u c e d p a t h o l o g i e s s h o u l d be directed t o w a r d specific s y m p t o m s c h a r a c t e r i z i n g the psychological pathology.
Acknowledgments G . D . held a scholarship f r o m le F o n d s de la R e c h e r c h e en Sant6 du Qu6bec ( F R S Q ) . A . L . O . H . held a fellowship f r o m T h e C a n a d i a n Institute of H e a l t h Research.
References Abbadie, C., Pan, Y., Drake, C. and Pasternak, G. (2000) Comparative immunohistochemical distributions of carboxy terminus epitopes from the mu-opioid receptor splice variants MOR-1D, MOR-1 and MOR-1C in the mouse and rat CNS. Neuroscience, 100: 141-153. Abramson, L.Y., Garber, J. and Edwards, N.B. (1978) Expectancy changes in depression and schizophrenia. J. Abnorm. Psychol., 87: 102-109. Adamec, R.E., Shallow, T. and Budgell, J. (1997) Blockade of CCK(B) but not CCK(A) receptors before and after the stress of predator exposure prevents lasting increases in anxiety-like behavior: implications for anxiety associated with posttraumatic stress disorder. Behav. Neurosci., 111: 435-449. Adamec, R., Kent, P., Anisman, H., Shallow, T. and Merali, Z. (1998) Neural plasticity, neuropeptides and anxiety in animals: implications for understanding and treating affective disorder following traumatic stress in humans. Neurosci. Biobehav. Rev., 23:301-318. Agmo, A. and Belzung, C. (1998) The role of subtypes of the opioid receptor in the anxiolytic action of chlordiazepoxide. Neuropharmacology, 37: 223-232. Agmo, A., Galvan, A., Heredia, A. and Morales, M. (1995) Naloxone blocks the antianxiety but not the motor effects of benzodiazepines and pentobarbitah experimental studies and literature review. Psychopharmacology (Berl), 120:186-194. Ahmed, S.H. and Koob, G.F. (1997) Cocaine- but not foodseeking behavior is reinstated by stress after extinction. Psychopharmacology (Berl), 132: 289-295. Akil, H., Watson, S.J., Young, E., Lewis, M.E., Khachaturian, H. and Walker, J.M. (1984) Endogenous opioids: biology and functions. Ann. Rev. Neurosci., 7: 223-255.
Akil, H., Owens, C., Gutstein, H., Taylor, L., Curran, E. and Watson, S. (1998) Endogenous opioids: overview and current issues. Drug Alcohol Depend., 51: 127-140. Albert, D.J. and Walsh, M.L. (1982) The inhibitory modulation of agonistic behavior in the rat brain: a review. Neurosci. Biobehav. Rev., 6: 125-143. Amit, Z. and Galina, Z.H. (1986) Stress-induced analgesia: adaptive pain suppression. Physiol. Rev., 66: 1091-1120. Angulo, J.A., Printz, D., Ledoux, M. and McEwen, B.S. (1991) Isolation stress increases tyrosine hydroxylase mRNA in the locus coeruleus and midbrain and decreases proenkephalin mRNA in the striatum and nucleus accumbens. Mol. Brain Res., 11: 301-308. Anisman, H. and Zacharko, R.M. (1990) Multiple neurochemical and behavioral consequences of stressors: implications for depression. Pharmacol. Ther., 46:119-136. Anisman, H. and Zacharko, R.M. (1992) Depression as a consequence of inadequate neurochemical adaptation in response to stressors. Br. J. Psychiatry, Suppl, 36-43. Arntz, A. (1993) Endorphins stimulate approach behaviour, but do not reduce subjective fear. A pilot study. Behav. Res. Ther., 31: 403-405. Baamonde, A., Dauge, V., Ruiz-Gayo, M., Fulga, I.G., Turcaud, S., Fournie-Zaluski, M.C. and Roques, B.P. (1992) Antidepressant-type effects of endogenous enkephalins protected by systemic RB 101 are mediated by opioid delta and dopamine D1 receptor stimulation. Eur. J. Pharmacol., 216:157-166. Baker, D.G., West, S.A., Orth, D.N., Hill, K.K., Nicholson, W.E., Ekhator, N.N., Bruce, A.B., Wortman, M.D., Keck, P.E., Jr. and Geracioti, T.D., Jr. (1997) Cerebrospinal fluid and plasma beta-endorphin in combat veterans with post-traumatic stress disorder. Psychoneuroendocrinology, 22: 517-529. Beaulieu, J., Champagne, D. and Drolet, G. (1996) Enkephalin innervation of the paraventricular nucleus of the hypothalamus: distribution of fibers and origins of input. J. Chem. Neuroanat., 10: 79-92. Belzung, C. and Agmo, A. (1997) Naloxone blocks anxiolyticlike effects of benzodiazepines in Swiss but not in Balb/c mice. Psychopharmacology (Berl), 132: 195-201. Benedetti, F. and Amanzio, M. (1997) The neurobiology of placebo analgesia: from endogenous opioids to cholecystokinin. Prog. Neurobiol., 52: 109-125. Bertrand, E., Smadja, C., Mauborgne, A., Roques, B.P. and Dauge, V. (1997) Social interaction increases the extracellular levels of [Met]enkephalin in the nucleus accumbens of control but not of chronic mild stressed rats. Neuroscience, 80: 17-20. Bitsios, P., Philpott, A., Langley, R.W., Bradshaw, C.M. and Szabadi, E. (1999) Comparison of the effects of diazepam on the fear-potentiated startle reflex and the fear-inhibited light reflex in man. J. Psychopharmacol., 13: 226-234.
574 Blackburn, T.P., Cross, A.J., Hille, C. and Slater, P. (1988) Autoradiographic localization of delta opiate receptors in rat and human brain. Neuroscience, 27: 497-506. Blanchard, D.C., Weatherspoon, A., Shepherd, J., Rodgers, R.J., Weiss, S.M. and Blanchard, R.J. (1991) "Paradoxical" effects of morphine on antipredator defense reactions in wild and laboratory rats. Pharmacol. Biochem. Behav., 40: 819-828. Blanchard, R.J., Shepherd, J.K., Rodgers, R.J., Magee, L. and Blanchard, D.C. (1993) Attenuation of antipredator defensive behavior in rats following chronic treatment with imipramine. Psychopharmacology (Berl), 110: 245-253. Blanchard, R.J., Nikulina, J.N., Sakai, R.R., McKittrick, C., McEwen, B. and Blanchard, D.C. (1998) Behavioral and endocrine change following chronic predatory stress. Physiol. Behav., 63: 561-569. Bodnar, R.J. and Hadjimarkou, M.M. (2002) Endogenous opiates and behavior: 2001. Peptides, 23: 2307-2365. Bolles, R.C. and Fanselow, M.S. (1982) Endorphins and behavior. Annu. Rev. Psychol., 33: 87-101. Breier, A. (1989) A.E. Bennett award paper. Experimental approaches to human stress research: assessment of neurobiological mechanisms of stress in volunteers and psychiatric patients. Biol. Psychiatry, 26: 438-462. Breier, A., Albus, M., Pickar, D., Zahn, T.P., Wolkowitz, O.M. and Paul, S.M. (1987) Controllable and uncontrollable stress in humans: alterations in mood and neuroendocrine and psychophysiological function. Am. J. Psychiatry, 144: 1419-1425. Bremner, J.D. (1999) Does stress damage the brain? Biol. Psychiatry, 45: 797-805. Brown, J.D. and Siegel, J.M. (1988) Attributions for negative life events and depression: the role of perceived control. J. Pers. Soc. Psychol., 54: 316-322. Cabib, S. and Puglisi-Allegra, S. (1996) Stress, depression and the mesolimbic dopamine system. Psychopharmacology (Berl), 128: 331-342. Calcagnetti, D.J. and Schechter, M.D. (1994) Deficits in shockinduced freezing and naltrexone enhancement of freezing in fawn hooded rats. Brain Res. Bull., 35: 37-40. Calenco-Choukroun, G., Dauge, V., Gacel, G., Feger, J. and Roques, B.P. (1991) Opioid delta agonists and endogenous enkephalins induce different emotional reactivity than mu agonists after injection in the rat ventral tegmental area. Psychopharmacology (Berl), 103: 493-502. Calvo-Torrent, A., Brain, P.F. and Martinez, M. (1999) Effect of predatory stress on sucrose intake and behavior on the plus-maze in male mice. Physiol. Behav., 67: 189-196. Cancela, L.M., Bregonzio, C. and Molina, V.A. (1995) Anxiolytic-like effect induced by chronic stress is reversed by naloxone pretreatment. Brain Res. Bull., 36: 209-213. Cannizzaro, C., Martire, M., Cannizzaro, E., Provenzano, G., Gagliano, M., Carollo, A., Mineo, A. and Steardo, L. (2001) Long-lasting handling affects behavioural reactivity in adult
rats of both sexes prenatally exposed to diazepam. Brain Res., 904: 225-233. Castilla-Cortazar, I., Castilla, A. and Gurpegui, M. (1998) Opioid peptides and immunodysfunction in patients with major depression and anxiety disorders. J. Physiol. Biochem., 54: 203-215. Ceccatelli, S. and Orazzo, C. (1993) Effect of different types of stressors on peptide messenger ribonucleic acids in the hypothalamic paraventricular nucleus. Acta. Endocrinol., 128: 485-492. Chamberlin, N.L., Mansour, A., Watson, S.J. and Saper, C.B. (1999) Localization of mu-opioid receptors on amygdaloid projection neurons in the parabrachial nucleus of the rat. Brain Res., 827: 198-204. Cohen, M.R., Pickar, D., Cohen, R.M., Wise, T.N. and Cooper, J.N. (1984) Plasma cortisol and beta-endorphin immunoreactivity in human obesity. Psychosom. Med., 46:454-462. Cuadra, G., Zurita, A., Lacerra, C. and Molina, V. (1999) Chronic stress sensitizes frontal cortex dopamine release in response to a subsequent novel stressor: reversal by naloxone. Brain Res. Bull., 48: 303-308. Cui, X.J. and Vaillant, G.E. (1997) Does depression generate negative life events? J. Nerv. Ment. Dis., 185: 145-150. Curtis, A.L., Bello, N.T. and Valentino, R.J. (2001) Evidence for functional release of endogenous opioids in the locus ceruleus during stress termination. J. Neurosci., 21: RC152. Darko, D.F., Risch, S.C., Gillin, J.C. and Golshan, S. (1992) Association of beta-endorphin with specific clinical symptoms of depression. Am. J. Psychiatry, 149:1162-1167. Davidson, R.J. and Irwin, W. (1999) The functional neuroanatomy of emotion and affective style. Trends Cogn. Sci., 3: 11-21.
Davis, M., Hitchcock, J.M., Bowers, M.B., Berridge, C.W., Melia, K.R. and Roth, R.H. (1994) Stress-induced activation of prefrontal cortex dopamine turnover: blockade by lesions of the amygdala. Brain Res., 664: 207-210. de Felipe, M.C., De Ceballos, M.L., Gil, C. and Fuentes, J.A. (1985) Chronic antidepressant treatment increases enkephalin levels in n. accumbens and striatum of the rat. Eur. J. Pharmacol., 112:119-122. de Felipe, M.C., Jimenez, I., Castro, A. and Fuentes, J.A. (1989) Antidepressant action of imipramine and iprindole in mice is enhanced by inhibitors of enkephalin-degrading peptidases. Eur. J. Pharmacol., 159: 175-180. de Gandarias, J.M., Echevarria, E., Acebes, I., Silio, M. and Casis, L. (1998) Effects of imipramine administration on mu-opioid receptor immunostaining in the rat forebrain. Arzneimittelforschung, 48: 717-719. de Gandarias, J.M., Irazusta, J., Varona, A., Gil, J., Fernandez, D. and Casis, L. (1999) Effect of imipramine on enkephalin-degrading peptidases. Eur. Neuropsychopharmacol., 9: 493-499.
575
Derrick, B.E., Weinberger, S.B. and Martinez, J.L., Jr. (1991) Opioid receptors are involved in an NMDA receptorindependent mechanism of LTP induction at hippocampal mossy fiber-CA3 synapses. Brain Res. Bull., 27: 219-223. Dielenberg, R.A., Carrive, P. and McGregor, I.S. (2001) The cardiovascular and behavioral response to cat odor in rats: unconditioned and conditioned effects. Brain Res., 897: 228-237. Diorio, D., Viau, V. and Meaney, M.J. (1993) The role of the medial prefrontal cortex (cingulate gyrus) in the regulation of hypothalamic-pituitary-adrenal responses to stress. J. Neurosci., 13: 3839-3847. Djurovic, D., Milic-Askrabic, J. and Majkic-Singh, N. (1999) Serum beta-endorphin level in patients with depression on fluvoxamine. Farmaco, 54: 130-133. Drevets, W.C. (1999) Prefrontal cortical-amygdalar metabolism in major depression. Ann. N.Y. Acad. Sci., 877: 614-637. Drevets, W.C., Ongur, D. and Price, J.L. (1998) Neuroimaging abnormalities in the subgenual prefrontal cortex: implications for the pathophysiology of familial mood disorders. Mol. Psychiatry, 3: 220-226, 190-221. Drolet, G., Morilak, D.A. and Chalmers, J. (1991 a) Endogenous opioid tonically inhibit the depressor neurons in the caudal ventrolateral medulla of rabbits: mediation through delta and kappa receptors. Neuropharmacology, 30: 383-390. Drolet, G., Morilak, D.A. and Chalmers, J. (1991b) Opioid innervation of the caudal ventrolateral medulla is not critical for the expression of the aortic depressor nerve response in the rabbit. J. Auton. Nerv. Syst., 32: 37-46. Drugan, R.C., Basile, A.S., Ha, J.H. and Ferland, R.J. (1994) The protective effects of stress control may be mediated by increased brain levels of benzodiazepine receptor agonists. Brain Res., 661: 127-136. Drugan, R.C., Basile, A.S., Ha, J.H., Healy, D. and Ferland, R.J. (1997) Analysis of the importance of controllable versus uncontrollable stress on subsequent behavioral and physiological functioning. Brain Res. Protoc., 2: 69-74. Duka, T., Millan, M.J., Ulsamer, B. and Doenicke, A. (1982) Naloxone attenuates the anxiolytic action of diazepam in man. Life Sci., 31:1833-1836. Dumont, E.C., Kinkead, R., Trottier, J., Gosselin, I. and Drolet, G. (2000) Effect of chronic psychogenic stress exposure on enkephalin neuronal activity and expression in the rat hypothalamic paraventricular nucleus. J. Neurochem., 75:2200-2211. Dziedzicka-Wasylewska, M. and Rogoz, R. (1995) The effect of prolonged treatment with imipramine and electroconvulsive shock on the levels of endogenous enkephalins in the nucleus accumbens and the ventral tegmentum of the rat. J. Neural. Transm. Gen. Sect., 102: 221-228. Dziedzicka-Wasylewska, M. and Papp, M. (1996) Effect of chronic mild stress and prolonged treatment with imipramine on the levels of endogenous Met-enkephalin in the rat
dopaminergic mesolimbic system. Pol. J. Pharmacol., 48: 53-56. Dziedzicka-Wasylewska, M., Dlaboga, D., PierzchalaKoziec, K. and Rogoz, Z. (2002) Effect of tianeptine and fluoxetine on the levels of Met-enkephalin and mRNA encoding proenkephalin in the rat. J. Physiol. Pharmacol., 53:117-125. Erb, S., Shaham, Y. and Stewart, J. (1998) The role of corticotropin-releasing factor and corticosterone in stressand cocaine-induced relapse to cocaine seeking in rats. J. Neurosci., 18: 5529-5536. Eriksson, E., Westberg, P., Thuresson, K., Modigh, K., Ekman, R. and Widerlov, E. (1989) Increased cerebrospinal fluid levels of endorphin immunoreactivity in panic disorder. Neuropsychopharmacology, 2: 225-228. Fallon, J.H. and Leslie, F.M. (1986) Distribution of dynorphin and enkephalin peptides in the rat brain. J. Comp. Neurol., 249: 293-336. Fanselow, M.S. (1986) Conditioned fear-induced opiate analgesia: a competing motivational state theory of stress analgesia. Ann. N.Y. Acad. Sci., 467: 40-54. Fernandez, B., Alberti, I., Kitchen, I. and Viveros, M.P. (1999) Neonatal naltrindole and handling differently affect morphine antinociception in male and female rats. Pharmacol. Biochem. Behav., 64: 851-855. Ferris, C.F., Melloni, R.H., Jr., Koppel, G., Perry, K.W., Fuller, R.W. and Delville, Y. (1997) Vasopressin/serotonin interactions in the anterior hypothalamus control aggressive behavior in golden hamsters. J. Neurosci., 17: 4331-4340. Ferris, C.F., Stolberg, T. and Delville, Y. (1999) Serotonin regulation of aggressive behavior in male golden hamsters (Mesocricetus auratus). Behav. Neurosci., 113: 804-815. Ferry, B., Roozendaal, B. and McGaugh, J. (1999) Role of norepinephrine in mediating stress hormone regulation of long-term memory storage: a critical involvement of the amygdala. Biological Psychiatry, 46: 1140-1152. File, S.E. and Rodgers, R.J. (1979) Partial anxiolytic action of morphine sulphate following microinjection into the central nucleus of the amygdala in rats. Pharmacol. Biochem. Behav., 11: 313-318. Filliol, D., Ghozland, S., Chluba, J., Martin, M., Matthes, H.W., Simonin, F., Befort, K., Gaveriaux-Ruff, C., Dierich, A., LeMeur, M., Valverde, O., Maldonado, R. and Kieffer, B.L. (2000) Mice deficient for delta- and mu-opioid receptors exhibit opposing alterations of emotional responses. Nat. Genet., 25: 195-200. Fontaine, R., Chouinard, G. and Annable, L. (1984) Rebound anxiety in anxious patients after abrupt withdrawal of benzodiazepine treatment. Am. J. Psychiatry, 141: 848-852. Fontana, F., Bernardi, P., Pich, E.M., Boschi, S., De Iasio, R., Spampinato, S. and Grossi, G. (1997) Opioid peptide modulation of circulatory and endocrine response to mental stress in humans. Peptides, 18: 169-175.
576 Fontana, F., Bernardi, P., Pich, E.M., Boschi, S., De Iasio, R. and Spampinato, S. (1998) Endogenous opioid peptides and mental stress in congestive heart failure patients. Peptides, 19: 21-26. Gallagher, M., Kapp, B.S. and Pascoe, J.P. (1982) Enkephalin analogue effects in the amygdala central nucleus on conditioned heart rate. Pharmacol. Biochem. Behav., 17: 217-222. Gallego, M., Casis, L. and Casis, O. (1998) Imipramine inhibits soluble enkephalin-degrading aminopeptidase activity in vitro. Eur. J. Pharmacol., 360:113-116. Ge, Y., Lundeberg, T. and Yu, L.C. (2002) Blockade effect of mu and kappa opioid antagonists on the anti-nociception induced by intra-periaqueductal grey injection of oxytocin in rats. Brain Res., 927: 204-207. Gelsema, A.J., McKitrick, D.J. and Calaresu, F.R. (1987) Cardiovascular responses to chemical and electrical stimulation of amygdala in rats. Am. J. Physiol., 253: R712-R718. Giardino, L., Bettelli, C., Pozza, M. and Calza, L. (1999) Regulation of CCK mRNA expression in the rat brain by stress and treatment with sertraline, a selective serotonin re-uptake inhibitor. Brain Res., 824: 304-307. Good, A.J. and Westbrook, R.F. (1995) Effects of a microinjection of morphine into the amygdala on the acquisition and expression of conditioned fear and hypoalgesia in rats. Behav. Neurosci., 109:631-641. Goodwin, G.A. and Barr, G.A. (1997) Evidence for opioid and nonopioid processes mediating adaptive responses of infant rats that are repeatedly isolated. Dev. Psychobiol., 31: 217-227. Goodwin, G.M., Austin, M.P., Curran, S.M., Ross, M., Murray, C., Prentice, N., Ebmeier, K.P., Bennie, J., Carroll, S. and Dick, H. et al. (1993) The elevation of plasma beta-endorphin levels in major depression. J. Affect. Disord., 29: 281-289. Grewal, S.S., Shepherd, J.K., Bill, D.J., Fletcher, A. and Dourish, C.T. (1997) Behavioural and pharmacological characterisation of the canopy stretched attend posture test as a model of anxiety in mice and rats. Psychopharmacology (Berl), 133: 29-38. Grossman, A. (1986) Opioids and stress: the role of ACTH and epinephrine. In: Tach6, Y. and Cantin, M. (Eds.), Neuropeptides and Stress. Springer-Verlag, New York, pp. 313-324. Guthrie, J. and Basbaum, A. (1984) Colocalization of immunoreactive proenkephalin and prodynorphin products in medullary neurons of the rat. Neuropeptides, 4: 437-445. Harbuz, M. and Lightman, S. (1989) Responses of hypothalamic and pituitary mRNA to physical and psychological stress in the rat. J. Endocrinol., 122: 705-711. Harbuz, M., Russell, J., Summer, B., Kawata, M. and Lightman, S. (1991) Rapid changes in the content of proenkephalin A and corticotrophin releasing hormone mRNAs in the paraventricular nucleus during morphine withdrawal in urethane-anaesthetized rats. Mol. Brain Res., 9: 285-291.
Harlan, R.E., Shivers, B.D., Romano, G.J., Howells, R.D. and Pfaff, D.W. (1987) Localization of proenkephalin mRNA in the rat brain and spinal cord by in situ hybridization. J. Comp. Neurol., 258: 159-184. Hayden-Hixson, D.M. and Nemeroff, C.B. (1993) Role(s) of neuropeptides in responding and adaptation to stress: a focus on corticotropin-releasing factor and opioid peptides. In: Stanford, S.C. and Salmon, P. (Eds.), Stress, from Stress to Syndrome. Academic Press, London, pp. 356-391. He, X.P., Chen, B.Y., Zhu, J.M. and Cao, X.D. (1989) Change of Leu-enkephalin- and B-endorphin-like immunoreactivity in the hippocampus after electroconvulsive shock and electroacupuncture. Acupunct. Electrother. Res., 14: 131-139. Hebb, A.L.O., Mendella, P.D. and Zacharko, R.M. (1997) Immediate delta-receptor activation following stressor imposition influences behavioral reactivity to a subsequent stressful encounter. Soc. Neurosci. Abst., 23: 1857. Hebb, A.L., Zacharko, R.M., Dominguez, H., Trudel, F., Laforest, S. and Drolet, G. (2002a) Odor-induced variation in anxiety-like behavior in mice is associated with discrete and differential effects on mesocorticolimbic cholecystokinin mRNA expression. Neuropsychopharmacology, 27: 744-755. Hebb, A.L.O., Lemerrer, J., Laforest, S., Zacharko, R.M. and Drolet, G. (2002b) Exposure to the predator odor (TMT) enhances mesocorticolimbic neuronal activation and enkephalin mRNA expression associated with anxiety in mice. In: Program No, 670.15. 2002 Abstract Viewer/Itinerary Planner. Society for Neuroscience, Washington, DC, Online. Heinsbroek, R.P., van Haaren, F. and van de Poll, N.E. (1988) Sex differences in passive avoidance behavior of rats: sexdependent susceptibility to shock-induced behavioral depression. Physiol. Behav., 43: 201-206. Hernandez, L.L. and Watson, K.L. (1997) Opioid modulation of attention-related responses: delta-receptors modulate habituation and conditioned bradycardia. Psychopharmacology (Berl), 131: 140-147. Hernandez, L.L., Watson, K.L., Fowler, B.M., Bair, K.D. and Singha, A.K. (1997)Opioid modulation of attention-related responses: peripheral-to-central progression and development of mu influence as learning occurs. Psychopharmacology (Berl), 132: 50-60. Holaday, J.W. (1983) Cardiovascular effects of endogenous opiate systems. Ann. Rev. Pharmacol. Toxicol., 23: 541-594. Holaday, J.W., Tortella, F.C., Long, J.B., Belenky, G.L. and Hitzemann, R.J. (1986) Endogenous opioids and their receptors. Evidence for involvement in the postictal effects of electroconvulsive shock. Ann. N.Y. Acad. Sci., 462: 124-139. Hong, J.S., Gillin, J.C., Yang, H.Y. and Costa, E. (1979) Repeated electroconvulsive shocks and the brain content of endorphins. Brain Res., 177: 273-278. Horvath, G. (2000) Endomorphin-1 and endomorphin-2: pharmacology of the selective endogenous mu-opioid receptor agonists. Pharmacol. Ther., 88: 437-463.
577 Hotsenpiller, G. and Williams, J.L. (1997) A synthetic predator odor (TMT) enhances condiotioned analgesia and fear when paired with a benzodiazepine receptor inverse agonist (FG-7142). Psychobiology, 25: 83-88. Howlett, T.A. and Rees, L.H. (1986) Endogenous opioid peptides and hypothalamo-pituitary function. Ann. Rev. Physiol., 48: 527-536. Hurd, Y.L. (1996) Differential messenger RNA expression of prodynorphin and proenkephalin in the humain brain. Neuroscience, 72: 767-783. Ibarra, P., Bruehl, S.P., McCubbin, J.A., Carlson, C.R., Wilson, J.F., Norton, J.A. and Montgomery, T.B. (1994) An unusual reaction to opioid blockade with naltrexone in a case of post-traumatic stress disorder. J. Trauma. Stress, 7: 303-309. Imperato, A., Puglisi-Allegra, S., Grazia Scrocco, M., Casolini, P., Bacchi, S. and Angelucci, L. (1992) Cortical and limbic dopamine and acetylcholine release as neurochemical correlates of emotional arousal in both aversive and non-aversive environmental changes. Neurochem. Int., 20 Suppl.: 265S-270S. Izumi, T. (1998) Behavioral and neurochemical study on the role of the brain cholecystokinin system in anxiety. Hokkaido Igaku Zasshi, 73: 463-473. Jamner, L.D. and Leigh, H. (1999) Repressive/defensive coping, endogenous opioids and health: how a life so perfect can make you sick. Psychiatry Res., 85:17-31. Janssen, S.A. and Arntz, A. (1996) Anxiety and pain: attentional and endorphinergic influences. Pain, 66: 145-150. Janssen, S.A. and Arntz, A. (1997) No evidence for opioidmediated analgesia induced by phobic fear. Behav. Res. Ther., 35: 823-830. Janssens, C.J.J.G., Helmond, F.A., Loyens, L.W.S., Schouten, W.G.P. and Wiegant, V.M. (1995) Chronic stress increases the opioid-mediated inhibition of the pituitaryadrenocortical response to acute stress in pigs. Endocrinology, 136: 1468-1473. Jinks, A.L. and McGregor, I.S. (1997) Modulation of anxietyrelated behaviours following lesions of the prelimbic or infralimbic cortex in the rat. Brain Res., 772: 181-190. Kalin, N.H., Shelton, S.E. and Barksdale, C.M. (1988) Opiate modulation of separation-induced distress in non-human primates. Brain Res., 440: 285-292. Kalivas, P.W. (1993) Neurotransmitter regulation of dopamine neurons in the ventral tegmental area. Brain Res. Rev., 18: 75-113. Kalivas, P.W., Duffy, P., Dilts, R. and Abhold, R. (1988) Enkephalin modulation of A10 dopamine neurons: a role in dopamine sensitization. Ann. N.Y. Acad. Sci., 537: 405-414. Kalyuzhny, A.E. and Wessendorf, M.W. (1997) CNS GABA neurons express the mu-opioid receptor: immunocytochemical studies. Neuroreport., 8: 3367-3372. Kang, W., Wilson, S.P. and Wilson, M.A. (1999) Changes in nociceptive and anxiolytic responses following herpes
virus-mediated preproenkephalin overexpression in rat amygdala are naloxone-reversible and transient. Ann. N.Y. Acad. Sci., 877: 751-755. Kang, W., Wilson, S.P. and Wilson, M.A. (2000) Overexpression of proenkephalin in the amygdala potentiates the anxiolytic effects of benzodiazepines. Neuropsychopharmacology, 22: 77-88. Katoh, A., Nabeshima, T. and Kameyama, T. (1990) Behavioral changes induced by stressful situations: effects of enkephalins, dynorphin, and their interactions. J. Pharmacol. Exp. Ther., 253: 600-607. Katoh, A., Nabeshima, T. and Kameyama, T. (1991) Interaction between enkephalinergic and dopaminergic systems in stressful situations. Eur. J. Pharmacol., 193: 95-99. Katoh, A., Nabeshima, T., Ukai, R. and Kameyama, T. (1992) Endorphins: do not affect behavioral stress responses in mice. Peptides, 13: 737-739. Kavaliers, M. (1988) Brief exposure to a natural predator, the short-tailed weasel, induces benzodiazepine-sensitive analgesia in white-footed mice. Physiol. Behav., 43: 187-193. Kavaliers, M. and Choleris, E. (1997) Sex differences in Nmethyl-D-aspartate involvement in kappa opioid and nonopioid predator-induced analgesia in mice. Brain Res., 768: 30-36. Kavaliers, M., Colwell, D.D. and Perrot-Sinal, T.S. (1997) Opioid and non-opioid NMDA-mediated predator-induced analgesia in mice and the effects of parasitic infection. Brain Res., 766:11-18. Kelly, P.A., Ford, I. and McCulloch, J. (1986) The effect of diazepam upon local cerebral glucose use in the conscious rat. Neuroscience, 19: 257-265. Kemble, E.D. and Bolwahnn, B.L. (1997) Immediate and longterm effects of novel odors on risk assessment in mice. Physiol. Behav., 61: 543-549. Kessing, L.V., Mortensen, P.B. and Bolwig, T.G. (1998) Clinical definitions of sensitisation in affective disorder: a case register study of prevalence and prediction. J. Affect. Disord., 47: 31-39. Khachaturian, H., Lewis, M. and Watson, S.J. (1983) Enkephalin systems in the diencephalon and brainstem of the rat. J. Comp. Neurol., 220: 310-320. Kieffer, B.L. (1999) Opioids: first lessons from knockout mice. Trends Pharmacol. Sci., 20: 19-26. Kim, J.J. and Yoon, K.S. (1998) Stress: metaplastic effects in the hippocampus. Trends Neurosci., 21: 505-509. Kiritsy-Roy, J., Appel, N., Bobbitt, F. and Van Loon, G. (1986) Effects of mu-opioid receptor stimulation in the hypothalamic paraventricular nucleus on basal and stressinduced catecholamine secretion and cardiovascular responses. J. Pharmacol. Exp. Toxicol., 239: 814-822. Klein, L.C., Popke, E.J. and Grunberg, N.E. (1998) Sex differences in effects of opioid blockade on stress-induced freezing behavior. Pharmacol. Biochem. Behav., 61: 413-417.
578 Kollack-Walker, S. and Newman, S.W. (1995) Mating and agonistic behavior produce different patterns of Fos immunolabeling in the male Syrian hamster brain. Neuroscience, 66:721-736. Konig, M., Zimmer, A.M., Steiner, H., Holmes, P.V., Crawley, J.N., Brownstein, M.J. and Zimmer, A. (1996) Pain responses, anxiety and aggression in mice deficient in pre- proenkephalin. Nature, 383: 535-538. Korn, M.L., Plutchik, R. and Van Praag, H.M. (1997) Panicassociated suicidal and aggressive ideation and behavior. J. Psychiatr. Res., 31: 481-487. Kunos, G. and Varga, K. (1995) The tachycardia associated with the defense reaction involves activation of both GABAA and GABAB receptors in the nucleus tractus solitarii. Clin. Exp. Hypertens., 17: 91-100. Kurumaji, A., Mitsushio, H. and Takashima, M. (1988) Chronic dietary treatment with antidepressants decrease brain Met-enkephalin-like immunoreactivity in the rat. Psychopharmacology (Berl), 94: 188-192. Lason, W., Przewlocka, B. and Przewlocki, R. (1987) Single and repeated electroconvulsive shock differentially affects the prodynorphin and pro-opiomelanocortin system in the rat. Brain Res., 403: 301-307. Lawrence, D. and Pittman, Q.J. (1985) Interaction between descending paraventricular neurons and vagal motor neurons. Brain Res., 332: 158-160. Lester, L.S. and Fanselow, M.S. (1985) Exposure to a cat produces opioid analgesia in rats. Behav. Neurosci., 99: 756-759. Liberzon, I., Zubieta, J.K., Fig, L.M., Phan, K.L., Koeppe, R.A. and Taylor, S.F. (2002) mu-Opioid receptors and limbic responses to aversive emotional stimuli. Proc. Natl. Acad. Sci. USA, 99: 7084-7089. Lichtman, A.H. and Fanselow, M.S. (1990) Cats produce analgesia in rats on the tail-flick test: naltrexone sensitivity is determined by the nociceptive test stimulus. Brain Res., 533: 91-94. Lightman, S.L. and Young, W.S.I. (1987a) Changes in hypothalamic preproenkephalin A mRNA following stress and opiate withdrawal. Nature, 328: 643-645. Lightman, S.L. and Young, W.S.I. (1987b) Vasopressin, oxytocin, dynorphin, enkephalin and corticotrophin-releasing factor mRNA stimulation in the rat. J. Physiol. (London), 394: 23-39. Lightman, S.L. and Young, W.S.I. (1988) Corticotrophinreleasing factor, vasopressin and pro-opiomelanocortin mRNA responses to stress and opiates in the rat. J. Physiol. (London), 403: 511-523. Lightman, S.L. and Young, W.S.I. (1989) Influence of steroids on the hypothalamic corticotropin-releasing factor and preproenkephalin mRNA responses to stress. Proc. Natl. Acta. Sci. USA, 86: 4306-4310. Loas, G. (1996) Vulnerability to depression: a model centered on anhedonia. J. Affect. Disord., 41: 39-53.
MacNeil, G., Sela, Y., McIntosh, J. and Zacharko, R.M. (1997) Anxiogenic behavior in the light-dark paradigm following intraventricular administration of cholecystokinin-8S, restraint stress, or uncontrollable footshock in the CD-1 mouse. Pharmacol. Biochem. Behav., 58: 737-746. Maier, S.F. (1984) Learned helplessness and animal models of depression. Prog. Neuropsychopharmacol. Biol. Psychiatry, 8:435-446. Mansi, J.A., Laforest, S. and Drolet, G. (2000) Effect of stress exposure on the activation pattern of enkephalin-containing perikarya in the rat ventral medulla. J. Neurochem., 74: 2568-2575. Mansour, A., Khachaturian, H., Lewis, M., Akil, H. and Watson, S. (1987) Autoradiographic differentiation of mu, delta, and kappa opioid receptors in the rat forebrain and midbrain. J. Neurosci., 7: 2445-2464. Mansour, A., Khachaturian, H., Lewis, M.E., Akil, H. and Watson, S.J. (1988) Anatomy of CNS opioid receptors. Trends Neurosci., 11:308-314. Mansour, A., Fox, C.A., Burke, S., Meng, F., Thompson, R.C., Akil, H. and Watson, S.J. (1994) Mu, delta and kappa opioid receptor mRNA expression in the rat CNS: an in situ hybridization study. J. Comp. Neurol., 350: 412-438. Mansour, A., Fox, C.A., Akil, H. and Watson, S.J. (1995) Opioid-receptor mRNA expression in the rat CNS: anatomical and functional implications. Trends Neurosci., 18: 22-29. Maremmani, I., Marini, G. and Fornai, F. (1998) Naltrexoneinduced panic attacks. Am. J. Psychiatry, 155: 447. Marson, L., JA, K.-R. and Van, L.G. (1989) ~t-Opioid peptide modulation of cardiovascular and sympathoadrenal responses to stress. Am. J. Physiol., 257: R901-R908. Martin-Schild, S., Gerall, A.A., Kastin, A.J. and Zadina, J.E. (1999) Differential distribution of endomorphin 1- and endomorphin 2-like immunoreactivities in the CNS of the rodent. J. Comp. Neurol., 405: 450-471. Martinez, J.L., Jr. and Weinberger, S.B. (1987) Memory traces: how to increase and decrease their strength. Int. J. Neurol., 21-22: 33-50. Martinez, J.L., Jr., Weinberger, S.B. and Schulteis, G. (1988) Enkephalins and learning and memory: a review of evidence for a site of action outside the blood-brain barrier. Behav. Neural Biol., 49:192-221. Martinez, M., Phillips, P.J. and Herbert, J. (1998) Adaptation in patterns of c-fos expression in the brain associated with exposure to either single or repeated social stress in male rats. Eur. J. Neurosci., 10: 20-33. Masure, C.M. (1994) Stress and Psychiatric Disorders. American Psychiatric Press, Washington. Matsuzawa, S., Suzuki, T., Misawa, M. and Nagase, H. (1999) Different roles of mu-, delta- and kappa-opioid receptors in ethanol-associated place preference in rats exposed to conditioned fear stress. Eur. J. Pharmacol., 368: 9-16.
579 McCubbin, J.A. (1993) Stress and endogenous opioids: behavioral and circulatory interactions. Biol. Psychol., 35: 91-122. McCubbin, J., Bruehl, S., Wilson, J., Sherman, J., Norton, J. and Colclough, G. (1998) Endogenous opioids inhibit ambulatory blood pressure during naturally occurring stress. Psychosomatic Medicine, 60: 227-231. McEwen, B.S. and Sapolsky, R.M. (1995) Stress and cognitive function. Curr. Opin. Neurobiol., 5: 205-216. McFall, M., Fontana, A., Raskind, M. and Rosenheck, R. (1999) Analysis of violent behavior in Vietnam combat veteran psychiatric inpatients with posttraumatic stress disorder. J. Trauma Stress, 12:501-517. McGivern, R.F., Mousa, S., Couri, D. and Berntson, G.G. (1983) Prolonged intermittent footshock stress decreases Met and Leu enkephalin levels in brain with concomitant decreases in pain threshold. Life Sci., 33: 47-54. McGregor, I.S. and Dielenberg, R.A. (1999) Differential anxiolytic efficacy of a benzodiazepine on first versus second exposure to a predatory odor in rats. Psychopharmacology (Berl), 147: 174-181. Menetrey, D. and Basbaum, A.I. (1987) The distribution of substance P-, enkephalin- and dynorphin-immunoreactive neurons in the medulla of the rat and their contribution to bulbospinal pathways. Neuroscience, 23: 173-187. Metalsky, G.I. and Joiner, T.E., Jr. (1992) Vulnerability to depressive symptomatology: a prospective test of the diathesis-stress and causal mediation components of the hopelessness theory of depression. J. Pets. Soc. Psychol., 63: 667-675. Meunier, J.C., Mollereau, C., Toll, L., Suaudeau, C., Moisand, C., Alvinerie, P., Butour, J.L., Guillemot, J.C., Ferrara, P. and Monsarrat, B. et al. (1995) Isolation and structure of the endogenous agonist of opioid receptor-like ORL1 receptor. Nature, 377: 532-535. Mogil, J.S. and Pasternak, G.W. (2001) The molecular and behavioral pharmacology of the orphanin FQ/nociceptin peptide and receptor family. Pharmacol. Rev., 53: 381-415. Molewijk, H.E., van der Poel, A.M. and Olivier, B. (1995) The ambivalent behaviour '~stretched approach posture" in the rat as a paradigm to characterize anxiolytic drugs. Psychopharmacology (Berl), 121: 81-90. Morgan, M.A. and LeDoux, J.E. (1995) Differential contribution of dorsal and ventral medial prefrontal cortex to the acquisition and extinction of conditioned fear in rats. Behav. Neurosci., 109:681-688. Morgan, M.A., Romanski, L.M. and LeDoux, J.E. (1993) Extinction of emotional learning: contribution of medial prefrontal cortex. Neurosci. Lett., 163: 109-113. Morilak, D.A., Drolet, G. and Chalmers, J. (1990a) Cardiovascular effects of the opioid antagonist naloxone in the rostral ventrolateral medulla of rabbits. Am. J. Physiol., 258:R325-R331.
Morilak, D.A., Drolet, G. and Chalmers, J. (1990b) Tonic opioid inhibition of the pressor region of the rostral ventrolateral medulla of rabbits is mediated by delta receptors. J. Pharmacol. Exp. Toxicol., 254: 671-676. Moynihan, J.A., Karp, J.D., Cohen, N. and Ader, R. (2000) Immune deviation following stress odor exposure: role of endogenous opioids. J. Neuroimmunol., 102: 145-153. Nagy, L.M., Krystal, J.H., Charney, D.S., Merikangas, K.R. and Woods, S.W. (1993) Long-term outcome of panic disorder after short-term imipramine and behavioral group treatment: 2.9-year naturalistic follow-up study. J. Clin. Psychopharmacol., 13: 16-24. Nikulina, E.M., Hammer, R.P., Jr., Miczek, K.A. and Kream, R.M. (1999) Social defeat stress increases expression of mu-opioid receptor mRNA in rat ventral tegmental area. Neuroreport, 10:3015-3019. Nobre, M.J., Ribeiro dos Santos, N., Aguiar, M.S. and Brandao, M.L. (2000) Blockade of mu- and activation of kappa-opioid receptors in the dorsal periaqueductal gray matter produce defensive behavior in rats tested in the elevated plus-maze. Eur. J. Pharmacol., 404: 145-151. Olson, G.A., Olson, R.D., Kastin, A.J. and Coy, D.H. (1979) Endogenous opiates: through 1978. Neurosci. Biobehav. Rev., 3: 285-299. Olson, G.A., Olson, R.D., Kastin, A.J. and Coy, D.H. (1980) Endogenous opiates: 1979. Peptides, 1: 365-379. Olson, G.A., Olson, R.D., Kastin, A.J. and Coy, D.H. (1981) Endogenous opiates: 1980. Peptides, 2: 349-369. Olson, G.A., Ol,on, R.D., Kastin, A.J. and Coy, D.H. (1982) Endogenous opiates: 1981. Peptides, 3: 1039-1072. Olson, G.A., Olson, R.D. and Kastin, A.J. (1983) Endogenous opiates: 1982. Peptides, 4: 563-576. Olson, G.A., Olson, R.D. and Kastin, A.J. (1984) Endogenous opiates: 1983. Peptides, 5: 975-992. Olson, G.A., Olson, R.D. and Kastin, A.J. (1985) Endogenous oDates: 1984. Peptides, 6: 769-791. Olson, G.A., Olson, R.D. and Kastin, A.J. (1986) Endogenous opiates: 1985. Peptides, 7: 907-933. Olson, G.A., Olson, R.D. and Kastin, A.J. (1987) Endogenous oDates: 1986. Peptides, 8:1135-1164. Olson, G.A., Olson, R.D. and Kastin, A.J. (1989a) Endogenous opiates: 1987. Peptides, 10: 205-236. Olson, G.A., Olson, R.D. and Kastin, A.J. (1989b) Endogenous opiates: 1988. Peptides, 10: 1253-1280. Olson, G.A., Olson, R.D. and Kastin, A.J. (1990) Endogenous opiates: 1989. Peptides, 11: 1277-1304. Olson, G.A., Olson, R.D. and Kastin, A.J. (1991) Endogenous oDates: 1990. Peptides, 12: 1407-1432. Olson, G.A., Olson, R.D. and Kastin, A.J. (1992) Endogenous opiates: 1991. Peptides, 13: 1247-1287. Olson, G.A., Olson, R.D. and Kastin, A.J. (1993) Endogenous opiates: 1992. Peptides, 14: 1339-1378. Olson, G.A., Olson, R.D. and Kastin, A.J. (1994) Endogenous opiates: 1993. Peptides, 15: 1513-1556.
580 Olson, G.A., Olson, R.D. and Kastin, A.J. (1995) Endogenous opiates: 1994. Peptides, 16: 1517-1555. Olson, G.A., Olson, R.D. and Kastin, A.J. (1996) Endogenous opiates: 1995. Peptides, 17:1421-1466. Olson, G., Olson, R. and Kastin, A. (1997) Endogenous opiates: 1996. Peptides, 18: 1651-1688. Olson, G.A., Olson, R.D., Vaccarino, A.L. and Kastin, A.J. (1998) Endogenous opiates: 1997. Peptides, 19: 1791-1843. Otto, M.W., Pollack, M.H., Sachs, G.S., Reiter, S.R., MeltzerBrody, S. and Rosenbaum, J.F. (1993) Discontinuation of benzodiazepine treatment: efficacy of cognitive-behavioral therapy for patients with panic disorder. Am. J. Psychiatry., 150:1485-1490. Palkovits, M. (1987) Organization of the stress response at the anatomical level. Prog. Brain Res., 72: 47-55. Palkovits, M., Mezey, E. and Eskay, R.L. (1987) Distribution and possible origin of b-endorphin and ACTH in discrete brainstem nuclei of rats. Neuropeptides, 9: 123-137. Pan, Z.Z. (1998) mu-Opposing actions of the kappa-opioid receptor. Trends Pharmacol. Sci., 19: 94-98. Pechnick, R.N. (1993) Effects of opioids on the hypothalamopituitary-adrenal axis. Ann. Rev. Pharmacol. Toxicol., 33: 353-382. Petrovich, G.D., Scicli, A.P., Thompson, R.F. and Swanson, L.W. (2000) Associative fear conditioning of enkephalin mRNA levels in central amygdalar neurons. Behav. Neurosci., 114: 681-686. Petrusz, P., Merchenthaler, I. and Maderdrut, J.L. (1985) Distribution of enkephalin-containing neurons in the central nervous system. In: Bj6rklund, A. and H6kfelt, T., (Eds.), Handbook of Chemical Neuroanatomy. Elsevier Science, Amsterdam, pp. 273-334. Piva, M.A., Medina, J.H., de Blas, A.L. and Pena, C. (1991) Formation of benzodiazepine-like molecules in rat brain. Biochem. Biophys. Res. Commun., 180:972-981. Plata-Salaman, C.R., Ilyin, S.E., Turrin, N.P., Gayle, D., Flynn, M.C., Bedard, T., Merali, Z. and Anisman, H. (2000) Neither acute nor chronic exposure to a naturalistic (predator) stressor influences the interleukin-lbeta system, tumor necrosis factor-alpha, transforming growth factorbeta l, and neuropeptide mRNAs in specific brain regions. Brain Res. Bull., 51: 187-193. Pohorecky, L.A., Skiandos, A., Zhang, X., Rice, K.C. and Benjamin, D. (1999) Effect of chronic social stress on deltaopioid receptor function in the rat. J. Pharmacol. Exp. Ther., 290: 196-206. Porteous, A. and Tyndall, J. (1994) Yes, I want to walk to the OR. Can. Oper. Room Nurs. J., 12: 15-25. Privette, T.H. and Terrian, D.M. (1995) Kappa opioid agonists produce anxiolytic-like behavior on the elevated plus-maze. Psychopharmacology (Berl), 118: 444-450. Przewlocki, R., Przewlocka, B. and Lason, W. (1991) Adaptation of opioid systems to stress. In: Almeida, O.F.X.
and Shippenberg, T.S. (Eds.), The Neurobiology of Opioid Systems to Stress. Springer-Verlag, Berlin, pp. 229-243. Przewlocki, R., Lason, W., Turchan, J. and Przewlocka, B. (1997) Imipramine induces alterations in proenkephalin and prodynorphin mRNAs level in the nucleus accumbens and striatum in the rat. Pol. J. Pharmacol., 49: 351-355. Quirion, R. and Pert, C.B. (1981) Dynorphins: similar relative potencies on mu, delta- and kappa-opiate receptors. Eur. J. Pharmacol., 76: 467-468. Ragnauth, A., Schuller, A., Morgan, M., Chan, J., Ogawa, S., Pintar, J., Bodnar, R.J. and Pfaff, D.W. (2001) Female preproenkephalin-knockout mice display altered emotional responses. Proc. Natl. Acad. Sci. USA, 98: 1958-1963. Ray, A. and Henke, P.G. (1990) Enkephalin-dopamine interactions in the central amygdalar nucleus during gastric stress ulcer formation in rats. Behav. Brain Res., 36: 179-183. Ray, A., Henke, P.G. and Sullivan, R.M. (1988) Opiate mechanisms in the central amygdala and gastric stress pathology in rats. Brain Research, 23: 195-198. Reinscheid, R.K. and Civelli, O. (2002) The orphanin FQ/ nociceptin knockout mouse: a behavioral model for stress responses. Neuropeptides, 36: 72-76. Reinscheid, R.K., Nothacker, H.P., Bourson, A., Ardati, A., Henningsen, R.A., Bunzow, J.R., Grandy, D.K., Langen, H., Monsma, F.J., Jr. and Civelli, O. (1995) Orphanin FQ: a neuropeptide that activates an opioidlike G protein-coupled receptor. Science, 270: 792-794. Risch, S.C. (1997) Recent advances in depression research: from stress to molecular biology and brain imaging. J. Clin. Psychiatry, 58 Suppl. 5: 3-6. Rodgers, R.J. and File, S.E. (1979) Exploratory behaviour and aversive thresholds following intra-amygdaloid application of opiates in rats. Pharmacol. Biochem. Behav., 11:505-511. Rodgers, R.J. and Randall, J.I. (1987) Social conflict analgesia: inhibition of early non-opioid component by diazepam or flumazepil fails to affect appearance of late opioid component. Brain Res. Bull., 19: 141-144. Roozendaal, B., Koolhaas, J.M. and Bohus, B. (1991) Attenuated cardiovascular, neuroendocrine, and behavioral responses after a single footshock in central amygdaloid lesioned male rats. Physiol. Behav., 50: 771-775. Rosen, J.B., Fanselow, M.S., Young, S.L., Sitcoske, M. and Maren, S. (1998) Immediate-early gene expression in the amygdala following footshock stress and contextual fear conditioning. Brain Res., 796: 132-142. Russell, J.A. and Douglas, A.J. (2000) Opioids. In: Fink, G., (Ed.), Encyclopedia of Stress. Academic Press, San Diego, pp. 86-98 Sante, A.B., Nobre, M.J. and Brandao, M.L. (2000) Place aversion induced by blockade of mu or activation of kappa opioid receptors in the dorsal periaqueductal gray matter. Behav. Pharmacol., 11: 583-589. Sapolsky, R.M. (1996) Why stress is bad for your brain. Science, 273: 749-750.
581 Sawchenko, P.E. (1986) Neuropeptides, the paraventricular nucleus and the integration of hypothalamic neuroendocrine and autonomic function. In: TachS, Y. and Cantin, M. (Eds.), Neuropeptides and Stress. Springer-Verlag, New York, pp. 73-91. Sawchenko, P.E., Imaki, T., Potter, E., Kovacs, K., Imaki, J. and Vale, W. (1993) The functional neuroanatomy of corticotropin-releasing factor. In: Chadwick, D.J., Marsh, J. and Ackrill, K. (Eds.), Corticotropin-Releasing Factor. John Wiley & Sons Ltd., Chichester, pp. 5-29. Schafer, M.K.-H., Day, R., Watson, S.J. and Akil, H. (1991) In: Almeida, O.F.X. and Shippenberg, T.S. (Eds.), Neurobiology of Opioids. Springer-Verlag, Berlin, pp. 53-71. Schulteis, G. and Martinez, J.L., Jr. (1992) Peripheral modulation of learning and memory: enkephalins as a model system. Psychopharmacology (Berl), 109: 347-364. Schulteis, G. and Martinez, J.L., Jr. (1993) Experiencedependent regulation of Leu-enkephalin hydrolysis in rat plasma. Peptides, 14: 161-167. Shaikh, M.B., Lu, C.L. and Siegel, A. (1991a) An enkephalinergic mechanism involved in amygdaloid suppression of affective defence behavior elicited from the midbrain periaqueductal gray in the cat. Brain Res., 559: 109-117. Shaikh, M.B., Lu, C.L. and Siegel, A. (1991b) Affective defense behavior elicited from the feline midbrain periqueductal gray is regulated by mu and delta opioid receptors. Brain Res., 557: 344-348. Shear, M.K., Ball, G., Fitzpatrick, M., Josephson, S., Klosko, J. and Frances, A. (1991) Cognitive-behavioral therapy for panic: an open study. J. Nerv. Ment. Dis., 179: 468-472. Sheline, Y.I., Gado, M.H. and Price, J.L. (1998) Amygdala core nuclei volumes are decreased in recurrent major depression. Neuroreport, 9: 2023-2028. Sheline, Y.I., Sanghavi, M., Mintun, M.A. and Gado, M.H. (1999) Depression duration but not age predicts hippocampal volume loss in medically healthy women with recurrent major depression. J. Neurosci., 19: 5034-5043. Shepherd, J.K., Blanchard, D.C., Weiss, S.M., Rodgers, R.J. and Blanchard, R.J. (1992) Morphine attenuates antipredator ultrasonic vocalizations in mixed-sex rat colonies. Pharmacol. Biochem. Behav., 41: 551-558. Siegel, R.A., Duker, E.M., Fuchs, E., Pahnke, U. and Wutte, W. (1984) Responsiveness of mesolimbic, mesocortical, septal, and hippocampal cholecystokinin and substance P neuronal systems to stress in the male rat. Neurochem. Int., 6: 783-789. Siegel, A., Schubert, K.L. and Shaikh, M.B. (1997) Neurotransmitters regulating defensive rage behavior in the cat. Neurosci. Biobehav. Rev., 21: 733-742. Soltis, R.P., Cook, J.C., Gregg, A.E. and Sanders, B.J. (1997) Interaction of GABA and excitatory amino acids in the basolateral amygdala: role in cardiovascular regulation. J. Neurosci., 17: 9367-9374.
Southwick, S.M., Paige, S., Morgan, C.A., 3rd, Bremner, J.D., Krystal, J.H. and Charney, D.S. (1999) Neurotransmitter alterations in PTSD: catecholamines and serotonin. Semin Clin. Neuropsychiatry, 4: 242-248. Spiegel, D.A., Bruce, T.J., Gregg, S.F. and Nuzzarello, A. (1994) Does cognitive behavior therapy assist slow-taper alprazolam discontinuation in panic disorder? Am. J. Psychiatry, 151:876-881. Sribanditmongkol, P., Sheu, M.J. and Tejwani, G.A. (1994) Inhibition of morphine tolerance and dependence by diazepam and its relation to the CNS Met-enkephalin levels. Brain Res., 645: 1-12. Stimmel, G.L. (1995) How to counsel patients about depression and its treatment. Pharmacotherapy, 15: 100S-104S. Strack, A.M., Sawyer, W.B., Platt, K.B. and Loewy, A.D. (1989a) CNS cell groups regulating the sympathetic outflow to adrenal gland as revealed by transneuronal cell body labeling with pseudorabies virus. Brain Res., 491: 274-296. Strack, A.M., Sawyer, W.B., Hughes, J.H., Platt, K.B. and Loewy, A.D. (1989b) A general pattern of CNS innervation of the sympathetic outflow demonstrated by transneuronal pseudorabies viral infections. Brain Res., 491:156-162. Stuckey, J., Marra, S., Minor, T. and Insel, T.R. (1989) Changes in mu opiate receptors following inescapable shock. Brain Res., 476: 167-169. Sun, S.Y., Liu, Z., Li, P. and Ingenito, A.J. (1996) Central effects of opioid agonists and naloxone on blood pressure and heart rate in normotensive and hypertensive rats. Gen. Pharmacol., 27:1187-1194. Sverko, V., Marotti, T., Rocic, B., Rabatic, S. and Hrsak, I. (1997) Influence of methionine-enkephalin on stress-induced parameters. Int. J. Immunopharmacology, 19: 691-698. Swanson, L.W., Sawchenko, P.E. and Lind, R.W. (1986) Regulation of multiple peptides in CRF parvocellular neurosecretory neurons: implications for the stress response. Prog. Brain Res., 169-190. Swanson, L.W., Sawchenko, P.E., Lind, R.W. and Rho, J.-H. (1987) The CRH motoneuron: differential peptide regulation in neurons with possible synaptic, paracrine and endocrine outputs. Ann. N.Y. Acad. Sci., 512: 12-23. Szekely, J.I. (1990) Opioid peptides and stress. Crit. Rev. Neurobiol., 6: 1-12. Tanaka, M., Ida, Y. and Tsuda, A. (1988) Naloxone, given before but not after stress exposure, enhances stress-induced increases in noradrenaline release in rats brain regions. Pharmacol. Biochem. Behav., 29:613-619. Tanaka, M., Ida, Y., Tsuda, A., Tsujimaru, S., Shiarao, I. and Oguchi, M. (1989) Met-enkephalin, injected during the early phase of stress, attenuates stress-induced increases in noradrenaline release in rat brain regions. Pharmacol. Biochem. Behav., 32: 791-795. Tanaka, M., Yoshida, M., Emoto, H. and Ishii, H. (2000) Noradrenaline systems in the hypothalamus, amygdala and
582 locus coeruleus are involved in the provocation of anxiety: basic studies. Eur. J. Pharmacol., 405: 397-406. Tejedor-Real, P., Mico, J.A., Maldonado, R., Roques, B.P. and Gibert-Rahola, J. (1993) Effect of mixed (RB 38A) and selective (RB 38B) inhibitors of enkephalin degrading enzymes on a model of depression in the rat. Biol. Psychiatry, 34: 100-107. Tejedor-Real, P., Mico, J.A., Maldonado, R., Roques, B.P. and Gibert-Rahola, J. (1995) Implication of endogenous opioid system in the learned helplessness model of depression. Pharmacol. Biochem. Behav., 52: 145-152. Tejedor-Real, P., Mico, J.A., Smadja, C., Maldonado, R., Roques, B.P. and Gilbert-Rahola, J. (1998) Involvement of delta-opioid receptors in the effects induced by endogenous enkephalins on learned helplessness model. Eur. J. Pharmacol., 354: 1-7. Tilson, H., McLamb, R. and Hong, J. (1986) Behavioral effects of centrally administered dynorphin and [D-ala2-D-leu] enkephalin (DADLE) in rats. Neuropeptides, 8: 193-206. Tsuda, M., Suzuki, T., Misawa, M. and Nagase, H. (1996) Involvement of the opioid system in the anxiolytic effect of diazepam in mice. Eur. J. Pharmacol., 307: 7-14. Uhl, G.R., Childers, S. and Pasternak, G. (1994) An opiatereceptor gene family reunion. Trends Neurosci., 17: 89-93. Ukai, M., Takada, A., Sasaki, Y. and Kameyama, T. (1997) Stimulation of delta l- and delta2-opioid receptors produces amnesia in mice. Eur. J. Pharmacol., 338: 1-6. Vaccarino, A.L. and Kastin, A.J. (2000) Endogenous opiates: 1999. Peptides, 21:1975-2034. Vaccarino, A.L. and Kastin, A.J. (2001) Endogenous opiates: 2000. Peptides, 22: 2257-2328. Vaccarino, A.L., Olson, G.A., Olson, R.D. and Kastin, A.J. (1999) Endogenous opiates: 1998. Peptides, 20: 1527-1574. VandenBerg, C., Lamberts, R., Wolterink, G., Wiegant, V. and VanRee, J. (1998) Emotional and footshock stimuli induce differential long-lasting behavioural effects in rats; involvement of opioids. Brain Research, 799: 6-15. van den Buuse, M. (1998) Role of the mesolimbic dopamine system in cardiovascular homeostasis. Stimulation of the ventral tegmental area modulates the effect of vasopressin on blood pressure in conscious rats. Clin. Exp. Pharmacol. Physiol., 25:661-668. Van Dijken, H.H., Van der Heyden, J.A., Mos, J. and Tilders, F.J. (1992) Inescapable footshocks induce progressive and long-lasting behavioural changes in male rats. Physiol. Behav., 51: 787-794. Vivian, J.A. and Miczek, K.A. (1998) Effects of mu and delta opioid agonists and antagonists on affective vocal and reflexive pain responses during social stress in rats. Psychopharmacology (Berl), 139: 364-375. Vochteloo, J.D. and Koolhaas, J.M. (1987) Medial amygdala lesions in male rats reduce aggressive behavior: interference with experience. Physiol. Behav., 41: 99-102.
Walker, D.L. and Davis, M. (1997) Double dissociation between the involvement of the bed nucleus of the stria terminalis and the central nucleus of the amygdala in startle increases produced by conditioned versus unconditioned fear. J. Neurosci., 17: 9375-9383. Wall, P.M. and Messier, C. (2000a) Concurrent modulation of anxiety and memory. Behav. Brain Res., 109: 229-241. Wall, P.M. and Messier, C. (2000b) U-69,593 microinjection in the infralimbic cortex reduces anxiety and enhances spontaneous alternation memory in mice. Brain Res., 856: 259-280. Watkins, L.R. and Mayer, D.J. (1986) Multiple endogenous opiate and non-opiate analgesia systems: evidence of their existence and clinical implications. Ann. N.Y. Acad. Sci., 467: 273-299. Watson, S.J., Khachaturian, H., Akil, H., Coy, D.H. and Goldstein, A. (1982) Comparaison of the distribution of dynorphin systems and enkephalin systems in brain. Science, 218: 1134-1136. Watts, A.G. (1991) Ether anesthesia differentially affects the content of prepro-corticotropin-releasing hormone, preproneurotensin/neuromedin N and prepro-enkephalin mRNAs in the hypothalamic paraventricular nucleus of the rat. Brain Res., 544: 353-357. Watts, A.G. (1992) Disturbance of fluid homeostasis leads to temporally and anatomically distinct responses in neuropeptide and tyrosine hydroxylase mRNA levels in the paraventricular and supraoptic nuclei of the rat. Neuroscience, 46: 859-879. Weiss, E.L., Longhurst, J.G. and Mazure, C.M. (1999) Childhood sexual abuse as a risk factor for depression in women: psychosocial and neurobiological correlates. Am. J. Psychiatry, 156:816-828. Westbrook, R.F., Good, A.J. and Kiernan, M.J. (1997) Microinjection of morphine into the nucleus accumbens impairs contextual learning in rats. Behav. Neurosci., 111: 996-1013. Westrin, A., Ekman, R. and Traskman-Bendz, L. (1999) Alterations of corticotropin releasing hormone (CRH) and neuropeptide Y (NPY) plasma levels in mood disorder patients with a recent suicide attempt. Eur. Neuropsychopharmacol., 9: 205-211. Wiedenmayer, C.P., Noailles, P.A., Angulo, J.A. and Barr, G.A. (2002) Stress-induced preproenkephalin mRNA expression in the amygdala changes during early ontogeny in the rat. Neuroscience, 114:7-11. Williams, S.L., Kinney, P.J. and Falbo, J. (1989) Generalization of therapeutic changes in agoraphobia: the role of perceived self-efficacy. J. Consult. Clin. Psychol., 57: 436-442. Wilson, M.A., Mascagni, F. and McDonald, A.J. (2002) Sex differences in delta opioid receptor immunoreactivity in rat medial amygdala. Neurosci. Lett., 328: 160-164. Wisniewska, R.J. and Wisniewski, K. (1996) The effect of cholecystokinin (CCK-33) and C-terminal fragments of
583 cholecystokinin: CCK-8 and CCK-4 on the cardiovascular system in rats. Gen. Pharmacol., 27: 159-163. Wuster, M., Rubini, P. and Schultz, R. (1981) The preference of putative pro-enkephalins for different types of opiate receptors. Life Sci., 29: 1219-1227. Yamada, K. and Nabeshima, T. (1995) Stress-induced behavioral responses and multiple opioid systems in the brain. Beh. Brain Res., 67: 133-145. Young, W.S.I. and Lightman, S.L. (1992) Chronic stress elevates enkephalin expression in the rat paraventricular and supraoptic nuclei. Mol. Brain Res., 13: 111-117. Zacharko, R.M. and Anisman, H. (1991) Stressor-induced anhedonia in the mesocorticolimbic system. Neurosci Biobehav. Rev., 15: 391-405. Zacharko, R.M., Koszycki, D., Mendella, P.D. and Bradwejn, J. (1995) Behavioral, neurochemical, anatomical and electrophysiological correlates of panic disorder: multiple transmitter interaction and neuropeptide colocalization. Prog. Neurobiol., 47: 371-423. Zacharko, R.M., Maddeaux, C., Hebb, A.L., Mendella, P.D. and Marsh, N.J. (1998) Vulnerability to stressor-induced disturbances in self-stimulation from the dorsal and ventral A10 area: differential effects of intraventricular
D-Ala2-Met5-enkephalinamide, D-Ala2, N-Me-Phe4, GlyO15-enkephalin, and D-Pen2, D-Pen5-enkephalin administration. Brain Res. Bull., 47: 237-248. Zadina, J.E., Hackler, L., Ge, L.J. and Kastin, A.J. (1997) A potent and selective endogenous agonist for the mu-opiate receptor. Nature, 386: 499-502. Zadina, J.E., Martin-Schild, S., Gerall, A.A., Kastin, A.J., Hackler, L., Ge, L.J. and Zhang, X. (1999) Endomorphins: novel endogenous mu-opiate receptor agonists in regions of high mu-opiate receptor density. Ann. N.Y. Acad. Sci., 897: 136-144. Zhang, H.T., Xu, Z.M., Luo, Z.P. and Qin, B.Y. (1996) Anxiogenic effect of naltrexone in social interaction test in rats. Zhongguo Yao Li Xue Bao, 17:314-317. Zis, A.P., Haskett, R.F., Albala, A.A., Carroll, B.J. and Lohr, N.E. (1985) Opioid regulation of hypothalamicpituitary-adrenal function in depression. Arch. Gen. Psychiatry, 42: 383-386. Zurita, A., Cuadra, G. and Molina, V.A. (1999) The involvement of an opiate mechanism in the sensitized behavioral deficit induced by early chronic variable stress: influence of desipramine. Behav. Brain Res., 100: 153-159.
This Page Intentionally Left Blank
T. Steckler, N.H. Kalin and J.M.H.M. Reul (Eds.) Handbook of Stress and the Brain, Vol. 15 ISBN 0-444-51173-3 Copyright 2005 Elsevier B.V. All rights reserved
CHAPTER 4.10
A c e t y l c h o l i n e s t e r a s e as a w i n d o w o n t o stress responses Hermona Soreq l'*, Raz Yirmiya 2, Osnat Cohen 3 and David Glick ~ 1Department of Biological Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel 2Department of Psychology, The Hebrew University of Jerusalem, Jerusalem, Israel 3Departments of Biological Chemistry and Psychology, The Hebrew University of Jerusalem, Jerusalem, Israel
Abstract: It has long been known that cholinergic neurotransmission is intimately associated with mammalian stress responses. Inhibition of acetylcholinesterase (ACHE), like stress, elevates the levels of acetylcholine (ACh) in the short term, and both conditions induce some common long-lasting behavioral symptoms. Therefore, AChE manipulations provide an interesting window onto stress responses. Like many other stimuli, both stress and inhibition of AChE cause an increase in AChE gene expression that is also associated with a shift in its pre-mRNA splicing pattern. Of the several variants of AChE that arise due to alternative splicing, it is specifically the usually rare, soluble AChE-R variant that is up-regulated. Transgenic mice that over-express AChE also show many of the same symptoms as stress: erratic behavior following circadian light/dark shift, progressive failure of learning and memory, intensified long-term potentiation (LTP), development of neuropathologies, progressive muscle fatigue and degeneration of neuromuscular junctions. Altered expression of other cholinergic proteins in these mice, e.g. protein kinase C13II choline acetyltransferase and the high affinity choline transporter, suggests chronic feedback responses to the cholinergic imbalance. Stress-associated characteristics can be ameliorated in mice and humans by treatment with antisense agents that induce selective destruction of AChE-R, which provides further support for changes in alternative splicing, and in particular the accumulation of this variant, having a role in the etiology of stress responses. Introduction
biological and behavioral approaches to cholinergic neurotransmission has contributed, we feel, to a deeper appreciation of the role of cholinergic neurotransmission, and of acetylcholinesterase (ACHE) in particular, in stress responses. This chapter, therefore, presents long-term cellular and behavioral stress responses as molecular and biochemical processes that are amenable to further study, and, we hope, to therapeutic intervention.
Acetylcholine (ACh), the modulator of numerous physiological reactions, also modulates mammalian stress responses. In particular, a striking similarity exists between reported long-term responses to anticholinesterase exposure and traumatic experiences (Fullerton and Ursano, 1990; Somani et al., 2000). Furthermore, anti-cholinesterases and stress both induce expression of the acetylcholinesterase gene, A C H E (Kaufer et al., 1998; Kaufer et al., 1999). The realization of this similarity has drawn us into stress studies from backgrounds in molecular biology and neuroimmunology. The melding of our molecular,
Cholinergic responses to stress It is well accepted that stress in mammals is rapidly followed by a pronounced activation of central cholinergic pathways that is correlated with transiently enhanced release of ACh (Imperato et al.,
*Corresponding author. Tel.: +972 2 658 5109; Fax: + 972 2 652 0258; E-mail:
[email protected] 585
586 1991; Dazzi et al., 1995). In addition to ACh release, exposure to stress induces short- and long-term alterations in various other components of the cholinergic system. These include the density and composition of cholinergic receptors, choline uptake and expression and activity of ACh-related enzymes. In rodents, exposure to acute stressors, such as restraint, foot-shock or swimming, produces a rapid but transient activation of central cholinergic systems in general and the septo-hippocampal system in particular. These effects of stress were measured by various methods, including the release of newly synthesized [3H]-ACh from hippocampal synaptosomal preparations (Finkelstein et al., 1985) or in vivo microdialysis (Tajima et al., 1996; Mizuno and Kimura, 1997). The changes in ACh release depend on the parameters of the stressor. For example, in several studies enhanced hippocampal ACh release could be observed as early as 10-15 min following the initiation of restraint stress. The cholinergic response declined after 50-60min of restraint, but when the animals were freed from the restraining tubes after 2 h, ACh levels increased again, possibly because of the additional stress (Imperato et al., 1989; Tajima et al., 1996). In another study, rats that were restrained and immersed in a 37~ water bath also showed stimulated release of hippocampal ACh, but only following removal from restraint. In contrast, rats that were restrained and immersed in a 20~ water bath displayed decreased ACh levels during cold exposure, which returned to near-baseline values following removal from cold water (Stillman et al., 1997). Other stressors produce similar effects, e.g., exposure to foot-shock stress, delivered for 8 min, induced a rapid and marked (75%) increase in hippocampal ACh output, which persisted for about 40min (Dazzi et al., 1995). Changes in number and affinity of cholinergic receptors were also demonstrated following exposure to stress. These changes depend on the parameters of the stressful stimulus, particularly its intensity and duration. For example, in one study, immobilization stress for 2hr, but not 10rain, caused a significant increase in the affinity of the muscarinic receptor in several brain areas, including the hippocampus and basal ganglia (Gonzalez and Pazos, 1992, but see Finkelstein et al., 1985). Similarly,
chronic exposure to short periods of immobilization (10 min/day for 3-21 days) had no effect on cholinergic receptors, whereas exposure to 2hr/day for 3-21 days resulted in significantly increased number (Gonzalez and Pazos, 1992) and affinity (Finkelstein et al., 1985) of muscarinic receptors. Changes in the sensitivity of cholinergic receptors have also been demonstrated pharmacologically: following chronic food-restriction stress, rats exhibited a greater behavioral response to a muscarinic agonist and a lower behavioral response to a muscarinic antagonist in modulation of passive avoidance learning (Orsini et al., 2001). In laboratory animals both stress and inhibition of AChE indirectly cause elevation of AChE mRNA and protein levels in the brain (Fullerton and Ursano, 1990; Somani et al., 2000), while suppressing levels of mRNAs that encode the ACh-synthesizing enzyme choline acetyltransferase (CHAT) and the vesicular acetylcholine transporter (vAChT) (Friedman et al., 1996; Kaufer et al., 1998). These observations indicate that a decrease in ACh, due to increased hydrolysis is re-enforced by reduced ACh-synthesis in cholinergic pathways. These complementary modulations of cholinergic gene expression ensure that transient acute cholinergic hyperactivation is followed by a persistent suppression of cholinergic neurotransmission in the mammalian brain. These clues have led us to the initiation of a systematic investigation of the role of AChE in stress responses in several experimental paradigms, including the human hematopoietic system (Grisaru et al., 2001), mouse hippocampal slices (Friedman et al., 1998) and intestinal epithelium (Shapira et al., 2000b), myasthenic rat muscle (Brenner et al., 2003) and testicular tubules (Mor et al., 2001). In all of these systems we found that various stressors induce expression of AChE-R mRNA, one of the AChE pre-mRNA splicing variants (Grisaru et al., 1999; Soreq and Glick, 2000). These findings highlight the role of the normally rare AChE-R in mammalian stress. The observations, in five major organ systems, suggest a central role for cholinergic pathways in mediating long-term stress responses. The effects of chronic stress exposure on cholinergic neurotransmission were examined by others, revealing long-term changes that were present even many days after termination of the exposure. For example, 10 days
587 following the termination of chronic stress (daily exposure to water immersion and 2-hr restraint for 4 weeks), rats exhibited markedly enhanced KC1evoked cholinergic response within the hippocampus, although basal ACh release was not changed (Mizoguchi et al., 2001). Several lines of evidence suggest that these initial events contribute to the behavioral consequences of stress. For example, scopolamine, a muscarinic antagonist, affects rodent responses to foot-shock stress (Kaneto, 1997), and long-term psychological disturbances, strikingly reminiscent of those that characterize post-traumatic stress disorder (PTSD), are associated with both acute (Burchfiel and Duffy, 1982; Rosenstock et al., 1991) and chronic (Li et al., 2000; Stephens et al., 1995) exposure to inhibitors of ACHE. The scope of these observations could have been expanded to include panic disorders, which have an important cholinergic component (Battaglia, 2002), and depression (Lupien et al., 1999), but this would take us beyond the mandate of this chapter.
Stillman et al., 1997) and the finding that exogenous administration of CRF activates cholinergic neurons and induces hippocampal ACh release (Day et al., 1998a; Day et al., 1998b; Stillman et al., 1997). Further studies demonstrated that the relationships between stress, adrenocortical activation and ACh release are not direct, because the time course of stress-induced plasma corticosterone elevation does not parallel that of ACh release. For example, during continuous restraint for 2 hr, ACh release gradually decreases following initial elevation, but it increases again when animals are released from the restraining tubes. In contrast, corticosterone levels remain elevated for the whole restraining period and decrease when animals are released (Imperato et al., 1991). Furthermore, stress-induced ACh release was found to be similar in adrenalectomized and sham-operated rats, and was not influenced by pre-treatment with the glucocorticoid antagonist RU38486 (Imperato et al., 1991), providing additional support for the hypothesis that the effects of stress on cholinergic systems are not mediated solely by adrenocortical activation.
The relationships between stress-induced pituitary-adrenocortical activation and ACh release The effects of the hypothalamo-pituitary-adrenal (HPA) axis on the cholinergic system and its role in stress-induced cholinergic activation have been intensively studied. The hypothalamus is the source of corticotropin-releasing factor (CRF); the pituitary of ACTH, and the adrenal of glucocorticoids. Initial studies demonstrated that large doses of either adrenocorticotropic hormone (ACTH) or corticosterone activate the septo-hippocampal cholinergic system similarly to the activation of this system by short-term stress (Gilad, 1987; Gilad et al., 1985). Choline uptake and ACh release were also elevated 2 days after adrenalectomy. The effects of adrenalectomy were attenuated by corticosterone, which reduces ACTH levels, but not by ACTH administration (Gilad et al., 1985). This effect may be the result of reduced feedback inhibition, which results in elevated CRF and ACTH levels. A role for CRF in cholinergic activation is also suggested by the presence of CRF receptors on cholinergic cells (Chen et al., 2000; Sauvage and Steckler, 2001;
Gulf War syndrome and penetrance of the blood-brain barrier under stress Cholinergic neurotransmission is probably involved in most of the known arousal-related sensory stimulation effects. For example, ACh release increases dramatically in the frontal cortex and hippocampus of rats introduced into a novel environment (Inglis et al., 1994), as well as following presentation of auditory, visual, olfactory and tactile sensory stimuli (Acquas et al., 1996). The cholinergic activation is restricted to presentation of novel stimuli, which may be regarded as mildly stressful. If a novel stimulus, such as tone or light, is followed by an unconditioned strong stressful stimulus (such as foot-shock), it will continue to elicit ACh release. However, presentation of a tone or light to habituated animals, i.e., following repetitive presentations of the same stimulus, will fail to elicit a cholinergic response (Acquas et al., 1996). These findings indicate that ACh release in the cortex and hippocampus are reliably activated only by
588 behaviorally relevant stimuli. During stressful conditions, the activation of the cholinergic system by novel, conditioned or the stressful stimulus itself provides an important mechanism for adaptive enhancement of arousal and/or attentional processes. Stress-induced ACh release is probably also involved in other cognitive functions, particularly learning and memory. The relationship between memory processes and cholinergic neurotransmission is well established in the literature. Early reports of the role of ACh in learning and memory (Drachman and Leavitt, 1974), and evidence of substantial reductions in neocortical cholinergic function in Alzheimer's disease (Bartus et al., 1982; Nilsson et al., 1986) provided evidence for the cholinergic hypothesis of memory. In particular, cholinergic stimulation is known to facilitate working memory performance (Ellis and Nathan, 2001), supposedly by influencing the signal-to-noise ratio for the response of single neurons in the cortex (Furey et al., 2000). This increases the selectivity of perceptual responses, possibly enlarging the response to afferent input and reducing background activity. Consistent with this notion, cholinergic antagonists, such as scopolamine, selectively interfere with encoding of new information, but not with its retrieval, whereas cholinesterase inhibitors, such as physostigmine or Alzheimer's disease drugs (Giacobini, 2002) improve working memory. It can be speculated that under conditions of acute stress, working memory is an important asset for survival. Thus, cholinergicmediated improvement in working memory performance might be adaptive. Stress-induced cholinergic activation may also play a role in other forms of memory, such as aversive conditioning. For example, enhancement of inescapable foot-shock stressinduced ACh transmission by pretreatment with an anticholinesterase (eserine sulfate) was found to increase the learned helplessness produced in this paradigm, reflected by increased escape deficits observed 3 days later (Kelsey, 1983). The mechanisms that mediate the effects of stress-induced cholinergic activation on learning and memory are not clear. It has been suggested that the CRF system may interact with cholinergic activation in memory modulation, but this hypothesis has not received much experimental support (Steckler and Holsboer, 2001).
Gulf War syndrome and penetrance of the blood-brain barrier under stress Cholinergic pathways are involved in controlling numerous peripheral functions, e.g. neuromuscular activity, salivation, intestinal functions and lacrymation. Over the years, this led to the development of drugs aimed at controlling these functions. To avoid unwanted effects over higher brain functions, these drugs were designed to remain in the circulation, where they are held, thanks to the blood-brain barrier (BBB) (Rubin and Staddon, 1999). A notable example is pyridostigmine bromide (PB), used for the past 40 years for treating patients with myasthenia gravis, an autoimmune syndrome that causes debilitating muscle fatigue (Berrouschot et al., 1997). During the 1991 Gulf War, injection of PB was a prophylaxic treatment of soldiers, in anticipation of chemical warfare, to transiently block AChE and protect it from permanent inhibition by nerve agents. This treatment was based on clinical studies that demonstrated its effectiveness and showed side effects that were strictly limited to peripheral symptoms (Beck et al., 2001). However, impairments in higher functions of treated soldiers and subsequent symptoms that appeared to originate in the CNS led to questions regarding the ability of PB to remain peripheral under stresses associated with war (Golomb, 1999). This, in turn, provoked further studies that explored behavioral changes under PB administration. It was thus found that PB modified the acoustic startle response (Servatius et al., 1998), locomotor activity in an open field (Kant et al., 2001), hand and eye coordination (Wolthuis et al., 1995), the capacity to press a lever in a delayed reinforced manner (van Haaren et al., 1999) and visual discrimination properties (Liu, 1992). A relatively simple explanation of these behavioral effects would be that the stress associated with PB injection modifies the properties of the BBB and enabled PB penetrance of the brain, in spite of the cationic group that had been introduced into this carbamate to prevent penetrance through the BBB. This was the working hypothesis of a study (Friedman et al., 1996) that reported efficient inhibition of brain AChE by intraperitoneally injected PB into forced swimstressed, but not naive, mice. However, the complete picture is far from simple, as other studies- with
589 other stressors and other rodent m o d e l s - reported no inhibition of brain AChE under systemic administration of PB (Cook et al., 1988; Stitcher et al., 1978). The later-reported transcriptional feedback response, including AChE overproduction under exposure to anti-cholinesterases (Kaufer et al., 1998) offers a tentative explanation of this inconsistency. Yet more recently, the lethality of PB in animals was reported to be increased (Chaney et al., 1999; Chaney et al., 2000) and its capacity to inhibit brain AChE facilitated, when co-injected with permethrin or N,N-diethyl-m-tolumide (Abou-Donia et al., 2001; Abou-Donia et al., 1996). While it is difficult to compare these studies to one another, because they used different stressors or animals, it seems possible that under some circumstances stress can cause permeabilization of the BBB. Corticotropin-releasing factor, a 41-residue peptide originating in the hypothalamus, apparently has a central role in response to stress. It has long been known to cause release of ACTH from the pituitary into the bloodstream, where it travels to the adrenal cortex to promote synthesis and secretion of cortisol, the source of many other stress responses, among them activation of ACHE gene expression. More recently, CRF, acting with mast cells, has been shown to increase permeability of the BBB (Esposito et al., 2001; Esposito et al., 2002). In a dramatic demonstration of the effect of stress, Relyea and Mills found that the anti-AChE pesticide, carbaryl, was 2 to 4 times more lethal to amphibians when they were exposed to the stress of predatory cues (Relyea and Mills, 2001). This may be an additional demonstration of the effect of stress on the BBB.
machines, under the direction of a number of sometimes competing small splicing factor proteins (Stamm, 2002; Zhou et al., 2002). The limited number of these factors, and the balance among them, would allow a concerted response to an external change by the generation of characteristic splicing variants of a potentially large number of genes. Thus, a stress, starvation for example, may initially bring about the up-regulation of a single splicing factor, which because of its involvement in splicing of a wide variety of pre-mRNAs, results in the biosynthesis of the proteins that can minimize the effects of starvation. The integration of the response is built into the participation of that one (or those few) splicing factor(s) in the synthesis of physiologically appropriate proteins by splicing of their gene transcripts. Similarly, a defect, inherited or acquired, in one of these splicing factors would affect a correspondingly wide variety of such events. Although the extent of alternative splicing as an integrating (or disintegrating) principle is still being explored, it is a fruitful source of hypotheses that may help to explain the many forms that responses to stress may take in different tissues, and the similarities among responses to various kinds of stresses. One example, taken from the neurosciences, is the stress-induced modification of K + channels (Xie and McCobb, 1998; Xie and Black, 2001), which is regulated by neuronal activity-dependent transcriptional changes in a number of splicing regulatory proteins (Daoud et al., 1999). Another example, is the stress-directed splicing of AChE pre-mRNA (Meshorer et al., 2002; Soreq and Glick, 2000; Meshorer and Soreq, 2002), which we explored.
mRNA splicing variations
Acetylcholinesterase and stress responses
One of the strategies by which Nature has greatly expanded the expression potential of the genome is by alternative splicing of pre-mRNAs. In approximately 20% of the expressed genes, the original transcript can generate a number of different mature mRNAs by selection of only some of the open reading frames and elimination of others. Thus, subtly or substantially different variant proteins may be generated from a single gene. The process of splicing is accomplished by complex molecular
The short-term stress response is typified by increases of epinephrine and glucocorticoids, which facilitate a mobilization of energy stores and a suppression of competing activities, such as growth, reproduction, immune response and tissue repair. Prolonged elevation of glucocorticoids by stress results in central nervous system (CNS) abnormalities, among them learning and memory defects, perhaps caused by decreased branching of dendrites, and a variety of psychiatric problems, including those associated with
590 PTSD. However, short-lived traumatic effects, which do not result in long-term activation of the HPA axis, can also result in PTSD (Sapolsky, 2002). It appears, therefore, that some stress responses are mediated by other, more persistent factors. While it is highly unlikely that AChE is the only mediator of HPA activation, the process leading to AChE-R production appears to fit the description of a prime suspect: the A C H E promoter contains glucocorticoid response elements, which increase AChE expression following stress (Grisaru et al., 1999; Shapira et al., 2000b). Following stress, the major newly-synthesized neuronal AChE variant is AChE-R. This implies that glucocorticoids and/or other stressinduced modulator(s) must also influence the splicing of the AChE pre-mRNA. AChE-R mRNA and AChE-R itself remain in neurons for weeks following stress (Meshorer et al., 2002) and the increased presence of (transgenic) AChE-R exerts a protective effect from the microanatomical neuropathologies that accumulate under excess of neuronal AChE-S (Sternfeld et al., 2000). Moreover, the suppression of AChE-R by a specific antisense agent (Galyam et al., 2001) alters neurite extension (Grifman et al., 1998), improves the outcome of closed head injuries (Shohami et al., 2000) and reverses myasthenic symptoms, which are associated with increased levels of circulating AChE-R (Argov et al., 2003; Brenner et al., 2003). A significant source of the variety of AChE isoforms is 3'-alternative pre-mRNA splicing (Fig. 1) which confers different C-terminal sequences on a 543-residue core protein. A single human A C H E gene gives rise to a wide variety of protein variants. The 543-residue core of human AChE is encoded by 3 exons, 2, 3 and 4, and by itself is catalytically competent; 3'alternative splicing of the pre-mRNA result in additional C-terminal sequences of the S (synaptic), R (readthrough) and E (erythrocytic) variants. Human AChE-S terminates with 40 residues (DTLDEAERQWKAEFHRWSSYMVHWKNQFDHYSKQDRCSDL); AChE-E with 14 residues (ASEAPSTCPGFTHG); and AChE-R with 26 residues (GMQGPAGSGWEEGSGSPPGVTPL FSP). The translation start codon is in exon 2, which encodes a leader sequence that does not appear in any of these mature proteins. In addition to a proximal
promoter next to exon 1 (Atanasova et al., 1999; Camp and Taylor, 1998; Chan et al., 1998), a far upstream distal enhancer region is rich in potential regulatory sequences. For instance, the A C H E transcriptional activation by cortisol (Grisaru et al., 2001) is likely due to the distal glucocorticoid response element (Meshorer et al., 2002). A deletion mutation in this region disrupts one of two HNF3 binding sites, which consequently activates transcription (Shapira et al., 2000b). Normally, much more AChE-S than AChE-R mRNA is produced, but under stress or inhibition of ACHE, alternative splicing produces much more of the AChE-R mRNA, likely under c-fos regulation (Kaufer et al., 1998). The C-terminal sequence of AChE-S enables it to form multimers that can then be joined to anchoring proteins (Ohno et al., 2000; Perrier et al., 2002) that attach them to the synaptic membrane; and AChE-E dimerizes and undergoes a transesterification reaction to replace its C-terminal sequence with a glycophosphatidylinositol group that can be embedded in the erythrocyte membrane (Silman and Futerman, 1987). AChE-R, however, cannot form multimers, and when secreted, it remains DNA
stress
c-los
............... ~>k'["~ -17 Kb
pre-mRNA
:1
2
mRNA
346
2
1'
1'
~2
3 2'
4 3'
5 6 4'
~3 i i i 4i ~5 6
2'
2
3"
4*
34 5
2
345
4'
AChE-S
AChE-R
AChE-E
site of action
additional functions cholinergic signalling
cell proliferation remodeling of dendrites
scavenging of inhibitors?
Fig. 1. The molecular biology of human ACHE.
591 soluble. Additional variation arises from posttranslational changes: glycosylation, which affects turnover (Kronman et al., 2000), formation of intermolecular disulfide bridges and attachment to a phospholipid or collagen-like protein anchor to synapse membranes (as mentioned), or intracellular interaction with partner proteins (Birikh et al., 2003).
Note on nomenclature Another nomenclature is based on physical rather than molecular biological relations, and names the synaptic and erythropoietic variants T (tailed), and H (hydrophobic), respectively (Massoulie, 2000). The many guises in which AChE-T occurs are further termed G, for globular, or A for asymmetric and G1, G2 etc. for monomer, dimer, etc.
Transcriptional feedback response to stress The functioning of the A C H E gene is subject to massive developmental pressures, yet retains a certain level of plasticity also in the adult. In both neocortical and hippocampal neurons, various external stimuli induce rapid, yet long-lasting A C H E gene expression. In fact, the ACHE gene responds with increased transcription to psychological stress (Kaufer et al., 1998), anti-AChE intoxication (Shapira et al., 2000b), closed head injury (Shohami et al., 2000) and autoimmune impairments of neuromuscular function (Brenner et al., 2003) and immobilization stress (Nijholt et al., 2003). It is presumed that psychological or physical stress induces cholinergic excitation via release of ACh. Elevated cortisol and the consequent feedback over-expression of AChE then act to dampen excessive neurotransmission towards its normal level (Kaufer et al., 1998). This is important both for cholinergic neurotransmission and for other neurotransmission circuits modulated by ACh, for example hippocampal glutamatergic activity (Gray et al., 1996; Meshorer et al., 2002), and dopaminergic circuits in the substantia nigra (Llinas and Greenfield, 1987). That over-produced AChE can also protect the organism from the toxicity of anti-AChEs, has been demonstrated in laboratory animals (Ashani et al., 1991; Wolfe et al., 1992; Doctor et al., 1993; Raveh et al., 1989).
Transcriptional activation is common to many genetically determined responses to pharmaceuticals, e.g. by cytochrome P-450 proteins (Evans and Relling, 1999). However, in addition to transcriptional activation, AChE mRNA transcripts in nerve, muscle and blood cells are subject to calcineurincontrolled differentiation-induced stabilization (Chart et al., 1998; Luo et al., 1999). Both these processes increase the amount of AChE when and where it is needed. As AChE-R mRNA is significantly less stable than AChE-S mRNA (Chart et al., 1998; Luo et al., 1999), any stabilizing effect should significantly favor the R-variant. This should be of particular interest in the context of the routine use of antiAChEs in the treatment of Alzheimer's disease (AD) patients (Palmer, 2002). Indeed, AChE-R was recently shown to accumulate in the cerebrospinal fluid of AD patients treated with anti-AChEs (Darreh-Shori et al., 2002, 2003). The variable efficacy of such agents among individuals may therefore reflect differential capacities to induce transcriptional activation and/or stabilization of AChE mRNA.
Transgenic overexpression o f neuronal A ChE-S recapitulates chronic stress effects We created a transgenic mouse model (TgS), in which overexpression of human synaptic AChE-S, limited to CNS neurons (Beeri et al., 1995), promotes a lateonset and progressive impairment in learning and memory that in humans is associated with PTSD (Kaufer and Soreq, 1999). The cognitive defects observed in these mice were assumed to reflect the physiological state induced by an excess of ACHE, but we cannot rule out the defects being secondary effects, resulting from adaptations to the increased levels of CNS ACHE. The possibility that modified regulation of A C H E gene expression may have imposed profound disruption of both central and peripheral cholinergic systems is strengthened by our more recent findings of multi-levelled impairments in these mice in neuromuscular junction (NMJ) structure and function (Farchi et al., 2003), which eventually lead to amyotrophy in TgS mice (Andres et al., 1997). However, there is a temporal gap between over-expression of AChE in young TgS mice
592 and the delayed onset of neurodegenerative and cognitive processes, perhaps reflecting the fact that excess AChE causes damage only when present for a long time.
Compensatory mechanisms as suppressors of stress symptoms If, indeed, AChE over-production is generally associated with long-term stress responses, the question arises, how does the brain handle the resultant state that is induced by elevated ACh hydrolysis? The obvious answer is, by initiating compensatory mechanisms that would elevate the cholinergic state to retrieve functional balance. Similar compensatory mechanisms are generally assumed to enable the extended pre-symptomatic stages that accompany neurodegenerative disease, e.g. AD and Parkinson's disease (Zigmond,1997). Neurodeterioration, in this view, would be due to inadequate compensation for consequences of both inherited defects and stress. Elevation of the cholinergic status would, however, be possible only if sufficient numbers of viable cholinergic neurons were available. Indeed, animal models are known that develop cognitive impairments at relatively early ages due to inherited loss of cholinergic forebrain neurons (Zigmond, 1997; Sago et al., 1998). Primary stress-related function subserved by the cholinergic system, particularly within the hypothalamus, is in regulation of the HPA axis. Indeed, the secretion of CRF can be also altered by ACh, although the direction of the cholinergic influence may vary in different brain areas: e.g. ACh stimulated CRF secretion from the hypothalamus, but inhibited its release from cortical tissues (Tizabi and Calogero, 1992). Moreover, microinjection of ACh into the hypothalamus induced the expression of CRF and pro-opiomelanocortin m R N A within the hypothalamus and pituitary, respectively (Ohmori et al., 1995). Both nicotinic and muscarinic receptors may be involved in HPA axis regulation. Nicotine was found to activate CRF neurons in various brain areas, including the hypothalamic paraventricular nucleus (Nilsson et al., 1986) and to induce CRF secretion in vitro (Calogero et al., 1988). The effects of nicotine on the release of ACTH from the pituitary may
reflect indirect noradrenergic pathways mediated via the brainstem (Fu et al., 1997; Matta et al., 1990). Muscarinic mechanisms are also involved: administration of the muscarinic agonist arecoline stimulates the HPA axis in the rat and this effect was found to be mediated mainly by the release of endogenous CRF (Calogero et al., 1989). Furthermore, blockade of hippocampal muscarinic receptors augmented ACTH and corticosterone responses to restraint without altering basal HPA activity (Bhatnagar et al., 1997). Together, these findings strongly suggest that cholinergic systems regulate stress-induced HPA activity and may serve to coordinate behavioral and neuroendocrine responses to stress.
Cholinesterase genetics and stress responses
AChE levels depend on multiple inherited and acquired elements, so that in some humans, there is a higher than usual basal level of A C H E expression (Silver, 1974). As in most circumstances the individual shows no ill effects, there is apparently an adaptation to this state. This may involve an increased level of ACh receptors, similar to TgS mice (Perry et al., 2000), or increased high affinity choline transporter, also shown in TgS mice (Erb et al., 2001). It was hypothesized that similar to TgS mice, individuals with constitutive AChE over-expression would be unable to respond appropriately to stress and that their A C H E gene would contain some clues as to the cause. Therefore the genomic DNA from 340 subjects was analyzed, with special attention to a region of the promoter sequence that was rich in transcription factor binding elements and which includes a glucocorticoid response element. Two adjacent mutations in this distal upstream enhancer domain of the human A C H E gene were discovered in heterozygous carriers (Shapira et al., 2000a): a 4-bp deletion and a single nucleotide substitution. The deletion, identified in a woman who presented acute hypersensitivity to pyridostigmine, was found in transfected cells to constitutively increase AChE expression by abolishing 1 of 2 adjacent HNF3 binding sites. Because the deletion confers a gain of function of ACHE, the trait is dominant; the substitution impairs the glucocorticoid receptor binding site in this region. Further studies will be
593 required to find whether this trait is also associated with increased risk for exaggerated stress responses.
Environmental stress on AChE mitigated by
butyrylcholinesterase Since its discovery, AChE has been known as the enzyme that hydrolyzes the neurotransmitter, ACh. The biological role of the AChE-homologous enzyme, butyrylcholinesterase (BuChE), has long remained elusive. It has been postulated that BuChE is a back-up for ACHE, and in the very special case of the AChE-knockout mouse, it may be BuChE that in fact performs ACh hydrolysis. Nevertheless, in the real world of Nature, there are no known cases of human AChE mutations that abolish its activity, which is a powerful message that AChE serves an irreplaceable function. It must be mentioned, however, that there are mutations of the AChE-Sanchoring protein, which result in end-plate AChE deficiencies and cause major neuromuscular defects (Ohno et al., 2000). Another role proposed for BuChE is as a molecular decoy that absorbs antiAChEs that may find their ways into the body and minimize this source of stress on the organ systems that depend on a functional ACHE. AChE and BuChE have overlapping specificities for substrates and inhibitors, with BuChE being somewhat more promiscuous. In consequence, just about every antiAChE is also an anti-BuChE. The environment contains many and varied anti-AChEs, ranging from the anatoxins, natural organophosphate poisons of blue-green algae (Carmichael, 1994) and the abundant glycoalkaloids of Solanaceae (potatoes, tomatoes, aubergines) (Friedman, 2002; McGehee et al., 2000; Roddick, 1989) to the toxins of snake venoms, e.g. fasciculin (Marchot et al., 1997) and natural medicines of plant origin, e.g. huperzine and physostigmine (Giacobini, 2000). An anti-AChE entering the body will react with serum BuChE (and, for that matter, AChE-E on erythrocyte membranes) before even coming into contact with AChE-S at neuromuscular junctions or brain synapses. The individual is thus protected by the ability of BuChE to adsorb AChE inhibitors. Consistent with its being a molecular decoy for ACHE, are the prominence of BuChE in the serum
and its capacity to react quickly with a wide spectrum of compounds. Furthermore, some polymorphisms of the BCHE gene render carriers increasingly susceptible to the ill effects of anti-AChE exposure. Polymorphism of BCHE has been extensively surveyed, originally by study of the variant-characteristic susceptibility to inhibitors of the serum activity (Kalow and Genest, 1957), more recently by molecular genotyping (La Du et al., 1990; Loewenstein et al., 1995). BCHE mutations are very unevenly distributed around the world, with dramatically high or low frequencies found especially in historically isolated ethnic groups, possibly reflecting genetic founder effects. The different BuChE variants and their frequencies may also reflect an evolutionary adaptation to local environmental factors. Because BuChE mutants offer varying protection against antiAChEs, carriers of these mutations may be more vulnerable than non-carriers to anti-AChEs and may show exaggerated responses when exposed. An extension of this idea, in conjunction with the similarity of chemical and other stressors, is increased vulnerability of BuChE mutation carriers to lateonset diseases. In the case of AD, some have found such an association (Lehmann et al., 1997; Lehmann et al., 2000), but others have not (Brindle et al., 1998).
Non-classical biological roles of acetylcholinesterase
Because stress events induce AChE overexpression, the concentration of the AChE protein will be higher following stress. This will effect both the hydrolytic and the non-classical actions of ACHE, both of which may be relevant to the organismal response to stress. Non-classical actions of ACHE, i.e. those not associated with hydrolysis of ACh at the synapse or NMJ, have been reported by a number of laboratories, among them, those of M.E. Appleyard (Appleyard, 1992), John Bigbee (Bigbee et al., 1999), W. Stephen Brimijoin (Koenigsberger et al., 1997), Susan Greenfield (Greenfield, 1996), Paul Layer (Layer, 1996) and David Small (Small et al., 1996). It is still a challenge to integrate the disparate, but not contradictory, findings of these research groups into a coherent view of the biology of ACHE. Recent discoveries may indicate a biochemical basis of at
594 least some of these seemingly anomalous effects. There is a homology among AChE and other members of the cz/[3 fold family of proteins, including the neurotactins, which are involved in cell-cell adhesion. The conserved domain of neurotactins may be exchanged for AChE and still retain cell-cell interaction (Darboux et al., 1996). Moreover, genetic inactivation of AChE does not prevent its neurite growth-promoting activity (Grifman et al., 1998; Sternfeld et al., 1998a). The mammalian non-catalytic AChE-homologous proteins, the neuroligins, reside in excitatory synapses (Song et al., 1999), and are known to bind neuronal cell surface proteins, the neurexins (Nguyen and Sudhof, 1997); neurexins, neurotactins and neuroligins are transmembrane proteins with C-terminal cytoplasmic tails which could enable signal transduction through the binding of PDZ domain proteins, e.g. membrane-associated guanylate kinases (MAGUKs; (Ponting et al., 1997), and thus provide the molecular basis of the complex consequences that are characteristic of these cellcell interactions. Many in the research community are now open to the recognition of non-cholinergic roles of AChE (Botti et al., 1998), and more such functions will doubtlessly be identified in the near future. Cell-cell interactions are mediated by the interaction of membrane-bound neuroligin-1 with membrane-bound [3-neurexin. If neuroligin-1 is displaced from [3-neurexin by the homologous (Tsigelny et al., 2000), soluble, AChE-R, the cell-cell interaction is broken. This is likely to modify the properties and/or intracellular signalling activities of PDZ domain proteins that interact with both neuroligin-1 and [3-neurexin, with predictable effects on excitatory synapse activities. When fully understood, this version (Fig. 2) may prove to have been too simplistic, as it cannot be readily reproduced in cultured neurons (Scheiffele et al., 2000), but it's elements seem safely established. The behavioral implications of neuroligins' expression have recently been demonstrated in that genetic polymorphisms in the human neuroligin gene were found to increase the risk for behavioral impairments and autism (Zoghbi, 2003; Jamain et al., 2003). This, in turn, highlights the potential importance that displacement of neuroligin with AChE-R may have on stereotypic behavior.
PDZ-domain protein_D [_~-neurexi~
(~ AChE-R
t~!l~
Fig. 2. Proposed molecular basis of non-catalytic properties of ACHE. Compensation for neuron loss by increased neurite outgrowth can delay the symptoms of neurodegeneration. Various studies support the notion of AChE's participation in such processes. Studies of the morphogenic roles of AChE have been facilitated by the construction of transgenic mouse lines that overexpress a specific AChE variant, AChE-S (TgS) (Beeri et al., 1995) or AChE-R (TgR) (Sternfeld et al., 1998b), by the use of stably transfected cell lines (Koenigsberger et al., 1997; Grifman et al., 1998; Bigbee et al., 1999) and in primary neurons that express and produce small quantities of a recombinant variant (Sternfeld et al., 1998a). In several of these model systems, human AChE-R emerged as having effects distinct from those of AChE-S. In cultured glioblastoma cells, over-expressed AChE-R confers a phenotype of small, round, rapidly dividing cells as opposed to the AChE-S phenotype of process growth (Karpel et al., 1996; Perry and Soreq, 2002). Antisense suppression of AChE mRNA in neuroblastoma cells was associated with complete loss of neuritogenesis, which was retrieved by re-transfection with AChE-S (Koenigsberger et al., 1997). Similar results were obtained in PC12 cells where either AChE-R or the non-catalytic homolog, neuroligin, retrieved neurite growth following antisense suppression of AChE-R (Grifman et al., 1998). The changes under stress in the levels of AChE variants further imply an altered ratio between AChE-R and AChE-S in the stressed nervous system. This highlights one of the key challenges in this field, namely the search for the physiological functions of the different splice variants. While previous theories
595 of AChE's involvement in neurophysiological activities were largely limited to cholinergic neurotransmission, its non-catalytic activities likely span many more circuits and brain regions. Moreover, the soluble AChE-R monomers secreted under stress were recently shown to modulate glutamatergic neurotransmission and affect the stress-induced changes in long-term potentiation (Vereker et al., 2000; Nijholt et al., 2003), a cellular function related to memory, or the facilitation of long-term depression (Xu et al., 1997), its opposite.
Acetylcholinesterase-R is overproduced under the influence of several stressors Facilitation of the capacity for ACh hydrolysis provides useful short-term suppression of the cholinergic hyperexcitation that is associated with stress responses. This can prevent epileptic seizures, a known consequence of anti-cholinesterase exposure (Blanchet et al., 1994; Shih and McDonough, 1997), and head injury (Shaw, 2002). Moreover, in the long run, these forms of stress or trauma - acute psychological stress, exposure to anti-AChEs, head i n j u r y - all can lead to delayed neurodeterioration. Further studies will be required to determine whether the association of AChE-R with these physiological conditions reflects a causal relationship to neurodeterioration, whether the expression of AChE-R is a protective mechanism that is not always sufficient to prevent the deterioration, or if both assumptions are correct, with the AChE-R concentration determining its effects. To begin to address these questions we examined stress-induced and stress-related neuropathological and behavioral parameters in mice with transgenic overexpression of various AChE isoforms (Table 1). As described below, TgS mice exhibit impairments in
spatial learning and memory (Beeri et al., 1995), which may be partially accounted for by the biochemical alterations in their brains. TgS mice adapt to the high levels of AChE by increased synthesis of high-affinity choline transporter (elevating pre-synaptic choline uptake) and acetyl cholinetransferase (facilitating ACh synthesis). The rather counter-intuitive result is an unchanged level of ACh in conscious mice; under halothane anesthesia the TgS ACh levels were lower than in parent strain mice, attesting to the transient nature of these compensatory effects (Erb et al., 2001). The modified AChE-R/ AChE-S ratio may induce persistent changes in the CNS. Exposure of TgS mice to acute levels of the anti-AChE, diisopropylfluorophosphonate (DFP), failed to induce AChE-R overproduction in their intestinal endothelium, an exposure response that occurred readily in the parent FVB/N strain (Shapira et al., 2000b). The high level of AChE in brains of TgS or the altered AChE-S/AChE-R ratio may render these mice particularly vulnerable to the longterm consequences of acute stress. The TgS mice make an intriguing model in which to study the roles of AChE and cholinergic signalling in mammalian stress responses, but because several components of the cholinergic system have been perturbed, a model with which it is necessary to perform a large number of control experiments.
Behavioral manifestations of stress in transgenic mice with cholinergic imbalances Behavioral differences between TgS and control mice were first sought by telemetric recording of locomotion patterns, both under basal conditions, as well as following the stress of a switch in the circadian light/ dark cycle. Under normal conditions, naive TgS mice displayed close to normal locomotion behavior,
Table 1. Stress-inducing physiological phenotypes associated with cholinergic impairments Insult
Outcome
Reference
Forced swim (psychological) Light/dark switch (physiological) Anti-cholinesterase exposure Head injury
Hippocampal hyperexcitation Enhanced locomotion Impaired working memory Impaired motor coordination
Friedman et al. (1998) Cohen et al. (2002) Kaufer et al. (1999) Shohami et al. (2000)
596 Table 2. Behavioral stress correlates in transgenic mice overexpressing distinct AChE variants. Transgene
Behavioraltest
Stress-relevant phenotype
Reference
S
Morris water maze
Impaired acquisition of spatial information
S S S R
Social exploration Elevated plus maze Locomotion Emergence into a new field
Working memory deficit (extended sniffing time) Reduced anxiety (prolonged open arm time) Aimless hyperactivity after light/dark switch Extended conflict behavior (delayed emergence)
Beeri et aI. (1995); Beeri et al. (1997) Cohen et al. (2002) Erb et al. (2001) Cohen et al. (2002) Birikh et al. (2003)
similar to that of naive mice from the non-transgenic strain. However, their locomotor response to stress, either in their cages or in an elevated plus maze, was distinct from that of the parent strain. We also assessed the effects of AChE overexpression in these mice on social memory functioning, which is known to be markedly affected by exposure to various physical and psychological stressors. This issue is summarized in Table 2 and detailed below.
Response to circadian shift Mammalian stress responses are known to be subject to circadian regulation. Circadian differences were reported in the stress-induced pressor activity in mice (Bernatova et al., 2002) and the circadian regulation of cortisol levels in human infants was found to include a significant genetic component (Bartels et al., 2003). Moreover, women with and without sexual abuse and post-traumatic stress disorder differ substantially in the diurnal pattern of their HPA axis activity as well as in their response to neuroendocrine challenges (Bremner et al., 2003). Cholinergic neurotransmission circuits are also known to be subject to circadian changes (Carlson, 1994) and control the sensorimotor cortical regions regulating such activity (Fibiger, 1991). Conversely, the stressful shift in circadian cycle is known to produce considerable impairment of the locomotor behavior of both animals and humans (Weibel et al., 1999; Lightman et al., 2000). To assess the general responsiveness of normal and TgS mice to this stressor, we recorded their locomotor activity, using telemetric transmitters, implanted in the peritoneal cavity. Movement was monitored by a sensor to
estimate the distance the animal moved over several days. Under routine conditions, both the parental strain FVB/N mice and TgS mice displayed similar home cage activity. Their circadian rhythms included, as expected, significantly more frequent and pronounced locomotor activity during the early part of the dark phase of the circadian cycle. Seventy-two hours following reversal of the light/dark phases, both genotypes lost most of the circadian rhythm in their locomotor activity. This response is common to most rodents, as reported by others (Hillegaart and Ahlenius, 1994) with a strong contribution of their background genotypes. However, FVB/N and TgS mice subjected to a circadian light/dark shift presented distinctive behavioral patterns. The parent strain, similarly to other mammals, presented general reduction in post-shift locomotor activity. In sharp contrast, TgS mice showed a general increase in postshift activity, both during the dark and the light phases. In particular, activity in the dark phase of the reversed cycle was significantly greater in TgS compared with control mice. These findings indicate that adjustment to the circadian insult was markedly impaired in TgS mice, suggesting that these mice display a genetic predisposition to abnormal responses to changes in the circadian rhythm, and perhaps other stresses, as well.
Impaired social interactions Decrements in social investigation following repeated contact between mice were reported by others to be subject to cholinergic regulation (Winslow and Camacho, 1995). The intensified response of TgS mice to the stress of a circadian switch therefore
597 suggested that the excess of AChE is the cause. However, in spite of their inbred genotype, individual TgS mice presented distinct patterns of social interaction, attesting to the acquired nature of much of the phenotype. Intriguingly, the inbred TgS mice presented relatively high variability in their locomotion activity, suggesting experience-derived contribution to this phenotype. The variable nature of the excessive locomotor activity in individual TgS mice indicated daily stress origin(s), variable in its extent and duration. The molecular origin of such heterogeneity could be the increased amount of neuronal vAChT, ChAT and mAChE-R mRNAs in the sensorimotor cortex and hippocampal neurons. While both psychological and physical stressors induce neuronal AChE-R over-production, the acquired feedback responses to their cholinergic imbalance may depend on individual experiences, explaining this variability. This conclusion further implies that transcriptional activation and shifted alternative splicing in the brain of mammals are most valuable, in that they prevent excess accumulation of AChE-S and its consequent behavioral damage, Recently, it was reported that the C-terminus of AChE-S includes a region with neurotoxic properties (Greenfield and Vaux, 2002), perhaps through its capacity to promote nuclear translocation of AChE-S (Perry et al., 2002). While the exaggerated stress responses, such as the intense locomotor response to the mild stress of a circadian switch can be expected to exacerbate the hypocholinergic state of these already compromised animals, it cannot compensate for the yet-unknown nuclear effects and/or neurotoxicity of the excess AChE-S in neurons. Transient antisense suppression of transgenics' locomotor activity further substantiates the involvement of AChE-R in the circadian light/dark shiftassociated hyperactive response. To assess this assumption, TgS mice were injected intraperitoneally with 50 g/kg of a 3'-terminally 2'-O-methyl protected antisense oligonucleotide, EN101, that suppresses de novo production of mouse AChE-R (Shohami et al., 2000; Cohen et al., 2002). The difference between pre- and post-treatment locomotor activity was calculated for each animal, following EN101 or saline injection. TgS mice presented only a transient decrease in locomotor activity following EN101 treatment. In myasthenic rats with impaired
locomotion, and massive AChE-R excess in their circulation, 500 mg/kg EN101 sufficed to improve locomotion for over 24h, demonstrating dose and time dependence for this effect (Brenner et al., 2003).
AChE overexpression and memory functioning Stress insults notably intensify fear memory (Nijholt et al., 2003). To explore the possibility that the modified expression pattern of AChE is causally involved in this phenomenon, immobilizationstressed wild-type mice were subjected to antisense suppression of their hippocampal AChE-R. Reduction of immunochemically detected AChE-R by 25% prevented the elevation of LTP and the characteristic freezing response to a foot-shock insult (ibid.). The finding that AD is associated with premature death of cholinergic neurons, initially within the basal forebrain, and the limited efficacy of anti-AChE drugs in ameliorating these deficits, raised the question whether cholinergic imbalance may produce cognitive impairments. Indeed, stress is also associated with cognitive disturbances (Lupien and Lepage, 2001). If so, the stress-induced increases in AChE expression and catalytic activity, may be causally involved with the stress component of cognitive impairment in the demented brain. In addition, AChE-S overexpression was found to enhance the formation of amyloid plaques in the brains of double transgenics, which express both human AChE-S and the Swedish double mutated amyloid protein, as compared with the parent strain, with amyloid mutation alone (Rees et al., 2003). This change in the properties of another neuroactive protein emphasizes the complexity of the feedback responses that may be induced under ACE-S overexpression and complicates the interpretation of the stress-induced changes.
Progressive decline in dendritic arbors Concomitant with the effects of stress on memory are remodelling changes in the dendritic arbors of neurons in various brain regions. These depend both on the type of stress and the brain region where they occur; for example, chronic immobilization
598 stress induces dendritic atrophy and debranching in CA3 pyramidal neurons of the hippocampus, while pyramidal and stellate neurons in the amygdala exhibit enhanced dendritic arborization in response to the same stress (Vyas et al., 2002). Chronic, unpredictable stress, however, had little effect on CA3 neurons, but induced atrophy in bipolar neurons of the basolateral complex in the amygdala (Vyas et al., 2002). Thus, the cellular and molecular differences among the responses to different kinds of stress are still largely unexplained. To examine their neurodeterioration, we compared the dendritic arbors in cortical neurons of TgS mice and wild-type controls and tested them in parallel in the Morris water maze. This test assesses spatial memory, which is known to deteriorate with age; however, the age-dependent deterioration in spatial memory was rapidly accelerated in TgS mice (Beeri et al., 1995), reaching a level of total failure in young adults (Beeri et al., 1997). The progressively reduced dendritic fields in the brains of these mice (Beeri et al., 1997) might represent the structural correlate of their spatial memory impairment. This supports the above summarized studies that have frequently revealed neuronal damage or atrophy, mirroring cognitive dysfunction in demented animals and humans. While it is not yet clear whether the limited dendritic arbors in TgS mice reflect enhanced pruning or a suppressed growth of neurites, the thinned neural network that this entails mimics a parallel phenomenon in the brain of AD patients (Ruppin and Reggia, 1995). The impairments in the Morris water maze performance in TgS mice thus suggest that prolonged AChE overexpression is associated with a progressive decline in learning and memory.
A ChE-R contribution to adverse stress responses is highly variable The delayed consequences of stress and neurodegeneration are both known to involve impairment of other aspects of learning and memory, in particular those that related to individual relationships. To test whether this aspect of behavior, as well, is associated with AChE regulation, we assessed the memory
functioning of TgS mice in the social recognition test, which is based on olfactory perception (Dantzer et al., 1990). To examine the effect of AChE-R overexpression on social recognition, and its correlation with pre-treatment symptom severity, TgS mice were divided into three groups, and those exhibiting short or long exploration of a familiar juvenile were selected based on a baseline social recognition test. AChE-R mRNA was then suppressed by EN101 (AS3) treatment. Social recognition was tested again 1, 3 and 6 days following two intracerebroventricular injections of EN101, separated by 24 h. As expected, there was a significant overall difference between the short and the long groups in exploration time. However, post-hoc tests revealed that these groups differed significantly only during the pre-treatment day, and not after the EN101 treatment. Furthermore, within the long, but not the short explorers group, social exploration of the 'same' juvenile was significantly reduced 1 day after the EN101 injection, (at the same dose as before), with progressive increases in social exploration time during the 5 subsequent days. This experiment thus demonstrated both the causal involvement and the reversibility of the AChE-R effect, especially in animals with severe pre-treatment impairments and in comparison to the short-term efficacy of tacrine (Cohen et al., 2002).
Antisense A ChE-R mRNA suppression selectively reduces brain A ChE-R protein To conceptually prove the putative role of AChE-R in mediating the impaired social recognition, we conducted a second experiment, in which mice completed a social recognition test before and after two injections with either EN101 or a sequence specificity control. The tested EN101 effect was central (due to the intracerebroventricular administration). Twenty-four hours following the second test, their brains were removed and AChE-R expression was assessed. As found in the first experiment, Tg mice with long pre-treatment explorative behavior displayed a significant improvement in social exploration of the 'same' juvenile 24 h following the second treatment with EN101, but not with the
599 irrelevant AS-ON targeted to BuChE mRNA (ASB). Control mice with either long or short pre-treatment social exploration showed no response to either EN101 or the irrelevant antisense oligonucleotide, perhaps reflecting a limitation in the resolution power of these behavioral tests. Immunodetected AChE-R protein levels were significantly lower in EN101 treated mice as compared with ASB-treated mice, regardless of their genotype or pre-treatment behavior pattern. In contrast, densitometric analysis of immunodetected total AChE protein (detected by an antibody targeted to the N-terminus, common to both isoforms) revealed essentially unchanged signals. Together, these findings attest to the selectivity of the antisense treatment for treating AChE-R over-expressing animals and its sequence-specificity in reversing the AChE-R induced impairment of behavior. The outcome of this second experiment has also provided a tentative explanation of the long duration of the antisense effect, in that disrupted function appears to be associated with higher levels of AChE-R. So long as AChE-R remains below a threshold level, function remains normal, even if the antisense agent is no longer present. AChE-R expression, thus, presents a relatively wide safety margins, above which it causes deleterious effects.
Peripheral/central interactions: transfer of stress signals through cholinergic pathways Recently, a "cholinergic anti-inflammatory pathway" has been identified in which cholinergic signalling through the efferent vagus nerve modulates the mammalian inflammatory response (Bernik et al., 2002; Borovikova et al., 2000; Tracey et al., 2001). ACh, the principal vagal neurotransmitter, significantly attenuated the release of the proinflammatory cytokines, tumor necrosis factor-0~ (TNF00, interleukin (IL)-I{3, IL-6 and IL-18 (but not the anti-inflammatory cytokine IL-10), in lipopolysaccharide-stimulated human macrophage cultures and in live rats. The signalling pathway involves the ~7 nicotinic receptor (Tracey, 2002). A parallel process in the brain, or indeed in other leukocytes, has not yet been explored. An interesting point of this
IL-1
~- A C h E t
= acetylcholine,~
Scheme 1 observation is that ACh in the blood is given a physiological role, and as a corollary, the AChE of blood, notably the AChE-E on the surface of erythrocytes is placed in the spotlight. However, the amount of blood AChE-E changes only very slowly, which seems to disqualify it from a regulatory role in inflammatory responses; AChE-S, which is expressed in multiple leukocyte lineages (Deutsch et al., 2002), as well as AChE-R, being soluble and having a short half-life, are more likely regulators of responses to inflammatory challenges, as well as controllers of the heightened anxiety that accompanies the inflammatory responses (Danzer, 1999; Reichenberg et al., 2001). In light of reports that stress, both in humans and in animals, involves increased production of proinflammatory cytokines, e.g. IL-1 (Maes et al., 1998; Nguyen et al., 1998; Spivak et al., 1997), and that IL-1 causes AChE over-production in PC12 cells (Li et al., 2000), we postulate the following relationship (Scheme 1): IL-1 induction of AChE over-expression suppresses ACh, ablating the interference by ACh in IL-1 production, a cycle that may explain prolongation of both the stress responses and the overexpression of AChE and cytokines (Scheme 1). Further experiments will be required for establishing the complete circle and demonstrating the causal involvement of AChE in regulating the brain levels of pro-inflammatory cytokines. A logical extension of this concept is that AChE-S and/or AChE-R levels should be pivotal for regulation of proinflammatory cytokines in both the peripheral and central nervous systems. Thus, stressinduced elevation of cortisol levels results in elevated neuronal AChE production (Meshorer et al., 2002). This would reduce ACh and elevate production of pro-inflammatory cytokines (Scheme 2). According to this model, in addition to the direct suppression of blood cytokine production by cortisol (Marx, 1995), cortisol would also activate brain cytokine production, by an indirect cholinergic-mediated mechanism. This indirect route may explain some of the permissive and pro-inflammatory actions of
600
stress
The future of acetylcholinesterase in stress studies
Anti-AChE therapies
Scheme 2 The cellular and biochemical events to which we attribute predicted stress-associated changes. glucocorticoids (Brooke and Sapolsky, 2002; Munck and Naray-Fejes-Toth, 1994; Wilckens and De Rijk, 1997). In support of this hypothesis, we have recently shown that the endotoxin-induced changes in human working and declarative memory associate both with degradation of plasma AChE-R and with the circulation levels of pro-inflammatory cytokines (Cohen et al., 2003). Stress induces the release of cytokines and cortisol. Cytokines elevation is associated with immune, neuroendocrine and behavioral responses (Konsman et al., 2002). Cortisol induces AChE-R production, which should elevate (A) AChE plasma activity. In the periphery, ACh released from neuronal vesicles into the synaptic cleft, acts to suppress (I?) cytokines production in macrophages (upward curved arrow). If this is also the case within the brain, stress-induced increase in AChE-R expression and activity will result in lower ACh levels, which may further enhance neuronal cytokine production. AChE-R accumulation is transient because this enzyme and its mRNA are relatively unstable. Therefore, the cognitive effects of inflammation would be expected to be limited to the duration of cortisol's presence in the circulation and perhaps several hours after, but not much longer. This theory is compatible with previous findings in humans (Yirmiya et al., 2000) and with our on-going studies.
As the role of AChE-R in stress responses is brought to light, it is becoming apparent that a key to regulating these responses is manipulation of the levels of this protein. It will be a challenge to devise therapeutic strategies that will regulate it, while leaving normal cholinergic neurotransmission unaffected. One such strategy uses an antisense reagent to specifically destroy the mRNA that encodes that variant. Although the antisense approach has been around for several decades, the mechanism of action of antisense oligonucleotides is incompletely understood (Opalinska and Gewirtz, 2002). Nevertheless, whatever the molecular mechanisms there have been experimental and even clinical successes in using this approach (Orr, 2001). In our hands, such an agent, EN101, has successfully aided recovery from closed head injury in mice (Shohami et al., 2000), retrieved functional working memory in TgS mice (Cohen et al., 2002) and reversed the symptoms of experimental autoimmune myasthenia gravis in rats (Brenner et al., 2003), EN101 is now being successfully tested in the clinic for treatment of human myasthenia gravis (Argov et al., 2003).
The cholinergic component of stress may confer bidirectional effects on cognitive functions Behind the conventional view of stress responses reflecting a complex syndrome or disease, lies the fact that acute stress represents an extreme example of a natural process, enabling the adjustment to changing environment through neuronal plasticity. This is consistent with the fact that acute glucocorticoid administration, while impairing retrieval of long-term declarative memory (Kirschbaum et al., 1996), improves the working memory in humans (de Quervain et al., 2000). It is also compatible with the reports that cholinergic enhancement facilitates the increased selectivity of perceptual processing during working memory (Furey et al., 2000), and that higher cortisol values facilitate spatial memory in toddlers (Stansbury et al., 2000). Thus, the stress-induced effects on memory functions are complex, and may
601 reflect both the intensity of the stressful experience and the type of memory function that was measured. This chapter would be incomplete without mentioning the complex signalling cascades which are pivotal for the described stress responses. These likely involve changes in, especially PCK[3II, which has been found essential for the contextual fear response, a characteristic consequence of acute stress (Weeber et al., 2000). PCKI3II is an alternative splicing product of the PCK[3 gene. A tentative link between this signaling cascade and the cholinergic feedback response to stress was indicated in a recent two-hybrid search for cellular-binding partners of AChE-R, which revealed association of AChE-R with the PKC[3II scaffold protein, RACK1 (Birikh et al., 2003). Compatible with this finding, inherited AChE-R overexpression in transgenic mice resulted in perikaryal clusters enriched with PKC[3II, accompanied by PKC-augmented LTP enhancement (Nijholt et al., 2003). The decreases in RACK1 in the brain of AD patients (Battaini et al., 1999) further supports the yet elusive link between the stress load with which one is confronted and the onset of neurodegeneration.
Abbreviations ACh ACTH ACHE AChE AD BuChE ChAT CNS CRF DFP HPA NMJ PB PTSD TgR
TgS vAChT
acetylcholine adrenocorticotropic hormone; a.k.a. corticotropin acetylcholinesterase gene acetylcholinesterase protein Alzheimer's disease butyrylcholinesterase choline acetyltransferase central nervous system corticotrophin releasing factor diisopropylfluorophosphonate hypothalamo-pituitary-adrenal (axis) neuromuscular junction pyridostigmine bromide post-traumatic stress disorder a transgenic mouse strain that expresses human AChE-R a transgenic mouse strain that expresses human AChE-S vesicular acetylcholine transporter
Acknowledgements Some of the work reported here was supported by the US Army Medical Research and Materiel C o m m a n d under grant No. DAMD17-99-1-9547, the Israel Science Foundation (618/02-1), the USIsrael Binational Science Foundation (1999-115), European Community Grant (LSHM-CT-2003503330) and Ester Neuroscience.
References Abou-Donia, M.B., Goldstein, L.B., Jones, K.H., AbdelRahman, A.A., Damodaran, T. Ca++-dependent protein kinase (PKC) activity and intracellular translocation (Young et al., 2002). V., Dechkovskaia, A.M., Bullman, S.L., Amir, B.E. and Khan, W.A. (2001) Locomotor and sensorimotor performance deficit in rats following exposure to pyridostigmine bromide, DEET, and permethrin, alone and in combination. Toxicol Sci, 60: 305-314. Abou-Donia, M.B., Wilmarth, K.R., Jensen, K.F., Oehme, F.W. and Kurt, T.L. (1996) Neurotoxicity resulting from coexposure to pyridostigmine bromide, deet, and permethrin: implications of Gulf War chemical exposures. J. Toxicol. Environ. Health, 48: 35-56. Acquas, E., Wilson, C. and Fibiger, H.C. (1996) Conditioned and unconditioned stimuli increase frontal cortical and hippocampal acetylcholine release: effects of novelty, habituation, and fear. J. Neurosci., 16: 3089-3096. Andres, C., Beeri, R., Friedman, A., Lev-Lehman, E., Henis, S., Timberg, R., Shani, M. and Soreq, H. (1997) Acetylcholinesterase-transgenic mice display embryonic modulations in spinal cord choline acetyltransferase and neurexin IB gene expression followed by late-onset neuromotor deterioration. Proc. Natl. Acad. Sci. USA, 94: 8173-8178. Appleyard, M.E. (1992) Secreted acetylcholinesterase: nonclassical aspects of a classical enzyme. Trends Neurosci., 15: 485-490. Argov, Z., McKee, D., Agus, S., Soreq, H., Ben-Yoseph, O., Brawer, S. and Sussman, J. (2003) EN101: a novel antisense therapeutic strategy for myasthenia gravis. Paper presented at: Fifty-fifth Annual Meeting of the American Academy of Neurology, 2003 (Honolulu). Ashani, Y., Shapira, S., Levy, D., Wolfe, A.D., Doctor, B.P. and Raveh, L. (1991) Butyrylcholinesterase and acetylcholinesterase prophylaxis against soman poisoning in mice. Biochem. Pharmacol., 41: 37-41. Atanasova, E., Chiappa, S., Wieben, E. and Brimijoin, S. (1999) Novel messenger RNA and alternative promoter for murine acetylcholinesterase. J. Biol. Chem., 274: 21078-21084.
602 Bartels, M., de Geus, E.J., Kirschbaum, C., Sluyter, F., and Boomsma, D.I. (2003) Heritability of daytime cortisol levels in children. Behav. Genet., 33: 421-433. Bartus, R.T., Dean, R.L., 3rd, Beer, B. and Lippa, A.S. (1982) The cholinergic hypothesis of geriatric memory dysfunction. Science, 217:408-4 14. Battaglia, M. (2002) Beyond the usual suspects: a cholinergic route for panic attacks. Mol Psychiatry, 7: 239-246. Battaini, F., Pascale, A., Lucchi, L., Pasinetti, G.M. and Govoni, S. (1999) Protein kinase C anchoring deficit in postmortem brains of Alzheimer's disease patients. Exp. Neurol., 159: 559-564. Beck, K.D., Zhu, G., Beldowicz, D., Brennan, F.X., Ottenweller, J.E., Moldow, R.L. and Servatius, R.J. (2001) Central nervous system effects from a peripherally acting cholinesterase inhibiting agent: interaction with stress or genetics. Ann. N. Y. Acad. Sci., 933: 310-314. Beeri, R., Andres, C., Lev-Lehman, E., Timberg, R., Huberman, T., Shani, M. and Soreq, H. (1995) Transgenic expression of human acetylcholinesterase induces progressive cognitive deterioration in mice. Curr. Biol., 5: 1063-1071. Beeri, R., Le Novere, N., Mervis, R., Huberman, T., Grauer, E., Changeux, J.P. and Soreq, H. (1997) Enhanced hemicholinium binding and attenuated dendrite branching in cognitively impaired acetylcholinesterase-transgenic mice. J. Neurochem., 69: 2441-2451. Bernatova, I., Key, M.P. Lucot, J.B. and Morris, M. (2002) Circadian differences in stress-induced pressor reactivity in mice. Hypertension, 40: 768-773. Bernik, T.R., Friedman, S.G., Ochani, M., DiRaimo, R., Ulloa, L., Yang, H., Sudan, S., Czura, C.J., Ivanova, S.M. and Tracey, K.J. (2002) Pharmacological stimulation of the cholinergic antiinflammatory pathway. J. Exp. Med., 195: 781-788. Berrouschot, J., Baumann, I., Kalischewski, P., Sterker, M. and Schneider, D. (1997) Therapy of myasthenic crisis. Crit. Care Med., 25: 1228-1235. Bhatnagar, S., Costall, B. and Smythe, J.W. (1997) Hippocampal cholinergic blockade enhances hypothalamicpituitary-adrenal responses to stress. Brain Res., 766: 244-248. Bigbee, J.W., Sharma, K.V., Gupta, J.J. and Dupree, J.L. (1999) Morphogenic role for acetylcholinesterase in axonal outgrowth during neural development. Environ. Health Perspect., 107: Suppl 1, 81-87. Birikh, K., Sklan, E., Shoham, S. and Soreq, H. (2003) Interaction of "readthrough" acetylcholinesterase with RACK1 and PKCbetaII correlates with intensified fearinduced conflict behavior. Proc. Natl. Acad. Sci. USA, 100: 283-288. Blanchet, G., Carpentier, P., Lallement, G. and SentenacRoumanou, H. (1994) Prevention and treatment of status epilepticus induced by soman. Ann. Pharm. Fr., 52:11-24.
Borovikova, L.V., Ivanova, S., Zhang, M., Yang, H., Botchkina, G.I., Watkins, L.R., Wang, H., Abumrad, N., Eaton, J.W. and Tracey, K.J. (2000) Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin. Nature, 405: 458-462. Botti, S.A., Felder, C.E., Sussman, J.L. and Silman, I. (1998) Electrotactins: a class of adhesion proteins with conserved electrostatic and structural motifs. Protein Eng., 11:415-420. Bremner, J.D., Vythilingam, M., Anderson, G., Vermetten, E., McGlashan, T., Heninger, G., Rasmusson, A., Southwick, S.M. and Charney, D.S. (2003) Assessment of the hypothalamic-pituitary-adrenal axis over a 24-hour diurnal period and in response to neuroendocrine challenges in women with and without childhood sexual abuse and posttraumatic stress disorder. Biol. Psychiatry, 54: 710-718. Brenner, T., Hamra-Amitay, Y., Evron, T., Boneva, N., Seidman, S. and Soreq, H. (2003) The role of readthrough acetylcholinesterase in the pathophysiology of myasthenia gravis. FASEB J., 17: 214-222. Brindle, N., Song, Y., Rogaeva, E., Premkumar, S., Levesque, G., Yu, G., Ikeda, M., Nishimura, M., Paterson, A., Sorbi, S. et al. (1998) Analysis of the butyrylcholinesterase gene and nearby chromosome 3 markers in Alzheimer disease. Hum. Mol. Genet., 7: 933-935. Brooke, S.M. and Sapolsky, R.M. (2002) Glucocorticoid Exacerbation of gpl20 Neurotoxicity: Role of Microglia. Exp. Neurol., 177:151-158. Burchfiel, J.L. and Duffy, F.H. (1982) Organophosphate neurotoxicity: chronic effects of sarin on the electroencephalogram of monkey and man. Neurobehav. Toxicol. Teratol., 4: 767-778. Calogero, A.E., Gallucci, W.T., Bernardini, R., Saoutis, C., Gold, P.W. and Chrousos, G.P. (1988) Effect of cholinergic agonists and antagonists on rat hypothalamic corticotropinreleasing hormone secretion in vitro. Neuroendocrinology, 47: 303-308. Calogero, A.E., Kamilaris, T.C., Gomez, M.T., Johnson, E.O., Tartaglia, M.E., Gold, P.W. and Chrousos, G.P. (1989) The muscarinic cholinergic agonist arecoline stimulates the rat hypothalamic-pituitary-adrenal axis through a centrally-mediated corticotropin-releasing hormone-dependent mechanism. Endocrinology, 125: 2445-2453. Camp, S. and Taylor, P. (1998) Intronic elements appear essential for the differentiation-specific expression of acetylcholinesterase in C2C12 myotubes. In: Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L. and Gentry, M.K. (Eds.), Structure and Function of Cholinesterases and Related Proteins. Plenum Press, New York, pp. 51-55. Carlson, N.R. (1994) Physiology of Behavior, Allyn and Bacon. Needham Heights, MA. Carmichael, W. (1994) The toxins of cyanobacteria. Sci. Amer., 270: 78-86. Chan, R.Y., Adatia, F.A., Krupa, A.M. and Jasmin, B.J. (1998) Increased expression of acetylcholinesterase T and R
603 transcripts during hematopoietic differentiation is accompanied by parallel elevations in the levels of their respective molecular forms. J. Biol. Chem., 273: 9727-9733. Chaney, L.A., Rockhold, R.W., Wineman, R.W., and Hume, A.S. (1999) Anticonvulsant-resistant seizures following pyridostigmine bromide (PB) and N,N-diethyl-m-toluamide (DEET) Toxicol. Sci., 49: 306-311. Chaney, L.A., Wineman, R.W., Rockhold, R.W. and Hume, A.S. (2000) Acute effects of an insect repellent, N,N-diethylm-toluamide, on cholinesterase inhibition induced by pyridostigmine bromide in rats. Toxicol. Appl. Pharmacol., 165: 107-114. Chen, Y., Brunson, K.L., Muller, M.B., Cariaga, W. and Baram, T.Z. (2000) Immunocytochemical distribution of corticotropin-releasing hormone receptor type-1 (CRF(1))like immunoreactivity in the mouse brain: light microscopy analysis using an antibody directed against the C-terminus. J. Comp. Neurol., 420: 305-323. Cohen, O., Erb, C., Ginzberg, D., Pollak, Y., Seidman, S., Shoham, S., Yirmiya, R. and Soreq, H. (2002) Neuronal overexpression of 'readthrough' acetylcholinesterase is associated with antisense-suppressible behavioral impairments. Mol. Psychiatry, 7: 874-885. Cohen, O., Reichenberg, A., Perry, C., Ginzberg, D., Pollmficher, T., Soreq, H. and Yirmiya, R. (2003) Endotoxin-induced changes in human working and declarative memory are associated with C - terminal cleavage of plasma "readthrough" acetylcholinesterase. J. Molec. Neurosci., 21: 199-212. Cook, W.O., Beasley, V.R., Dahlem, A.M., Dellinger, J.A., Harlin, K.S. and Carmichael, W.W. (1988) Comparison of effects of anatoxin-a(s) and paraoxon, physostigmine and pyridostigmine on mouse brain cholinesterase activity. Toxicon., 26: 750-753. Dantzer, R., Tazi, A. and Bluthe, R.M. (1990) Cerebral lateralization of olfactory-mediated affective processes in rats. Behav. Brain Res., 40: 53-60. Danzer, R., Whollman, E.E. and Yirmiya, R. (1999) Cytokines, Stress and Depression. Kluwer Academic/Plenum, New York. Daoud, R., Da Penha Berzaghi, M., Siedler, F., Hubener, M. and Stamm, S. (1999) Activity-dependent regulation of alternative splicing patterns in the rat brain. Eur. J. Neurosci., 11: 788-802. Darboux, I., Barthalay, Y., Piovant, M. and Hipeau Jacquotte, R. (1996) The structure-function relationships in Drosophila neurotactin show that cholinesterasic domains may have adhesive properties. EMBO J., 15: 4835-4843. Darreh-Shori, T., Almkvist, O., Guan, Z.Z., Garlind, A., Strandberg, B., Svensson, A.L., Soreq, H., Hellstrom-Lindahl, E. and Nordberg, A. (2002) Sustained cholinesterase inhibition in AD patients receiving rivastigmine for 12 months. Neurology, 59: 563-572.
Darreh-Shori, T., Hellstr6m-Lindahl, E., Flores-Flores, C, Guan, Z., Soreq, H. and Nordberg, A. (2004) Long-lasting acetylcholinesterase splice variations in anticholinesterasetreated Alzheimer's disease patients. J. Neurochem., 88: 1102-1113. Day, J.C., Koehl, M., Deroche, V., Le Moal, M. and Maccari, S. (1998a) Prenatal stress enhances stress- and corticotropin-releasing factor-induced stimulation of hippocampal acetylcholine release in adult rats. J. Neurosci., 18: 1886-1892. Day, J.C., Koehl, M., Le Moal, M. and Maccari, S. (1998b) Corticotropin-releasing factor administered centrally, but not peripherally, stimulates hippocampal acetylcholine release. J. Neurochem., 71: 622-629. Dazzi, L., Motzo, C., Imperato, A., Serra, M., Gessa, G.L. and Biggio, G. (1995) Modulation of basal and stress-induced release of ace@choline and dopamine in rat brain by abecarnil and imidazenil, two anxioselective gamma-aminobutyric acidA receptor modulators. J. Pharmacol. Exp. Ther., 273: 241-247. de Quervain, D.J., Roozendaal, B., Nitsch, R.M., McGaugh, J.L., and Hock, C. (2000) Acute cortisone administration impairs retrieval of long-term declarative memory in humans. Nat. Neurosci., 3: 313-314. Deutsch, V.R., Pick, M., Perry, C., Grisaru, D., Hemo, Y., Golan-Hadari, D., Grant, A., Eldor, A. and Soreq, H. (2002) The stress-associated acetylcholinesterase variant AChE-R is expressed in human CD34(+) hematopoietic progenitors and its C-terminal peptide ARP promotes their proliferation. Exp. Hematol., 30:1153-1161. Doctor, B.P., Blick, D.W., Caranto, G., Castro, C.A., Gentry, M.K., Larrison, R., Maxwell, D.M., Murphy, M.R., Schutz, M., Waibel, K. et al. (1993) Cholinesterases as scavengers for organophosphorus compounds: protection of primate performance against soman toxicity. Chem. Biol. Interact, 87: 285-293. Drachman, D.A. and Leavitt, J. (1974) Human memory and the cholinergic system. A relationship to aging? Arch. Neurol., 30: 113-121. Ellis, K.A. and Nathan, P.J. (2001) The pharmacology of human working memory. Int. J. Neuropsychopharmacol., 4:299-313. Erb, C., Troost, J., Kopf, S., Schmitt, U., Loffelholz, K., Soreq, H. and Klein, J. (2001) Compensatory mechanisms enhance hippocampal acetylcholine release in transgenic mice expressing human acetylcholinesterase. J. Neurochem., 77: 638-646. Esposito, P., Chandler, N., Kandere, K., Basu, S., Jacobson, S., Connolly, R., Tutor, D. and Yheoharides, T.C. (2002) Corticotropin-releasing hormone and brain mast cells regulate blood-brain-barrier permeability induced by acute stress. J. Pharmacol. Exp. Ther., 303: 1061-1066. Esposito, P., Gheorghe, D., Kandere, K., Pang, X., Connolly, R., Jacobson, S. and Theoharides, T.C. (2001) Acute stress increases permeability of the blood-brain-barrier
604 through activation of brain mast cells. Brain Res., 888:117-127. Evans, W.E. and Relling, M.V. (1999) Pharmacogenomics: translating functional genomics into rational therapeutics. Science, 286: 487-491. Farchi, N., Soreq, H. and Hochner, B. (2003) Chronic acetylcholinesterase overexpression induces multilevelled aberrations in mouse neuromuscular physiology. J. Physiol., 546: 165-173. Fibiger, H.C. (1991) Cholinergic mechanisms in learning, memory and dementia: a review of recent evidence. Trends Neurosci., 14: 220-223. Finkelstein, Y., Koffler, B., Rabey, J.M. and Gilad, G.M. (1985) Dynamics of cholinergic synaptic mechanisms in rat hippocampus after stress. Brain Res., 343: 314-319. Friedman, A., Kaufer, D., Pavlovsky, L. and Soreq, H. (1998) Cholinergic excitation induces activity-dependent electrophysiological and transcriptional responses in hippocampal slices. J. Physiol. Paris, 92: 329-335. Friedman, A., Kaufer, D., Shemer, J., Hendler, I., Soreq, H. and Tur-Kaspa, I. (1996) Pyridostigmine brain penetration under stress enhances neuronal excitability and induces early immediate transcriptional response. Nat. Med., 2: 1382-1385. Friedman, M. (2002) Tomato glycoalkaloids: role in the plant and in the diet. J. Agric. Food Chem., 50: 5751-5780. Fu, Y., Matta, S.G., Valentine, J.D., and Sharp, B.M. (1997) Adrenocorticotropin response and nicotine-induced norepinephrine secretion in the rat paraventricular nucleus are mediated through brainstem receptors. Endocrinology, 138: 1935-1943. Fullerton, C.S., and Ursano, R.J. (1990) Behavioral and psychological responses to chemical and biological warfare. Mil. Med., 155: 54-59. Furey, M.L., Pietrini, P., and Haxby, J.V. (2000) Cholinergic enhancement and increased selectivity of perceptual processing during working memory. Science, 290:2315-2319. Galyam, N., Grisaru, D., Grifman, M., Melamed-Book, N., Eckstein, F., Seidman, S., Eldor, A. and Soreq, H. (2001) Complex host cell responses to antisense suppression of ACHE gene expression. Antisense Nucl. Acid Drug Dev., 11: 51-57. Giacobini, E. (2000) Cholinesterase inhibitors: from the Calabar bean to Alzheimer therapy. In: Giacobini, E. (ed.), Cholinesterases and Cholinesterase Inhibitors, Martin Dunitz, London, pp. 181-226. Giacobini, E. (2002) Long-term stabilizing effect of cholinesterase inhibitors in the therapy of Alzheimer' disease. J. Neural. Transm. Suppl., 62:181-187. Gilad, G.M. (1987) The stress-induced response of the septo-hippocampal cholinergic system. A vectorial outcome of psychoneuroendocrinological interactions. Psychoneuroendocrinology, 12: 167-184.
Gilad, G.M., Mahon, B.D., Finkelstein, Y., Koffler, B. and Gilad, V.H. (1985) Stress-induced activation of the hippocampal cholinergic system and the pituitary-adrenocortical axis. Brain Res., 347: 404-408. Golomb, B. (1999) A Review of the Scientific Literature as it Pertains to Gulf War Illnesses, Vol 2 Santa Monica, Rand Corp. Gonzalez, A.M., and Pazos, A. (1992) Modification of muscarinic acetylcholine receptors in the rat brain following chronic immobilization stress: an autoradiographic study. Eur. J. Pharmacol., 223: 25-31. Gray, R., Rajan, A.S., Radcliffe, K.A., Yakehiro, M., and Dani, J.A. (1996) Hippocampal synaptic transmission enhanced by low concentrations of nicotine. Nature, 383: 713-716. Greenfield, S. (1996) Non-classical actions of cholinesterases: role in cellular differentiation, tumorigenesis and Alzheimer's disease. Neurochem. Int., 28: 485-490. Greenfield, S. and Vaux, D.J. (2002) Parkinson's disease, Alzheimer's disease and motor neurone disease: identifying a common mechanism. Neuroscience, 113: 485-492. Grifman, M., Galyam, N., Seidman, S. and Soreq, H. (1998) Functional redundancy of acetylcholinesterase and neuroligin in mammalian neuritogenesis. Proc. Natl. Acad. Sci. USA, 95: 13935-13940. Grisaru, D., Deutch, V., Shapira, M., Galyam, N., Lessing, B., Eldor, A. and Soreq, H. (2001) ARP, a peptide derived from the stress-associated acetylcholinesterase variant, has hematopoietic growth promoting activities. Mol. Med., 7: 93-105. Grisaru, D., Sternfeld, M., Eldor, A., Glick, D. and Soreq, H. (1999) Structural roles of acetylcholinesterase variants in biology and pathology. Eur. J. Biochem., 264: 672-686. Hillegaart, V. and Ahlenius, S. (1994) Time course for synchronization of spontaneous locomotor activity in the rat following reversal of the daylight (12:12 h) cycle. Physiol. Behav., 55: 73-75. Imperato, A., Puglisi-Allegra, S., Casolini, P. and Angelucci, L. (1991) Changes in brain dopamine and acetylcholine release during and following stress are independent of the pituitaryadrenocortical axis. Brain Res., 538:111-I 17. Imperato, A., Puglisi-Allegra, S., Casolini, P., Zocchi, A. and Angelucci, L. (1989) Stress-induced enhancement of dopamine and acetylcholine release in limbic structures: role of corticosterone. Eur. J. Pharmacol., 165: 337-338. Inglis, F.M., Day, J.C. and Fibiger, H.C. (1994) Enhanced acetylcholine release in hippocampus and cortex during the anticipation and consumption of a palatable meal. Neuroscience, 62: 1049-1056. Jamain, S., Quach, H., Betancur, C., Rastam, M., Colineaux, C., Gillberg, I.C., Soderstrom, H., Giros, B., Leboyer, M., Gillberg, C., et al. (2003) Mutations of the X-linked genes encoding neuroligins NLGN3 and NLGN4 are associated with autism. Nat. Genet., 34: 27-29.
605
Kalow, W. and Genest, K. (1957) A method for detection of atypical forms of human serum cholinesterase: Determination of dibucaine numbers. Can. J. Biochem., 35: 339-346. Kaneto, H. (1997) Learning/memory processes under stress conditions. Behav. Brain Res., 83: 71-74. Kant, G.J., Bauman, R.A., Feaster, S.R., Anderson, S.M., Saviolakis, G.A. and Garcia, G.E. (2001) The combined effects of pyridostigmine and chronic stress on brain cortical and blood acetylcholinesterase, corticosterone, prolactin and alternation performance in rats. Pharmacol. Biochem. Behav., 70:209-218. Karpel, R., Sternfeld, M., Ginzberg, D., Guhl, E., Graessmann, A. and Soreq, H. (1996) Overexpression of alternative human acetylcholinesterase forms modulates process extensions in cultured glioma cells. J. Neurochem., 66:114-123. Kaufer, D., Friedman, A., Seidman, S. and Soreq, H. (1998) Acute stress facilitates long-lasting changes in cholinergic gene expression. Nature, 393: 373-377. Kaufer, D., Friedman, A., Seidman, S. and Soreq, H. (1999) Anticholinesterases induce multigenic transcriptional feedback response suppressing cholinergic neurotransmission. Chem. Biol. Interact., 119-120: 349-360. Kaufer, D. and Soreq, H. (1999) Tracking cholinergic pathways from psychological and chemical stressors to variable neurodeterioration paradigms. Curr. Opin. Neurol., 12: 739-743. Kelsey, J.E. (1983) The role of norepinephrine and acetylcholine in mediating escape deficits produced by inescapable shocks. Behav. Neural. Biol., 37: 326-331. Kirschbaum, C., Wolf, O.T., May, M., WippicK W. and Hellhammer, D.H. (1996) Stress- and treatment-induced elevations of cortisol levels associated with impaired declarative memory in healthy adults. Life Sci., 58: 1475-1483. Koenigsberger, C., Chiappa, S. and Brimijoin, S. (1997) Neurite differentiation is modulated in neuroblastoma cells engineered for altered acetylcholinesterase expression. J. Neurochem., 69:1389-1397. Konsman, J.P., Parnet, P. and Dantzer, R. (2002) Cytokineinduced sickness behaviour: mechanisms and implications. Trends Neurosci., 25: 154-159. Kronman, C., Chitlaru, T., Elhanany, E., Velan, B. and Shafferman, A. (2000) Hierarchy of post-translation modifications involved in the circulatory longevity of glycoproteins. Demonstration of concerted contributions of glycan sialylation and subunit assembly to the pharmacokinetic behavior of acetylcholinesterase. J. Biol. Chem., 275: 29488-29502. La Du~ B.N., Bartels, C.F., Nogueira, C.P., Hajra, A., Lightstone, H., Van der Spek, A. and Lockridge, O. (1990) Phenotypic and molecular biological analysis of human butyrylcholinesterase variants. Clin. Biochem., 23: 423-431.
Layer, P.G. (1996) Non-classical actions of cholinesterases: role in cellular differentiation, tumorigenesis and Alzheimer's disease. Neurochem. Int., 28: 491~,95. Lehmann, D.J., Johnston, C. and Smith, A.D. (1997) Synergy between the genes for butyrylcholinesterase K variant and apolipoprotein E4 in late-onset confirmed Alzheimer's disease. Hum. Mol. Genet., 6: 1933-1936. Lehmann, D.J., Nagy, Z., Litchfield, S., Borja, M.C. and Smith, A.D. (2000) Association of butyrylcholinesterase K variant with cholinesterase-positive neuritic plaques in the temporal cortex in late-onset Alzheimer's disease. Hum. Genet., 106: 447-452. Li, Y., Liu, L., Kang, J., Sheng, J.G., Barger, S.W., Mrak, R.E. and Griffin, W.S. (2000) Neuronal-glial interactions mediated by interleukin-1 enhance neuronal acetylcholinesterase activity and mRNA expression. J. Neurosci., 20: 149-155. Lightman, S.L., Windle, R.J., Julian, M.D., Harbuz, M.S., Shanks, N., Wood, S.A., Kershaw, Y.M. and Ingram, C.D. (2000) Significance of pulsatility in the HPA axis. Novartis Found Symp., 227: 244-257. Liu, W.F. (1992) Acute effects of oral low doses of pyridostigmine on simple visual discrimination and unconditioned consummatory acts in rats. Pharmacol. Biochem. Behav., 41: 251-254. Llinas, R.R. and Greenfield, S.A. (1987) On-line visualization of dendritic release of acetylcholinesterase from mammalian substantia nigra neurons. Proc. Natl. Acad. Sci. USA, 84: 3047-3050. Luo, Z.D., Wang, Y., Werlen, G., Camp, S., Chien, K.R. and Taylor, P. (1999) Calcineurin enhances acetylcholinesterase mRNA stability during C2-C12 muscle cell differentiation. Mol. Pharmacol., 56: 886-894. Lupien, S.J. and Lepage, M. (2001) Stress, memory, and the hippocampus: can't live with it, can't live without it. Behav. Brain Res., 127: 137-158. Lupien, S.J., Maheu, F., Tu, M.T., Lemay, M., McEwen, B.S. and Meaney, M.J. (1999) Increased cortisol levels and impaired cognition in human aging: implication for depression and dementia in later life. Rev. Neurosci., 10: 117-139. Maes, M., Song, C., Lin, A., De Jongh, R., Van Gastel, A., Kenis, G., Bosmans, E., De Meester, I., Benoy, I., Neels, H., Demedts, P., Janca, A., Scharpe, S. and Smith, R.S. (1998) The effects of psychological stress on humans: increased production of pro-inflammatory cytokines and a Thl-like response in stress-induced anxiety. Cytokine, 10:313-318. Marchot, P., Prowse, C.N., Kanter, J., Camp, S., Ackermann, E.J., Radic, Z., Bougis, P.E., and Taylor, P. (1997) Expression and activity of mutants of fasciculin, a peptidic acetylcholinesterase inhibitor from mamba venom. J. Biol. Chem., 272:3502-3510. Marx, J. (1995) How the glucocorticoids suppress immunity. Science, 270: 232. Massoulie, J. (2000) Molecular forms and anchoring of acetylcholinesterase. In: E. Giacobini, (Ed.), Cholinesterases
606 and Cholinesterase Inhibitors. Martin Dunitz, London, pp. 81-101. Matta, S.G., Singh, J. and Sharp, B.M. (1990) Catecholamines mediate nicotine-induced adrenocorticotropin secretion via alpha-adrenergic receptors. Endocrinology, 127: 1646-1655. McGehee, D.S., Krasowski, M.D., Fung, D.L., Wilson, B., Gronert, G.A. and Moss, J. (2000) Cholinesterase inhibition by potato glycoalkaloids slows mivacurium metabolism. Anesthesiology, 93: 510-519. Meshorer, E., Erb, C., Gazit, R., Pavlovsky, L., Kaufer, D., Friedman, A., Glick, D., Ben-Arie, N. and Soreq, H. (2002) Alternative splicing and neuritic mRNA translocation under long-term neuronal hypersensitivity. Science, 295: 508-512. Meshorer, E. and Soreq, H. (2002) Pre-mRNA splicing modulations in senescence Aging Cell, 1(1): 10-16. Mizoguchi, K., Yuzurihara, M., Ishige, A., Sasaki, H. and Tabira, T. (2001) Effect of chronic stress on cholinergic transmission in rat hippocampus. Brain Res., 915:108-111. Mizuno, T. and Kimura, F. (1997) Attenuated stress response of hippocampal acetylcholine release and adrenocortical secretion in aged rats. Neurosci. Lett., 222: 49-52. Mor, I., Grisaru, D., Titelbaum, L., Evron, T., Richler, C., Wahrman, J., Sternfeld, M., Yogev, L., Meiri, N., Seidman, S. and Soreq, H. (2001) Modified testicular expression of stressassociated "readthrough" acetylcholiensterase predicts male infertility. FASEB J., 15: 2039-2041. Munck, A. and Naray-Fejes-Toth, A. (1994) Glucocorticoids and stress: permissive and suppressive actions. Ann. N. Y. Acad. Sci., 746: 115-130. Nguyen, K.T., Deak, T., Owens, S.M., Kohno, T., Fleshner, M., Watkins, L.R., and Maier, S.F. (1998) Exposure to acute stress induces brain interleukin-lbeta protein in the rat. J. Neurosci., 18: 2239-2246. Nguyen, T. and Sudhof, T.C. (1997) Binding properties of neuroligin 1 and neurexin 113 reveal function as heterophilic cell adhesion molecules. J Biol Chem, 272: 26032-26039. Nijholt, I., Farchi, N., Kye, M.-J., Sklan, E., Shoham, S., Verbeurre, B., Owen D., Hochner, B., Spiess, J., Soreq, H. and Blank, T. (2003) Alternative splicing modulation of hippocampal long-term potentiation and fear memory. Mol. Psych., in press. Nilsson, L., Nordberg, A., Hardy, J., Wester, P. and Winblad, B. (1986) Physostigmine restores 3H-acetylcholine efflux from Alzheimer brain slices to normal level. J. Neural. Transm., 67: 275-285. Ohmori, N., Itoi, K., Tozawa, F., Sakai, Y., Sakai, K., Horiba, N., Demura, H. and Suda, T. (1995) Effect of acetylcholine on corticotropin-releasing factor gene expression in the hypothalamic paraventricular nucleus of conscious rats. Endocrinology, 136: 4858-4863. Ohno, K., Engel, A.G., Brengman, J.M., Shen, X.M., Heidenreich, F., Vincent, A., Milone, M., Tan, E., Demirci, M., Walsh, P., et al. (2000) The spectrum of
mutations causing end-plate acetylcholinesterase deficiency. Ann. Neurol., 47: 162-170. Opalinska, J.B. and Gewirtz, A.M. (2002) Nucleic-acid therapeutics: basic principles and recent applications. Nat. Rev. Drug Discov., 1: 503-514. Orr, R.M. (2001) Technology evaluation: fomivirsen, Isis Pharmaceuticals Inc/CIBA vision. Curr. Opin. Mol. Ther., 3: 288-294. Orsini, C., Castellano, C. and Cabib, S. (2001) Pharmacological evidence of muscarinic-cholinergic sensitization following chronic stress. Psychopharmacology (Berl), 155: 144-147. Palmer, A.M. (2002) Pharmacotherapy for Alzheimer's disease: progress and prospects. Trends Pharmacol. Sci., 23: 426-433. Perrier, A.L., Massoulie, J. and Krejci, E. (2002) PRIMA: the membrane anchor of acetylcholinesterase in the brain. Neuron, 33: 275-285. Perry, C., Sklan, E.H., Birikh, K., Shapira, M., Trejo, L., Eldor, A. and Soreq, H. (2002) Complex regulation of acetylcholinesterase gene expression in human brain tumors. Oncogene, 21: 8428-8441. Perry, C. and Soreq, H. (2002) Transcriptional regulation of erythropoiesis. Fine tuning of combinatorial multi-domain elements. Eur. J. Biochem., 269:3607-3618. Perry, E., Martin-Ruiz, C., Lee, M., Griffiths, M., Johnson, M., Piggott, M., Haroutunian, V., Buxbaum, J.D., Nasland, J., Davis, K., Gotti, C., Clementi, F., Tzartos, S., Cohen, O., Soreq, H., Jaros, E., Perry, R., BaUard, C., McKeith, I. and Court, J. (2000) Nicotinic receptor subtypes in human brain ageing, Alzheimer and Lewy body diseases. Eur. J. Pharmacol., 393: 215-222. Ponting, C., Phillips, C., Davies, K. and Blake, D. (1997) PDZ domains: targeting signalling molecules to sub-membranous sites. Bioessays., 19: 469-479. Raveh, L., Ashani, Y., Levy, D., De La Hoz, D., Wolfe, A.D. and Doctor, B.P. (1989) Acetylcholinesterase prophylaxis against organophosphate poisoning. Quantitative correlation between protection and blood-enzyme level in mice. Biochem. Pharmacol., 38: 529-534. Rees, T., Hammond, P.I., Soreq, H., Younkin, S. and Brimijoin, S. (2003) Acetylcholinesterase promotes betaamyloid plaques in cerebral cortex. Neurobiol. Aging., 24: 777-787. Rees, T., Hammond, P.I., Soreq, H., Younkin, S. and Brimijoin, S. (2003) Acetylcholinesterase promotes betaamyloid plaques in cerebral cortex. Neurobiol. Aging, 24: 777-787 Reichenberg, A., Yirmiya, R., Schuld, A., Kraus, T., Haack, M., Morag, A. and Pollmacher, T. (2001) Cytokine-associated emotional and cognitive disturbances in humans. Arch. Gen. Psychiatry., 58: 445-452. Relyea, R.A. and Mills, N. (2001) Predator-induced stress makes the pesticide carbaryl more deadly to gray treefrog tadpoles (Hyla versicolor) Proc. Natl. Acad. Sci. USA, 98:2491-2496.
607
Roddick, J.G. (1989) The acetylcholinesterase-inhibitory activity of steroidal glycoalkaloids and their aglycones. Phytochemistry, 28: 2631-2634. Rosenstock, L., Keifer, M., Daniell, W., McConnell, R. and Claypoole, K. (1991) Chronic central nervous system effects of acute organophosphate pesticide intoxication. The Pesticide Health Effects Study Group. Lancet., 338: 223-227. Rubin, L.L. and Staddon, J.M. (1999) The cell biology of the blood-brain barrier. Annu. Rev. Neurosci., 22:11-28. Ruppin, E. and Reggia, J.A. (1995) Patterns of functional damage in neural network models of associative memory. Neural Comput., 7:1105-1127. Sago, H., Carlson, E.J., Smith, D.J., Kilbridge, J., Rubin, E.M., Mobley, W.C., Epstein, C.J. and Huang, T.T. (1998) TslCje, a partial trisomy 16 mouse model for Down syndrome, exhibits learning and behavioral abnormalities. Proc. Natl. Acad. Sci. USA, 95: 6256-6261. Sapolsky, R.M. (2002) Chickens, eggs and hippocampal atrophy. Nat. Neurosci., 5:1111-1113. Sauvage, M. and Steckler, T. (2001) Detection of corticotropinreleasing hormone receptor 1 immunoreactivity in cholinergic, dopaminergic and noradrenergic neurons of the murine basal forebrain and brainstem nuclei-potential implication for arousal and attention. Neurosci., 104: 643-652. Scheiffele, P., Fan, J., Choih, J., Fetter, R. and Serafini, T. (2000) Neuroligin expressed in nonneuronal cells triggers presynaptic development in contacting axons. Cell, 101: 657-669. Schwarz, M., Glick, D., Loewenstein, Y. and Soreq, H. (1995) Engineering of human cholinesterases explains and predicts diverse consequences of administration of various drugs and poisons. Pharmacol. Ther., 67: 283-322. Servatius, R.J., Ottenweller, J.E., Beldowicz, D., Guo, W., Zhu, G. and Natelson, B.H. (1998) Persistently exaggerated startle responses in rats treated with pyridostigmine bromide. J. Pharmacol. Exp. Ther., 287: 1020-1028. Shapira, M., Grant, A., Korner, M. and Soreq, H. (2000a) Genomic and transcriptional characterization of the human ACHE locus: complex involvement with acquired and inherited diseases. Isr. Med. Assoc. J., 2: 470-473. Shapira, M., Tur-Kaspa, I., Bosgraaf, L., Livni, N., Grant, A.D., Grisaru, D., Korner, M., Ebstein, R.P. and Soreq, H. (2000b) A transcription-activating polymorphism in the ACHE promoter associated with acute sensitivity to antiacetylcholinesterases. Hum. Mol. Genet., 9: 1273-1281. Shaw, N. (2002) The neurophysiology of concussion. Prog. Neurobiol., 67, 281. Shih, T.M. and McDonough, J.H., Jr. (1997) Neurochemical mechanisms in soman-induced seizures. J. Appl. Toxicol., 17: 255-264. Shohami, E., Kaufer, D., Chen, Y., Seidman, S., Cohen, O., Ginzberg, D., Melamed-Book, N., Yirmiya, R. and Soreq, H. (2000) Antisense prevention of neuronal damages following head injury in mice. J. Mol. Med., 78: 228-236.
Silman, I. and Futerman, A.H. (1987) Modes of attachment of acetylcholinesterase to the surface membrane. Eur. J. Biochem., 170:11-22. Silver, A. (1974) The Biology of Cholinesterases. Amsterdam, North Holland. Small, D.H., Michaelson, S. and Sberna, G. (1996) Nonclassical actions of cholinesterases: role in cellular differentiation, tumorigenesis and Alzheimer's disease. Neurochem. Int., 28: 453-483. Somani, S.M., Husain, K., Asha, T. and Helfert, R. (2000) Interactive and delayed effects of pyridostigmine and physical stress on biochemical and histological changes in peripheral tissues of mice. J. Appl. Toxicol., 20: 327-334. Song, J.Y., Ichtchenko, K., Sudhof, T.C. and Brose, N. (1999) Neuroligin 1 is a postsynaptic cell-adhesion molecule of excitatory synapses. Proc. Natl. Acad. Sci. USA, 96: 1100-1105.
Soreq, H. and Glick, D. (2000) Novel roles for cholinesterases in stress and inhibitor responses. In: Giacobini, E. (Ed.), Cholinesterases and Cholinesterase Inhibitors: Basic, Preclinical and Clinical Aspects. Martin Dunitz, London, pp. 47-61. Spivak, B., Shohat, B., Mester, R., Avraham, S., Gil-Ad, I., Bleich, A., Valevski, A. and Weizman, A. (1997) Elevated levels of serum interleukin-1 beta in combat-related posttraumatic stress disorder. Biol. Psychiatry., 42: 345-348. Stamm, S. (2002) Signals and their transduction pathways regulating alternative splicing: a new dimension of the human genome. Hum. Mol. Genet., 11: 2409-2416. Stansbury, K., Haley, D. and Koeneker, A. (2000) Higher cortisol values facilitate spatial memory in toddlers. Brief report. Ann. N. Y. Acad. Sci., 911: 456-458. Steckler, T. and Holsboer, F. (2001) Interaction between the cholinergic system and CRF in the modulation of spatial discrimination learning in mice. Brain Res., 906: 46-59. Stephens, R., Spurgeon, A., Calvert, I.A., Beach, J., Levy, L.S., Berry, H. and Harrington, J.M. (1995) Neuropsychological effects of long-term exposure to organophosphates in sheep dip. Lancet, 345:1135-1139. Sternfeld, M., Ming, G., Song, H., Sela, K., Timberg, R., Poo, M. and Soreq, H. (1998a) Acetylcholinesterase enhances neurite growth and synapse development through alternative contributions of its hydrolytic capacity, core protein, and variable C termini. J. Neurosci., 18: 1240-1249. Sternfeld, M., Patrick, J.D. and Soreq, H. (1998b) Position effect variegations and brain-specific silencing in transgenic mice overexpressing human acetylcholinesterase variants. J. Physiol. Paris, 92: 249-255. Sternfeld, M., Shoham, S., Klein, O., Flores-Flores, C., Evron, T., Idelson, G.H., Kitsberg, D., Patrick, J.W. and Soreq, H. (2000) Excess "readthrough" acetylcholinesterase attenuates but the "synaptic" variant intensifies neurodeterioration correlates. Proc. Natl. Acad. Sci. USA, 97: 8647-8652.
608 Stillman, M.J., Shukitt-Hale, B., Coffey, B.P., Levy, A. and Lieberman, H.R. (1997) In vivo hippocampal acetylcholine release during exposure to acute stress. Stress 1, 191-200. Stitcher, D.L., Harris, L.W., Heyl, W.C., and Alter, S.C. (1978) Effects of pyridostigmine and cholinolytics on cholinesterase and acetylcholine in Soman poisoned rats. Drug Chem. Toxicol., 1: 355-362. Tajima, T., Endo, H., Suzuki, Y., Ikari, H., Gotoh, M. and Iguchi, A. (1996) Immobilization stress-induced increase of hippocampal acetylcholine and of plasma epinephrine, norepinephrine and glucose in rats. Brain Res., 720: 155-158. Tizabi, Y. and Calogero, A.E. (1992) Effect of various neurotransmitters and neuropeptides on the release of corticotropin-releasing hormone from the rat cortex in vitro. Synapse, 10: 341-348. Tracey, K.J. (2002) The inflammatory reflex. Nature, 420: 853-859. Tracey, K.J., Czura, C.J. and Ivanova, S. (2001) Mind over immunity. FASEB J., 15: 1575-1576. Tsigelny, I., Shindyalov, I.N., Bourne, P.E., Sudhof, T.C. and Taylor, P. (2000) Common EF-hand motifs in cholinesterases and neuroligins suggest a role for Ca2 + binding in cell surface associations. Protein Sci., 9: 180-185. van Haaren, F., De Jongh, R., Hoy, J.B., Karlix, J.L., Schmidt, C.J., Tebbett, I.R. and Wielbo, D. (1999) The effects of acute and repeated pyridostigmine bromide administration on response acquisition with immediate and delayed reinforcement. Pharmacol. Biochem. Behav., 62: 389-394. Vereker, E., O'Donnell, E. and Lynch, M.A. (2000) The inhibitory effect of interleukin-lbeta on long-term potentiation is coupled with increased activity of stress-activated protein kinases. J. Neurosci., 20:6811-6819. Vyas, A., Mitra, R., Shankaranarayana Rao, B.S. and Chattarji, S. (2002) Chronic stress induces contrasting patterns of dendritic remodeling in hippocampal and amygdaloid neurons. J. Neurosci., 22:6810-6818. Weeber, E.J., Atkins, C.M., Selcher, J.C., Varga, A.W., Mirnikjoo, B., Paylor, R., Leitges, M. and Sweatt, J.D. (2000) A role for the beta isoform of protein kinase C in fear conditioning. J. Neurosci., 20:5906-5914.
Weibel, L., Follenius, M. and Brandenberger, G. (1999) Biologic rhythms: their changes in night-shift workers. Presse Med., 28: 252-258. Wilckens, T. and De Rijk, R. (1997) Glucocorticoids and immune function: unknown dimensions and new frontiers. Immunol. Today, 18:418-424. Winslow, J.T. and Camacho, F. (1995) Cholinergic modulation of a decrement in social investigation following repeated contacts between mice. Psychopharmacology (Berl), 121: 164-172. Wolfe, A.D., Blick, D.W., Murphy, M.R., Miller, S.A., Gentry, M.K., Hartgraves, S.L., and Doctor, B.P. (1992) Use of cholinesterases as pretreatment drugs for the protection of rhesus monkeys against soman toxicity. Toxicol. Appl. Pharmacol., 117: 189-193. Wolthuis, O.L., Groen, B., Busker, R.W. and van Helden, H.P. (1995) Effects of low doses of cholinesterase inhibitors on behavioral performance of robot-tested marmosets. Pharmacol. Biochem. Behav., 51: 443-456. Xie, J. and Black, D.L. (2001) A CaMK IV responsive RNA element mediates depolarization-induced alternative splicing of ion channels. Nature, 410: 936-939. Xie, J. and McCobb, D.P. (1998) Control of alternative splicing of potassium channels by stress hormones. Science, 280: 443-446. Xu, L., Anwyl, R. and Rowan, M.J. (1997) Behavioural stress facilitates the induction of long-term depression in the hippocampus. Nature, 387: 497-500. Yirmiya, R., Pollak, Y., Morag, M., Reichenberg, A., Barak, O., Avitsur, R., Shavit, Y., Ovadia, H., Weidenfeld, J., Morag, A., Newman, M.E. and Pollmacher, T. (2000) Illness, cytokines, and depression. Ann. N. Y. Acad. Sci., 917: 478-487. Zhou, Z., Licklider, L.J., Gygi, S.P. and Reed, R. (2002) Comprehensive proteomic analysis of the human spliceosome. Nature, 419: 182-185. Zigmond, M.J. (1997) Do compensatory processes underlie the preclinical phase of neurodegenerative disease? Insights from an animal model of parkinsonism. Neurobiol. Dis., 4: 247-253. Zoghbi H.Y. (2003) Postnatal neurodevelopmental disorders: meeting at the synapse? Science, 302: 826-830.
T. Steckler, N.H. Kalin and J.M.H.M. Reul (Eds.) Handbook of Stress and the Brain, gol. 15
ISBN 0-444-51173-3 Copyright 2005 Elsevier B.V. All rights reserved CHAPTER 4.11
Pathways and transmitter interactions mediating an integrated stress response Colin D. Ingram* Psychobiology Research Group, School of Neurology, Neurobiology and Psychiatry, University of Newcastle, Royal Victoria Infirmary, Newcastle NE1 4LP, UK
Abstract: Stress evokes a number of coincident neuroendocrine, autonomic, and behavioral responses which serve to avoid the damaging effects of the threat and restore homeostasis. Various techniques, notably those using immediateearly gene expression (IEG), have helped to define the neural networks which are activated by different types of stress stimuli and which serve to coordinate the different elements of the response. Although different stress modalities utilize distinct neural pathways each can be generalized to comprise: (i) a cognitive or sensory system which responds to introceptive or exteroceptive signals and includes a "threshold detector mechanism" which, when exceeded, will activate, (ii) a "response activating network" which distributes a stress signal to the various output systems to generate an appropriate response. Diversity and integration within this network is achieved through a hierarchy of overlapping pathways, with some having the capacity for direct, rapid activation in response to an immediate threat to homeostasis, while higher order pathways provide a distributed signal to several output systems. Certain transmitters have a preeminent role in regulating diverse aspects of the stress response (notable corticotropin-releasing factor) and it is suggested that the organization of the response activating network enables these transmitters to fulfil important roles in integrating the stress response.
diversity of processes that need to be accommodated by any integrated system. In addition, the list of neurotransmitters and neuromodulators for which there is evidence for involvement in stress grows ever longer. The foregoing chapters in this section have provided in-depth reviews of the evidence for specific transmitter involvement in the response to stress, and a number of other recent reviews have provided insight into the overall organization of stress-related systems (Lopez et al., 1999; Sawchenko et al., 2000; Pacfik and Palkovits, 2001; Vermetten and Bremner, 2002; Carrasco and van de Kar, 2003; Herman et al., 2003; Millan, 2003; Phillips et al., 2003). However, whilst these have considered the evidence that one or other particular pathway or transmitter is either affected by or affects the stress response, there remains a need to provide a conceptual framework for how stress systems operate and how different transmitters and pathways might contribute to an
This book is testament to the fact that in recent years the application of new pharmacological, neurochemical, anatomical, and molecular technologies to systems level neuroscience has dramatically advanced our understanding of the field of stress neurobiology. The availability of pharmacological tools to selectively modulate synaptic transmission and the ability to identify specific neurons activated by stressors, combined with our growing knowledge of neuroanatomical relations, has led to the point where it is possible to begin to provide an overall description of the neural circuits which serve to integrate the stress response. However, with understanding has also come complexity. The multimodal nature of the stress response and the increasing number of factors shown to modulate this response have added to the
*Tel.: +44 191 282 5678; Fax: +44 191 282 5108; E-mail:
[email protected] 609
610 overall integrated response. In the introductory chapter, Herman et al. (Chapter 4.1) provide an excellent overview of the current status of neural circuitry relevant to the stress activation of the hypothalamo-pituitary-adrenal (HPA) axis. Because of the well understood target for this circuitry (the paraventricular nucleus (PVN)) and the easily measurable index of response (plasma ACTH or corticosterone), this is perhaps the stress-activated system which is currently best understood. Once similar understanding is available for the other behavioral and physiological responses to stress it may be possible to map out the complete integrated system, which generates a coordinated response to stressful stimuli. Since we are not yet at this stage, this chapter will not adopt a strictly hodological (brain connectivity) approach to integration between the various neural systems subserving the response to stress, but will discuss some generalized models for integration. Furthermore, this consideration will be restricted to the response to acute stress stimuli, rather than chronic or repeated stresses which can lead to adaptive processes in stress circuits. Whilst this approach runs the risk of being too simplistic, it serves to highlight some basic attributes of stressresponsive circuits and the role that particular transmitters may have. These models will consider the hierarchical nature of different circuits and will propose the possible organization of a network that would account for the apparent coordinating role of one particular transmitter, corticotropin-releasing factor (CRF).
Defining the stress response and determining the underlying circuits
Stress-response systems At a systems level the central stress response can be considered in terms of a classical reflex arc: the afferent limb comprising a variety of sensory modalities (nociceptors, chemoreceptors, auditory and visual systems, etc.), as well as cognitive neural circuits that underlie emotional processes, while the efferent or effector limb comprises appropriate behavioral, physiological, and autonomic responses that subserve a number of defensive, protective, and
adaptive functions (Fig. 1). In this respect, stress may be considered an extension of homeostasis and all homeostatic pathways potentially can contribute to the integrated stress system. Whilst this view touches on the much debated boundary between Cannon's concepts of homeostasis and Selye's "stress syndrome" considered earlier in this volume (Chapters 1.1 and 1.2) and elsewhere (Pacfik and Palkovits, 2001), it does serve to highlight two important features of an integrated stress-response system. Firstly, there are no specific "stress receptors" and thus the neural origin of any stress response are normal sensory and emotional pathways. However, within these pathways there may be thresholds that determine whether a specific level of activity, or condition that is perceived to endanger the wellbeing of the individual, alters the stimulus so that it takes on a "stressful" quality. Secondly, the output is not through a single effector system. Therefore, an integrated stress-response system should not be defined on the basis of either a generalised, nonspecific activation (Selye), or of one particular behavioral, autonomic, or neuroendocrine measure (often determined by the specialism of the researcher). With this in mind it is possible to construct a simple integrated stress-response system in which sensory and cognitive/emotional inputs feed into a central "response activating network" that, in turn, provides the appropriate signals to generate a coordinated output (Fig. 1). The output or effector limb of the stress response involves widespread effects on endocrine systems which can signal a whole body reaction. These include changes in the hypothalamo-pituitarygonadal axis, oxytocin, prolactin, growth hormone, and renin-angiotensin, as well as HPA activation (van de Kar and Blair, 1999; Tsigos and Chrousos, 2002; and see Chapter 1.4). In this respect there is considerable participation of hypothalamic tuberoinfundibular neurons, which synthesize and secrete hormones or regulatory factors (e.g. CRF, GnRH, TRH, GHRH, dopamine). Stress also modulates autonomic outflow, including sympathetic changes to cardiovascular, respiratory, metabolic, renal, and sweat gland function, and vagal and sacral parasympathetic efferents, which control gut motility. The sympathoexcitatory response will involve activation of the supraspinal innervation of the
611
Fig. 1. Model of the basic organization of the central stress-responsive network. Three main input systems provide the trigger for stress responses: emotional/cognitive inputs reflect the psychological state of the individual, chemoreceptor/mechanoreceptor inputs provide signals about the internal environment, and sensory inputs respond to various external stimuli. Three major output systems encompass those regulating behavior, neuroendocrine, and autonomic activity. The inputs can interact directly with the outputs as part of normal function in the form of a reflex arc. However, when the inputs pass a threshold level to trigger a stress reaction a central response activating network activates the different output systems to achieve a coordinated response.
cholinergic preganglionic neurons which principally arises from the ventrolateral and ventromedial areas of the rostral medulla, the spinal projections of the raphe nuclei, the A5 (ventrolateral pons) noradrenergic neurons, and a population of dorsomedial parvocellular neurons in the PVN (Senba et al., 1993; Pacfik and Palkovits, 2001; Sved et al., 2001). Lesser hypothalamic pathways also arise in the lateral hypothalamus and arcuate, perifornical, and dorsomedial hypothalamic (DMH) nuclei. Many of these descending projections are peptidergic in phenotype (CRF, vasopressin (AVP), oxytocin, somatostatin, enkephalin, and atrial natriuretic peptide) and relay via the catecholaminergic neurons in the medulla or terminate directly on the preganglionic neurons in the thoracolumbar intermediolateral cell column (Palkovits, 1999). Although stressors may cause simultaneous activation of many of these descending pathways, anatomical tract tracing and immediate-early gene (IEG) expression studies indicate that functional subgroups exist within this network, allowing for variations in the response.
The final effector system of the stress response is that of behavior. Of all the components of the stress response, behavior is the most species specific and, like the majority of the data in this review, what is considered here are data mainly obtained from the rat. However, although the expression of stress behavior may vary in other species, the underlying circuitry is likely to be common. This circuitry generates an appropriate emotional coping strategy. These may be divided into "active" coping strategies (confrontation, flight, flight) that are particularly adaptive if the stress is escapable and are characterized by coincident sympathoexcitation (hypertension, tachycardia), and "passive" coping strategies (immobility, hyporeactivity) appropriate for inescapable stress and characterized by coincident sympathoinhibition. Either strategy also encompasses a number of displacement activities which, in the rat, involve considerable orofacial activity (grooming, chewing). The medial hypothalamus, amygdala, and midbrain periaqueductal gray (PAG) constitute the main neural substrates for the integration of aversive states, and distinct divisions mediate the different forms of
612 emotional coping and their associated patterns of autonomic activity. For example, within the PAG, excitation of the lateral region generates active coping, while the ventrolateral region generates passive coping (Bandler et al., 2000). Recent evidence has also shown that both these regions of the PAG communicate with the medial prefrontal cortex (mPFC) and parabrachial area in topographically distinct parallel circuits to generate these different behavioral coping strategies (Bernard and Bandler, 1998). Importantly the parabrachial area is also a major relay for viscerosensory information from the nucleus of the tractus solitarius (NTS) and spinal cord going to the central amygdala (CeA) and bed nuclei of the stria terminalis (BNST) and, thus, may provide the link between behavioral and autonomic responses. The appropriate combination of these varied adaptive outputs allows the animal to resist the effects of stress. In this respect although activation of the HPA axis outside the normal daily pattern has been considered to be the principal characteristic of a stress response, it should not be forgotten that there are instances in which stress does not result in increased HPA activity. For example, certain acute stressors like hypercapnia can cause acute anxiety and sympathoactivation with no HPA response, and repeated restraint stress results in dissociation of the various outputs, with habituation of both the HPA response and associated the expression of Fos in the PVN, while the autonomic response (tachycardia) persists, indicating the continued stressful quality of the stimulus (Stamp and Herbert, 1999). Furthermore, any stress-responsive system needs to be sufficiently flexible to accommodate the varying patterns of neuroendocrine activity in response to different stimuli; e.g. ether stress will increase ACTH, AVP, and oxytocin, while restraint will increase ACTH and oxytocin but not AVP, and cold stress affects ACTH neither oxytocin nor AVP (Gibbs, 1984). Thus, whilst the response activating network which orchestrates the appropriate output may be illustrated by a single box in a model of integration (Fig. 1), this does not mean that it exists as a unitary mechanism. Indeed, considerable evidence argues against the concept of a unitary stress system, most notable being the evidence of different stress categories or modalities (Li, et al., 1996; Dayas et al., 2001 a; Pac~tk and Palkovits, 2001; Herman et al., 2003).
Stress modalities
As well as the separation of stressors into escapable and inescapable described above, a wealth of data support the division of stress stimuli into two broad categories. The first category has been variously termed physical, systemic, introceptive, or reactive, and covers stimuli such as hemorrhage or other cardiovascular challenges, challenges to the immune system (infection), pain, respiratory stimuli (e.g. CO2 or ether), and heat stress. Broadly speaking these stimuli require an immediate response and the underlying neural pathways are not dissimilar to a fast reflex arc, with little dependence on higher order processing. The second category of stimuli has been termed emotional, psychological/psychogenic, processive, exteroceptive, or anticipatory, and includes restraint, fear (conditioned avoidance), novel environment (e.g. open field or plus maze), swim stress, noise, motion stress, visual, predator stimuli, psychosocial stress, and conflict. The important feature of these stressors is the dependence on information processing and on prior experience, and thus the underlying neural pathways incorporate several cognitive areas of the telencephalon. Obviously stressors may convey varying proportions of these different modalities (e.g. footshock not only has a painful physical component but also an emotional reaction to the unpredictable stimulus from the animal's environment) and so the boundary may not appear precise. This spectrum has led others to include further subdivisions of stress responses (e.g. physical, psychological, social and cardiovascular/metabolic: Pac~tk and Palkovits, 2001), however, a binary division remains the best validated. Until recently this categorization was based on subjective and anthropomorphic views as to the attributes of the stress. However, an increasing body of objective measures confirm this distinction, including neurotransmitter responses to different stressors (e.g. Inoue et al., 1994; Adell et al., 1997), the regional pattern of activation of different brain areas (e.g. Li et al., 1996; Emmert and Herman, 1999; Dayas et al., 2001a; Herman et al., 2003), and the selective effects of different stressors on physiological and neuroendocrine responses (e.g. Gibbs, 1984; Sawchenko et al., 2000; Pac~k and Palkovits, 2001). The following sections will consider the identity of the neural
613 systems which may integrate responses to these different stress modalities.
Immediate-early gene (lEG) mapping of stress-responsive circuits Since the early 1990s, studies of the patterns of cellular activation using the expression of IEGs (e.g. c-fos, fos-b, jun b, NGFI-A, NGFI-B, fra-2) and their protein products have provided considerable insight into the pathways activated by a wide range of stressful stimuli. However, before considering how these data have contributed to understanding an integrated stress-responsive circuit, it is important to note a few technical limitations. Firstly, for practical reasons, the areas of the brain analyzed are frequently preselected. Thus, the majority of studies have focused on the activation of areas with wellestablished roles in stress neurobiology (e.g. PVN, amygdala, locus coeruleus (LC)), although a few have mapped wider regions of the brain, allowing for the identification of novel responsive areas (e.g. Smith et al., 1992; Senba et al., 1993; Cullinan et al., 1995; Elmquist et al., 1996; Campeau and Watson, 1997; Emmert and Herman, 1999; Palkovits, 1999; Windle et al., 2004). Secondly, most studies have examined the c-fos gene or its product Fos. Whilst comparisons with other IEGs have shown similar patterns of activation (e.g. Stamp and Herbert, 1999), the c-fos gene may not be always activated by stress. For example, swim stress increases NGFI-A (zif/268) but not c-fos m R N A in the lateral BNST and peripeduncular nucleus (Cullinan et al., 1995). Thirdly, only a minority of studies have determined either the neurochemical identity or afferent connections of the neurons activated by stress, and where this has happened most studies have focussed on a limited number of transmitters, such as CRF, noradrenaline and serotonin (5-HT) (e.g. Ceccatelli et al., 1989; Pezzone et al., 1993; Grahn et al., 1999; Ishida et al., 2002; Helfferich and Palkovits, 2003; see also Chapters 4.1, 4.4, and 4.6). However, accepting these limitations, studies of IEG expression have established a number of important characteristics of the stress-responsive network.
One of the most important features of stress circuitry established by IEG mapping is the existence of modality-specific pathways. Thus, while some areas exhibit consistent activation by stress and may be considered to be the signature of a stressful stimulus (e.g. PVN), others show dependence on the two major categories of stressors described above. This is particularly the case for telencephalic regions and reflects the varying contribution of cognitive processing to the response. Therefore, while two psychological stressors, swim stress and restraint, evoke largely similar regional patterns of gene expression (Cullinan et al., 1995), exposure to an open field (psychological stress) evokes much greater activation of the medial amygdala (MeA) and lateral septum than ether vapour (physical stress), with the reverse being the case in the parietal cortex (Emmert and Herman, 1999). Indeed the amygdala displays a high degree of modality dependence, in that physical stressors (hemorrhage and immune challenge) elicit Fos expression in neurons of the CeA while psychological stressors (noise, restraint and forced swim) primarily have effects on neurons of the MeA (Dayas et al., 2001a; Herman et al., 2003). Thus, it has been well established that the psychological stress of restraint (as distinct from complete immobilization) will induce Fos protein or c-los m R N A expression in the MeA rather than CeA (e.g. da Costa et al., 1996; Dayas et al., 1999), and ibotenic acid lesions of the MeA (but not CeA) will reduce restraint-induced activation of the PVN (Dayas et al., 1999). However, this dichotomous circuitry is not limited to the amygdala. For example, although all stressors recruit both A1 and A2 (noradrenergic) and C1 and C2 (adrenergic) neurons, physical stressors appear to target a more rostral population of A1 and A2 neurons than psychological stressors (Dayas et al., 2001a). Likewise, within the PAG physical stressors (hemorrhage and pain) increase Fos in the ventrolateral area, consistent with involvement in a passive coping strategy, while psychological stressors (restraint and swim stress) increase Fos in the lateral area, consistent with its role in active coping (Bernard and Bandler, 1998). These differential patterns confirm the plurality of the circuits which serve to integrate the appropriate stress response. A second feature of stress circuits revealed by lEG mapping is the differentiation between those areas
614 which are activated by the sensory stimulus (i.e. the afferent pathway), and those which are only activated when there is a stress response (i.e. the response activating network) (Fig. 1). In this respect it should be remembered that there are no specific stress receptors and it is necessary to consider the mechanisms by which a normal sensory (or cognitive) pathway may lead to the activation of the stress response. This is likely to involve a threshold detection mechanism. For example, auditory stimuli will not activate a stress response until the intensity (volume), frequency, and/or duration of the stimulus reaches a threshold when it is considered to be stressful. The pathways underlying this transition have been elegantly illustrated by Campeau and Watson (1997) using measures of plasma corticosterone and regional c-fos mRNA expression following exposure of rats to differing intensities of white noise. Areas of the brain in which c-fos mRNA indicated activation could be classified into three categories: (i) those which were part of a general arousal system and which were similarly activated by the experimental conditions, irrespective of the intensity of the noise (e.g. anterior cortical amygdala, basolateral amygdala (BLA), and MeA, anteroventral and mediodorsal thalamus, cingulate and piriform cortices); (ii) those which were part of the normal auditory pathway and which showed graded increases in gene expression as a function of the noise intensity, irrespective of whether the intensity was below or above the threshold required for HPA activation (e.g. cochlear nuclei, nuclei of the inferior colliculus; medial geniculate nucleus, the superior olivary complex, auditory cortex); and (iii) those areas considered to be part of the stress processing pathway which were only excited at intensities of 90dB and above, at which point plasma corticosterone levels indicated that the HPA axis had become activated. These areas included the medial and ventral nuclei of the BNST and adjacent septohypothalamic area/ventrolateral septum (SHy/VLS), the ventral dentate gyrus, lateral and medial preoptic areas (MPOA), median raphe nucleus (MRN), pedunculopontine tegmentum (PPTg), and PVN. Although the neurochemical identities of the neurons comprising this latter category were not determined, it is likely that the PVN neurons will have included CRF neurons driving the HPA response.
Furthermore, it is interesting that in this and studies of other stressors (e.g. Arnold et al., 1992; Senba et al., 1993; Chen and Herbert, 1995; Cullinan et al., 1995; da Costa et al., 1996; Stamp and Herbert, 1999; Windle et al., 2004) the BNST and SHy/VLS regions have been shown to be activated, since these regions are the origin of GABAergic relays to the PVN (Sawchenko and Swanson, 1983; Herman et al., 2002) and may serve as important stress processing areas, integrating inputs from the amygdala (Weller and Smith, 1982). The BNST is also a site that projects to autonomic centers in the brainstem and may be important for coordinating limbic control of cardiovascular function (Loewy, 1991). Activation of the M R N suggests the involvement of serotonergic neurons, while the PPTg neurons are likely to be cholinergic, both of which have been implicated in regulating arousal. Thus, each of these areas may constitute a part of the network coordinating the different components of the stress response. Although Campeau and Watson (1997) provided a schematic of the connections between these different auditory-, arousal-, and stress-related systems, it was not possible to conclude at what point in this putative stress circuit the normal response to an auditory signal had been converted to a stress response and, therefore, the identity of the threshold detector mechanism remains to be determined.
Elements of the response activating network Evidence from a large number of anatomical, recording, and lesioning studies have identified areas which may be considered to be the principal constituents of the response activating network. Each of these areas contributes to the network function by integrating specific types of signals, by distributing signals throughout the network, or by contributing to the output function. The following sections briefly summarize these different integrative functions.
Medial hypothalamus: P VN and D M H The PVN is often considered to be the principal nucleus mediating efferent responses to stress, as both the HPA axis and autonomic responses originate here (see Chapter 4.1 for detailed review). Indeed
615 activation of the stress-related neurons of PVN may be regarded as the signature without which a stimulus cannot be considered stressful. Within the PVN CRF-containing parvocellular neurons are activated by both physical and psychological stress stimuli, reflecting the neuroendocrine component of the response (Ceccatelli et al., 1989; Helfferich and Palkovits, 2003). In addition, efferents containing oxytocin, AVP, and possibly neuropeptide Y (NPY) arising from the dorsal parvocellular region project both to the brainstem and directly to the spinal preganglionic neurons in the intermediolateral cell column, where they are involved in integrating autonomic (sympathetic) responses (Coote et al., 1998). Further integration of autonomic, neuroendocrine, and behavioral responses to emotional stress may also occur in the adjacent area of the DMH (DiMicco et al., 2002), and both the PVN and DMH constitute part of the hypothalamic defense area which interconnects with other medial hypothalamic areas to generate appropriate responses to threatening stimuli (Canteras, 2002). Many of the afferents to the PVN (at least those subserving HPA activation) relay in a ring of GABAergic neurons in the sub-PVN region, peri-PVN, BNST, MPOA, ventromedial hypothalamus, and DMH (Herman et al., 2002). However, the PVN also receives direct inputs from circulating factors in the blood and cerebrospinal fluid and in this respect some factors which induce stress responses (e.g. immune factors or glucoprivation) may involve a very short neural pathway.
Extended amygdala As noted above and reviewed in Chapter 6.2, the CeA and MeA are involved in processing different stress modalities, and for relaying stress signals to other limbic and brainstem areas (Davis et al., 1994). Electrical stimulation of the CeA evokes both signs of arousal and sympathetic activation, and lesions of the CeA decreases HPA activation during immobilization and blocks fear conditioning (Hitchcock and Davis, 1986). While the MeA and CeA are involved in amygdaloid output, the BLA is involved in processing afferent sensory information and plays an important role in integrating inputs from various cortical and thalamic sites which are relayed via
intra-amygdaloid connections to other subnuclei. The BLA may also play an important role in associative learning which underlies fear conditioning and anxiety. This integrative role is illustrated by the ability of neurotoxic lesions of the BLA and CeA to block the effect of a conditioned fear stress on dopamine, noradrenaline, and 5-HT transmission in the mPFC and stress-induced freezing, defecation, and HPA activation (Goldstein et al., 1996). The BNST is considered to be part of the extended amygdala and, as such, there are marked nucleusspecific associations: i.e. the CeA is connected to lateral BNST (oval nucleus), while the MeA is connected to the anterodorsal/medial nuclei of the BNST, both through the stria terminalis and ventral amygdalofugal pathways. Importantly the BNST appears to be the relay for inputs to the PVNcontrolling tonic HPA activity (Herman et al., 1994) and in this respect plays an important role in determining basal HPA activity on which stress acts. The anterolateral BNST is also involved both in higher order activation of stress-responsive circuits and in the generation of coping behaviors.
Hippocampus The precise functions of the hippocampus in stress responding are somewhat controversial, but are principally twofold. Firstly, it is an area important for learning and memory, and thereby contributes to the processing of stressful information, The hippocampus has a particularly important role in episodic memory for the emotional context of memories and plays a role in regulating behavioral responses to threatening environmental contexts (Phillips and LeDoux, 1992). In this respect, although Fos expression indicates that the hippocampus is activated by various stress conditions, it appears to be principally reactive to exteroceptive (psychogenic) stress and not the interoceptive (physical) stresses. Secondly, the hippocampus provides inhibitory control over the HPA axis and may mediate its effects on the PVN via inhibitory connections to subcortical areas (Herman et al., 2002, 2003). There has also been considerable emphasis placed on the hippocampus as a site for glucocorticoid negative feedback (see Chapter 3 of this volume). This
616 inhibition involves both basal (tonic) control and the termination of responses to stress. Indeed, lesions of the hippocampus result in prolonged HPA responses to psychological (processive) stressors, but not physical (reactive) stressors (Herman et al., 2003), consistent with its involvement in cognitive processing. However, the role of the hippocampus and subiculum in regulating basal and stress-induced HPA activity has been questioned (e.g. Tuvnes et al., 2003). In part this may relate to misperceptions of the hippocampus being a unitary structure with little regional specialization. In this respect, within the septohippocampal complex it is an area in the ventral subiculum that appears to provide the main output, at least in respect of regulating the PVN and HPA axis activity (Herman et al., 1998). The outflow from this ventral subicular area is glutamatergic and innervates a number of areas including the mPFC and the GABAergic neurons in the peri-PVN region, BNST, and hypothalamus (MPOA and DMH).
Septum The septal area has two major functions in stress responding. Firstly, it regulates hippocampal function through the septohippocampal pathway and this connection is important in controlling the termination of stress responses. Secondly, it functions as a relay for limbic afferents that pass through the SHy/VLS, an area which is consistently activated by the psychological stress of restraint, noise, or novelty (da Costa et al., 1996; Campeau and Watson, 1997; Emmert and Herman, 1999), but not by physical stressors.
Prefrontal cortex (cingulate cortex) Various cortical areas display increased Fos expression following psychological stress, but not after physical stress, most important among which is the mPFC (see Chapter 6.3). Thus, expression of Fos in this region occurs in response to restraint (Chen and Herbert, 1995; Cullinan et al., 1995) and noise (Campeau and Watson, 1997), but not in response to other stressors, like lipopolysaccharide (LPS) (Elmquist et al., 1996; Yokoyama and Sasaki, 1999) or osmotic challenge (Sharp et al., 1991). There is
increasing interest in the PFC as an area integrating both the stress-induced activation of the HPA axis and glucocorticoid negative feedback with executive function and cognitive processing (Sullivan and Gratton, 2002). Furthermore, the PFC plays an important role in generating an active coping strategy in response to a stressful stimulus (Giorgi et al., 2003), and lesions of the mPFC markedly alter stress responses, primarily affecting responses to emotional stressors (Diorio et al., 1993; Sullivan and Gratton, 1999). Indeed lesions of the mPFC increase both the HPA response to restraint stress and the expression of c-fos mRNA in various areas including PVN, MeA, ventrolateral BNST, and piriform and dorsal endopiriform cortices (Figueiredo et al., 2003), consistent with the mPFC being a higher order area providing inhibitory control over pathways mediating psychological stress. In this respect the PFC has major connections to several areas involved in stress responding, including the amygdala, paraventricular thalamic nucleus, raphe nuclei, NTS, and the GABAergic interneurons in the BNST and hypothalamus that project to the PVN. It may, therefore, have a higher order function to regulate the activity of much of the response activating network, perhaps setting an emotional tone to information processing.
Locus coeruleus (LC) and AI[A2 medullary nuclei Numerous studies have demonstrated the activation of brainstem catecholaminergic neurons in the integrated response to stress and this is extensively reviewed in Chapters 4.3, 4.4, and 4.5 of this volume. The ventrolateral medulla (A1), dorsomedial medulla/NTS (A2), ventrolateral pons (A5), and LC (A6) all display increased IEG expression following stress. Increased Fos immunoreactivity or c-fos mRNA is observed in response to psychological stressors, such as restraint, noise, and forced swim (Cullinan et al., 1995; Campeau and Watson, 1997; Stamp and Herbert, 1999; Dayas et al., 2001b; Helfferich and Palkovits, 2003), or physical stressors, such as intraperitoneal injection of hypertonic saline, hypercapnia, and hemorrhage (Ceccatelli et al., 1989; Haxhiu et al., 1996; Dayas et al., 2001b), as well as following the mixed stimulus of footshock
617 (Pezzone et al., 1993) or the fear conditioned by exposure to footshock (Ishida et al., 2002). Combined immunocytochemistry for tyrosine hydroxylase has confirmed the noradrenergic phenotype of many of these neurons, although other noncatecholaminergic neurons are also activated, including GABA interneurons (Ishida et al., 2002). The known connectivity of these nuclei have implicated them in several integrative functions, particularly in relation to relaying primary viscerosensory information (A1/A2) and in sympathetic activation (A1, A2, and A5). The LC-noradrenaline system also has major roles in arousal and in the initiation and maintenance of forebrain neuronal activity appropriate for the collection and processing of salient stress-related information through diverse sensory, attention, and memory circuits (Berridge and Waterhouse, 2003).
Raphe Nuclei As reviewed extensively in Chapter 4.6, both the M R N and dorsal raphe nucleus (DRN) show responses to stress stimuli and, since these nuclei provide the majority of 5-HT innervation to the brain, increased serotonergic neurotransmission is implicit in their activation. However, the attributes of the stress stimuli have major effects on the pattern of responses in the two nuclei. Stressors, which are uncontrollable or which evoke conditioned fear, cause activation of DRN neurons. For example, inescapable tailshock or footshock leads to an increased expression of Fos in 5-HT-immunoreactive DRN neurons (Pezzone et al., 1993; Grahn et al., 1999), and fear conditioned by exposure to footshock induced Fos expression in both 5-HT and GABAergic neurons in the DRN (Ishida et al., 2002). In contrast, swim stress may reduce forebrain 5-HT release through inhibition of raphe neurons, and this is consistent with the fact that the increased Fos expression in the DRN occurs primarily in neurons immunoreactive for GABA in the dorsolateral subdivision and few have a 5-HT phenotype (Roche et al., 2003). Thus, while psychological stressors are effective in increasing Fos expression in both the raphe and mPFC, an immune stress (LPS) which has little or no psychological component, has no effect in either structure (Elmquist et al., 1996).
However, despite this apparent lack of Fos expression in the raphe, LPS does cause a marked increase in 5-HT neurotransmission in the hippocampus (Linthorst et al., 1995), suggesting either that the increase in transmission arises from an activity independent mechanism, or that the c-fos gene is not induced by this particular stimulus, or that a selective subpopulation of raphe neurons is affected. Indeed different stress modalities may selectively affect median versus dorsal raphe, or specific subpopulations of DRN neurons. For example, inescapable sound stress activates serotonergic neurons in the MRN, but not DRN, and selectively increases 5-HT turnover in M R N projection areas (hippocampus, nucleus accumbens (NAc), and cortex) but not in the caudate nucleus which receives input from the D RN (Daugherty et al., 2001). This functional separation may be important for integrating appropriate behavioral responses. It has been suggested that activation of DRN in response to acute threatening situations facilitate cognitive processes in the amygdala that evaluate the threat, whilst inhibiting escape behavior mediated via the PAG (Graeff et al., 1996). In contrast, the M R N hippocampal system may promote resistance to unavoidable stress by disconnecting aversive events from other cognitive and behavioral processes. A recent review of these functional subpopulations suggested that 5-HT neurons within the middle and caudal regions of the DRN may project to multiple components of the extended amygdala to facilitate various components of the stress response, while connections of the M R N with structures involved in the efferent pathway from the ventral subiculum may provide the control over the adaptation or coping in response to stress (Lowry, 2002).
A hierarchical model of integration within the stress-responsive network With increasing knowledge of areas activated by stress it has become possible to define the specific pathways underlying the response to individual stressors, including immobilization, cold, hemorrhage, hypoglycemia, and pain (Pacfik and Palkovits, 2001), and auditory stress (Campeau and Watson, 1997). However, from the foregoing
618 description of the major elements of the stressresponsive network, it is clear that many areas fulfil several functions or are only recruited under particular conditions. Therefore, simple static pathways, akin to fixed electrical circuits, may not be appropriate to explain the organization of a network which can respond to different stressors and which generates an integrated stress response. It is perhaps more appropriate to consider stress responses as generated by a number of parallel or overlapping pathways. Figure 2 illustrates a hypothetical model of this form of organization. Within this model, intrinsic and extrinsic signals are processed by cognitive and sensory systems, but input to the response activating network does not occur until they reach a threshold for activation. Output from the network is generated by well-defined motor neurons that generate specific behavioral and/or physiological effects (e.g. CRF neurons driving HPA activity or sympathetic cholinergic neurons regulating cardiovascular activity). Processing between these two occurs by at least three parallel streams: -
-
-
This model, in which integration is achieved through a combination of divergent and convergent streams, is compatible with the principles of integration, which Herman and colleagues have suggested might regulate the HPA axis. In this they propose that circuits subserving "anticipatory" responses are superimposed on the direct "reactive" (reflex) pathways to converge on the PVN, and have applied the term "hierarchical" to describe this pattern of organization (Herman et al., 2003; Chapter 4.1). This is a highly appropriate term, since,
Pathway (1) represents a simple, direct pathway where output is principally focussed on one (or a few) specific output system(s) that has high homeostatic value, although other outputs may play a minor role. These pathways are similar to Threshold Detector Mechanism
Stress Responses
Response Activating Network
~ ', [
Intrinsic Stimuli
reflex arcs and are particularly common for rapid responses to physical stressors. Pathway (2) represents a more complex distributed network which provides coordinate activation of several output systems. This organization is common for the higher order pathways subserving psychological stressors. Pathway (3) represent the highest order pathway which has important modulatory effects on the output systems, but may not transmit the primary stress signal. The overlapping of these higher order pathways generates the diversity of potential responses, and explains the existence of convergence on output systems which has been extensively reported by studies of retrograde labelling of stress-activated pathways.
~
~
~
v~
-
Escape behavior
w,- Freezing behavior Cognitive or Sensory Systems
lf~
I
r- HPA response
~(,~/~r
.........~..".
...............\
.
r
Neuroendocrine response
h~
,,- Adrenomedullary response Extrinsic Stimuli
[
(~)
w
9,- Cardiovascular response
Fig. 2. Hierarchical model for integrated responses to stressful stimuli. Cognitive and sensory systems function normally to respond to intrinsic and extrinsic signals but without generating a stress response. However, a detector mechanism operates to establish a threshold above which a signal is passed to a response activating network to generate an appropriate stress response. The response activating network comprises proximal components which provide fast input to systems that generate specific physiological or behavioral responses, and distributed networks which function to integrate a number of different responses. Depending on the nature of the stressful stimulus the threshold detector mechanism may either directly activate the proximal system (e.g. (1)) or may have a more indirect effect, activating overlapping higher order systems that generate the appropriate repertoire of responses (e.g. (2) and (3)).
619 while the model illustrated in Fig. 2 shows only a relatively small number of layers, the situation in reality may involve a more complex hierarchy. For example, it has been suggested that emotional perception of stress is processed through at least two parallel systems: a ventral system, comprising amygdala, insula, cingulate gyrus, and PFC, which is important for identifying the emotional significance of stimuli and the production of affective states and the accompanying autonomic responses; and a dorsal system, comprising the hippocampus and dorsal areas of the cingulate and PFC, which is responsible for executive functions, selective attention, and effortful regulation of affect (Phillips et al., 2003). Importantly, within this model the overall integrated response to a stress is not predetermined but is a function of the number and intensity of the different overlapping systems which are recruited. Furthermore, convergence may alter the pattern of stress responding at a molecular level. In this respect, it is interesting to note that recent data using oligonucleotide microarrays to compare the gene expression profiles in the PVN evoked by LPS (physiological stress) and restraint (psychological stress) have shown that, while several neuropeptides (e.g. orexin, preproenkephalin, NPY) show common changes in expression, many of the transcription factors induced were stimulus specific (Reyes et al., 2003). Thus, while the distinct pathways which convey the different stress modalities may evoke apparently similar HPA and sympathoadrenal activation, differences in the postreceptor signalling cascade evoked by the different convergent pathways generates separate molecular responses. As previously mentioned, a critical regulatory point in any model of stress responding is the gating or threshold detector mechanism that allows signals from normal sensory or cognitive pathways to enter the response activating network. This mechanism is likely to differ for the different processing streams. For responses to physical stressors, this gating could be a simple electrophysiological or signalling threshold which determines whether or not a neuron receiving the sensory information will fire and transmit the signal into the stress circuit. However, it is important to note that this threshold is likely to: (i) vary between pathways allowing for differential recruitment; (ii) show graded signal transduction
allowing for the variable levels of output observed for many stressors; and (iii) vary between individuals and between different circumstances, leading to observed differences in responses. For pathways that respond to psychological stressors, threshold detection will involve more extensive circuitry that engages cognitive processing which determines whether or not a stimulus constitutes a threat to the wellbeing of the individual and which ensures the response is contextually appropriate (Phillips et al., 2003). Therefore, gating cannot be a simple threshold but involves appraisal and identification of the stimulus salience. In respect of many sensory stimuli the threshold detector is likely to reside either in the thalamus or in the brainstem reticular activating system, which act as relays for pathways either to the sensory cortex for normal processing, or to the amygdala for emotional responses.
Transmitter involvement in stress-reactive circuits The areal (or regional) approach to determining integration within stress reactive circuits, considered above, is based primarily on known connectivity between and functionality of various activated brain nuclei. However, an alternative view to integration within stress circuits is based on the multiple functions of specific transmitters. Whilst these complementary hodological (connectivity) and neurochemical approaches have considerable overlap, there are a number of reasons for considering them separately. This is particularly the case where a transmitter shows widespread and synchronous release, or where a transmitter participates in several components of the stress response and, therefore, it is not possible to ascribe a specific site of action. Furthermore, for many transmitters their precise anatomical role in the pathways between stress stimulus and stress response remains to be determined. At least four approaches have been successfully used to define transmitter involvement in the integration of stress responses. These are: (i) pharmacological approaches based primarily on whether agonists or antagonists of particular transmitter pathways selectively modify the various measures of stress; (ii) neurochemical approaches,
620 which include measurement of changes in transmitter release either by in vivo or ex vivo methodology during the course of stress. This approach has particularly benefited from the development of realtime measurements that can be made simultaneously with behavior or other physiological responses, including microdialysis, voltammetry, and immunosensors; (iii) neuroanatomical approaches that determine the chemical identity of neurons activated by stressful stimuli, such as combined I E G and i m m u n o cytochemistry; and (iv) genetic approaches, which include identifying genes which show changes in expression during stress, or demonstrating the effect that transgenic methods of under- or over-expression have on the stress response.
The ways in which a particular transmitter may be considered to be "involved" in the functioning of an integrated stress-responsive network are varied (Fig. 3). Whilst the involvement usually implies a contribution to direct transduction pathways, transmitters may also fulfil i m p o r t a n t m o d u l a t o r y or permissive functions (see 6 and 7 in Fig. 3). Furthermore, it is important to distinguish between neurotransmitter responses which are integral to driving the stress response and those which arise from the effects of stress (i.e. secondary to autonomic, behavioral, or neuroendocrine activation; transmitter 8 in Fig. 3). This is particularly i m p o r t a n t in respect of the effects of glucocorticoids which themselves may have an integrating role (see Chapter 3). Thus, while the
?
?
Transmitter 1
Transmitter 1
T~ah:~ ivati~ Tr=:um~et%~vati~ ~A (~
m
Transmitter8
o
|
~ ~
ransmitter7 0
Transmitter 5
Transmitter3
CommonResponseProfile e.g.HPAactivation Fig. 3. Examples of the varied transmitter involvement in stress-responsive networks. In this theoretical model acute exposure to a first stress (stressor 1) activates a pathway utilizing transmitter 1 which in turn activates transmitter 2 and finally transmitter 3 which controls some of the common output, such as HPA and autonomic activation. If stressor 1 is chronically activated, then transmitter 4 becomes activated and may assume some of the function of transmitter 2. The effects of a second stressor may also be mediated through transmitter 1 but in a separate neuronal population. In this case it is able to activate transmitter 3 and the common response profile through transmitter 5, the function of which has a dependence on the activity of transmitter 6. Neurons producing transmitter 6 are not themselves activated by the stress but have a permissive role. This second stress pathway is suppressed by the activity of transmitter 7 (an endogenous antistress factor). Finally, the activation of the common stress response leads to activation of transmitter 8 which has no function in either pathway.
621 majority of secondary effects of corticosteroids are long term and contribute to the adaptive phenomena of habituation, cross-sensitization, and priming which modify subsequent responses to stimuli, some corticosteroid effects are sufficiently rapid to be considered part of the acute response to stress. Examples of this are the rise in hippocampal glutamate release following ether stress which parallel the rise in corticosterone and is abolished in adrenalectomized, corticosterone-replaced animals (Abraham et al., 1998), or the secondary increase in amygdaloid C R F and GABA release, which parallels the rise in corticosterone (Fig. 4; Cook, 2001). In addition to the effects of steroids, a wide variety of other factors may have an impact on the way a transmitter is involved in stress pathways. These include:
processes of habituation and sensitization, and the factor of chronicity will all alter stress pathways by adaptation of central transmitter, receptor, and/or transporter involvement (e.g. Fuchs and Flfigge, 2003). Indeed a single stressful event can have persistent effects that alter several neuroendocrine systems for days and weeks (Servatius et al., 2000; Valles et al., 2002), and this most likely involves reorganization of the neurocircuitry or transmitter expression levels. Furthermore, early life stress leads to life-long programming of transmitter involvement (see Part II: Chapters 1.1 and 1.2). This plasticity of transmitter function is important for ensuring maintained or appropriate responses to potentially life threatening stimuli.
Genotype and gender Stress history Aside from the obvious differences between species, marked differences in stress responses occur between strains (see Part II: Chapter 1.4) and this may have a
Whether previously exposed to the same (homotypic) or a different (heterotypic) stress stimulus, the 500
400
14
L
---!1--- PVN CRH AmygdalaCRH - -0- - AmygdalaGABA
i";
12 lO
m
300
8
m.
6
200 ! /
100
/
"2 lii~iii~i!Ji!iii!iiiiii~iii~iiiiiii!~iiii~ii~iiiiiii~iiiiii~i!i~iiii~i~ii~i~ii!~iIi~iiiii~!ii~i!~iIi~i~ii~i!i~iiiiiiiiii~iiiii!ii~i~Iii~iiiii~ii! i
l
!
!
l
0
10
20
30
40
-0
Time (rain) Fig. 4. Simultaneous real-time measurement of dynamic changes in extracellular CRF and GABA in the PVN and amygdala in response to psychological stress in a female sheep. The shaded bar represents 20 min during which the animal was placed in a padded cradle off the ground and exposed to the sound of a dog sporadically barking. CRF was measured from a single chamber microdialysis immunosensor located in the PVN and sampled at 2 min intervals. A second probe located in the amygdala had a double chamber allowing simultaneous measurement of both GABA and CRF. Data kindly provided by Dr. C.J. Cook, HortResearch, Hamilton, New Zealand. For methodology see Cook (1998, 2001).
622 transmitter basis. For example, the Maudsley Reactive and Nonreactive strains display marked differences in emotionality, possibly as a result of altered noradrenergic responses to stress (Blizard and Adams, 2002), and the High and Low anxiety-related (HAB and LAB) rats differ in their stress coping strategies (active vs. passive), possibly as a result of the differential expression and release of AVP in the PVN arising from single nucleotide polymorphisms in the gene promotor (Landgraf and Wigger, 2002). Furthermore, sexually diergic responses to stress may have their basis either in the effects of ovarian steroids on transmitter expression (Young, 1998; McCormick et al., 2002) or in sexually dimorphic transmitter pathways (Rhodes and Rubin, 1999; Figueiredo et al., 2002).
Physiological or pathological status Various physiological and pathological conditions will also alter the contribution a transmitter plays in stress pathways. For example, reproductive status has a major effect on stress responses, such that lactating rats display less anxious behavior and both pregnant and lactating rats show reduced responsiveness of the HPA axis to various physical or emotional stimuli (Lightman et al., 2001; Neumann, 2001), which correlates with attenuated activation of central stressreactive nuclei (da Costa et al., 1996). This reduced emotionality may be controlled, in part, by the increased central expression of oxytocin and prolactin at this time.
Transmitter systems mediating integrated stress responses While the diversity of transmitters implicated in one or other aspect of stress-reactive pathways continues to expand, relatively few hold pre-eminent roles in integrating multiple aspects of the response. These are primarily the amines, noradrenaline, 5-HT, and dopamine, and the peptides, CRF, and vasopressin. Other sections of this book provide comprehensive reviews of many of these transmitters, but here a brief overview of their organization and functions is
considered in respect of their possible contribution toward an integrated stress system.
Noradrenaline The involvement of central noradrenaline pathways in stress is well documented (see Chapters 4.3, 4.4, and 4.5 for reviews) and many areas implicated in stress processing receive noradrenaline innervation. This innervation arises either from the ventrolateral (A1) and dorsomedial (A2) medullary regions, which innervate the hypothalamus and amygdala via the ventral noradrenergic bundle, or from the LC (A6), which innervates mainly limbic and cortical areas via the dorsal noradrenergic bundle (Cunningham and Sawchenko, 1988). Furthermore, noradrenaline neurons of both the A5 and LC/subcoeruleus areas send descending projections to the intermediolateral cell column to regulate sympathetic activity. As a result of this widespread innervation many sensory stimuli, whether aversive or not, will stimulate noradrenergic activity in stress-related areas (Pac/tk et al., 1995). For example, noradrenaline release occurs in the hippocampus in response to both psychological stress (restraint; Vahabzadeh and Fillenz, 1992) and physical stress (intraperitoneal LPS; Linthorst et al., 1996), and several stress modalities will increase noradrenaline release in the mPFC, including immobilization, novel environment, or conditioned fear (McQuade and Stanford, 2000; Swanson et al., 2004). The dorsal noradrefiergic bundle is a major source of these mPFC afferents (Nakane et al., 1994) and simultaneous microdialysis in the mPFC and LC has shown synchronous release of noradrenaline in response to hypotension or handling stress (Kawahara et al., 1999), consistent with its origin in the LC. Furthermore, consistent with a widely distributed signalling function, any one particular stressor will cause noradrenaline release across several stress-related areas. For example, microdialysis studies have shown that electric footshock induces noradrenaline release in the PVN (Yokoo et al., 1990; Ishizuka et al., 2000), amygdala (Quirarte et al., 1998; Williams et al., 1998), hippocampus (Hajos-Korcsok et al., 2003), and mPFC (Ishizuka et al., 2000). However, noradrenaline release does show a certain degree of regional specificity, as
623 application of mild stressors (handling and tail pinch) will stimulate noradrenaline release in the mPFC and NAc, but not in the caudate-putamen (Cenci et al., 1992). Interestingly, the magnitude of this noradrenaline release has been shown to be directly proportional to the stimulus intensity (Pacfik, 2000) suggesting that, unlike other transmitters, the pathway mediating this response may not incorporate a threshold detection mechanism. Coupled with its widespread release, noradrenaline appears to play a role in many aspects of the stress response and, therefore, can be considered to subserve an important integrative function. However, this role may be largely restricted to that of activating or initiating a response rather than sustaining it, as measures of hippocampal noradrenaline release have shown that release does not persist during a sustained stress while behavioral and HPA components of the response do (Britton et al., 1992). In respect of innervation of the PVN, noradrenaline appears to have involvement in HPA regulation as CZlbreceptor m R N A colocalizes with CRF m R N A in the PVN (Day et al., 1999) and the ~1 antagonist prazosin will attenuate ACTH response to ether stress (Szafarczyk et al., 1987). However, noradrenaline involvement in mediating HPA responses to stress is strongly modality dependent. Using a wide variety of introceptive and exteroceptive stressors, Pacfik (2000) showed that noradrenaline release in the PVN varied in a way that did not correlate with the plasma ACTH response, suggesting that the relative contribution of noradrenaline to HPA activation was stressor specific. Indeed evidence from a range of lesion and retrograde labelling studies has indicated that direct noradrenaline projections to the PVN contribute to the HPA response to physical stressors, while responses to psychological stress may involve indirect noradrenaline connections with higher centers, possibly including relays in the MeA (Dayas et al., 2001a). Thus, 6-hydroxydopamine lesion of the ventral noradrenergic bundle, which carries noradrenaline afferents from the A 1/A2 to the PVN, does not affect the HPA response to novel environment (Castagn~ et al., 1990), but attenuates the response to ether stress (Szafarczyk et al., 1985). Furthermore, recent evidence has shown that injection of an anti-dopamine-13-hydroxylase-saporin conjugate into the PVN, which causes almost complete
ablation of noradrenaline terminals in the PVN and their associated cell bodies, profoundly impaired HPA activation and PVN Fos expression in response to glucoprivation (insulin-induced hypoglycemia) but was without effect on the corticosterone response to forced swim (Ritter et al., 2003). This modalityspecific differentiation is consistent with the involvement of different subgroups of A1/A2 neurons in the response to emotional and physical stressors (Dayas et al., 2001 a). As well as affecting neuroendocrine function, noradrenaline release in the amygdala may be involved in regulating memory storage and behavior. Evidence from region-specific administration of the ~l-adrenoceptor antagonist benoxathian has suggested that the noradrenaline innervation of the lateral BNST and CeA are respectively involved in aspects of anxiety-like behavior measured by the elevated plus maze and social interaction test (Cecchi et al., 2002). Furthermore, within cortical areas noradrenaline regulates variations in arousal during the sleep-wake cycle and may facilitate arousal in response to a number of physiological stimuli that have the potential to take on stressful properties (e.g. sensory stimuli from the viscera and cardiovascular system). In this respect noradrenaline pathways may play an important role in orientating attention toward salient environmental stimuli and facilitation of the cognitive processes that underlie stress responding. The control of ascending noradrenaline systems under both stressful and nonstressful conditions arises from a limited number of limbic and brainstem regions (Singewald and Philippu, 1998; Berridge and Waterhouse, 2003). Inputs to the A1, A2, and LC arise in the dorsal PVN, CeA, and BNST, while the A1/A2 also receive major innervation from viscerosensory areas in the NTS. Importantly many of the forebrain descending afferents arise from CRFcontaining regions. In this respect, Valentino and van Bockstaele suggest that stimulus evoked bursts of LC activity, possibly mediated by glutamate, may be important for selective attention, while sustained LC activation mediated by CRF may cause a shift from stimulus specific responding to a general arousal state which is important for scanning of multiple stimuli appropriate for the response to threatening situations (see Chapter 4.4).
624
Serotonin (5-HT) In a similar arrangement to noradrenaline, 5-HT is present in a widely distributed network. This arises from the midbrain raphe (DRN and MRN) and appears to contribute both to direct pathways activating components of the stress response and to higher order coordinated activation of a number of different areas. Direct serotonergic projections to the PVN have been demonstrated (Carasco and van der Kar, 2003; Herman et al., 2003), where they form synapses with CRF, oxytocin, and AVP neurons. In addition, 5-HTIA and 5-HTzA receptors are present on neurons of the PVN (Wright et al., 1995; Zhang et al., 2002), where they may contribute to activation of the HPA axis. Indeed the 5HTzA agonist DOI causes neuroendocrine responses similar to stress and induces Fos expression in CRF and oxytocin neurons of the PVN (van de Kar et al., 2001). 5-HT projections also go to most other nuclei activated by stress and a variety of stressors can evoke release or turnover of 5-HT in the PFC, hippocampus, amygdala, NAc, and LC (Pei et al., 1990; Shimizu et al., 1992; Kawahara et al., 1993; Inoue et al., 1994; Linthorst et al., 1995; Adell et al., 1997; Amat et al., 1998; Kaehler et al., 2000; Funada and Hara, 2001). However, whilst the serotonergic system provides widespread projections to the forebrain, it is perhaps a misconception that 5-HT generates a diffuse and nonspecific stress signal, as recent evidence has demonstrated the existence of functional subsets of raphe 5-HT neurons, which respond to stress in a selective manner (see above). In addition, microdialysis data obtained from forced swim or conditioned fear, suggest that 5-HT responses may require very specific behavioral reactivity and/or stimulus conditions, with responses dependent on the escape potential of the model. In this respect serotonergic antagonists will potentiate and SSRIs attenuate the expression of learned helplessness, suggesting that 5-HT plays an important part in stress coping strategies. Within the hierarchical model of stress responding (Fig. 2) 5-HT may have a relatively minor role in direct activation of output neurons (such as HPA activation), but primarily function to modulate the appropriate level of neuroendocrine and autonomic activity and coping strategy, whether it be through
facilitation of motor output or facilitation of freezing behavior (equivalent to pathway (3)). Regional differences in the levels of 5-HT attained during specific stressful stimuli may alter this pattern of coping. In this respect it should not be forgotten that the serotonergic system is under important control by noradrenaline afferents and there is accumulating evidence for an important innervation by CRF (see Linthorst, Chapter 4.6). Differential sensitivity of subpopulations of serotonergic neurons to these inputs will alter the balance of transmission to the respective projection regions.
Dopamine The role of dopamine in responses to stress are largely restricted to a subset of neurons in the ventral tegmental area (VTA) projecting to the PFC and NAc, with the former projection being the more stress sensitive (Roth et al., 1988; Pani et al., 2000). In this respect, handing stress and hypotension strongly stimulate the release of dopamine in the mPFC (Kawahara et al., 1999), while the acute stress of restraint or footshock will activate the mesolimbic dopamine system (Puglisi-Allegra et al., 1991). Conditioned stress causes selective increase in dopamine utilization in medial and lateral PFC and NAc, but not in perirhinal or cingulate cortices, BLA, or caudate-putamen (Goldstein et al., 1994), and likewise handling and tail pinch stimulates DA release in the mPFC, but has only small effects in NAc and no effects in caudate-putamen (Cenci et al., 1992). Consistent with this pattern of release, footshock increases Fos expression in prelimbic and infralimbic cortices and in tyrosine hydroxylaselabeled neurons of the VTA (Morrow et al., 2000), and restraint stress increases the concentrations of the dopamine metabolite DOPAC in the PFC and NAc, and induces Fos immunoreactivity in dopamine neurons of the VTA but not the substantia nigra (Deutch et al., 1991). Furthermore, retrograde tracer studies confirm the activation of a distinct subset of VTA dopamine neurons, which project to the PFC. Thus, from a large number of studies it has been established that acute stress evokes a greater increase in dopamine metabolism and release within the PFC than other subcortical areas (Finlay and
625 Zigmond, 1997). Indeed the mesoprefrontal system appears to be particularly responsive to low intensity stresses that do not affect other ascending dopaminergic systems (Horger and Roth, 1996), and the basis of this differential sensitivity may relate to the relative lack of D2 inhibitory autoreceptors in the mesoprefrontal pathway, coupled to the presence of extensive excitatory inputs to the VTA. Interestingly the effect of stress on dopamine-dependent behaviors and on the activation of afferents to the NAc appears to depend on the chronicity and degree of control that the animal can exert on the stress stimulus (Cabib and Puglisi-Allegra, 1996). In this respect dopamine in the mPFC may normally act to suppress mesolimbic dopamine transmission, but this fails in conditions of extreme or unpredictable stress. Dopamine innervation also appears to be important for stress-induced activation of neurons in the anterolateral BNST (Kozicz, 2002), which are involved both in higher order activation of stressresponsive circuits and in the generation of coping behaviors (see above). Dopamine plays a role in the hedonic and reward aspects of stress, and the effects of stress on sexual activity and appetite and on the sensitivity to drugs of abuse are possibly mediated through the dopamine system. Dopamine also has the capacity to increase the gain of neuronal information processing and, therefore, can affect the learning and information processing associated with future stress. Finally it is notable that the CeA plays an important role in dopamine neurotransmission in the PFC, such that lesions of CeA block stress-induced dopamine release in the PFC and infusion of AMPA into the CeA evokes both a rapid increase in PFC dopamine release and an increase in arousal state (Stalnaker and Berridge, 2003). This is consistent with CeA involvement in coordination of neural systems regulating the behavioral state during stress.
Acetylcholine (A Ch) Acetylcholine is released in the hippocampus and cortex in response to a number of stressful stimuli, including footshock (Dazzi et al., 1995) and immobilization (Tajima et al., 1996), and this response may have dependence on serotonergic
inputs from the DRN. However, it is unclear whether it is stress itself which triggers ACh release or whether it is novelty, since the responses are transient and appear to occur with a variety of novel stimuli (Acquas et al., 1996). In this regard cholinergic neurons in the PPTg have an important role in controlling arousal and attention and, since forebrain cholinergic innervation is known to facilitate working memory performance by increasing the discriminatory capacity of cortical neurons, the role of acute changes in ACh may have particular relevance to the acquisition of memories. However, there is evidence that acute hippocampal release of ACh may also be involved in the termination of the effects of stress. Blocking hippocampal muscarinic receptors has the effect of augmenting restraint-induced release of ACTH and corticosterone (Bhatnagar et al., 1997), and septohippocampal cholinergic inputs may be essential for the hippocampus to express its normal inhibitory effect on the HPA axis (Han et al., 2002). Interestingly, the expression of certain variants of the metabolic enzyme, acetylcholinesterase, is highly sensitive to stress, and differences in both basal and stress-induced levels of this enzyme may underlie inter-individual and adaptive responses to stress (see Soreq et al., Chapter 4.10),
Amino acids (glutamate and GABA) Despite (or possibly due to) the importance of glutamate as a central transmitter, its role in mediating the various aspects of the stress response have received relatively scant attention. Both restraint and swim stress evoke widespread release of glutamate in mPFC, hippocampus, striatum, and NAc, with the largest increase in mPFC (Moghaddam, 1993). However, whether glutamate released in response to various noxious and nonnoxious stimuli is functional has been questioned (Timmerman et al., 1999). Nevertheless, because of its well-established role in cognitive processes, it is likely that glutamate will modulate responses to psychological stressors which utilize cognitive circuits. Indeed hippocampal (ventral subicular) efferents are glutamatergic, and glutamate appears to have an important role in modifying stress-induced activation of the mPFC, particularly the mesoprefrontal
626 dopamine system. For example, conditioned stress (tone paired with footshock) causes increased mPFC dopamine and 5-HT turnover, and increased serum corticosterone and freezing behavior. Freezing behavior and dopamine utilization in the PFC could be blocked by an NMDA glycine site antagonist, but HPA responses and other neurochemical changes were unaffected, indicating functional selectivity (Goldstein et al., 1994). Handling stress also increases extracellular dopamine and its metabolites in the PFC, but does not modify glutamate. Retrodialysis of ionotropic glutamate receptor agonists, N M D A and AMPA, significantly attenuates this handling-induced dopamine release (del Arco and Mora, 2001), while retrodialysis of a metabotropic glutamate (mGlu2/3) receptor agonist significantly attenuated immobilization-induced noradrenaline release (but not DA) (Swanson et al., 2004). The near ubiquitous inhibitory effect of GABA on neural activity means that it is hard to attribute the effects of GABAergic agonists and antagonists to specific aspects of the stress response. Furthermore, for technical reasons relatively few studies have measured regional stress-induced GABA release. However, increased GABA release has been measured in the LC in response to immobilization or hypovolemia (Singewald et al., 1995), and in the amygdala in response to noise (Singewald et al., 2000) or psychogenic stress (Fig. 4). This release may modulate transmission through the activated response network. Furthermore, many PVN afferents are immunoreactive for glutamic acid decarboxylase and these GABAergic afferents appear to play a major role in regulating CRF neurons and activity of the HPA axis (Boudaba et al., 1996; Herman et al., 2002; see Chapter 4.1). Evidence from organotypic culture has shown that tonic GABAergic inhibition suppresses CRF neurons and withdrawal of this tone (either by antagonist or appropriate afferent input) unmasks a glutamatergic AMPA/kainate receptor mediated excitation, which drives CRF neuron activity (Bartanusz et al., 2004). The presence of a GABAergic tone has been demonstrated in vivo by the fact that local application of bicuculline into the PVN increases Fos expression and HPA activity (Cole and Sawchenko, 2002). However, while glutamate has a role in regulating PVN CRF neurons
(see Chapter 4.7), no study has yet directly addressed its involvement in stress-induced HPA activation. Finally, one should remember that corticosteroids have major effects on glutamate and GABA neurotransmission and, therefore, one cannot exclude possible indirect effects during stress (see Chapter 4.7).
Nitric oxide (NO) There is increasing evidence that NO plays an important regulatory role in many parts of the stress activating network. Restraint stress activates putative NO-producing neurons in many autonomic centers (including medial septum, amygdala, PVN, raphe nuclei, NTS, and ventrolateral medulla) and may function to decrease sympathetic output (Krukoff, 1998). NO appears to be particularly important for regulating the PVN outflow which regulates sympathetic activity (Kenney et al., 2003). Indeed many PVN neurons that express Fos following immobilization stress also contain NO synthase, and treatment with competitive NO synthase inhibitors blocks stress-induced Fos expression in the PVN (Amir et al., 1997). The levels of NO metabolites in the PVN region also increase after intraperitoneal administration of interleukin-ll3, but not after footshock (Ishizuka et al., 2000). However, footshock will increase NO in the mPFC, indicating regional selectivity. Interestingly, many (if not all) cholinergic neurons in the PPTg colocalize NO synthase (Sugaya and McKinney, 1995), suggesting NO may also contribute to the arousal and attentional aspects of the stress response.
Vasopressin (A VP) and oxytocin Along with CRF, AVP is released from parvocellular PVN neurons during a stress response and plays a key role in the synergistic activation of pituitary corticotrophs (see Chapter 1.3). However, in addition to this peripheral action, AVP may have important central effects. Microdialysis within the PVN shows that AVP is released in response to the emotional stress of social defeat, but not by exposure to a novel cage (Wotjak et al., 1996). In contrast, within the septum and amygdala, social defeat (a purely
627 emotional stress) does not evoke AVP release (Engelmann et al., 2000) despite the ability of this stress to evoke powerful behavioral responses and activation of the HPA axis (Ebner et al., 2000). However, forced swim stress (a combined physical and emotional stress) will induce the release of AVP (Ebner et al., 2002). The function of this centrally released AVP may relate to acute stresscoping strategies, anxiety-related behavior, and/or learning and memory processes (see Chapter 2.6). Retrodialysis of a V1 antagonist in the amygdala demonstrates that AVP regulates coping strategy during forced swim, reducing active coping (struggling) and increasing passive coping (floating) (Ebner et al., 2002). Furthermore, recent data suggests that differences in central AVP pathways correlates with anxiety-like behavior (Landgraf and Wigger, 2002). AVP is also important in the adaptive responses to persistent or repeated stress, since the increased expression by the hypophysiotrophic neurons of the PVN may play a role in driving corticotroph activity when the levels of CRF are reduced by the feedback effects of glucocorticoids (Ma and Lightman, 1998). Oxytocin is also released into the circulation from magnocellular neurons of the supraoptic nucleus and PVN in response to stressful stimuli, such as ether, restraint, swim stress, motion stress, novel environment, or fear conditioned by footshocks (Gibbs, 1984; Nishioka et al., 1998; Zou et al., 1998; Neumann, 2002). The function of this neuroendocrine response is unclear, but may contribute to hepatic glycogenolysis or cardiovascular regulation. In addition to this endocrine function, oxytocin may also be released centrally. This can occur either coincident with peripheral release, as for the release into the PVN during motion stress (Nishioka et al., 1998), or in the absence of peripheral release, as for the release into the supraoptic nucleus and septum following social defeat (Ebner et al., 2000; Engelmann et al., 2000). This centrally released oxytocin may play an important integrative function. For example, oxytocin is expressed by a number of descending PVN efferents and is implicated in the regulation of sympathetic outflow (Coote et al., 1998). In addition, intracerebroventricular administration of oxytocin will attenuate the HPA responses to noise
or restraint, and will attenuate stress-induced c-fos mRNA expression in the PVN and SHy/VLS (Windle et al., 1997, 2004). While infusion of an antagonist indicates that endogenous oxytocin modulates the HPA response to a novel environment (Neumann, 2002), it appears not to regulate coping strategy during the conflict test (Ebner et al., 2000).
Other neuropeptides with potential integrative roles in stress responding It would be beyond the scope of this review to consider all the transmitters which have the potential to contribute to the integrated stress response, however, it is worth mentioning a few for which recent evidence has suggested an important role.
Substance P Central substance P pathways may be important in the process of terminating the activation of the stressactivated circuits, particularly those controlling the HPA axis (Jessop et al., 2000). They may also contribute to the activation of the sympathetic system during acute stress response (Ku et al., 1998), e.g. pressor responses involve the CeA, ventromedial hypothalamus, PVN, and rostral ventrolateral medulla (A1). Microinjection of substance P into these areas will induce pressor responses and substance P has been suggested to mediate the pressor response to CeA activation (Wu et al., 1999). Furthermore there is increasing evidence that substance P plays a role in modulating anxiety-like behavior (Aguiar and Brandao, 1996; Boyce et al., 2001; Santarelli et al., 2001) and studies of the effects of NK-1 antagonists have demonstrate important behavioral fupctions (see Part II: Chapter 4.3). ' t~ii~_
Galanin Galanin may be involved in the stress coping generated by the BNST-amygdala complex. Injection of galanin into the CeA has an anxiolytic effect and recent evidence has suggested that
628 noradrenaline neurotransmission can stimulate stress-induced release of galanin which acts to attenuate anxiety behavior during acute stress (Khoshbouei et al., 2002). This galanin may arise from the closely associated lateral BNST, where dense noradrenergic and dopaminergic fibers contact galanin neurons (Kozicz, 2001). However, galanin administered into the lateral BNST appears to facilitate behavioral responses to stress (Morilak et al., 2003). Whether this is due to an autoinhibitory effect on efferents to the CeA is not clear.
Neuropeptide Y (NP Y) Stress has marked effects on regional levels of expression of NPY (Thorsell et al., 1998; Krukoff et al., 1999; Makino et al., 2000). Interestingly, intraamygdaloid injections of NPY have anxiolytic effects (Heilig et al., 1993) and microinjection of NPY receptor ligands has suggested that NPY Y1 receptors may mediate these effects. Furthermore, recent evidence has shown that increased emotionality is observed in mice with an homologous recombination knockout of the preproNPY gene, while rats overexpressing NPY in the hippocampus display no overt behavioral responses to stress (Heilig and Thorsell, 2002). However, it is not clear whether this is a direct effect on stress circuits or on the acquisition and processing of memories necessary for responses to emotional stressors.
Integration through the coordinating effect of specific transmitters: role for CRF The large body of evidence, provided above and elsewhere in this volume, has shown that individual transmitters can have multiple involvement in generating the behavioral and physiological responses to stress, or can evoke responses which have a superficial resemblance to those occurring during stress. This has led to the popular concept that specific transmitters may fulfil a function to integrate or coordinate the stress response. This integration may occur either early in the process of perception, at the point at which the sensory or cognitive process
passes a threshold to become a stress response, or at a later stage in the pathway, where a distributed network synchronously contacts several output systems (cf. Pathways (2) and (3) in Fig. 2). However, while either of these arrangements would confer integration on a stress-response network, it is important that this should be distinguished from the situation where the same transmitter contributes to different stress pathways which does not confer integration (cf. Transmitter 1 in Fig. 3). Indeed, evidence based on global changes in transmitter function (e.g. agonist/antagonist administration or knockout/overexpression studies) may not make this important distinction. Nevertheless, it is clear that certain transmitters not only possess the appropriate anatomical arrangement to generate integration within a stress-responsive network, but to do so in a way that is selective for stress responding. One transmitter which has been widely accepted to fulfil this role is CRF, which is both the most important neuroendocrine factor controlling HPA activity, and acts as a central transmitter controlling aspects of behavior and autonomic/physiological activation (Brown and Fisher, 1985; Dunn and Berridge, 1990; Owens and Nemeroff, 1991; de Souza, 1995; Bakshi and Kalin, 2000; Smagin and Dunn, 2000; Smagin et al., 2001). Some of the features of these roles are briefly addressed here.
Role of CRF in mediating the stress response The functions of central CRF and related peptides (e.g. urocortins) are extensively reviewed elsewhere in this volume and it is unnecessary to repeat the evidence here (see Chapters 2.3, 2.4, 4.4, 4.6, Part II: 1.3, and 4.1). However, it is worth noting that there is an extensive network of CRF-containing neurons that originate in several stress-responsive areas of the brain (PVN, BNST, CeA, LC, parabrachial area, hippocampus, and NAc) (Sawchenko and Swanson, 1985). Furthermore, the increase in CRF m R N A in response to a wide variety of stressors of different modalities (Bakshi and Kalin, 2000) indicates that these CRF neurons form part of a common responsive system. However, consistent with a more selective role in regulating transmission, intracerebral
629 injection of the CRF antagonist, s-helical CRF9_41, has been shown to decrease restraint-induced c-fos m R N A expression in the PVN without affecting IEG induction in the VLS or LC (Imaki et al., 1995), suggesting that not all of the network activated by stress is dependent upon CRF neurotransmission. Shortly after its identification as a regulator of the HPA axis, CRF was shown to induce behavioral activation which resembled some of the features seen during stress (Sutton et al., 1982; Dunn and Berridge, 1990). Importantly the behavioral effects of intracerebrally administered CRF depend upon context: testing in a familiar environment or low arousal state evoked locomotor activity, exploration (rearing), displacement (grooming), and attenuated feeding, while prominent behavioral suppression occurred when CRF was tested in an unfamiliar state under conditions of arousal (Koob, 1999; Smagin et al., 2001). This suggests that CRF acts to modulate the response of a pathway rather than having a predefined effect. Lesions of the CeA will block some of the behavioral effects of centrally administered CRF, and this may be mediated through the reciprocal excitatory connections with LC-noradrenaline system (see Valentino and van Bockstaele, Chapter 4.4). Beyond this pharmacological effect of exogenous CRF, the role of endogenous CRF in response to stress is demonstrated by the ability of antagonists to attenuate the behavioral (e.g. Hotta et al., 1999; Smagin et al., 2001; see Part II: Chapters 1.3 and 4.1 for review) and sympathoexcitatory responses (Jezova et al., 1999) to stress, as well as the stressinduced increase in Fos expression in the PVN (Imaki et al., 1995). Furthermore, genetic modification of the CRF system evokes marked differences in stress- or anxiety-like behaviors consistent with its involvement (Bakshi and Kalin, 2000; Coste et al., 2001; Mfiller et al., 2003; and see Part II: Chapter 1.3 for review). The pharmacology of these responses and the relative contribution of CRF and urocortins has been reviewed elsewhere (Smagin et al., 2001), but CRF-R1 has been particularly associated with the cognitive aspects of stress behavior, including emotionality, attention, and executive function (Steckler and Holsboer, 1999; Bakshi and Kalin, 2000). Consistent with its potential role in coordinating multiple aspects of the stress response, CRF receptors are distributed in many of the areas of the brain
which exhibit stress-induced IEG expression. CRF-R1 receptors are located in the pituitary, PVN, hippocampus, amygdala, and neocortex (Wong et al., 1994; Chalmers et al., 1995; Sauvage and Steckler, 2001), while CRF-R2~ receptors are found in the lateral septum, amygdala, and ventromedial hypothalamus (Lovenberg et al., 1995; Sanchez et al., 1999). These two receptors appear to have opposing actions, since mice deficient for CRF-R1 display markedly attenuated responses to stress (Smith et al., 1998; Coste et al., 2001), while deficiency for CRF-R2 leads to anxiety-like behavior and exaggerated stress responses (Bale et al., 2000). However, recent evidence suggests that the coordinating role of CRF is not straightforward, since, while CRF-R1 knockout mice show reduced stress and anxiety levels, conditional knockout mutants, in which CRF-R1 function is selectively inactivated in the anterior forebrain and limbic brain structures but which leave HPA activity is intact, display hypersensitivity to stress (Mfiller et al., 2003). CRF is able to generate these responses through interactions with other transmitter systems. One of the best characterized transmitter interactions is the ability of CRF to regulate the activity of the noradrenergic system arising from the LC (Koob, 1999; Valentino and van Bockstaele, Chapter 4.4). CRF also evokes the release of 5-HT (or its metabolite) in the hippocampus (Linthorst et al., 2002), striatum (Price et al., 1998), and medial hypothalamic area (Lavicky and Dunn, 1993; for review see Linthorst, Chapter 4.6). This release is consistent with excitatory effects on the neurons of the raphe (Price et al., 1998; Lowry et al., 2000), although it may also be driven by increased afferent effects of noradrenaline. Intracerebroventricular administration of CRF will evoke release of hippocampal ACh similar to that evoked by stress (Day et al., 1998), and this may arise from the presence of CRF-R1 on the cholinergic neurons of the PPTg (Sauvage and Steckler, 2001). Exogenous CRF also increases dopamine turnover (measured by tissue DOPAC levels) in a number of hypothalamic and limbic areas, including the NAc, PVN, and periventricular nuclei (Pan et al., 1995). Thus, these widespread transmitter effects, coupled to the distribution of receptors in stress-related areas, are consistent with an integrative function for CRF.
630
CRF involvement in an integrated stress-response Despite the evidence for C R F involvement in multiple aspects of the stress response, it is not clear that this supports an integrative function within a hierarchical model of stress pathways (Fig. 2). While it may be conceptually useful to consider C R F as a single integrating system underlying the stress response, several lines of evidence suggest a more complex organization. Firstly, the distribution of C R F - c o n t a i n i n g neurons is such that they cannot operate as a single network which distributes a stress signal to coordinate several downstream response systems (i.e. similar to Pathways (2) or (3) in Fig. 2). A l t h o u g h it is possible that this network of C R F neurons may be innervated by a c o m m o n transmitter system, such as noradrenaline, to evoke coordinated activation, in this case C R F is not acting as the integrating factor. Secondly, while it is clear that C R F is released into both the P V N and amygdala
in response to a stressful stimulus and thereby might coordinate neuroendocrine and behavioral responses, recent technical advances have shown that the temporal profiles may be quite distinct (Fig. 4), arguing against a single system coordinating the responses. Thirdly, C R F administration, either intracerebrally (Arnold et al., 1992; Imaki et al., 1993; da Costa et al., 1997) or directly into the LC (Rassnick et al., 1998), produces a regional pattern of Fos expression that does not mirror the pattern induced by stress, and probably is a reflection of the distribution of the C R F - R 1 receptor and downstream innervated areas (Bittencourt and Sawchenko, 2000). While there may be significant overlap between these two stimuli, it does not prove the integrative function of C R F . F o r example, both intracerebral C R F and immobilization stress increase Fos in the PVN, MeA, and LC, but only C R F increases Fos in the BNST, CeA, and NTS, while only stress has an effect in the SHy (Imaki et al., 1993).
INPUT_ ,, .." . . . . "",,
-.
."''---"".
"--..;_.-""
//'"-----
|
|
...
LEUS 2
NUCLEUS 3
OUTPUT Fig. 5. Possible organization of CRF neurons within a distributed stress-responsive circuit. Neurons (1) in nucleus 1 receive inputs which may potentially be considered stressful. Output from these neurons goes to neurons (3) in nucleus 2 and also to local CRF neurons (2). Above a particular threshold for activation these CRF neurons are also activated and the stimulus takes on a stressful quality, signalling this to nucleus 2 by a convergent pathway. A population of CRF neurons (4) in nucleus 2 receives an input which is direct and does not rely on intranuclear connections. Once again a threshold for activation determines whether or not these neurones are activated and pass a stress signal to neurons (5) in nucleus 3. CRF neurons (6) in or close to nucleus 3 receive their inputs via collaterals from nucleus 2. When this input exceeds a threshold for activation, intrinsic connections within or close to nucleus 3 signal this as stressful. Output (7) from nucleus 3 is regulated by activity within the whole network. CRF may influence the activity of the network at several points through gain control and/or parallel processing. The overall contribution of CRF to transmission within this network may depend upon the relative number of CRF neurons and their different thresholds for activation.
631 Indeed since IEG expression displays at least two modality-specific patterns this unlikely to arise from a single CRF system. So how might stress pathways be organized in order for CRF to fulfil a pivotal role in integrating the activation of several transmitter systems and the expression of various aspects of the stress response? In the models described above, one or more discrete regulatory center(s) was considered to project to the various effector systems. However, while this distributed organization may be appropriate for transmitters that have a relatively restricted origin (e.g. noradrenaline in the LC or 5-HT in the DRN) this does not hold for CRF, which is both synthesized by neurons within many stress-responsive areas and has receptors within the same areas (see above and Chapter 4.4). However, transmitters with discrete origins but distributed projection sites, such as noradrenaline and 5HT, fulfil many functions in addition to contributing to the stress response and so cannot be considered to carry the stress signal alone. Thus, it may be more appropriate to consider the stress response as generated by a number of sensory and cognitive networks which variably employ noradrenaline, 5-HT, dopamine, ACh, and glutamate, but that, throughout these networks, CRF functions to modulate the signal in order to attribute a "stressful" characteristic. Using this concept of parallel processing, CRF neurons might function both as a threshold detector mechanism and as a mechanism to modulate the nature of signals passing through the network by changes in gain control (Fig. 5). This may operate through a number of different connections of CRF neurons which are known to exist within the network of stressresponsive areas. This model allows for the circuit to fulfil functions unrelated to stress, but to switch to a stress-responsive mode through the action of CRF at any of a number of different sites. This model incorporates the concept of threshold detection and distributed activation, and the integrative role of CRF can be conceived not simply as its contribution to one specific pathway, but by its ability to change the nature of the signal passing through the network. Importantly the CRF system may be activated by a wide range of stress stimuli but may only modulate transmission through pathways which are activated by specific stimuli, and therefore the model also
incorporates responding.
aspects
of
the
modality
selective
Conclusions So, from the foregoing account is it possible to provide an integrated view of pathway and transmitter involvement in stress? The cautious response to this is, "to some extent." Stress is a term which encompasses diverse range of complex responses and while technical advances have helped to identify many of the key areas and transmitters that contribute to generating these responses, we are still a long way from fully determining the way these join up to generate an integrated behavioral, neuroendocrine, and autonomic response. Nevertheless, certain principles have emerged which allow the construction of theoretical models. The future challenge will be to refine these models in order to eventually determine the neural basis of stress.
Abbreviations 5-HT ACh AVP BLA BNST CeA CRF CRF-R1/ CRF-R2 DMH DRN HPA IEG LC LPS MeA (m)PFC MPOA MRN NAc NO NPY NTS PAG
5-hydroxytryptamine (serotonin) acetylcholine vasopressin basolateral amygdala bed nuclei of the stria terminalis central nucleus of the amygdala corticotropin-releasing factor type 1/2 CRF receptor dorsomedial hypothalamus dorsal raphe nucleus hypothalamo-pituitary-adrenal immediate-early gene locus coeruleus lipopolysaccharide medial nucleus of the amygdala (medial) Prefrontal cortex medial preoptic area median raphe nucleus nucleus accumbens nitric oxide neuropeptide Y nucleus tractus solitarius periqueductal gray of the midbrain
632
PPTg PVN SHy VLS VTA
pedunculopontine tegmentum p a r a v e n t r i c u l a r nucleus of the hypothalamus s e p t o h y p o t h a l a m i c area ventrolateral septum ventral t e g m e n t a l area
Acknowledgments T h e a u t h o r is grateful to the m e m b e r s of his g r o u p a n d to colleagues, with w h o m discussions have h e l p e d to s h a p e these ideas. H e is especially grateful to Dr. C h r i s t i a n C o o k for p r o v i d i n g Fig. 4.
References Abraham, I., Juhasz, G., Kekesi, K.A. and Kovacs, K.J. (1998) Corticosterone peak is responsible for stress-induced elevation of glutamate in the hippocampus. Stress, 2:171-181. Acquas, E., Wilson, C. and Fibiger, H.C. (1996) Conditioned and unconditioned stimuli increase frontal cortical and hippocampal acetylcholine release: effects of novelty, habituation, and fear. J. Neurosci., I6: 3089-3096. Adell, A., Casanovas, J.M. and Artigas, F. (1997) Comparative study in the rat of the actions of different types of stress on the release of 5-HT in raphe nuclei and forebrain areas. Neuropharmacology, 36: 735-741. Aguiar, M.S. and Brandao, M.L. (1996) Effects of microinjections of the neuropeptides substance P in the dorsal periaqueductal gray on the behaviour of rats in the plusmaze test. Physiol. Behav., 60:1183-1186. Amat, L., Matus-Amat, P., Watkins, L.R. and Maier, S.F. (1998) Escapable and inescapable stress differentially and selectively alter extracellular levels of 5-HT in the ventral hippocampus and dorsal periaqueductal gray of the rat. Brain Res., 797: 12-22. Amir, S., Rackover, M. and Funk, D. (1997) Blockers of nitric oxide synthase inhibit stress activation of c-fos expression in neurons of the hypothalamic paraventricular nucleus in the rat. Neuroscience, 77: 623-627. Arnold, F.J.L., de Lucas Bueno, M., Shiers, H., Hancock, D.C., Evans, G.I. and Herbert, J. (1992) Expression of c-fos in regions of the basal limbic forebrain following intracerebroventricular corticotropin-releasing factor (CRF) in unstressed and stressed male rats. Neuroscience, 51: 377-390. Bakshi, V.P. and Kalin, N.H. (2000) Corticotropin-releasing hormone and animal models of anxiety: Gene-environment interactions. Biol. Psychiatry, 48:1175-1198. Bale, T.L., Contarino, A., Smith, G.W., Chan, R., Gold, L.H., Sawchenko, P.E., Koob, G.F., Vale, W.W. and Lee, K.F. (2000) Mice deficient for corticotropin-releasing hormone,
receptor-2 display anxiety-like behaviour and are hypersensitive to stress. Nat. Genet., 24: 410-414. Bandler, R., Keay, K.A., Floyd, N. and Price, J. (2000) Central circuits mediating patterned autonomic activity during active vs. passive emotional coping. Brain Res. Bull., 53: 95-104. Bartanusz, V., Muller, D., Gaillard, R.C., Streit, P., Vutskits, L. and Kiss, J.Z. (2004) Local ~,-aminobutyric acid and glutamate circuit control of hypophyseotrophic corticotropin-releasing factor neuron activity in the paraventricular nucleus of the hypothalamus. Eur. J. Neurosci., 19: 777-782. Bernard, J.F. and Bandler, R. (1998) Parallel circuits for emotional coping behaviour: new pieces in the puzzle. J. Comp. Neurol., 401: 429-436. Berridge, C.W. and Waterhouse, B.D. (2003) The locus coeruleus-noradrenergic system: modulation of behavioral state and state-dependent cognitive processes. Brain Res. Rev., 42: 33-84. Bhatnagar, S., Costall, B. and Smythe, J.W. (1997) Hippocampal cholinergic blockade enhances hypothalamicpituitary-adrenal responses to stress. Brain Res., 766: 244-248. Bittencourt, J.C. and Sawchenko, P.E. (2000) Do centrally administered neuropeptides access cognate receptors?: an analysis in the central corticotropin-releasing factor system. J. Neurosci., 20:1142-1156. Blizard, D.A. and Adams, N. (2002) The Maudsley Reactive and Nonreactive strains: a new perspective. Behav. Genet., 32: 277-299. Boudaba, C., Szabo, K. and Tasker, J.G. (1996) Physiological mapping of local inhibitory inputs to the hypothalamic paraventricular nucleus. J. Neurosci., 16:7151-7160. Boyce, S., Smith D., Carlson, E., Hewson, L., Rigby, M., O'Donnell, R., Harrison, T. and Rupniak, N.M. (2001) Intra-amygdala injection of the substance P [NK(1) receptor] antagonist L-760735 inhibits neonatal vocalisations in guinea pigs. Neuropharmacology, 41: 130-137. Britton, K.T., Segal, D.S., Kuczenski, R. and Hauger, R. (1992) Dissociation between in vivo hippocampal norepinephrine response and behavioral/neuroendocrine responses to noise stress in rats. Brain Res., 574: 125-130. Brown, M.R. and Fisher, L.A. (1985) Corticotropin-releasing factor: effects on the autonomic nervous system and visceral systems. Fed. Proc., 44: 243-248. Cabib, S. and Puglisi-Allegra, S. (1996) Stress, depression and the mesolimbic dopamine system. Psychopharmacology, 128: 331-342. Campeau, S. and Watson, S.J. (1997) Neuroendocrine and behavioral responses and brain pattern of c-fos induction associated with audiogenic stress. J. Neuroendocrinol., 9: 577-588. Canteras, N.S. (2002) The medial hypothalamic defense system: hodological organization and functional implications. Pharmacol. Biochem. Behav., 71: 481-491.
633 Carrasco, G.A. and van de Kar, L.D. (2003) Neuroendocrine pharmacology of stress. Eur. J. Pharmacol., 463: 235-272. Castagn~, V., Rivet, J.-M. and Morm~de, P. (1990) The integrity of the ventral noradrenergic bundle (VNAB) is not necessary for neuroendocrine stress response. Brain Res., 511: 349-352. Ceccatelli, S., Villar, M.J., Goldstein, M. and H6kfelt, T. (1989) Expression of c-fos immunoreactivity in transmitter-characterized neurons after stress. Proc. Natl. Acad. Sci. USA, 86: 9569-9573. Cecchi, M., Khoshbouei, H., Javors, M. and Morilak, D.A. (2002) Modulatory effects of norepinephrine in the lateral bed nucleus of the stria terminalis on behavioral and neuroendocrine responses to acute stress. Neuroscience, 112: 13-21. Cenci, M.A., Kalen, P., Mandel, R.J. and Bj6rklund, A. (1992) Regional differences in the regulation of dopamine and noradrenaline release in medial frontal cortex, nucleus accumbens and caudate-putamen: a microdialysis study in the rat. Brain Res., 581: 217-228. Chalmers, D.T., Lovenberg, T.W. and de Souza, E.B. (1995) Localization of novel corticotropin-releasing factor 2 (CRF(2)) mRNA expression to specific subcortical nuclei in rat b r a i n - comparison with CRF(1) receptor mRNA expression. J. Neurosci., 15: 6340-6350. Chen, X. and Herbert, J. (1995) Regional changes in c-fos expression in the basal forebrain and brainstem during adaptation to repeated stress: correlations with cardiovascular, hypothermic and endocrine responses. Neuroscience, 64: 675-685. Cole, R.L. and Sawchenko, P.E. (2002) Neurotransmitter regulation of cellular activation and neuropeptide gene expression in the paraventricular nucleus of the hypothalamus. J. Neurosci., 22: 959-969. Cook, C.J. (1998) Monitoring on-line of extracellular gammaamino-4-butyric acid using microdialysis coupled to immunosensor analysis. J. Neurosci. Meth., 82: 145-150. Cook, C.J. (2001) Measuring of extracellular cortisol and corticotropin-releasing hormone in the amygdala using immunosensor coupled microdialysis. J. Neurosci. Meth., 110: 95-101. Coote, J.H., Yang, Z., Pyner, S. and Deering, J. (1998) Control of sympathetic outflows by the hypothalamic paraventricular nucleus. Clin. Exp. Pharmacol. Physiol., 25: 461-463. Coste, S.C., Murray, S.E. and Stenzel-Poore, M.P. (2001) Animal models of CRH excess and CRH receptor deficiency display altered adaptations to stress. Peptides, 22: 733-741. Cullinan, W.E., Herman, J.P., Battaglia, D.F., Akil, H. and Watson, S.J. (1995) Pattern and time course of immediate early gene expression in the rat brain following acute stress. Neuroscience, 64: 477-505. Cunningham, E.T. and Sawchenko, P.E. (1988) Anatomical specificity of noradrenergic inputs to the paraventricular and
supraoptic nuclei of the rat hypothalamus. J Comp. Neurol., 274: 60-76. da Costa, A.P.C., Wood, S., Ingram, C.D. and Lightman, S.L. (1996) Region-specific reduction in stress-induced c-fos mRNA expression during pregnancy and lactation. Brain Res., 743: 177-184. da Costa, A.P.C., Kampa, R.J., Windle, R.J., Ingram, C.D. and Lightman, S.L. (1997) Region-specific immediate-early gene expression following the administration of corticotropinreleasing hormone in virgin and lactating rats. Brain Res., 770:151-162. Daugherty, W.P., Corley, K.C., Phan, T.H. and Boadle-Biber, M.C. (2001) Further studies on the activation of rat median raphe serotonergic neurons by inescapable sound stress. Brain Res., 923:103-111. Davis, M., Rainnie, D. and Cassell, M. (1994) Neurotransmission in the rat amygdala related to fear and anxiety. Trends Neurosci., 17: 208-214. Day, H.E.W., Campeau, S., Watson, S.J. Jr and Akil, H. (1999) Expression of ~lb adrenoceptor mRNA in corticotropinreleasing hormone-containing cells of the rat hypothalamus and its regulation by corticosterone. J. Neurosci., 19: 10098-10106. Day, J.C., Koehl, M., Le Moal, M. and Maccari, S. (1998) Corticotropin-releasing factor administered centrally, but not peripherally, stimulates hippocampal acetylcholine release. J. Neurochem., 71: 622-629. Dayas, C.V., Buller, K.M., Crane, J.W., Xu, Y. and Day, T.A. (2001a) Stressor categorization: acute physical and psychological stressors elicit distinctive recruitment patterns in the amygdala and in medullary noradrenergic cell groups. Eur. J. Neurosci., 14:1143-1152. Dayas, C.V., Bullet, K.M. and Day, T.A. (1999) Neuroendocrine responses to an emotional stressor: evidence for involvement of the medial but not the central amygdala. Eur. J. Neurosci., 11: 2312-2322. Dayas, C.V., Buller, K.M. and Day, T.A. (2001b) Medullary neurones regulate hypothalamic corticotropin-releasing factor cell responses to an emotional stressor. Neuroscience, 105: 707-719. Dazzi, L., Motzo, C., Imperato, A., Serra, M., Gessa, G.L. and Biggio, G. (1995) Modulation of basal and stress-induced release of acetylcholine and dopamine in rat brain by abecarnil and imidazenil, two anxioselective gamma-aminobutyric acid A receptor modulators. J. Pharmacol. Exp. Ther., 273: 241-247. de Souza, E.B. (1995) Corticotropin-releasing factor receptors: physiology, pharmacology, biochemistry and role in central nervous system and immune disorders. Psychoneuroendocrinology, 20:789-819. del Arco, A. and Mora, F. (2001) Dopamine release in the prefrontal cortex during stress is reduced by the local activation of glutamate receptors. Brain Res. Bull., 56: 125-130.
634 Deutch, A.Y., Lee, M.C., Gillham, M.H., Cameron, D.A., Goldstein, M. and Iadarola, M.J. (1991) Stress selectively increases fos protein in dopamine neurons innervating the prefrontal cortex. Cereb. Cortex, 1: 273-292. DiMicco, J.A., Samuels, B.C., Zaretskaia, M.V. and Zaretsky, D.V. (2002) The dorsomedial hypothalamus and the response to stress: part renaissance, part revolution. Pharmacol. Biochem. Behav., 71: 469-480. Diorio, D., Viau, V. and Meaney, M.J. (1993) The role of the medial prefrontal cortex (cingulate gyrus) in the regulation of hypothalamic-pituitary-adrenal responses to stress. J. Neurosci., 13: 3839-3847. Dunn, A.J. and Berridge, C.W. (1990) Physiological and behavioral responses to corticotropin-releasing factor administration: is CRF a mediator of anxiety or stress responses? Brain Res. Rev., 15: 71-100. Ebner, K., Wotjak, C.T., Landgraf, R. and Engelmann, M. (2000) A single social defeat experience selectively stimulates the release of oxytocin, but not vasopressin, within the septal brain area of male rats. Brain Res., 872: 87-92. Ebner, K., Wotjak, C.T., Landgraf, R. and Engelmann, M. (2002) Forced swimming triggers vasopressin release within the amygdala to modulate stress-coping strategies in rats. Eur. J. Neurosci., 15: 384-388. Elmquist, J.K., Scammell, T.E., Jacobson, C.D. and Saper, C.B. (1996) Distribution of Fos-like immunoreactivity in the rat brain following intravenous lipopolysaccharide administration. J. Comp. Neurol., 371: 85-103. Emmert, M.H. and Herman, J.P. (1999) Differential forebrain c-fos mRNA induction by ether inhalation and novelty: evidence for distinctive stress pathways. Brain Res., 845: 60-67. Engelmann, M., Wotjak, C.T., Ebner, K. and Landgraf, R. (2000) Behavioural impact of intraseptally released vasopressin and oxytocin in rats. Exp. Physiol., 85: 125S-130S. Figueiredo, H.F., Bruestle, A., Bodie, B., Dolgas, C.M. and Herman, J.P. (2003) The medial prefrontal cortex differentially regulates stress-induced c-fos expression in the forebrain depending on type of stressor. Eur. J. Neurosci., 18: 2357-2364. Figueiredo, H.F., Dolgas, C.M. and Herman, J.P. (2002) Stress activation of cortex and hippocampus is modulated by sex and stage of estrus. Endocrinology, 143: 2534-2540. Finlay, J.M. and Zigmond, M.J. (1997) The effects of stress on central dopaminergic neurons: possible clinical implications. Neurochem. Res., 22: 1387-1394. Fuchs, E. and Fltigge, G. (2003) Chronic social stress: effects on limbic brain structures. Physiol. Behav., 79: 417-427. Funada, M. and Hara, C. (2001) Differential effects of psychological stress on activation of the 5-hydroxytrptamineand dopamine-containing neurons in the brain of freely moving rats. Brain Res., 901: 247-251.
Gibbs, D.M. (1984) Dissociation of oxytocin, vasopressin and corticotropin secretion during different types of stress. Life Sci., 35: 487-491. Giorgi, O., Lecca, D., Piras, G., Driscoll, P. and Corda, M.G. (2003) Dissociation between mesocortical dopamine release and fear-related behaviours in two psychogenetically selected lines of rats that differ in coping strategies to aversive conditions. Eur. J. Neurosci., 17: 2716-2726. Goldstein, L.E., Rasmusson, A.M., Bunney, B.S. and Roth, R.H. (1994) The NMDA glycine site antagonist (+)-HA-966 selectively regulates conditioned stress-induced metabolic activation of the mesoprefrontal cortical dopamine but not serotonin systems: a behavioral, neuroendocrine, and neurochemical study in the rat. J. Neurosci., 14: 4937-4950. Goldstein, L.E., Rasmusson, A.M., Bunney, B.S. and Roth, R.H. (1996) Role of the amygdala in the coordination of behavioral, neuroendocrine, and prefrontal cortical monoamine responses to psychological stress in the rat. J. Neurosci., 16: 4787-4798. Graeff, F.G., Guimar~es, F.S., de Andrade, T.G.C.S. and Deakin, J.F.W. (1996) Role of 5-HT in stress, anxiety, and depression. Pharmacol. Biochem. Behav., 54: 129-141. Grahn, R.E., Will, M.J., Hammack, S.E., Maswood, S., McQueen, M.B., Watkins, L.R. and Maier, S.F. (1999) Activation of serotonin-immunoreactive cells in the dorsal raphe nucleus in rats exposed to an uncontrollable stressor. Brain Res., 826: 35-43. Hajos-Korcsok, E., Robinson, D.D., Yu, J.H., Fitch, C.S., Walker, E. and Merchant, K.M. (2003) Rapid habituation of hippocampal serotonin and norepinephrine release and anxiety-related behaviors, but not plasma corticosterone levels, to repeated footshock stress in rats. Pharmacol. Biochem. Behav., 74: 609-616. Han, J.S., Bizon, J.L., Chun, H.J., Maus, C.E. and Gallagher, M. (2002) Decreased glucocorticoid receptor mRNA and dysfunction of HPA axis in rats after removal of the cholinergic innervation to the hippocampus. Eur. J. Neurosci., 16:1399-1404. Haxhiu, M.A., Yung, K., Erokwu, B. and Cherniack, N.S. (1996) CO2-induced c-fos expression in the CNS catecholaminergic neurons. Respir. Physiol., 105: 35-45. Heilig, M., McLeod, S., Brot, M., Heinrichs, S.C., Menzaghi, F., Koob, G.F. and Britton, K.T. (1993) Anxiolytic-like action of neuropeptide Y: mediation by Y1 receptors in the amygdala and dissociation from food intake effects. Neuropsychopharmacology, 8: 357-363. Heilig, M. and Thorsell, A. (2002) Brain neuropeptide Y (NPY) in stress and alcohol dependence. Rev. Neurosci., 13: 85-94. Helfferich, F. and Palkovits, M. (2003) Acute audiogenic stressinduced activation of CRH neurons in the hypothalamic paraventricular nucleus and catecholaminergic neurons in the medulla oblongata. Brain Res., 975: 1-9. Herman, J.P., Cullinan, W.E. and Watson, S.J. (1994) Involvement of the bed nucleus of the stria terminalis in
635 tonic regulation of paraventricular hypothalamic CRH and AVP mRNA expression. J. Neuroendocrinol., 6: 433-442. Herman, J.P., Dolgas, C.M. and Carlson, S.C. (1998) Ventral subiculum coodinates situation-specific neuroendocrine and behavioral stress responses. Neuroscience, 86: 449-459. Herman, J.P., Figueiredo, H., Mueller, N.K., Ulrich-Lai, Y., Ostrander, M.M., Choi, D.C. and Cullinan, W.E. (2003) Central mechanisms of stress integration: hierarchical circuitry controlling hypothalamo-pituitary-adrenocortical responsiveness. Front. Neuroendocrinol., 24: 151-180. Herman, J.P., Tasker, J.G., Ziegler, D.R. and Cullinan, W.E. (2002) Local circuit regulation of paraventricular nucleus stress integration: Glutamate-GABA connections. Pharmacol. Biochem. Behav., 71: 457-468. Hitchcock, J. and Davis, M. (1986) Lesions of the amygdala, but not of the cerebellum or red nucleus, block conditioned fear as measured with the potentiated startle paradigm. Behav. Neurosci., 100:11-22. Horger, B.A. and Roth, R.H. (1996) The role of mesoprefrontal dopamine neurons in stress. Crit. Rev. Neurobiol., 10: 395-418. Hotta, M., Shibasaki, T., Arai, K. and Demura, H. (1999) Corticotropin-releasing factor receptor type 1 mediates emotional stress-induced inhibition of food intake and behavioral changes in rats. Brain Res., 823: 221-225. Imaki, T., Shibasaki, T., Hotta, M. and Demura, H. (1993) Intracerebroventricular administration of corticotropinreleasing factor induces c-fos mRNA expression in brain regions related to stress responses: comparison with pattern of c-fos mRNA induction after stress. Brain Res., 616: 114-125. Imaki, T., Shibasaki, T., Wang, X.Q. and Demura, H. (1995) Intracerebroventricular administration of corticotropinreleasing factor antagonist attenuates c-los mRNA expression in the paraventricular nucleus after stress. Neuroendocrinology, 61: 445-452. Inoue, T., Tsucgiya, K. and Koyama, T. (1994) Regional changes in dopamine and serotonin activation with various intensity of physical and psychological stress in the rat brain. Pharmacol. Biochem. Behav., 49: 911-920. Ishida, Y., Hashiguchi, H., Takeda, R., Ishizuka, Y., Mitsuyama, Y., Kannan, H., Nishimori, T. and Nakahara, D. (2002) Conditioned-fear stress increases Fos expression in monoaminergic and GABAergic neurons of the locus coeruleus and dorsal raphe nuclei. Synapse, 45: 46-51. Ishizuka, Y., Ishida, Y., Jin, Q., Kato, K., Kunitake, T., Mitsuyama, Y. and Kannan, H. (2000) Differential profiles of nitric oxide and norepinephrine releases in the paraventricular nucleus region in response to mild footshock in rats. Brain Res., 862: 17-25. Jessop, D.S., Renshaw, D., Larsen, P.J., Chowdrey, H.S. and Harbuz, M.S. (2000) Substance P is involved in terminating the hypothalamic-pituitary-adrenal axis response to acute
stress through centrally located neurokinin-1 receptors. Stress, 3: 209-220. Jezova, D., Ochedalski, T., Glickman, M., Kiss, A. and Aguitera, G. (1999) Central corticotropin-releasing hormone receptors modulate hypothalamic-pituitary-adrenocortical and sympathoadrenal activity during stress. Neuroscience, 94: 797-802. Kaehler, S.T., Singewald, N., Sinner, C., Thurnher, C. and Philippu, A. (2000) Conditioned fear and inescapable shock modify the release of serotonin in the locus coeruleus. Brain Res., 859: 249-254. Kawahara, Y., Kawahara, H. and Westerink, B.H. (1999) Comparison of effects of hypotension and handling stress on the release of noradrenaline and dopamine in the locus coeruleus and medial prefrontal cortex of the rat. Naunyn Schmiedebergs Arch Pharmacol., 360: 42-49. Kawahara, H., Yoshida, M., Yokoo, H., Nishi, M. and Tanaka, M. (1993) Psychological stress increases serotonin release in the rat amygdala and prefrontal cortex assessed by in vivo microdialysis. Neurosci. Lett., 162: 81-84. Kenney, M.J., Weiss, M.L. and Haywood, J.R. (2003) The paraventricular nucleus: an important component of the central neurocircuitry regulating sympathetic nerve outflow. Acta Physiol. Scand., 177: 7-15. Khoshbouei, H., Cecchi, M., Dove, S., Javors, M. and Morilak, D.A. (2002) Behavioral reactivity to stress: amplification of stress-induced noradrenergic activation elicits a galanin-mediated anxiolytic effect in central amygdala. Pharmacol. Biochem. Behav., 71: 407-4 17. Koob, G.F. (1999) Corticotropin-releasing factor, norepinephrine, and stress. Biol. Psychiatry, 46:1167-1180. Kozicz, T. (2001) Axon terminals containing tyrosine hydroxylase- and dopamine-[3-hydroxylase immunoreactivity form synapses with galanin immunoreactive neurons in the lateral division of the bed nucleus of the stria terminalis in the rat. Brain Res., 914: 23-33. Kozicz, T. (2002) Met-enkephalin immunoreactive neurons recruited by acute stress are innervated by axon terminals immunopositive for tyrosine hydroxylase and dopaminealpha-hydroxylase in the anterolateral division of bed nuclei of the stria terminalis in the rat. Eur. J. Neurosci., 16: 823-835. Krukoff, T.L. (1998) Central regulation of autonomic function: no brakes? Clin. Exp. Pharmacol. Physiol., 25: 474--478. Krukoff, T.L., MacTavish, D. and Jhamandas, J.H. (1999) Effects of restraint stress and spontaneous hypertension on neuropeptide Y neurones in the brainstem and arcuate nucleus. J. Neuroendocrinol., 11: 715-723. Ku, Y.-H., Tan, L., Li, L.-S. and Ding, X. (1998) Role of corticotropin-releasing factor and substance P in pressor responses of nuclei controlling emotion and stress. Peptides, 19: 677-682.
636 Landgraf, R. and Wigger, A. (2002) High vs low anxiety-related behavior rats: an animal model of extremes in trait anxiety. Behav. Genet., 32: 301-314. Lavicky, J. and Dunn, A.J. (1993) Corticotropin-releasing factor stimulates catecholamine release in hypothalamus and prefrontal cortex in freely moving rats as assessed by microdialysis. J. Neurochem., 60: 602-612. Li, H.Y., Ericsson, A. and Sawchenko, P.E. (1996) Distinct mechanisms underlie activation of hypothalamic neurosecretory neurons and their medullary catecholaminergic afferents in categorically different stress paradigms. Proc. Natl. Acad. Sci. USA, 93: 2359-2364. Lightman, S.L., Windle, R.J., Wood, S.A., Kershaw, Y.M., Shanks, N. and Ingram, C.D. (2001) Peripartum plasticity within the hypothalamo-pituitary-adrenal axis. Prog. Brain Res., 133:111-129. Linthorst, A.C.E., Flachskamm, C., Holsboer, F. and Reul, J.M.H.M. (1996) Activation of serotonergic and noradrenergic neurotransmission in the rat hippocampus after peripheral administration of bacterial endotoxin: involvement of the cyclo-oxygenase pathway. Neuroscience, 72: 989-997. Linthorst, A.C.E., Flachskamm, C., Mtiller-Preuss, P., Holsboer, F. and Reul, J.M.H.M. (1995) Effect of bacterial endotoxin and interleukin-l[3 on hippocampal serotonergic neurotransmission, behavioral activity, and free corticosterone levels: an in vivo microdialysis study. J. Neurosci., 15: 2920-2934. Linthorst, A.C.E., Panalva, R.G., Flachskamm, C., Holsboer, F. and Reul, J.M.H.M. (2002) Forced swim activates rat hippocampal serotonergic neurotransmission involving a corticotropin-releasing hormone-dependent mechanism. Eur. J. Neurosci., 16: 2441-2452. Loewy, A.D. (1991) Forebrain nuclei involved in autonomic control. Prog. Brain Res., 87: 253-268. Lopez, J.F., Akil, H. and Watson, S.J. (1999) Neural circuits mediating stress. Biol. Psychiat., 46: 1461-1471. Lovenberg, T.W., Liaw, C.W., Grigoriadis, D.E., Clevenger, W., Chalmers, D.T., de Souza, E.B. and Oltersdorf, T. (1995) Cloning and characterization of a functionally distinct corticotropin-releasing factor receptor subtype from rat brain. Proc. Natl. Acad. Sci. USA, 92: 836-840. Lowry, C.A. (2002) Functional subsets of serotonergic neurones: implications for control of the hypothalamic-pituitaryadrenal axis. J. Neuroendocrinol., 14: 911-923. Lowry, C.A., Rodda, J.E., Lightman, S.L. and Ingram, C.D. (2000) Corticotropin-releasing factor increases in vitro firing rates of serotonergic neurons in the rat dorsal raphe nucleus: evidence for activation of a topographically organized mesolimbocortical serotonergic system. J. Neurosci., 20: 7728-7736. Ma, X.M. and Lightman, S.L. (1998) The arginine vasopressin and corticotrophin-releasing hormone gene transcription
responses to varied frequencies of repeated stress in rats. J. Physiol., 510:605-614. Makino, S., Baker, R.A., Smith, M.A. and Gold, P.W. (2000) Differential regulation of neuropeptide Y mRNA expression in the arcuate nucleus and locus coeruleus by stress and antidepressants. J. Neuroendocrinol., 12: 387-395. McCormick, C.M., Linkroum, W., Sallinen, B.J. and Miller, N.W. (2002) Peripheral and central sex steroids have differential effects on the HPA axis of male and female rats. Stress, 5: 235-247. McQuade, R. and Stanford, S.C. (2000) A microdialysis study of the noradrenergic response in rat frontal cortex and hypothalamus to a conditioned cue for aversive, naturalistic environmental stimuli. Psychopharmacology, 148: 201-208. Millan, M.J. (2003) The neurobiology and control of anxious states. Prog. Neurobiol., 70: 83-244. Moghaddam, B. (1993) Stress preferentially increases extraneuronal levels of excitatory amino acids in the prefrontal cortex: comparison to hippocampus and basal ganglia. J. Neurochem., 60: 1650-1657. Morilak, D.A., Cecchi, M. and Khoshbouei, H. (2003) Interactions of norepinephrine and galanin in the central amygdala and lateral bed nucleus of the stria terminalis modulate the behavioral response to acute stress. Life Sci., 73: 715-726. Morrow, B.A., Elsworth, J.D., Lee, E.J. and Roth, R.H. (2000) Divergent effects of putative anxiolytics on stress-induced Fos expression in the mesoprefrontal system of the rat. Synapse, 36:143-154. Mfiller, M.B., Zimmermann, S., Sillaber, I., Hagemeyer, T.P., Deussing, J.M., Timpl, P., Kormann, M.S.D., Droste, S.K., Kfihn, R., Reul, J.M.H.M., Holsboer, F. and Wurst, W. (2003) Limbic corticotropin-releasing hormone receptor 1 mediates anxiety-related behavior and hormonal adaptation to stress. Nat. Neurosci., 6:1100-1107. Nakane, H., Shimizu, N. and Hori, T. (1994) Stress-induced norepinephrine release in the rat prefrontal cortex measured by microdialysis. Am. J. Physiol., 267: R1559-R1566. Neumann, I.D. (2001) Alterations in behavioral and neuroendocrine stress coping strategies in pregnant, parturient and lactating rats. Prog. Brain Res., 133: 143-152. Neumann, I.D. (2002) Involvement of the brain oxytocin system in stress coping: interactions with the hypothalamopituitary-adrenal axis. Prog. Brain Res., 139: 147-162. Nishioka, T., Anselmo-Franci, J.A., Li, P., Callahan, M.F. and Morris, M. (1998) Stress increases oxytocin release within the hypothalamic paraventricular nucleus. Brain Res., 781: 57-61. Owens, M.J. and Nemeroff, C.B. (1991) Physiology and pharmacology of corticotropin-releasing factor. Pharmacol. Rev., 43: 425-473. Pac/tk, K. (2000) Stressor-specific activation of the hypothalamic-pituitary-adrenocortical axis. Physiol. Res., 49: Sl1-S17.
637
Pacfik, K. and Palkovits, M. (2001) Stressor specificity of central neuroendocrine responses: implications for stressrelated disorders. Endocr. Rev., 22: 502-548. Pacfik, K., Palkovits, M., Kopin, I.J. and Goldstein, D.S. (1995) Stress-induced norepinephrine release in the hypothalamic paraventricular nucleus and pituitary-adrenocortical and sympathoadrenal activity: in vivo microdialysis studies. Front. Neuroendocrinol., 16:89-150. Palkovits, M. (1999) Interconnections between the neuroendocrine hypothalamus and the central autonomic system. Front Neuroendocrinol., 20: 270-295. Pan, J.T., Lookingland, K.J. and Moore, K.E. (1995) Differential effects of corticotropin-releasing hormone on central dopaminergic and noradrenergic neurons. J. Biomed. Sci., 2: 50-56. Pani, L., Porcella, A. and Gessa, G.L. (2000) The role of stress in the pathophysiology of the dopaminergic system. Mol. Psychiatry, 5: 133-138. Pei, Q., Zetterstr6m, T. and Fillenz, M. (1990) Tail pinchinduced changes in the turnover and release of dopamine and 5-hydroxytryptamine in different brain regions of the rat. Neuroscience, 35: 133-138. Pezzone, M.A., Lee, W.S., Hoffman, G.E., Pezzone, K.M. and Rabin, B.S. (1993) Activation of brainstem catecholaminergic neurons by conditioned and unconditioned aversive stimuli as revealed by c-Fos immunoreactivity. Brain Res., 608: 310-318. Phillips, M.L., Drevets, W.C., Rauch, S.L. and Lane, R. (2003) Neurobiology of emotion perception I: the neural basis of normal emotion perception. Biol. Psychiat., 54: 504-514. Phillips, R.G. and LeDoux, J.E. (1992) Differential contribution of amygdala and hippocampus to cued and contextual fear conditioning. Behav. Neurosci., 106: 274-285. Price, M.L., Curtis, A.L., Kirby, L.G., Valentino, R.J. and Lucki, I. (1998) Effect of corticotropin-releasing factor on brain serotonergic activity. Neuropsychopharmacology, 18: 492-502. Puglisi-Allegra, S., Imperato, A., Angelucci, L. and Cabib, S. (1991) Acute stress induces time-dependent responses in dopamine mesolimbic system. Brain Res., 217-222. Quirarte, G.L., Galvez, R., Roozendaal, B. and McGaugh, J.L. (1998) Norepinephrine release in the amygdala in response to footshock and opioid peptidergic drugs. Brain Res., 808: 134-140.
Rassnick, S., Hoffman, G.E., Rabin, B.S. and Sved, A.F. (1998) Injection of corticotropin-releasing hormone into the locus coeruleus or footshock increases neuronal Fos expression. Neuroscience, 85: 259-268. Reyes, T.M., Walker, J.R., DeCino, C., Hogenesch, J.B. and Sawchenko, P.E. (2003) Categorically distinct acute stressors elicit dissimilar transcriptional profiles in the paraventricular nucleus of the hypothalamus. J. Neurosci., 23:5607-5616.
Rhodes, M.E. and Rubin, R.T. (1999) Functional sex differences ('sexual diergism') of central nervous system cholinergic systems, vasopressin, and hypothalamicpituitary-adrenal axis activity in mammals: a selective review. Brain Res. Rev., 30: 135-152. Ritter, S., Watts, A.G., Dinh, T.T., Sanchez-Watts, G. and Pedrow, C. (2003) Immunotoxin lesion of hypothalamically projecting norepinephrine and epinephrine neurons differentially affects circadian and stressor-stimulated corticosterone secretion. Endocrinology, 144: 1357-1367. Roche, M., Commons, K.G., Peoples, A. and Valentino, R.J. (2003) Circuitry underlying regulation of the serotonergic system by swim stress. J. Neurosci., 23: 970-977. Roth, R.H., Tam, S.Y., Ida, Y., Yang, J.X. and Deutch, A.Y. (1988) Stress and the mesocorticolimbic dopamine systems. Ann. N.Y. Acad. Sci., 537: 138-147. Sanchez, M.M., Young, L.J., Plotsky, P.M. and Insel, T.R. (1999) Autoradiographic and in situ hybridization localization of corticotropin-releasing factor 1 and 2 receptors in nonhuman primate brain. J. Comp. Neurol., 408: 365-377. Santarelli, L., Gobbi, G., Debs, P.C., Sibille, E.L., Blier, P., Hen, R. and Heath, M.J.S. (2001) Genetic and pharmacological disruption of neurokinin 1 receptor function decreases anxiety-related behaviors and increases serotonergic function. Proc. Natl. Acad. Sci. USA, 98: 1912-1917. Sauvage, M. and Steckler, T. (2001) Detection of corticotropinreleasing hormone receptor 1 immunoreactivity in cholinergic, dopaminergic, and noradrenergic neurons of the murine basal forebrain and brainstem nuclei. Potential implications for arousal and attention. Neuroscience, 104: 643-652. Sawchenko, P.E., Li, H.Y. and Ericsson, A. (2000) Circuits and mechanisms governing hypothalamic responses to stress: a tale of two paradigms. Prog. Brain Res., 122: 61-78. Sawchenko, P.E. and Swanson, L.W. (1983) The organization of the forebrain afferents to the paraventricular and supraoptic nuclei. J. Comp. Neurol., 186: 621-656. Sawchenko, P.E. and Swanson, L.W. (1985) Localization, colocalization, and plasticity of corticotropin-releasing factor immunoreactivity in rat brain. Fed. Proc., 44: 221-227. Senba, E., Matsunaga, K., Tohyama, M. and Noguchi, K. (1993) Stress-induced c-fos expression in the rat brain: activation mechanism of sympathetic pathway. Brain Res. Bull., 31: 329-344. Servatius, R.J., Natelson, B.H., Moldow, R., Pogach, L., Brennan, F.X. and Ottenweller, J.E. (2000) Persistent neuroendocrine changes in multiple hormonal axes after a single or repeated stressor exposures. Stress, 3: 263-274. Sharp, F.R., Sagar, S.M., Hicks, K., Lowenstein, D. and Hisanga, K. (1991) C-fos mRNA, Fos, and Fos-related antigen induction by hypertonic saline and stress. J. Neurosci., 11: 2321-2331. Shimizu, N., Take, S., Hori, T. and Oomura, Y. (1992) In vivo measurement of hypothalamic serotonin release by
638 intracerebral microdialysis: significant enhancement by immobilization stress in rats. Brain Res. Bull., 28: 727-734. Singewald, N., Kouvelas, D., Mostafa, A., Sinner, C. and Philippu, A. (2000) Release of glutamate and GABA in the amygdala of conscious rats by acute stress and baroreceptor activation: differences between SHR and WKY rats. Brain Res., 864: 138-141. Singewald, N., Zhou, G.Y. and Schneider, C. (1995) Release of excitatory and inhibitory amino acids from the locus coeruleus of conscious rats by cardiovascular stimuli and various forms of acute stress. Brain Res., 704: 42-50. Singewald, N. and Philippu, A. (1998) Release of neurotransmitters in the locus coeruleus. Prog. Neurobiol., 56: 237-267. Smagin, G.N. and Dunn, A.J. (2000) The role of CRF receptor subtypes in stress-induced behavioural responses. Eur. J. Pharmacol., 405: 199-206. Smagin, G.N., Heinrichs, S.C. and Dunn, A.J. (2001) The role of CRH in behavioral responses to stress. Peptides, 22:713-724. Smith, G.W., Aubry, J.M., Dellu, F., Contarino, A., Bilezigian, L.M., Gold, L.H., Chen, R., Marchuk, Y., Hauser, C., Bentley, C.A., Sawchenko, P.E., Koob, G.F., Vale, W. and Lee, K.F. (1998) Corticotropin releasing factor receptor 1-deficient mice display decreased anxiety, impaired stress response, and aberrant neuroendocrine development. Neuron, 20:1093-1102. Smith, M.A., Banerjee, S., Gold, P.W. and Glowa, J. (1992) Induction of c-fos mRNA in rat brain by conditioned and unconditioned stressors. Brain Res., 578: 135-141. Stalnaker, T.A. and Berridge, C.W. (2003) AMPA receptor stimulation within the central nucleus of the amygdala elicits a differential activation of central dopaminergic systems. Neuropsychopharmacology, 28: 1923-1934. Stamp, J.A. and Herbert, J. (1999) Multiple immediate-early gene expression during physiological and endocrine adaptation to repeated stress. Neuroscience, 94: 1313-1322. Steckler, T. and Holsboer, F. (1999) Corticotropin-releasing hormone receptor subtypes and emotion. Biol. Psychiatry, 46: 1480-1508. Sugaya, K. and McKinney, M. (1994) Nitric oxide synthase gene expression in cholinergic neurons in the rat brain examined by combined immunocytochemistry and in situ hybridization histochemistry. Mol. Brain Res., 23:111-125. Sullivan, R.M. and Gratton, A. (1999) Lateralized effects of medial prefrontal cortex lesions on neuroendocrine and autonomic responses in rats. J. Neurosci., 19: 2834-2840. Sullivan, R.M. and Gratton, A. (2002) Prefrontal cortical regulation of hypothalamic-pituitary-adrenal function in the rat and implications for psychopathology: side matters. Psychoneuroendocrinology, 27: 99-114. Sutton, R.E., Koob, G.F., Le Moal, M., Rivier, C. and Vale, W.W. (1982) Corticotropin releasing factor produces behavioural activation in rats. Nature, 297: 331-333.
Sved, A.F., Cano, G. and Card, J.P. (2001) Neuroanatomical specificity of the circuits controlling sympathetic outflow to different targets. Clin. Exp. Pharmacol. Physiol., 28:115-119. Swanson, C.J., Perry, K.W. and Schoepp, D.D. (2004) The mGlu2/3 receptor agonist, LY354740, blocks immobilization-induced increases in noradrenaline and dopamine release in the rat medial prefrontal cortex. J. Neurochem., 88: 194-202. Szafarczyk, A., Alonso, G., Ixart, G., Malaval, F. and Assenmacher, I. (1985) Diurnal stimulated and stressinduced ACTH release in rats is mediated by ventral noradrenergic bundle. Am. J. Physiol., 249: E219-E226. Szafarczyk, A., Malaval, F., Laurent, A., Gibaud, R. and Assenmacher, I. (1987) Further evidence for a central stimulatory action of catecholamines on adrenocorticotropin release in the rat. Endocrinology, 121: 883-892. Tajima, T., Endo, H., Suzuki, Y., Ikari, H., Gotoh, M. and Iguchi, A. (1996) Immobilization stress-induced increase of hippocampal acetylcholine and of plasma epinephrine, norepinephrine and glucose in rats. Brain Res., 720:155-158. Thorsell, A., Svensson, P., Wiklund, L., Sommer, W., Ekman, R. and Heilig, M. (1998) Suppressed neuropeptide Y (NPY) mRNA in rat amygdala following restraint stress. Regul. Pept., 75/76: 247-254. Timmerman, W., Cisci, G., Nap, A., de Vries, J.B. and Westerink, B.H. (1999) Effects of handling on extracellular levels of glutamate and other amino acids in various areas of the brain measured by microdialysis. Brain Res., 833: 150-160.
Tsigos, C. and Chrousos, G. (2002) Hypothalamic-pituitaryadrenal axis, neuroendocrine factors and stress. J. Psychosom. Res., 53: 865-871. Tuvnes, F.A., Steffenach, H.A., Murison, R., Moser, M.B. and Moser, E.I. (2003) Selective hippocampal lesions do not increase adrenocortical activity. J. Neurosci., 23: 4345-4354. Vahabzadeh, A. and Fillenz, M. (1994) Comparison of stressinduced changes in noradrenergic and serotonergic neurons in the rat hippocampus using microdialysis. Eur. J. Neurosci., 6: 1205-1212. Valles, A., Marti, O., Harbuz, M.S. and Armario, A. (2002) A single lipopolysaccharide administration is sufficient to induce a long-term desensitization of the hypothalamicpituitary-adrenal axis. Neuroscience, 112: 383-389. van de Kar, L.D. and Blair, M.L. (1999) Forebrain pathways mediating stress-induced hormone secretion. Front. Neuroendocrinol., 20: 1-48. van de Kar, L.D., Javed, A., Zhang, Y.H., Serres, F., Raap, D.K. and Gray, T.S. (2001) 5-HT2A receptors stimulate ACTH, corticosterone, oxytocin, renin, and prolactin release and activate hypothalamic CRF and oxytocinexpressing cells. J. Neurosci., 21: 3572-3579. Vermetten, E. and Bremner, J.D. (2002) Circuits and systems in stress. I. Preclinical studies. Depress. Anxiety, 15: 126-147.
639 Weller, K.L. and Smith, D.A. (1982) Afferent connections to the bed nucleus of the stria terminalis. Brain Res., 232: 255-270. Williams, C.L., Men, D., Clayton, E.C. and Gold, P.E. (1998) Norepinephrine release in the amygdala after systemic injection of epinephrine or escapable footshock: contribution of the nucleus of the solitary tract. Behav. Neurosci., 112: 1414-1422. Windle, R.J., Kershaw, Y.M., Shanks, N., Wood, S.A., Lightman, S.L. and Ingrain, C.D. (2004) Oxytocin attenuates stress-induced c-fos mRNA expression in specific forebrain regions associated with modulation of hypothalamopituitary-adrenal activity. J. Neurosci., 24: 2974-2982. Windle, R.J., Shanks, N., Lightman, S.L. and Ingram, C.D. (1997) Central oxytocin administration reduces stressinduced corticosterone release and anxiety behavior in rats. Endocrinology, 138: 2829-2834. Wong, M.L., Licinio, J., Pasternak, K.I. and Gold, P.W. (1994) Localization of corticotropin-releasing hormone (CRH) receptor mRNA in adult rat brain by in situ hybridization histochemistry. Endocrinology, 135: 2275-2278. Wotjak, C.T., Kubota, M., Liebsch, G., Montkowski, A., Holsboer, F., Neumann, I. and Landgraf, R. (1996) Release of vasopressin within the rat paraventricular nucleus in response to emotional stress: a novel mechanism of regulating adrenocorticotropic hormone secretion? J. Neurosci., 16: 7725-7732. Wright, D.E., Seroogy, K.B., Lundgren, K.H., Davis, B.M. and Jennes, L. (1995) Comparative localization of serotonin 1A,
1C, and 2 receptor subtype mRNAs in rat brain. J. Comp. Neurol., 351: 357-373. Wu, J.S., Ku, Y.H., Li, L.S., Lu, Y.C., Ding, X. and Wang, Y.G. (1999) Corticotropin releasing factor and substance P mediate the nucleus amygdaloideus centralis-nucleus ventromedialis-nucleus dorsomedialis pressor system. Brain Res., 842: 392-398. Yokoo, H., Tanaka, M., Yoshida, M., Tsuda, A., Tanaka, T. and Mizoguchi, K. (1990) Direct evidence of conditioned fear-elicited enhancement of noradrenaline release in the rat hypothalamus assessed by intracranial microdialysis. Brain Res., 536: 305-308. Yokoyama, C. and Sasaki, K. (1999) Regional expressions of Fos-like immunoreactivity in rat cerebral cortex after stress; restraint and intraperitoneal lipopolysaccharide. Brain Res., 816: 267-275. Young, E.A. (1998) Sex differences and the HPA axis: implications for psychiatric disease. J. Gend. Specif. Med., 1: 21-27. Zhang, Y., Damjanoska, K.J., Carrasco, G.A., Dudas, B., D'Souza, D.N., Tetzlaff, J., Garcia, F., Hanley, N.R., Scripathirathan, K., Petersen, B.R., Gray, T.S., Battaglia, G., Muma, N.A. and van de Kar, L.D. (2002) Evidence that 5-HT2A receptors in the hypothalamic paraventricular nucleus mediate neuroendocrine responses to (-)DOI. J. Neurosci., 22: 9635-9642. Zou, C.J., Onaka, T. and Yagi, K. (1998) Role of adrenoceptors in vasopressin, oxytocin, and prolactin responses to conditioned fear stimuli in the rat. J. Neuroendocrinol., 10: 905-910.
This Page Intentionally Left Blank
SECTION 5
Neuroplasticity and Stress
This Page Intentionally Left Blank
T. Steckler, N.H. Kalin and J.M.H.M. Reul (Eds.) Handbook of Stress and the Brain, Vol. 15
ISBN 0-444-51173-3 Copyright 2005 Elsevier B.V. All rights reserved CHAPTER 5.1
The intracellular signaling cascade and stress Yogesh Dwivedi* and Ghanshyam N. Pandey Psychiatric Institute, Department of Psychiatry, University of Illinois at Chicago, 1601 W. Taylor St., Chicago IL, 60612, USA
Abstract: Exposure of stress precipitates a coordinated series of responses which may lead to changes in brain functions. Most of the changes in brain function are mediated by stress-induced activation of neurotransmitter systems in the brain, which include neurotransmitter receptors and these receptors-mediated signal transduction pathways. The signal transduction pathways are ultimately responsible for stress-induced changes in neuronal functions. Some of these changes are beneficial, however, depending upon the nature and duration of stressful stimuli, the changes in neuronal functions may be harmful, which may lead to many disorders. In the present chapter, we describe comprehensively the effects of stress on neurotransmitter receptors and various molecules of signal transduction pathways in brain.
Introduction
disorders such as depression and other psychiatric disorders. Intracellular signaling coordinates the behavior of individual cells within the brain in various physiological processes. This signaling requires three essential components: a molecular signal, also known as a neurotransmitter, that sends the information from one cell to another, a receptor that receives the signal and transmits the information provided by the signal, and a target molecule that mediates the cellular responses. A large number of receptors have been identified and grouped into three different families depending upon the mechanism used to transduce signal binding into a cellular response. These families are (1) receptors linked to ligand-gated ion channels, (2) receptors that have intrinsic enzymatic properties, and (3) G protein-coupled receptors. The rapid action of stress is mediated by neurotransmitters that regulate neuronal activity via the gating of ion channels, which causes ion influx and changes the membrane potential or can lead to the entry of Ca 2+ ions, which serves as a second messenger signal within the cell. On the other hand, most of the chronic effects of stress are mediated by G protein-coupled receptors or receptors that contain intrinsic enzymatic properties. Most neurotransmitter receptors belong to the
It is well established that several types of stressors, including psychosocial, social, or environmental stress, may lead to changes in brain function. Some of the changes due to acute stress are beneficial for the ability to deal with everyday challenges; however, failure to cope with the response to acute stress may lead to the syndrome of chronic stress, which is associated with alterations in the hypothalamicpituitary-adrenal (HPA) axis and with elevated secretion of glucocorticoids, with consequent illnesses including immunosuppression and psychiatric disorders. One of the mechanisms by which these changes occur could be by influencing the activation of neurotransmitters, their receptors, and these receptor-mediated intracellular signaling. Several studies demonstrate that chronic stress may cause abnormalities in intracellular signaling that may lead to changes in the functional properties of various important proteins or in the expression of certain genes, which may in turn participate in stress-related
*Corresponding author. Tel.: (312) 413-4557; Fax: (312) 355-3857; Email:
[email protected] 643
644 G protein-coupled receptor family. These receptors regulate intracellular reactions by an indirect mechanism involving an intermediate transducing molecule known as guanosine 5'-triphosphate (GTP) binding protein or G protein. Activated G proteins alter the functions of many downstream effectors. Most of these effectors are enzymes that produce intracellular second messengers. The second messengers then trigger the signaling cascade further downstream and cause a cellular response. Stress may affect intracellular signaling at multiple levels. In the present chapter, the focus is primarily on the stress modulation of G protein-coupled neurotransmitter receptors, G proteins, second messengers, and second messenger-dependent and -independent effector molecules.
Stress and G protein-coupled receptors The two most studied G protein-coupled receptors with respect to stress regulation are the serotonin (5HT) and adrenergic receptors. We will briefly describe the current state of knowledge of the effects of stress on these receptors. Stress is known to alter the brain monoaminergic system. Several studies indicate that serotonergic neurons are especially affected by stress and related disorders (reviewed by Chaouloff, 1993). A marked increase in 5HT synthesis and release has been observed in various brain areas of experimental animals in response to different stressful conditions such as electrical footshock, tail pinches, immobilization stress, or a cold environment (Curzon et al., 1972; Joseph and Kennett, 1983; Pei et al., 1990; Clement et al., 1993). Since corticosterone (or cortisol in humans) is released after stress-induced activation of HPA axis, many studies have demonstrated that adrenalectomy and replacement of corticosterone or the administration of corticosterone exogenously influences tryptophan hydroxylase activity and 5HT turnover in the brain (Azmitia and McEwen, 1974; Singh et al., 1990).
Serotonin receptors The neurotransmitter activity of 5HT has been attributed to its binding to 5HT receptors. 5HT
receptors have been classified into three different families on the basis of their signal transduction mechanism association (Barnes and Sharp, 1999; Albert and Tiberi, 2001; Hoyer et al., 2002). Class I are those linked to the adenylyl cyclase-cyclic AMP signaling system, such as 5HTIA, 5HT1B, 5HT1D, and 5HT4; class II are those linked to the phosphoinositide (PI) hydrolysis signaling system, such as 5HTzA, 5HTzB, and 5HT2c; and class III are those linked to ion channels such as 5HT3. Cloning studies have also revealed the existence of 5HT1E, 5HTIF, 5HTzF, 5HTs, 5HT6, and 5HT7 receptor subtypes. Of these 5HT1E, 5HT1F, 5HTs, 5HT6, and 5HT7 are linked to the adenylyl cyclase-cAMP and 5HTzF is linked to the PI signaling system. The specificity of the serotonergic responses to stress is made evident by the interactions with pre- and postsynaptic 5HT receptor subtypes. Since 5HT1A receptors are inhibitory somatodendritic receptor in raphe serotonergic cells and a postsynaptic receptor in selective serotonergic terminal fields, and since they play a key role in nerve firing activity and/or release of 5HT, many studies have focused on the role of 5HT1A in stress and stressrelated disorders. For example, chronic unpredictable stress significantly decreases the expression and number of binding sites for 5HT1A receptors across all hippocampal subfields (L6pez et al., 1998). Similar effects have been found following chronic restraint stress (Watanabe et al., 1993). Several studies also have demonstrated that administration of corticosterone or adrenalectomy can cause changes in the number or expression of 5HT1A receptors (reviewed by Chaouloff, 1993; 1995). These receptors show differences in their degree of sensitivity in various brain regions toward lower-versus highercirculating corticosterone levels. For example, continuous exposure to corticosterone decreases 5HT1A receptor binding in the dentate gyrus and the CA4 areas of the hippocampus (Mendelson and McEwen, 1992a, 1992b). On the other hand, upon removal of corticosterone 3 weeks prior to sacrifice of the rat, 5HT1A receptors increase in these areas of the hippocampus. A negative correlation between corticosterone treatment and 5HT1A receptor mRNA in rat dentate gyrus has also been reported (Meijer and de Kloet, 1994). Cortical areas are also sensitive to glucocorticoid treatment toward 5HT1A receptors.
645 This is evident from a study showing that corticosterone treatment to rats significantly decreases the number of 5HT1A binding sites in frontal cortex (Crayton et al., 1996). Studies of the effects of adrenalectomy and replacement with corticosterone show that adrenalectomy increases 5HT1A receptors in the CA1 area of the hippocampus, with parallel trends in CA4 and the dentate gyrus. These changes are reversed by the administration of corticosterone (Biegon et al., 1985; Rostene et al., 1985; Huang and Azmitia, 1999). Similar effects have been observed by many investigators who found an increase in 5HT1A receptor binding in CA1, 2, 3, and the dentate gyrus after adrenalectomy, which was restored to control levels 7-28 days after corticosterone replacement (Burnet et al., 1992; Chalmers et al., 1994; Kuroda et al., 1994; Tijani-Butt and Labow, 1994; Zong and Ciarnello, 1995; Holmes et al., 1995a). Another 5HT receptor subtype which has been studied the most with respect to stress and the HPA axis is 5HT2A. 5HT2A receptors have been widely implicated in the pathophysiology of depression and other psychiatric disorders (Graeff, 1997; Bell and Nutt, 1998; van Veelen and Kahn, 1999; Mann, 1999; 2003). Initial studies on the effects of corticosterone or ACTH treatment revealed a significant increase in 5HT2A receptor density in rat forebrain and adrenalectomy prevented this increase (Kuroda et al., 1992). Dexamethasone, an agonist to glucocorticoid receptors, also causes similar effects on 5HT2A receptors in rat brain (Kuroda et al., 1993). Consistent with these observations, several other studies have also demonstrated that corticosterone treatment to rats increases 5HT2A receptors in the frontal and parietal cortices (Fernandes et al., 1997; Takao et al., 1997). In our own studies, we observed that corticosterone treatment increases 5HT2A receptors in both cerebral cortex and hippocampus (Pandey and Dwivedi, 1999). The effects of adrenalectomy alone on 5HT2A receptors, on the other hand, have been inconsistent. Few studies demonstrate that adrenalectomy, like corticosterone treatment, increases 5HT2A receptors in the hippocampus (Martire et al., 1989; Jitsuiki et al., 2000; Katagiri et al., 2001), whereas several other studies show that adrenalectomy does not affect 5HT2A receptor density in frontal cortex of rats (Kuroda et al., 1992, 1994; Chaouloff, 1993).
Besides 5HT1A and 5HT2 receptors several other 5HT receptor subtypes are also regulated by stress and glucocorticoids. 5HTzc receptors, which have been involved in anxiety and various autonomic and neuroendocrine functions, have been shown to be increased after chronic stress in the CA2 area of the rat hippocampus. Depletion of cortisol by adrenalectomy also increases 5HT2c receptor mRNA in posterior CA1 and CA3 areas, and this effect is reversed by glucocorticoid replacement (Holmes et al., 1995b). The newly discovered 5HT6 and 5HT7 receptors have also been the subjects of study in the regulation of stress. This is mainly because of their similarities with 5HT1A receptors. Both 5HT6 and 5HT7 receptors are expressed in hippocampal and other limbic brain regions where 5HT1A receptors are highly enriched (Ward et al., 1995; Gerard et al., 1996; Gustafson et al., 1996; Stowe and Barnes, 1998). But 5HT6 and 5HT7 receptors are coupled to adenylyl cyclase-cAMP signal transduction system in a positive manner (Bard et al., 1993; Plassat et al., 1993; Kohen et al., 1996; Hirst et al., 1997; Sleight et al., 1998), in contrast to 5HT1A receptors, which are linked to this signaling system in a negative manner. The pharmacological properties of 5HT6 and 5HT7 receptors are also quite similar to those of 5HT1A receptor (Barnes and Sharp, 1999). Using in situ hybridization studies, it has been shown that mRNA expression of 5HT7 receptors is increased in CA1 and CA3 areas of the hippocampus and in the retrosplenal cortex after adrenalectomy (Le Corre et al., 1997). Another study suggests that adrenalectomy not only increases the mRNA expression of 5HT7 receptors, but also increases the mRNA expression of 5HT6 receptor in hippocampus and these increases are partially reversed by corticosterone replacement (Yau et al., 1997).
Adrenergic receptors Stress-induced disruption of central nervous noradrenergic activity is a well known phenomenon. An extensive body of literature has documented that there is increased sensitivity of the noradrenergic system in people under stress or suffering from depression (Bremner et al., 1996; Heninger et al.,
646 1998; Sullivan et al., 1999; Southwick et al., 1999a, 1999b, 1999c), in stressed nonhuman primates (Rosenblum et al., 1994), and in rats (Pacak et al., 1995; Dalley et al., 1996; Hellriegel and D'Mello, 1997). Many studies have also shown enhanced release of noradrenaline during stress exposure (Finlay et al., 1995; Goldstein et al., 1996; Birnbaum et al., 1999). The activity of the noradrenergic and adrenergic system is regulated via adrenoreceptors. Various responses to stress reactions are known to involve changes in the sensitivity and number of adrenoreceptors. Among the various subtypes of adrenergic receptors, modulation in ~1, ~2, and ~3-adrenoreceptors plays an important role in stressful conditions (Stone et al., 1985; Weiss et al., 1994; Stone et al., 1996; Stone and Quartermain, 1999). Single-stress challenges cause downregulation of [3-adrenergic receptors in the hypothalamus after footshock (Cohen et al., 1986) and in the cerebellum after immobilization stress (U'Prichard and Kvetnansky, 1980). The effect of repeated stress on [3-adrenergic receptors, on the other hand, depends upon the paradigm used. In stress-induced behavioral depression (also known as learned helplessness) higher [3adrenergic receptor density has been reported in the hippocampus but not in the cortex or the hypothalamus of rats (Martin et al., 1990). In our study, we found higher [3-adrenergic receptor density in hippocampus of learned helpless rats as compared with tested controls (Pandey et al., 1995). However, studies using other behavioral paradigm did not find significant differences in 13-adrenergic receptor density in frontal cortex, hippocampus, or hypothalamus of learned helpless rats (Brannan et al., 1995; Gurguis et al., 1996). In contrast to these studies, immobilization stress combined with immersion of rats in cold water cause decrease in ~-adrenergic receptor in the neurohypophysis and intermediate lobes of pituitary gland (Klenerova and Sida, 1994). Effect of a previous experience of stress by brief swim stress or daily injections of saline although do not affect [3-adrenergic receptors; however, swim stress followed by a course of saline injections causes downregulation of [3-adrenergic receptors (Davis et al., 1994). As far as ~ adrenergic receptors are concerned, most studies show that a single-stress challenge does
not cause any significant change in the number of binding sites for ~1 adrenergic receptors in rat brain (U'Prichard and Kvetnansky, 1980; Lynch et al., 1983; Cohen et al., 1986). However, a recent study suggests that restraint stress may alter expression of ~1 adrenergic receptors in a brain-region and timedependent manner. In hypothalamus, 30 and 60min restraint stress results in a significant decrease in ~1 adrenergic receptor mRNA. On the other hand, a significant increase in ~ adrenergic receptor mRNA has been noted after 60, 120, and 240 rain of restraint stress in mid brain (Miyahara, 1999). Additional studies have shown that the mRNA expression of a subtype of ~a adrenergic receptor, i.e., ~lb is increased in the paraventricular nucleus of the hypothalamus after adrenalectomy and that corticosterone replacement reduces this increase (Day et al., 1999). Similar t o ~1 adrenergic receptors, many studies have shown that 52 adrenergic receptors are resistant to change after single-stress challenge (reviewed by Stanford, 1995). However, downregulation in the mid-brain and the brain stem after immobilization stress (U'Prichand and Kvetnansky, 1980; Weiss, 1994) and upregulation in the hypothalamus (Nukina et al., 1987) and the cortex (U'Prichand and Kvetnansky, 1980; Cohen et al., 1986) of (~2 adrenergic receptors after several types of single-stress challenge have also been reported. When the same stress is given repeatedly, ~ adrenergic receptor binding does not change (reviewed by Stanford, 1995) but the effect of repeated stress on 52 adrenergic receptors is more labile, however, is quite inconsistent. For example, there are reports which suggest upregulation (U'Prichand and Kvetnansky, 1980), no change (Yamanaka et al., 1987), or even downregulation (Torda et al., 1981; Lynch et al., 1983) of 5 2 adrenergic receptors in cerebral cortex after repeated stress. Chronic psychosocial stress, on the other hand, induces dynamic changes in region-specific upor downregulation of 52 adrenergic receptors (Flugge, 1996). A recent study suggests that chronic stress as well as short-term treatment with corticosterone downregulate 52 adrenergic receptors in several brain regions of male tree shrews whereas long-term treatment with corticosterone upregulates 52 adrenergic receptors in these animals (Flugge, 1999).
647
Stress and G proteins Guanine nucleotide binding proteins (G proteins) occupy a central position and play a critical role in transducing extracellular signals to cellular targets, thus transmitting messages from cell surface receptors to cellular effectors (Neer, 1995; Clapham and Neer, 1997; Hamm, 1998; Freissmuth et al., 1999; Neves et al., 2002). About 80% of the receptors for neurotransmitters, hormones, and neuromodulators have been shown to elicit their responses through G proteins. G proteins are heterotrimers consisting of 3 subunits, a, [3, and 7, each encoded by a specific gene. [3 and Y subunits bind tightly to each other, whereas the 13 subunit also contains a common binding site for subunit recognition. The a subunit binds to guanosine triphosphate (GTP) and confers receptor-effector specificity to G proteins. The 7 subunit has been reported to have a G protein-specific recognition site. In the resting state, guanosine diphosphate (GDP) is bound to the a subunit, and the three subunits (a, 13, and Y) are associated as a trimer. Receptor-mediated activation of G proteins causes the release of GDP from the a subunit, allowing GTP to bind and induce the dissociation of the G protein cz subunit from the [37 subunits. The a and 13Y subunits can then activate various effectors to modulate cellular responses. The free to release the fatty acid arachidonic acid and lysophospholipids (Farooqui et al., 1997; Kudo and Murakami, 2002;
654 Brown et al., 2003). These lipid products may serve as second messengers. The arachidonic acid is then metabolized, either by the lipooxygenase pathway, forming products like leukotrienes or the cyclooxygenase pathway, leading to the formation of prostaglandins and thromboxanes. Arachidonic acid modulates neuronal signaling by direct effects on ion channels or indirectly through the activation of PKC and through the activation of metabolites (Fig. 1). Many glucocorticoids have been shown to inhibit prostaglandin formation by preventing arachidonic acid release from phospholipids as well as the induction of cyclooxygenase (Blackwell et al., 1980; Croxtall et al., 2002) suggesting that elevation of glucocorticoids in response to stress can affect signaling mechanism mediated by phospholipase A2.
Stress and nitric oxide Another important G protein-coupled receptor signaling pathway is mediated through nitric oxide (Bredt et al., 1990; Forstermann et al., 1995). Nitric oxide is produced from arginine by nitric oxide synthase and acts as a transmitter by diffusing from the cytosol and activating guanylyl cyclase. Receptors such as glutamate or N-methyl-D-aspartate (NMDA) receptors lead to the activation of PLC, which results in the formation of IP3 and the release of Ca 2+ from intracellular sources. Ca 2+ combines with calmodulin and activates nitric oxide synthase, producing nitric oxide. Nitric oxide then stimulates guanylyl cyclase, causing an increase in cyclic guanine monophosphate (GMP), which in turn activates cGMP-dependent protein kinases. This results in the phosphorylation of proteins and subsequently in the modulation of K + and Ca 2+ channels (Fig. 1). Numerous studies show that nitric oxide participates in the control of many neurosecretory processes, including the corticotropin-releasing factor (CRF) neurosecretory system (Costa et al., 1993; Sandi and Guaza, 1995; Mcleod et al., 2001). Stress may influence neuronal excitability, in which nitric oxide functions as an amplifier or as a feedback regulator of neuronal excitation or inhibition, thus altering the homeostasis of the neurosecretory system (reviewed by Riedel, 2000). Long-term stress has been shown to increase the activity of CaZ+-dependent
nitric oxide synthase and induces the expression of inducible nitric oxide synthase in the rat cortex (Lorenzo Fernandez, 1999). It has been shown that inducible nitric oxide synthase production may be responsible for the neurodegenerative diseases caused by stress. Recently it has been reported that nitric oxide synthase may be responsible for the glucocorticoid-induced atrophy of hippocampal neurons (Regan et al., 1999). Several other studies also demonstrate that stress and corticosterone can regulate nitric oxide synthase expression in the brain (Ceccatelli and Orazzo, 1993; Weber et al., 1994). Removal of glucocorticoids by adrenalectomy increases the expression of nitric oxide synthase in the hippocampus, and administration of glucocorticoids attenuates the adrenalectomy-induced increase in nitric oxide synthase expression (Lopez-Figueroa et al., 1998). More evidence for the role of nitric oxide in stress comes from studies showing the prevention of behavioral effects of corticosterone (Sandi et al., 1996) and of the stress-induced adrenocorticotropic hormone and corticosterone response (Tsuchiya et al., 1997) by nitric oxide synthase inhibitors.
Stress and protein tyrosine kinase-mediated signaling There are two types of protein tyrosine kinases: (1) those that contain a receptor domain as well as a kinase domain, and thus cause self-phosphorylation and activation, and (2) those that do not contain a receptor domain but can phosphorylate substrate proteins. The category 1 protein tyrosine kinases are known as tyrosine receptor kinase or Trk (Patapoutian and Reichardt, 2001). Trks mediate the signaling systems of growth factors and neurotrophins and have been divided into three families: TrkA, TrkB, and TrkC. TrkA mediates the signaling of nerve growth factors, whereas TrkB is responsible for brain derived neurotrophic factor (BDNF)- and neurotrophin (NT) 4-induced signaling. TrkC mediates the functions of NT3. NT3 can also bind to and mediate functions through TrkA and TrkB. The binding of neurotrophins/growth factors to the appropriate Trk receptor leads to the dimerization and transphosphorylation of tyrosine residues in the intracellular domain of Trk receptors
655 and the subsequent activation of cytoplasmic signaling pathways (Huang and Reichardt, 2003). The signaling systems that are activated by Trk are extracellular-signaling-regulated mitogen-activated protein kinase (ERK-MAP kinase) and phosphoinositide (PI) 3-kinase/Akt. The ERK-mediated signaling cascade is an evolutionary conserved signaling mechanism that is involved in signal transduction from cell surface receptors to the nucleus (Lewis et al., 1998; Cobb, 1999). In this pathway, dimerization and activation of Trk receptors causes the recruitment of coupling proteins such as ShC, growth factor receptor bound protein (GRB2), and coupling protein son of sevenless (SOS). This in turn causes the activation of a small G protein, Ras (Nimnual et al., 1998). Ras then recruits another kinase, Raf to the membrane, where it is phosphorylated and activated by serine/threonine/tyrosine kinases. This leads to the activation of another kinase known as ERK kinase (also known as MEK). M E K then phosphorylates and activates ERK on serine/threonine residues (English et al., 1999) (Fig. 2). Once activated, ERK can phosphorylate many substrate proteins in the cytosol or translocate to the nucleus, where they regulate the activity of transcription factors (Chen et al., 1992; Lin et al., 1993; Marais et al., 1993). This leads to specific biological responses, including cell proliferation, differentiation, survival, gene expression, and cell cycle response of neurons to neural activity (Segar and Krebs, 1995; Lewis et al., 1998; Grewal et al., 1999; Kolkova et al., 2000). Because of the role played by ERK signaling in cell survival, recent studies are focusing on the role of the ERK signaling pathway in the stress response. For example, recently, it has been reported that chronic stress initiated by mild footshock for 21 days causes pronounced and persistent hyperphosphorylation and therefore activation of ERK isoforms, i.e., ERK1 and ERK2, in dendrites of the rat prefrontal cortex (Trentani et al., 2002). This hyperphosphorylation of ERK1 and 2 may represent a specific path by which chronic stress may affect the functioning of cortical structures and cause defects in the neural networks involved in elaboration of sensory stimuli. Cell culture studies also show that corticosterone activates ERK1 and ERK2 in PC 12 cells, which suggests that glucocorticoids mediate the activation of ERK1 and ERK2 signaling (Qiu et al., 2001).
A
A
Phosphorylation of Substrate Proteins Regulation of Neuronal Functions Fig. 2. Schematic representation of signal transduction mechanisms of extracellular signal-regulated (ERK) and phosphoinositide (PI) 3-kinase pathways. In the ERK pathway, the binding of trophic factors/growth factors to the tyrosine receptor kinase (Trk) causes its dimerization and activation. This results in the activation of Raf-1 through Shc, Grb2, Sos, and Ras. Activation of Raf-1 leads to the activation of ERK kinase (MEK) and of ERK-1/2 in a sequential manner. ERK-1 and ERK-2 then phosphorylate substrate proteins in cytosol or transcription factors in nucleus after translocation. In the PI3-kinase pathway, binding of growth factors to Trk receptors causes dimerization and activation of Trk receptors. PI3-kinase is activated following binding to Trk receptors through the p85 regulatory domain. This causes the recruitment of PI3-kinase (PI3K) from cytosol to the plasma membrane, where the catalytic domain of PI3-kinase, i.e., p110, phosphorylates PIP2 into PIP3. PIP3 then activates phosphoinositide-dependent kinase (PDK)-I, which phosphorylates and activates Akt. Akt then phosphorylates and activates or inactivates substrate proteins in cytosol or transcription factors in nucleus after translocation.
Other members of the MAP kinase family are c-Jun N-terminal kinase (JNK) and p38. Stress responses may also lead to the activation of these two kinases. Another signal transduction system that is activated by growth factors or neurotrophins and can be affected by stress is PI3 kinase/Akt. In this signaling pathway, activation of Trk receptors elicits the recruitment of the catalytic subunit of PI3 kinase to the vicinity of the plasma membrane. The catalytic subunit of PI3 kinase then phosphorylates phosphatidylinositol 4,5 biphosphate to phosphatidylinositol (3,4,5)-triphosphate. Phosphatidylinositol (3,4,5)triphosphate then activates the phosphorylating enzyme Akt through PI-dependent protein kinase-1
656 (Downward, 1998; Duronio et al., 1998). Akt then phosphorylates several proteins in the cytosol or transcription factors in the nucleus after translocation (Holgado-Madruga et al., 1997; Vaillant et al., 1999) (Fig. 2). Akt plays an important role in cell survival, which is mainly achieved by phosphorylating and therefore inhibiting a variety of substrates that affect apoptosis directly (Yao and Cooper, 1995; Dudek et al., 1997; Vanhaesebroeck et al., 1997; Eves et al., 1998; Datta et al., 1999; Hetman et al., 1999, Yuan and Yanker, 2000). In addition, the signaling pathways that are activated by protein tyrosine kinase are PLC 7-mediated signaling and STAT (signal transducers and activators of transcription) (Darnell, 1997; Pellegrini and Dusanter-Fourt, 1997; Leonard and O'shea, 1998; Kaplan and Miller, 2000; Huang and Reichardt, 2003). The elevation of cytosolic Ca 2+ following activation of PLC 7 results in a widespread protein phosphorylation by serine/threonine protein kinases. These include CaZ+-calmodulin dependent kinase, phosphorylase kinase, and elongation factor-2. The nonreceptor protein tyrosine kinases are a large group of signaling proteins that have diverse roles in cell proliferation, differentiation, and cell death (Gomperts et al., 2002). This family of proteins posseses no intrinsic catalytic activity, although they produce a response similar to that of receptors containing intrinsic catalytic activity. These protein tyrosine kinases recruit catalytic subunits within the cell in the form of one or more nonreceptor protein tyrosine kinases. This family of receptors is divided into ten different subfamilies: Src, Syk, Btk, Csk, Abl, JAK, Fak, Brk, Fes, and Ack.
Conclusion The data reviewed in this chapter lead one to conclude that stress may influence neurotransmitter receptors and a receptor-mediated signal transduction systems in the brain. Since altered HPA function and release of cortisol are well known phenomena during stress, most of the studies have focused on the regulation of neurotransmitter receptors and intracellular signaling by glucocorticoids. Some of the effects of stress on intracellular signaling may be receptor mediated, and some could be related to direct effects on individual
components of signaling pathways. Furthermore, some of the effects could be indirect, as many of the effects of glucocorticoids on PKA and PKC are evident only after chronic administration of glucocorticoids. These effects could be secondary to the changes in neurotransmitter receptors or components of intracellular signaling, or as a compensatory response to such changes. Nonetheless, stress alters the intracellular cascade at multiple levels. The final outcome of the alterations in the intracellular cascade is alterations in the functional properties of certain proteins, either through genomic or nongenomic action. Most of the protein kinases alter the functional properties of various proteins by phosphorylating them or by altering the transcription of genes, thereby modifying the expression of the proteins. The regulation of the gene expression of critical proteins by stress is described comprehensively in another chapter of this book ('Transcription factors as modulators of stress responsivity'). As stated earlier in this chapter, some of the acute effects of stress are beneficial; however, the chronic effects of stress may lead to abnormalities in the brain, including altered brain structures. Several studies indicate atrophy of the hippocampus and loss of neurons and/or glial cells in several structures of the brain. These deleterious effects of stress may be associated with psychiatric disorders such as posttraumatic stress disorder (PTSD) or depression. The findings of stress-induced modulation of neurotransmitter receptors and of the intracellular signaling cascade may be quite relevant in the pathophysiology of these psychiatric disorders, as similar findings, particularly those associated with 5HT2A, PKA, PKC, IP3, and G proteins, have been reported in the blood cells of patients with affective disorders and in postmortem brain of depressed or suicide victims.
References Albert, P.R. and Tiberi, M. (2001) Receptor signaling and structure: insights from serotoninl receptors. TRENDS in Endocrol. Metab., 12: 453-460. Azmitia, E.C. and McEwen, B.S. (1974) Adrenocortical influence on rat brain tryptophan hydroxylase activity. Brain Res., 78: 291-302. Barbacid, M. (1987) RAS genes. Annu. Rev. Biochem., 56: 779-827.
657 Bard, J.A., Zgombick, J., Adham, N., Vaysse, P., Branchek, T.A. and Weinshank, R.L. (1993) Cloning of a novel human serotonin receptor (5HT7) positively linked to adenylate cyclase. J. Biol. Chem., 268: 23422-23426. Barnes, N.M. and Sharp, T. (1999) A review of central 5HT receptors and their function. Neuropharmacol., 38: 1083-1152. Bell, C.J. and Nutt, D.J. (1998) Serotonin and panic. Br. J. Psychiatry., 172:465-471. Berridge, M.J. and Irvine, R.F. (1989) Inositol phosphates and cell signaling. Nature, 341: 197-205. Biegon, A., Rainbow, T.C. and McEwen, B.S. (1985) Corticosterone modulation of neurotransmitter receptors in rat hippocampus: A quantitative autoradiographic study. Brain Res., 332:309-314. Birnbaum, S., Gobeske, K.T., Auerbach, J., Taylor, J.R. and Arnsten, A.F.T. (1999) A role for norepinephrine in stressinduced cognitive deficits: ~-l-adrenoceptor mediation in the prefrontal cortex. Biol. Psychiatry, 46: 1266-1274. Blackwell, G.J., Carnucio, R., Di Rosa, M., Flower, R.J., Parente, L. and Persico, P. (1980) Macrocortin: A polypeptide causing the anti-phospholipase effect of glucocorticoids. Nature, 287: 147-149. Borrelli, E., Montmayeur, J.P. and Foulkes, N.S. (1992) Sassone-Corsi P. Signal transduction and gene control: The cAMP pathway. Crit. Rev. Oncog., 3: 321-338. Brannan, S.K., Miller, A., Jones, D.J., Kramer, G.L. and Petty, F. (1995) Beta-adrenergic receptor changes in learned helplessness may depend on stress and test parameters. Pharmacol. Biochem. Behav., 51: 553-556. Bredt, D.S., Hwang, P.M. and Snyder, S.H. (1990) Localization of nitric oxide synthase indicating a neural role for nitric oxide. Nature, 347: 768-770. Bremner, J.D., Krystal, J.H., Southwick, S.M. and Charney, D.S. (1996) Noradrenergic mechanism in stress and anxiety: II. Clinical studies. Synapse, 23: 39-51. Brown, W.J., Chambers, K. and Doody, A. (2003) PhospholipaseA2 (PLA2) enzymes in membrane trafficking: mediators of membrane shape and function. Traffic, 4: 214-221. Burnet, P.W.J., Mefford, I.N., Smith, C.C., Gold, P.W. and Sternberg, E.M. (1992) Hippocampal 8-[3H]hydroxy-2-(di-n propylamino) tetralin binding site densities, serotonin receptor (5HT1A) messenger ribonucleic acid abundance, and serotonin levels parallel the activity of the hypothalamopituitary-adrenal axis in rat. J. Neurchem., 59: 1062-1070. Cabrera-Vera, T.M., Vanhauwe, J., Thomas, T.O., Medkova, M., Preininger, A., Mazzoni, M.R. and Hamm, E. (2003) Insights into G protein structure, function, and regulation. Endocrine Rev., 24: 765-781. Campbell, S.L., Khosravi-Far, R., Rossman, K.L., Clark, G.J. and Der, C.J. (1998) Increasing complexity of RAS signaling. Oncogene, 17: 1395-1413.
Ceccatelli, S. and Orazzo, C. (1993) Effect of different types of stressors on peptide messenger ribonucleic acids in the hypothalamic paraventricular nucleus. Acta Endocrinol., 128: 485-492. Chalmers, D.T., Ldpez, J.F., Vfizquez, D.M., Akil, H. and Watson, S.J. (1994) Regulation of hippocampal 5HT1A receptor gene expression by dexamethasone. Neuropsychopharmacology, 10:215-222. Chang, F.-H. and Bourne, H.R. (1987) Dexamethasone increases adenylyl cyclase activity and expression of the cz subunit of Gs in GH3 cells. Endocrinology, 121: 1711-1715. Chaouloff, F. (1993) Physiopharmacological interactions between stress hormones and central serotonergic systems. Brain Res. Rev., 18: 1-32. Chaouloff, F. (1995) Regulation of 5HT receptors by corticosteroids: where do we stand? Fundam. Clin. Pharmacol., 9: 219-333. Chert, R.H., Sarnecki, C. and Blenis, J. (1992) Nuclear localization and regulation of erk- and rsk-encoded protein kinases. Mol. Cell. Biol., 12: 915-927. Clapham, D.E. and Neer, E.J. (1997) G protein beta gamma subunits. Annu. Rev. Pharmacol. Toxicol., 37: 167-203. Clegg, C.H. and McKnight, G.S. (1988) Genetic characterization of a brain-specific form of the type I regulatory subunit of cAMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA, 85: 3703-3707. Clement, H.W., Schfifer, F., Rowe, C., Gensa, D. and Wesemann, W. (1993) Stress-induced changes of extracellular 5-hydroxyindoleacetic acid concentrations followed in the nucleus raphe dorsalis and the frontal cortex of the rat. Brain Res., 64:117-124. Cobb, M.H. (1999) MAP kinase pathways. Prog. Biophys. Mol. Biol., 71: 479-500. Cockcroft, S. and Thomas, G.M.H. (1992) Inositol-lipidspecific phospholipase C isoenzymes and their differential regulation by receptors. Biochem. J., 288: 1-14. Cohen, R.M., Cohen, M.R. and McLellan, C.A. (1986) Foot shock induces time and region specific adrenergic receptor changes in rat brain. Pharmacol. Biochem. Behav., 24: 1587-1592. Costa, A., Trainer, P., Besser, M. and Grossman, A. (1993) Nitric oxide modulates the release of corticotropin-releasing hormone from the rat hypothalamus in vitro. Brain Res., 605: 187-192. Crayton, J.W., Joshi, I., Gulati, A., Arora, R.C. and Wolf, W.A. (1996) Effect of corticosterone on serotonin and catecholamine receptors and uptake sites in rat frontal cortex. Brain Res., 728: 260-262. Croxtall, J.D., van Hal, P.T., Choudhury, Q., Gilroy, D.W. and Flower, R.J. (2002) Different glucocorticoids vary in their genomic and non-genomic mechanism of action in A549 cells. Br. J. Pharmacology., 135: 511-519.
658 Curzon, G., Joseph, M.H. and Knott, P.J. (1972) Effects of immobilization and food deprivation on rat brain tryptophan metabolism. J. Neurochem., 19: 1967-1974. Czyrak, A. (1996) Modulation of the forskolin-induced cyclic AMP accumulation by corticosterone. Pol. J. Pharmacol., 48: 595-599. Daaka, Y., Luttrell, L.M. and Lefkowtiz, R.J. (1997) Switching of the coupling of the beta2-adrenergic receptor to different G proteins by protein kinase A. Nature, 390: 88-91. Dalley, J.W., Mason, K. and Stanford, S.C. (1996) Increased levels of extracellular noradrenaline in the frontal cortex of rats exposed to naturalistic environmental stimuli: Modulation by acute systemic administration of diazepam or buspirone. Psychopharmacology, 127: 47-54. Darnell, J.E. Jr. (1997) STATs and gene regulation. Science, 227: 1630-1635. Datta, S.R., Brunet, A. and Greenberg, M.E. (1999) Cellular survival: a play in three Akts. Genes. Dev., 13: 2905-2927. Davis, S., Heal, D.J. and Stanford, S.C. (1994) Sensitization to [3-adrenoceptor downregulation in mouse cerebral cortex in response to a brief swim stress. Br. J. Pharmacol., 12: 157P. Day, H.E.W., Campeau, S., Watson, S.J. Jr. and Akil, H. (1999) Expression of ~IB adrenoceptor mRNA in corticotropin-releasing hormone-containing cells of the rat hypothalamus and its regulation by corticosterone. J. Neurosci., 19: 10098-10106. de Kloet, E.R. (2000) Stress in the brain. Eur. J. Pharmacol., 405: 187-198. Downward, J. (1998) Mechanisms and consequences of activation of protein kinase B/AKT. Curr. Opin. Neurobiol., 10: 262-267. Downward, J. (1998) RAS signaling and apoptosis. Curr. Opin. Genet. Dev., 8: 49-54. Downward, J. (2003) Targeting RAS signaling pathways in cancer therapy. Nat. Rev. Cancer, 3:11-22. Dudek, H., Datta, S.R., Franke, T.F., Bimbaum, M.J., Yao, R., Cooper, G.M., Segal, R.A., Kaplan, D.R. and Greenberg, M.E. (1997) Regulation of neuronal survival by the serinethreonine protein kinase Akt. Science., 275: 661-665. Duman, R.S., Strada, S.J. and Enna, S.J. (1989) Glucocorticoid administration increases receptor-mediated and forskolinstimulated cyclic AMP accumulation in rat brain cerebral cortical slices. Brain Res., 477: 166-171. Duronio, V., Scheid, M.P. and Ettinger, S. (1998) Downstream signaling events regulated by phosphatidylinositol-3 kinase activity. Cell Signal, 10: 233-239. Dwivedi, Y. and Pandey, G.N. (1999a) Administration of dexamethasone upregulates protein kinase C activity and the expression of 7 and ~ protein kinase C isozymes in the rat brain. J. Neurochem., 72: 380-387. Dwivedi, Y. and Pandey, G.N. (1999b) Repeated administration of dexamethasone increases phosphoinositide-specific phospholipase C activity and mRNA and protein
expression of the phospholipase C ~1 isozyme in rat brain. J. Neurochem., 73: 780-790. Dwivedi, Y. and Pandey, G.N. (2000a) Adrenal glucocorticoids modulate [3H]cyclic AMP binding to protein kinase A (PKA), cyclic AMP-dependent PKA activity, and protein levels of selective regulatory and catalytic subunit isoforms of PKA in rat brain. J. Pharmacol. Exp. Ther., 294: 103-116. Dwivedi, Y., Rizavi, H.S., Rao, J.S. and Pandey, G.N. (2000b) Modifications in the phosphoinositide signaling pathway by adrenal glucocorticoids in rat brain: Focus on phosphoinositide-specific phospholipase C and inositol 1,4,5-trisphosphate. J. Pharmacol. Exp. Ther., 295: 244-254. English, J., Pearson, G., Wilsbacher, J., Swantek, J., Karandikar, M., Xu, S. and Cobb, M.H. (1999) New insights into the control of MAP kinase pathways. Exp. Cell. Res., 253: 255-270. Eves, E.M., Xiong, W., Bellacosa, A., Kennedy, S.G., Tsichlis, P.N., Rosner, M.R. and Hay, N. (1998) Akt, a target of phosphatidylinositol-3 kinase, inhibits apoptosis in a differentiating neuronal cell line. Mol. Cell. Biol., 18: 2143-2152. Exton, J.H. (1994) Phosphoinositide phospholipases and G proteins in hormone action. Annu Rev Physiol, 56: 349-369. Farooqui, A.A., Yang, H.C., Rosenberger, T.A. and Horrocks, L.A. (1997) Phospholipase A 2 and its role in brain tissue. J. Neurochem., 69: 889-901. Fernandes, C., McKittrick, C.R., File, S.E. and McEwen, B.S. (1997) Decreased 5HT|A and increased 5HT2A receptor binding after chronic corticosterone associated with a behavioral indication of depression but not anxiety. Psychoneuroendocrinology, 22: 477-491. Ffrench-Mullen, J.M. (1995) Cortisol inhibition of calcium currents in guinea pig hippocampal CA1 neurons via G-protein-coupled activation of protein kinase C. J. Neurosci., 15:903-911. Finlay, J.M., Zigmond, M.J. and Abercrombie, E.D. (1995) Increased dopamine and norepinephrine release in medial prefrontal cortex induced by acute and chronic stress: Effects of diazepam. Neuroscience, 64: 619-628. Flugge, G. (1996) Alterations in the central nervous alpha2adrenoceptor system under chronic psychosocial stress. Neuroscience, 75: 187-196. Flugge, G. (1999) Effects of cortisol on brain alpha2adrenoceptors: Potential role in stress. Neurosci. Biobehav. Rev., 23: 949-956. Forstemann, U., Gath, I., Schwartz, P., Closs, E.I. and Kleiner, H. (1995) Isoforms of nitric oxide synthase. Properties, cellular distribution and expressional control. Biochem. Pharmacol., 50: 1321-1332. Freissmuth, M., Waldhoer, M., Bofill-Cardoba, E. and Nanoff, C. (1999) G protein antagonists. Trends Pharmacol. Sci., 20: 237-245. Gannon, M.N., Brinton, R.E., Sakai, R.R. and McEwen, B.S. (1991) Diurnal differences and adrenal involvement in
659 calmodulin stimulation of hippocampal adenylyl cyclase activity. J. Neuroendocrinol., 3: 37-41. Gannon, M.N. and McEwen, B.S. (1990) Calmodulin involvement in stress- and corticosterone-induced regulation of cyclic AMP-generating systems in brain. J. Neurochem., 55: 276-284. Gerard, C., el Mestikawy, S., Lebrand, C., Adrien, J., Ruat, M., Traiffort, E., Hamon, M. and Martres, M.P. (1996) Quantitative RT-PCR distribution of serotonin 5HT6 receptor mRNA in the central nervous system of control or 5,7dihydroxytryptamine-treated rats. Synapse, 23: 164-173. Gilman, A.G. (1987) G proteins: transducers of receptorgenerated signals. Annu. Rev. Biochem., 56:515-649. Goldstein, L.E., Rasmusson, A.M., Bunney, S.B. and Roth, R.H. (1996) Role of the amygdala in the coordination of behavioral, neuroendocrine and prefrontal cortical monoamine responses to psychological stress in the rat. J. Neurosci., 16: 4787-4798. Gomperts, B.D., Tatham, P.E.R. and Kramer, I.M. (2002) Signal Transduction. Signaling pathways operated by non-receptor protein tyrosine kinases. Academic Press, pp. 283-297 Graeff, F.G. (1997) Serotonergic systems. Psychiatr. Clin. North Am., 20: 723-739. Grewal, S.S., York, R.D. and Stork, P.J. (1999) Extracellular signal-regulated kinase signaling in neurons. Curr. Opin. Neurobiol., 9: 544-553. Gurguis, G.N.M., Kramer, G. and Petty, F. (1996) Indices of brain beta-adrenergic receptor signal transduction in the learned helplessness animal model of depression. J. Psychiatr. Res., 30:135-146. Gustafson, E.L., Durkin, M.M., Bard, J.A., Zgombick, J. and Branchek, T.A. (1996) A receptor autoradiographic and in situ hybridization analysis of the distribution of the 5HT7 receptor in rat brain. Br. J. Pharmacol., 117: 657-666. Hamm, H.E. (1998) The many faces of G protein signaling. J. Biol. Chem., 273: 669-672. Harrelson, A. and McEwen, B.S. (1987) Steroid hormone influences on cyclic AMP-generating systems. Curr. Topics Membr. Transport., 31: 217-247. Harrelson, A.L., Rostene. W. and McEwen. B.S. (1987) Adrenocortical steroids modify neurotransmitter-stimulated cyclic AMP accumulation in the hippocampus and limbic brain of the rat. J. Neurochem., 48: 1648-1655. Hellriegel, E.T. and D'Mello, A.P. (1997) The effect of acute, chronic and chronic intermittent stress on the central noradrenergic system. Pharmacol. Biochem. Behav., 57: 207-214. Heninger, G.R., Charney, D.S. and Price, L.H. (1988) Noradrenergic and serotonergic receptor system function in panic disorder and depression. Acta Psychiatr. Scand. Suppl., 341: 138-150. Hepler, J.R. (1999) Emerging roles for RGS proteins in cell signaling. Trends in Pharmacol. Sci., 20: 376-382.
Hetman, M., Kanning, K., Cavanaugh, J.E. and Xia, Z. (1999) Neuroprotection by brain-derived neurotrophic factor is mediated by extracellular signal-regulated kinase and phosphatidylinositol-3 kinase. J. Biol. Chem., 274: 22569-22580. Hildebrandt, J.D. (1997) Role of subunit diversity in signaling by heterotrimeric G proteins. Biochem. Pharmacol., 54: 325-339. Hirst, W.D., Price, G.W., Rattray, M. and Wilkin, G.P. (1997) Identification of 5-hydroxytryptamine receptors positively coupled to adenylyl cyclase in rat cultured astrocytes. Br. J. Pharamcol., 120:509-515. Holgado-Madruga, M., Moscatello, D.K., Emlet, D.R., Dieterich, R. and Wong, A.J. (1997) Grb2-associated binder-1 mediates phosphatidylinositol 3-kinase activation and the promotion of cell survival by nerve growth factor. Proc. Natl. Acad. Sci. USA, 94: 12419-12424. Holmes, M.C., French, K.L. and Seckl, J.R. (1995a) Modulation of serotonin and corticosteroid receptor gene expression in the rat hippocampus with circadian rhythm and stress. Brain Res. Mol. Brain Res., 28: 186-192. Holmes, M.C., Yau, J.L.W., French, K.L. and Seckl, J.R. (1995b) The effect of adrenalectomy on 5HT and corticosteroid receptor subtype mRNA expression in rat hippocampus. Neuroscience, 64: 327-337. Hoyer, D., Hannon, J.P. and Martin, G.R. (2002) Molecular, pharmacological and functional diversity of 5HT receptors. Pharmacol. Biochem. Behav., 71: 533-554. Huang, E.J. and Reichardt, L.F. (2001) Neurotrophins: roles in neuronal development and function. Annu. Rev. Neurosci. 24: 677-736. Huang, E.J. and Reichardt, L.F. (2003) Trk receptors: roles in neuronal signal transduction. Annu. Rev. Biochem., 72: 609-642. Huang, J. and Azmitia, E.C. (1999) Homologous regulation of 5HT~A receptor mRNA in adult rat hippocampal dentate gyrus. Neurosci. Lett., 270: 5-8. Ishii, M. and Kurachi, Y. (2003) Physiological actions of regulators of G protein signaling (RGS) proteins. Life Sci., 74: 163-171. Jitsuiki, H., Kagaya, A., Goto, S., Horiguchi, J. and Yamawaki, S. (2000) Effect of lithium carbonate on the enhancement of serotonin2A receptor elicited by dexamethasone. Neuropsychobiology, 41: 55-61. Joseph, M.H. and Kennett, G.A. (1983) Stress-induced release of 5HT in hippocampus and its dependence on increased tryptophan availability: an in vivo electrochemical study. Brain Res., 270: 251-257. Joseph, S.K. and Williamson, J.R. (1989) Inositol polyphosphates and intracellular calcium release. Arch. Biochem. Biophys. 273: 1-15. Kaplan, D.R. and Miller, F.D. (2000) Neurotrophin signal transduction in the nervous system. Curr. Opin. Neurobiol., 10: 381-391. Katagiri, H., Kagaya, A., Nakae, S., Morinobu, S. and Yamawaki, S. (2001) Modulation of serotonin2a receptor
660 function in rats after repeated treatment with dexamethasone and L-type calcium channel antagonist nimodipine. Prog. NeuroPsychopharmacol. Biol. Psychiatry, 25: 1269-1281. Kim, D.-H., Jung, J.-S., Kim, H.-S., Suh, H.-W., Son, B.-K., Kim, Y.-H. and Song, D.-K. (2000) Inhibition of brain protein kinase C attenuates immobilization stress-induced plasma corticosterone levels in mice. Neurosci. Lett., 291: 69-72. Klenerova, V. and Sida, P. (1994) Changes in beta-adrenergic receptors in the neurohypophysis and intermediate lobe or rat hypophysis exposed to stress. Physiol. Res., 43: 289-292. Kohen, R., Metcalf, M.A., Khan, N., Druck, T., Huebner, K., Lachowicz, J.E., Meltzer, H.Y., Sibley, D.R., Roth, B.L. and Hamblin, M.W. (1996) Cloning, characterization, and chromosomal localization of a human 5HT6 serotonin receptor. Neurochem., 66: 47-56. Kolasa, K., Song, L. and Jope, R.S. (1992) Adrenalectomy increases phosphoinositide hydrolysis induced by norepinephrine or excitatory amino acids in rat hippocampal slices. Brain Res., 579: 128-134. Kolkova, K., Novitskaya, V., Pedersen, N., Berezin, V. and Bock, E. (2000) Neural cell adhesion molecule-stimulated neurite outgrowth depends on activation of protein kinase C and the RAS-mitogen-activated protein kinase pathway. J. Neurosci.., 20: 2238-2246. Kudo, I. and Murakami, M. (2002) Phospholipase A2 enzymes. Prostaglandis Other Lipid Mediat., 68-69: 3-58. Kuroda, Y., Mikuni, M., Nomura, N. and Takahashi, K. (1993) Differential effect of subchronic dexamethasone treatment on serotonin2 and 13 adrenergic receptors in the rat cortex and hippocampus. Neurosci. Lett., 195-198. Kuroda, Y., Mikuni, M., Ogawa, T. and Takahashi, K. (1992) Effect of ACTH, adrenalectomy and the combination treatment on the density of 5HT2 receptor binding sites in neocortex of rat forebrain and 5HT2 receptor-mediated wetdog shake behaviors. Psychopharmacology, 108: 27-32. Kuroda, Y., Watanabe, Y., Albeck, D.S., Hastings, N.B. and McEwen, B.S. (1994) Effects of adrenalectomy and Type I or Type II glucocorticoid receptor activation on 5HT~A and 5HT2 receptor binding and 5HT transporter mRNA expression in rat brain. Brain Res., 648: 157-161. Le Corre, S., Sharp, T., Young, A.H. and Harrison, P.J. (1997) Increase of 5HT7 (serotonin-7) and 5HTIA (serotonin-lA) receptor mRNA expression in rat hippocampus after adrenalectomy. Psychopharmacology, 130: 368-374. Lefkowitz, R.J. (1998) G protein-coupled receptors. III. New roles for receptor kinases and beta-arrestins in receptor signaling and desensitization. J. Biol. Chem., 273: 18677-18680. Leonard, W.J. and O'Shea, J.J. (1998) JAKs and STATs: biological implications. Annu. Rev. Immunol., 16: 293-322. Lewis, T.S., Shapiro, P.S. and Ahn, N.G. (1998) Signal transduction through MAP kinase cascades. Adv. Cancer Res., 49-139.
Lewis, T.S., Shapiro, P.S. and Ahn, N.G. (1998) Signal transduction through MAP kinase cascades. Adv. Cancer Res., 74: 49-139. Lin, L.L., Watermann, M., Lin, A.Y., Knopf, J.L., Seth, A., Davis, R.J. (1993) cPLA2 is phosphorylated and activated by MAP kinase. Cell 72: 269-278. L6pez, J.F., Chalmers, D.T., Little, K.Y. and Watson, S.J. (1998) Regulation of serotoninlA, glucocorticoid, and mineralocorticoid receptor in rat and human hippocampus: implications for the neurobiology of depression. Biol. Psychiatry., 43: 547-573. Lopez-Figueroa, M.O., Itoi, K. and Watson, S.J. (1998) Regulation of nitric oxide synthase messenger RNA expression in the rat hippocampus by glucocorticoids. Neuroscience, 87: 439-446. Lorenzo Fernandez, P. (1999) Stress and neurodegeneration: pharmacologic strategies. Ann. Rev. Acad. Nac. Med. (Madr)., 116:417-428. Lynch, M., Littleton, J., McKernan, R.M., Durcan, M.J., McMillian, T. and Campbell, I.C. (1983) ~-adrenoceptor number and function in rat cortex after ethanol and immobilization stress. Brain Res., 288: 145-149. Mann, J.J. (1999) Role of the serotonergic system in the pathogenesis of major depression and suicidal behavior. Neuropsychopharmacology, Suppl. 1: $99-S105. Mann, J.J. (2003) Neurobiology of suicidal behavior. Nat. Rev. Neurosci., 4: 819-828. Marais, R., Wynne, J. and Treisman, R. (1993) The SRF accessory protein E lk-1 contains a growth factor-regulated transcriptional activation domain. Cell, 73: 381-393. Marinissen, M.J. and Gutkind, J.S. (2001) G-protein-coupled receptors and signaling networks: Emerging paradigms. Trends Pharmacol. Sci., 22: 368-376. Martin, J.V., Edwards, E., Johnson, J.O. and Henn, F.A. (1990) Monoamine receptors in an animal model of affective disorder. J. Neurochem., 55:1142-1148. Martire, M., Pistritto, G. and Prexiosi, P. (1989) Different regulation of serotonin receptors following adrenal hormone imbalance in the rat hippocampus and hypothalamus. Neural Transm., 78: 109-120. McEwen, B.S. (1991) Stress and hippocampus. An update on current knowledge. Presse. Med., 20: 1801-1806. McLeod, T.M., Lopez-Figueroa, A.L. and Lopez-Figueroa, M.O. (2001) Nitric oxide, stress, and depression. Psychopharmacol. Bull., 35: 2441. Meijer, O.C. and de Kloet, E.R. (1994) Corticosterone suppresses the expression of 5HT1A receptor mRNA in rat dentate gyrus. Eur. J. Pharmacol., 266: 255-261. Meijer, O.C., de Lange, E.C., Breimer, D.D., de Boer, A.G., Workel, J.O. and de Kloet, E.R. (1998) Penetration of dexamethasone into brain glucocorticoid targets is enhanced in mdrlA P-glycoprotein knockout mice. Endocrinology, 139: 1789-1793.
661 Mellor, H. and Parker, P.J. (1998) The extended protein kinase C superfamily. Biochem. J., 332: 281-292. Mendelson, S.D. and McEwen, B.S. (1992a) Autoradiographic analyses of the effects of adrenalectomy and corticosterone on 5HTIA and 5HT~B receptors in the dorsal hippocampus and cortex of the rat. Neuroendocrinology, 55: 444-450. Mendelson, S.D. and McEwen, B.S. (1992b) Quantitative autoradiographic analyses of the time course and reversibility of corticosterone-induced decreases in binding at 5HTIA receptors in rat forebrain. Neuroendocrinology, 56: 881-888. Mitchell, J. and Bansal, A. (1997) Dexamethasone increases G~q/11 expression and hormone-stimulated phospholipase C activity in UMR-106-01 cells. Am. J. Physiol., 273: E528-E535. Miyahara, S., Komori, T., Fujiwara, R., Shizuya, K., Yamamoto, M., Ohmori, M. and Okazaki, Y. (1999) Effects of restraint stress on ~ adrenoceptor mRNA expression in the hypothalamus and midbrain of the rat. Brain Res., 843: 130-135. MoNey, P.L., Manier, D.H. and Sulser, F. (1983) Norepinephrine-sensitive adenylate cyclase system in rat brain: Role of adrenal corticosteroids. J. Pharmacol. Exp. Ther., 226: 71-77. Mobley, P.L. and Sulser, F. (1980) Adrenal corticoids regulate sensitivity of noradrenaline receptor-coupled adenylate cyclase in brain. Nature, 286: 608-609. MoNey, P.L., Sulser, F. (1980) Adrenal steroids affect the norepinephrine sensitive adenylate cyclase system in the rat limbic forebrain. Eur. J. Pharmacol., 65: 321-322. Muraoka, S.-I., Mikuni, M., Kagaya, A., Saitoh, K. and Takahashi, K. (1993) Dexamethasone potentiates serotonin2 receptor-mediated intracellular Ca 2+ mobilization in C6 glioma cells. Neuroendocrinology, 57: 322-329. Neer, E.J. (1995) Heterotrimeric G proteins: organizers of transmembrane signals. Cell, 80: 249-257. Nestler, E.J. and Greengard, P. (1984) Protein phosphorylation and the regulation of neuronal function. In: Siegel, G.H., Albers, R.W., Agranoff, B.W. and Molioff, P. (Eds.), Basic Neurochemistry: Molecular, Cellular, and Medical Aspects, Little Brown, Boston, pp. 449-474. Nestler, E.J., Terwilliger, R.Z. and Halm, E. (1989) Corticosterone increases protein tyrosine kinase activity in the locus coeruleus and other monoaminergic nuclei of rat brain. Mol. Pharmacol., 35: 265-270. Neves, S.R., Ram, P.T. and Iyengar, R. (2002) G protein pathways. Science, 296: 1636-1639. Newton, A.C. (2001) Protein kinase C: structural and spatial regulation by phosphorylation, cofactors, and macromolecular interactions. Chem. Rev., 101: 2353-2364. Ni, Y.G., Gold, S.J., Iredale, P.A., Terwilliger, R.Z., Duman, R.S. and Nestler, E.J. (1999) Region-specific regulation of RGS4 (Regulator of G protein-signaling protein Type 4) in brain by stress and glucocorticoids: In vivo and in vitro studies. J. Neurosci., 19: 3674-3680.
Nimnual, A.S., Yatsula, B.A. and Bar-Sagi, D. (1998) Coupling of RAS and RAC guanosine triphosphatases through the RAS exchanger SOS. Science, 279: 560-563. Nishizuka, Y. (1988) The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature, 334: 661-665. Nukina, I., Glavin, G.B. and LaBella, F.S. (1987) Acute coldrestraint stress affects alpha 2-adrenoceptors in specific brain regions of the rat. Brain Res., 401: 30-33. Okuhara, D.Y., Beck, S.G. and Muma, N.A. (1997) Corticosterone alters G protein ~-subunit levels in the rat hippocampus. Brain Res., 745: 144-151. Okuhara, D.Y. and Beck, S.G. (1998) Corticosteroids alter 5-hydroxytryptaminela receptor-effector pathway in hippocampal subfield CA3 pyramidal cells. J. Pharmacol. Exp. Ther., 284: 1227-1233. Pacak, K., McCarty, R., Palkovits, M., Kopin, I.J. and Goldstein, D.S. (1995) Effects of immobilization on in vivo release of norepinephrine in the bed nucleus of the stria terminalis in conscious rats. Brain Res., 688: 242-246. Pandey, G.N. and Dwivedi, Y. (1999) Effects of adrenal glucocorticoids on protein kinase C (PKC) binding sites, PKC activity, and expression of PKC isozymes in the rat brain. Soc. Neuroscience Abstr., 25: 1812. Pandey, S.C., Ren, X., Sagen, J. and Pandey, G.N. (1995) Betaadrenergic receptor subtypes in stress-induced behavioral depression. Pharmacol. Biochem. Behav., 51: 339-344. Patapoutian, A. and Reichardt, L.F. (2001) Trk receptors: mediators of neurotrophin action. Curt. Opin. Neurobiol., 11: 272-280. Pei, Q., Zetterstr6m, T. and Fillenz, M. (1990) Tail pinchinduced changes in the turnover and release of dopamine and 5-hydroxytryptamine in different brain regions of the rat. Neuroscience, 35: 133-138. Pellegrini, S. and Dusanter-Fourt, I. (1997) The structure, regulation and function of the Janus kinases (JAKs) and the signal transducers and activators of transcription (STATs). Eur. J. Biochem., 248:615-633. Plassat, J.-E., Amlaiky, N. and Hen, R. (1993) Molecular cloning of a mammalian serotonin receptor that activates adenylate cyclase. Mol. Pharmacol., 44: 229-236. Qiu, J., Lou, L.G., Huang, X.Y., Lou, S.J., Pei, G. and Chen, Y.Z. (i998) Nongenomic mechanisms of glucocorticoid inhibition of nicotine-induced calcium influx in PC12 cells: involvement of protein kinase C. Endocrinology, 139: 5103-5108. Qiu, J., Wang, P., Jing, Q., Zhang, W., Eix, X., Zhong, Y., Sun, G., Pei, G. and Chen, Y. (2001) Rapid activation of ERK1/2 mitogen-activated protein kinase by corticosterone in PC12 cells. Biochem. Biphys. Res. Commun. 287: 1017-1024. Reagan, L.P., McKittrick, C.R. and McEwen, B.S. (1999) Corticosterone and phenytoin reduce neuronal nitric oxide
662 synthase messenger RNA expression in rat hippocampus. Neuroscience, 91 (1): 211-219. Rhee, S.G. and Choi, K.D. (1992) Regulation of inositol phospholipid-specific phospholipase C isozymes. J. Biol. Chem., 267: 12393-12396. Riedel, W. (2000) Role of nitric oxide in the control of the hypothalamic-pituitary-adrenocortical axis. Z Rheumatol., 59 (Suppl. 2): 36-42. Roberts, V.J., Singhal, R.L. and Roberts, D.C. (1984) Corticosterone prevents the increase in noradrenaline-stimulated adenylyl cyclase activity in rat hippocampus following adrenalectomy or metopirone. Eur. J. Pharmacol., 103: 235-240. Rodan, S. and Rodan, G. (1986) Dexamethasone effects of [3-adrenergic receptors and adenylate cyclase regulatory proteins Gs and GI in ROS 17/2.8 cells. Endocrinology, 118: 2510-2518. Rosenblum, L.A., Coplan, J.D., Friedman, S., Bassoff, T., Gorman, J.M. and Andrews, M.W. (1994) Adverse early experiences affect noradrenergic and serotonergic functioning in adult primates. Biol. Psychiatry., 35: 221-227. Ross, E.M. and Wilkie, T.M. (2000) GTPase-activating proteins for heterotrimeric G proteins: regulators of G protein signaling (RGS) and RGS-like proteins. Annu. Rev. Biochem., 69: 795-827. Rostene, W.H., Rischette, C.T., Dussaillant, M. and McEwen, B.S. (1985) Adrenal steroid modulation of vasactive intestinal peptide effect on serotoninl binding sites in the rat brain shown by in vitro quantitative autoradiography. Neuroendocrinology, 40: 129-134. Saito, N., Guitart, X., Hayward, M., Tallman, J.F., Duman, R.S. and Nestler, E.J. (1989) Coritcosterone differentially regulates the expression of Gs~ and Giczmessenger RNA and protein in rat cerebral cortex. Proc. Natl. Acad. Sci. USA, 86: 3906-3910. Sandi, C. and Guaza, C. (1995) Evidence for a role of nitric oxide in the corticotrophin-releasing factor release induced by interleukin-l[3. Eur. J. Pharmacol., 274: 17-23. Sandi, C., Venero, C. and Guaza, C. (1996) Nitric oxide synthase inhibitors prevent rapid behavioral effects of corticosterone in rats. Neuroendocrinology, 63: 446-453. Sapolsky, R.M. (1987) Glucocorticoids and hippocampal damage. Trends Neurosci., 10: 346-349. Schinkel, A.H., Wagenaar, E., Mol, C.A. and van Deemter, L. (1996) P-glycoprotein in the blood-brain barrier of mice influces the brain pentration and pharmacological activity of many drugs. J. Clin. Invest., 97: 2517-2524. Scott, J.D., Glaccum, M.B., Zoller, M.J., Uhler, M.D., Helfman, D.M., McKnight, G.S. and Krebs, E.G. (1987) The molecular cloning of a type II regulatory subunit of the cAMP-dependent protein kinase from rat skeletal muscle and mouse brain. Proc. Natl. Acad. Sci. USA, 84: 5192-5196.
Seger, R. and Krebs, E.G. (1995) The MAPK signaling cascade. FASEB J., 9: 726-735. Simon, M.I., Strathmann, M.P. and Gautam, N. (1991) Diversity of G proteins in signal transduction. Science, 252: 802-808. Singh, V.B., Onaivi, E.S., Phan, T.H. and Boadle-Biber, M.C. (1990) The increases in rat cortical and midbrain tryptophan hydroxylase activity in response to acute or repeated sound stress are blocked by biolateral lesions to the central nucleus of the amygdala. Brain Res., 530: 49-53. Sleight, A.J., Boess, F.G., Bos, M. and Bourson, A. (1998) The putative 5HT6 receptor: localization and function. Ann. NY Acad. Sci., 861: 91-96. Southwick, S.M., Bremner, J.D., Rasmusson, A., Morgan, C.A. III, Arnsten, A. and Charney, D.S. (1999a) Role of norepinephrine in the pathophysiology and treatment of posttraumatic stress disorder. Biol. Psychiatry., 46: 1192-1204. Southwick, S.M., Morgan, C.A. III, Charney, D.S. and High, J.R. (1999b) Yohimbine use in a natural setting: Effects on posttraumatic stress disorder. Biol. Psychiatry., 46: 442-444. Southwick, S.M., Paige, S., Morgan, C.A. III, Bremner, J.D., Krystal, J.H. and Charney, D.S. (1999c) Neurotransmitter alterations in PTSD: Catecholamines and serotonin. Semin. Clin. Neuropsychiatry, 4: 242-248. Spaulding, S.W. (1993) The ways in which hormones change cyclic adenosine 3'5'-monophosphate-dependent protein kinase subunits, and how such changes affect cell behavior. Endocr. Rev., 14: 632-560. Stanford, S.C. (1995) Central noradrenergic neurones and stress. Pharmacol. Ther., 68: 297-342. Stone, E.A., Platt, J.E., Trullas, R. and Slucky, A.V. (1984) Reduction of the cAMP response to norepinephrine in rat cerebral cortex following repeated restraint stress. Psychopharmacology, 82: 403-405. Stone, E.A. and Quartermain, D. (1999) Alpha-1-noradrenergic neurotransmission, corticosterone, and behavioral depression. Biol. Psychiatry., 46: 1287-300. Stone, E.A., Rhee, J. and Quartermain, D. (1996) Blockade of effect of stress on risk assessment behavior in mice by a beta-1 adrenoceptor antagonist. Pharmacol. Bechem. Behav., 55: 215-217. Stone, E.A., Slucky, A.V., Platt, J.E. and Trullas, R. (1985) Reduction of the cyclic adenosine 3'5'-monophosphate response to catecholamines in rat brain slices after repeated restraint stress. J. Pharmacol. Exp. Ther., 233: 382-388. Stone, E.A. (1979) Reduction by stress of norepinephrinestimulated accumulation of cyclic AMP in rat cerebral cortex. J. Neurochem., 32: 1335-1337. Stowe, R.L. and Barnes, N.M. (1998) Cellular distribution of 5HT7 receptor mRNA in rat brain. Br. J. Pharmacol., 123: 229P. Sullivan, G.M., Coplan, J.D., Kent, J.M. and Gorman, J.M. (1999) The noradrengeric system in pathological anxiety: A
663
focus on panic with relevance to generalized anxiety and phobias. Biol. Psychiatry, 46: 1205-1218. Sze, P.Y. and Iqbal, Z. (1994) Glucocorticoid action on depolarization-dependent calcium influx in brain synaptosomes. Neuroendocrinology, 59: 457-465. Takahashi, M., M orinobu, S., Totsuka, S. and Endoh, M. (1996) Chronic dexamethasone administration decreases noradrenaline-stimulated, but not serotonin-stimulated, phosphoinositide metabolism in the rat brain. Naunyn. Schmiedebergs Arch. Pharmacol., 353: 616-620. Takao, K., Nagatani, T., Kitamura, Y. and Yamawaki, S. (1997) Effects of corticosterone on 5HT1A and 5HT2 receptor binding and on the receptor-mediated behavioral responses of rats. Eur. J. Pharmacol., 123-128. Talmi, M., Carlier, E., Rey, M. and Soumireu-Mourat, B. (1992) Modulation of the vitro electrophysiological effect of corticosterone by extracellular calcium in the hippocampus. Neuroendocrinology, 55: 257-263. Tang, W.J. and Gilman, A.G. (1992) Adenylyl cyclase. Cell, 70: 869-872. Tijani-Butt, S.M. and Labow, D.M. (1994) Time course of the effects of adrenalectomy and corticosterone replacement on 5HT1A receptors and 5HT uptake sites in the hippocampus and dorsal raphe nucleus of the rat brain: an autoradiographic analysis. Psychopharmacology, 113:481-486. Torda, T., Yamaguchi, I., Hirata, F., Kopin, I.J. and Axelrod, J. (1981) Mepacrine treatment prevents immobilization-induced desensitization of beta-adrenergic receptors in rat hypothalamus and brain stem. Brain Res., 205: 441-444. Trentani, A., Kuipers, S.D., Ter Horst, G.J. and Den Boer, J.A. (2002) Selective chronic stress-induced in vivo ER1/2 hyperphosphorylation in medial prefrontocortical dendrites: Implications for stress-related cortical pathology? Eur. J. Neurosci.., 15: 1681-1691. Tsuchiya, T., Kishimoto, J., Koyama, J. and Ozawa, T. (1997) Modulatory effect of L-NAME, a specific nitric oxide synthase (NOS) inhibitor, on stress-induced changes in plasma adrenocorticotropic hormone (ACTH) and corticosterone levels in rats: physiological significance of stressinduced NOS activation in hypothalamic-pituitary-adrenal axis. Brian Res., 776: 68-74. U'Prichard, D.C. and Kvetnansky, R. (1980) Central and peripheral adrenergic receptors in acute and repeated immobilization stress. In: Usdin, E., Kvetnansky, R., Kopin, I.J. (Eds.), Catecholamines and stress: Recent Advances, Elsevier, Amsterdam, pp. 293-308.
Vaillant, A.R., Mazzoni, I., Tudan, C., Boudreau, M., Kaplan, D.R. and Miller, F.D. (1999) Depolarization and neurotrophins converge on the phosphatidylinositol-3 kinase-Akt pathway to synergistically regulate neuronal survival. J. Cell. Biol., 146: 955-966. van Veelen, N.M. and Kahn, R.S. (1999) Dopamine, serotonin, and schizophrenia. Adv. Neurol., 80: 425429. Vanhaesebroeck, B., Leevers, S.J., Panayaton, G. and Waterfield, M.D. (1997) The phosphoinositide-3 kinase: a conserved family of signal transducers. Trend Biochem. Sci., 22: 267-272. Ward, R.P., Hamblin, M.W., Lachowicz, J.E., Hoffman, B.J., Sibley, D.R. and Dorsa, D.M. (1995) Localization of serotonin subtype 6 receptor messenger RNA in the rat brain by in situ hybridization histochemistry. Neurosci., 64: 1105-1111. Watanabe, Y., Sakai, R.R., MeEwen, B.S. and Mendelson, S. (1993) Stress and antidepressant effects on hippocampal and cortical 5HTIA and 5HT2 receptors and transport sites for serotonin. Brain Res., 615: 87-94. Weber, C.M., Eke, B.C. and Maines, M.D. (1994) Corticosterone regulates heine oxygenase-2 and NO synthase transcription and protein expression in rat brain. J. Neurochem., 63: 953-962. Weiss, G.K., Ratner, A., Voltura, A., Savage, D., Lucero, K. and Castillo, N. (1994) The effect of two different types of stress on locus coeruleus alpha2 receptor binding. Brain Res. Bull., 33: 219-221. Wolfgang, D., Chen, I. and Wand, G.S. (1995) Effects of restraint stress on components of adenylyl cyclase signal tranduction in the rat hippocampus. Neuropsychopharmacology, ll: 187-193. Yamanaka, K., Muramatsu, I. and Kigoshi, S. (1987) Effect of chronic nicotine treatment against repeated immobilization stress. Pharamcol. Biochem. Behav., 26: 259-263. Yao, R. and Cooper, G.M. (1995) Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor. Science, 267: 2003-2006. Yau, J.L.W., Noble, J., Widdowson, J. and Seckl, J.R. (1997) Impact of adrenalectomy on 5HT6 and 5HT7 receptor gene expression in the rat hippocampus. Mol. Brain Res., 45: 182-186. Yuan, J. and Yankner, B.A. (2000) Apoptosis in the nervous system. Nature, 407: 802-809. Zhong, P. and Ciarnello, R.D. (1995) Transcriptional regulation of hippocampal 5HT1A receptors by corticosteroid hormones. Mol. Brain Res., 29: 23-34.
This Page Intentionally Left Blank
T. Steckler, N.H. Kalin and J.M.H.M. Reul (Eds.) Handbook of Stress and the Brain, Vol. 15
ISBN 0-444-51173-3 Copyright 2005 Elsevier B.V. All rights reserved CHAPTER 5.2
The role of neurotrophic factors in the stress response Marco A. Riva* Center for Neuropharmacology, Department of Pharmacological Sciences, University of Milan, Milan 20133, Italy
Abstract: Neurotrophic factors are important regulators of cell function and participate in activity-dependent synaptic plasticity. Herein these proteins may represent an ideal candidate to mediate short- and long-term effects of stress on brain function and structure. The modulation of different neurotrophic molecules, including neurotrophins and fibroblast growth factors, is rather specific from the anatomical and temporal point of view. Acute stress can upregulate the expression of NGF, NT-3 as well as FGF-2, in different brain structures including the hippocampal formation and hypothalamus, suggesting the possibility that such a rapid effect may represent a protective mechanism to cope with stressful situations. Conversely the regulation of brain-derived neurotrophic factor (BDNF) expression appears to be more complex and region specific. Acute or chronic exposure to different types of stress downregulates BDNF expression in the hippocampus, an effect that may contribute to stress-induced impairment in brain function leading to cellular atrophy and cognitive deterioration. It is believed that pharmacological intervention aimed at normalizing the biosynthesis of neurotrophic factors may represent a novel and valuable strategy to ameliorate defects associated with psychiatric disorders where stress represents a major element of vulnerability.
Introduction
deleterious for neuronal cells and may play a relevant role in several psychiatric disorders, such as depression, schizophrenia, and posttraumatic stress disorders (PTSD), which are characterized by complex changes in brain plasticity (Sapolsky, 2000; Kim and Diamond, 2002). The brain can be considered a major player for the effects exerted by stress on body function. In fact the brain is not only responsible for the interpretation of the stressful experience and the development of proper coping strategies, but it is crucial for the memory of the stressful event. Depending on the type of stress, it has been postulated that the brain can set in motion different coping strategies. First of all, we should bear in mind that the response to psychological stressors is determined by the way an organism perceives and reacts to the stimulus. This individual susceptibility represents a major challenge in the identification of molecular substrate for the effects determined by stress on brain function.
The stress response can be considered a general reaction of an organism, which can monitor internal and external conditions in order to develop proper coping strategies that will allow survival. The stress response occurs through multiple pathways involving hormones and neurotransmitters. The hypothalamicpituitary-adrenal (HPA) system operates to control glucocorticoid hormones secreted by the adrenal glands, which are the most important steroid hormones secreted during stress. Glucocorticoids mobilize energy, increase cardiovascular tone, reinforce aspects of the immune system and modulate different systems of the body. However glucocorticoid hormones exert multiple effects on brain function and neuronal viability: excessive glucocorticoids are *Tel.: +39-02-50318334; Fax: +39-02-50318278; E-mail:
[email protected] 665
666 Second, the response of the brain to stress depends very much upon the timing and the duration of stress exposure. It has been well documented that stress during prenatal and early postnatal development may leave permanent traces on brain function determining changes in the activity of the HPA axis and interfering with the process of neuronal maturation (Ladd et al., 2000; McEwen, 2000a; Meaney, 2001). Later in life, the effects exerted by stress on brain function will depend very much on the type of stress and the length of exposure. On the short-term (acute effects) stress may impact neurotransmission and alter the performance in specific brain functions, including learning abilities (McEwen and Sapolsky, 1995; de Kloet et al., 1999): it is expected that such changes will be finalized to coping and survival. Conversely, prolonged exposure to stress can alter brain activity and function through changes, which usually lead to reduced cellular plasticity and enhanced neuronal vulnerability (Sapolsky, 2000; Kim and Diamond, 2002). Indeed structural alteration and reduced neurogenesis in selected brain regions have been reported to occur following prolonged stress exposure (Gould and Tanapat, 1999; McEwen, 2000a). During aging, a progressive deterioration of the mechanisms governing the function of the HPA axis can be observed and this, together with a "physiological" reduction in neuroplasticity, may lead to a higher susceptibility to stressful events. On this basis, during the last few years, several investigators have begun to address the question of how stress can alter brain function and what are the cellular and molecular correlates of the changes observed following stress exposure. Such strategy would ultimately lead to the identification of "vulnerability" factors, which might become the target of drugs aimed at reinstating normal brain activity. This scenario appears to be of great interest for several neuropsychiatric disorders where stress represents, if not the primary cause, an important vulnerability element. It is becoming widely accepted that the interaction between genetic and environmental factors, stress among the others, is a prerequisite for disease onset. For example, gene polymorphisms have been associated to several neuropsychiatric disorders without necessarily yielding a pathological phenotype, which instead can be
revealed following stress exposure at a certain stage of life. Neurotrophic factors appear to be good candidates for mediating short- and long-term effects of stress on brain function. In fact, this large and heterogeneous class of proteins is not only important in neuronal maturation and survival but plays a more complex role in controlling neuronal function and cellular resilience (Huang and Reichardt, 2001; Poo, 2001). I will therefore review and discuss the data supporting a role of different neurotrophic molecules in the response of the brain to stress, with a specific focus on the anatomical and temporal pattern of these responses. I will deal with two families of neurotrophic molecules, namely the neurotrophins and fibroblast growth factors, since their modulation by stress has been characterized in detail in more recent years.
Neurotrophic factors and brain plasticity Neurotrophic factors represent a wide and heterogeneous class of polypeptides exerting a complex array of activities on different cellular phenotypes. They were originally discovered for their role in development, as target-derived factors, but it is now generally agreed that neurotrophic factors, such as neurotrophins, are molecular effectors of several aspects of brain plasticity: they promote growth and health of neurons, modulate neurotransmitter function and are integral to the modifiability of the central nervous system. Several classes of neurotrophic factors for central and peripheral nervous system have been identified including the neurotrophins, the transforming growth factor 13 superfamily and the fibroblast growth factors. The neurotrophin family of neurotrophic factors comprises several polypeptides including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), NT-4/5, and NT-6. NGF was the first trophic factor to be discovered more than 50 years ago as target-derived trophic molecules that regulates the survival and maturation of developing neurons in the peripheral
667 nervous system (Levi-Montalcini, 1987). However during the last 10-15 years it has become clear that the neurotrophins not only support the survival of postmitotic neurons (Lewin and Barde, 1996), but also regulate other neuronal functions, including axon growth and synaptic plasticity (Black, 1999; McAllister, 1999; Lu and Gottschalk, 2000; Thoenen, 2000). Neurotrophins are synthesized as precursors (proneurotrophins) that are proteolytically cleaved to mature, biologically active neurotrophins (Edwards et al., 1988). It is assumed that they are packaged into vesicles in the soma in direct proportion to the level of their mRNA's, and that they are then transported to either presynaptic axon terminals or postsynaptic dendrites for local secretion. Proneurotrophins, including proNGF and proBDNF, can also be secreted and appear to have an opposite function with respect to their mature forms (Chao and Bothwell, 2002). Neurotrophins activate two different classes of receptors, the tropornyosin-related kinase (trk) family of receptor tyrosine kinases and the p75 receptor, a member of the tumor necrosis factor (TNF) receptor superfamily. Trk receptors appear to mediate almost all the survival-promoting activities of neurotrophins, with NGF activating TrkA, BDNF and NT4/5 activating TrkB, and NT-3 activating TrkC. In addition, NT-3 can also activate, although with lower affinity, TrkA and TrkB. These receptors exist as different splicing variants in their extracellular domain, which may affect ligand interaction (Patapoutian and Reichardt, 2001). The binding of the neurotrophin to Trk dimers initiates trans-autophosphorylation of specific tyrosine residues on the intracellular domain of the receptor (Kaplan and Miller, 2000). These phosphotyrosine residues serve as docking sites for elements of intracellular signaling cascades. Activated receptors initiate several signal transduction cascades, including mitogen-activated protein kinase (MAPK) pathways, the phosphatidylinositol 3-kinase (PI3K) pathway, and the phospholipase C-?, pathway (Kaplan and Miller, 2000; Huang and Reichardt, 2001). There is an extensive literature on the role of neurotrophin in synaptic plasticity. (Thoenen, 1995; Schinder and Poo, 2000; Poo, 2001). High levels of
neurotrophins may induce modifications of synaptic function and the formation of new synaptic contacts. The neurotrophin BDNF has been shown to regulate synaptic transmission across a broad temporal spectrum ranging from short-term modulation, which occurs in the order of seconds to minutes (Kang and Schuman, 1995), to prolonged effects that persist for many hours, such as the long-term potentiation (LTP) (Korte et al., 1995; Patterson et al., 1996) or long-term depression (LTD) response (Linden and Connor, 1995). Furthermore, the activity-dependent secretion of neurotrophins and their acute modulatory effects on synaptic efficacy suggest that they may be responsible for activity-induced LTP or LTD. Genetic deletion of BDNF in mice disrupted normal induction of LTP in the CA1 region of the hippocampus, which was rescued by exogenous administration of the neurotrophin (Korte et al., 1995; Patterson et al., 1996). In cultures of hippocampal neurones, the application of BDNF induces a rapid potentiation of glutamate-mediated synaptic transmission (Schinder et al., 2000), but reduces inhibitory transmission. Most synaptic effects of neurotrophins are accounted for by presynaptic modification of transmitter secretion, which may be triggered by a BDNFinduced increase in cytosolic calcium (Berninger et al., 1993). A recent report has demonstrated that the activity of BDNF at presynaptic sites may involve the phosphorylation of synapsin I (Jovanovic et al., 2000). Further evidence has indicated that BDNF may regulate the number of docked vesicle at CA1 synapses as well as the expression of proteins associated with synaptic vesicles (synapsins, synaptobrevin, synaptophysin and synaptotagmin) (PozzoMiller et al., 1999). The regulation of neurotrophin can occur at several levels. As we will discuss further in detail, a major mechanism is the regulation of their biosynthesis, which mainly occurs through changes in gene transcription. The modulation of neurotrophin biosynthesis is activity-dependent and can involve a complex interplay between neurotransmitters and hormones. However, in the case of BDNF, neuronal activity may also modulate the subcellular targeting of the neurotrophin to distal dendrites (Tongiorgi
668 et al., 1997) as well as its secretion from pre- and postsynaptic compartments (Canossa et al., 1997). Based on these observations neurotrophins, among other trophic molecules, may represent a key factor to link neuronal activity and synaptic plasticity. It is expected that changes in different aspects of the neurotrophin "system" represent one of the mechanisms through which environmental manipulation, such as stress, can alter cellular resilience and modify brain structure. Since the threshold of cellular vulnerability can be determined by the expression and activity of neurotrophic factors, a functional reduction of these elements would be harmful for specific cellular population and may increase the vulnerability of selected cellular phenotypes under challenging situations.
The modulation of neurotrophic factors in response to stress When analyzing the role of neurotrophic factors in the stress response, different aspects must be taken into consideration. The first element is the type of stressor employed. In fact, depending on the experimental paradigm, activation of different brain regions may occur. Although some brain regions, such as the hippocampus, express high levels of glucocorticoid and mineralocorticoid receptors, these hormones do not represent the only player in the stress response, which may also involve enhanced release of several neurotransmitters including noradrenaline, dopamine, and glutamate (Jedema and Moghaddam, 1994; Finlay et al., 1995; Moghaddam, 2002). Hence the modulation of neurotrophic factors following stress may represent one of the mechanisms through which different extracellular signals can integrate to regulate cell function. It can be hypothesized that these responses will depend upon the "state" of these systems and may be altered by pathologic situations, which affect the function of the HPA axis as well as neurotransmitter responses. The second important element is the duration of stress. As we will discuss later on, the functional implication of acute changes may be quite different, even opposed, to the changes observed after prolonged exposure to stress. Acutely, stress can
suppress specific functions via a rapid modulation of neurotrophic factors, but it may also contribute to the activation of protective/defensive mechanisms, which tend to counteract the adverse effects deriving from stress exposure. On the contrary the significance of the changes occurring after chronic stress points toward a reduction and long-term impairment in brain function, characterized by neuronal atrophy and decreased neurogenesis (Gould and Tanapat, 1999; McEwen, 2000a), and may be relevant for diseases that are characterized by enhanced vulnerability to stress. A third aspect is the timing of stress exposure: increasing evidences suggest that adverse life events during brain maturation can increase the vulnerability to psychiatric disorders (Meaney, 2001) and that one important feature of these conditions is a reduced cellular resilience (Duman et al., 2000; Manji et al., 2000). Stressful events during pregnancy or early postnatal life can alter the normal program of neuronal maturation, a highly regulated process that requires the timely expression of different neurotrophic molecules. Hence, stressful experience during specific developmental periods may alter the expression and function of neurotrophic factors, an event that will affect the proper maturation of specific neuronal circuitry. Finally stress-related changes may be qualitatively and quantitatively different in aging, when normal homeostatic mechanisms may be less functional.
Neurotrophins Nerve growth factor Nerve growth factor (NGF) was discovered 50 years ago as a molecule regulating the development and differentiation of peripheral nervous system (PNS) neurons. It has been classically considered a targetderived trophic molecule although this does not appear to be the only mode of action for this neurotrophin (Levi-Montalcini, 1987; Thoenen et al., 1987; Thoenen et al., 1988). NGF is expressed in the CNS, mainly in the hippocampus, cortex and olfactory bulb, where it is developmentally regulated and represents a major trophic support for cholinergic neurons of the basal forebrain (Hefti and
669 Weiner, 1986). N G F can be upregulated in response to injury within peripheral and central nervous system, and endogenous N G F signaling in neuronal and nonneuronal cells is believed to subserve neuroprotective function and facilitate neural repair (Sofroniew et al., 2001). In rodents, an increase in circulating and central levels of N G F has been reported to occur following stress exposure. Social stress, including intermale fighting and maternal aggression, results in an increase of N G F in the blood stream and in the CNS (Lakshmanan, 1986; Spillantini et al., 1989). The raise in serum levels of N G F is rapid, reaching a peak during the first 2-3 h following the fighting session. The source of N G F appears to be the submaxillary salivary gland where the trophic factor is present at very high levels. Interestingly, increased levels of N G F are not necessarily linked to fighting but appear to be related to the social status of the animals. Serum N G F levels of mice with repeated defeat and submission experience showed an increase double that is seen in dominant, attacking mice (Aloe et al., 1986). Moreover, a subordinate-like profile can be induced by systemic N G F administration (Bigi et al., 1992). It has been hypothesized (Alleva and Santucci, 2001) that N G F may exert a physiological role via the adrenal glands and could affect behavior also via changes in the production of adrenal hormones. In fact exogenous administration of N G F results in marked adrenal gland hypertrophy (Aloe et al., 1986; Bigi et al., 1992), an effect more pronounced in the medulla rather in the cortex. A stress-related increase of plasma N G F levels was also reported to occur in humans: an elevation of circulating neurotrophin was indeed observed during the first parachute jump from an airplane by young soldiers (Aloe et al., 1994). This effect appears to be due to the anticipation of the jump, but does not correlate with the activity of the HPA axis. Moreover, an increase was also observed in the high (Trk-A) and low (p75) affinity receptors for NGF, present in peripheral blood lymphocytes of the soldiers. Since N G F induces the expression of interleukin-2 receptors in these cells, it could be hypothesized that the neurotrophin might act on peripheral targets: more specifically, N G F released under a stressful situation may contribute to the reinforcement of the immune responses of the organism.
Stress can also modulate the expression of brain NGF, although the anatomical specificity of this effect depends upon the type of stressor. Aggressive behavior rapidly upregulates N G F m R N A in the hypothalamus, an effect that is not evident in other CNS or peripheral areas such as cerebral cortex, hippocampus and heart (Spillantini et al., 1989). The elevation of hypothalamic N G F m R N A was accompanied by an increase of biologically active N G F (Spillantini et al., 1989), which was not abolished by sialoadenectomy suggesting that the N G F found in the hypothalamus is not of salivary origin (Aloe et al., 1986). Several findings support the hypothesis that hypothalamic N G F might be involved in neuroendocrine function, thereby regulating behavioral outcome. Furthermore, N G F can facilitate structural changes and might affect the levels of other peptides or hormones present in the hypothalamus. Conversely, changes in hippocampal N G F appears to be less reproducible: a long immobilization (8 h) reduces N G F m R N A levels (Ueyama et al., 1997), but a shorter exposure to restraint stress does not alter its expression, although it reduces N G F m R N A levels in basal forebrain (Scaccianoce et al., 2000). An open question is the mechanism whereby stress regulates brain NGF. There is no direct relationship between these changes and variations in plasma corticosterone. Nevertheless, adrenal hormones appear to modulate the expression of the neurotrophin in several brain regions and within selected cellular phenotypes. Removal of adrenal glands reduces the levels of N G F in hippocampus and cerebral cortex (Barbany and Persson, 1992), which is in line with the observation that administration of the synthetic glucocorticoid dexamethasone increase the expression of the neurotrophin in these brain regions, without affecting its levels in hypothalamus, striatum, and cerebellum (Mocchetti et al., 1996). This regulation appears to occur mainly in neurons, although direct exposure of cultured astroglial cells to the synthetic glucocorticoid dexamethasone produces a marked reduction of N G F m R N A contents (Riva et al., 1995b). Furthermore, adrenalectomy reduces, at least in part, the upregulation of N G F expression following kainic acid injection, suggesting that stress and the enhanced release of adrenal hormones, occurring
670 during seizure activity, can also contribute to adaptive changes involving the modulation of this neurotrophin (Barbany and Persson, 1993). The stress sensitivity to NGF is also demonstrated by the effects produced by environmental manipulation occurring during development. An elevation of NGF expression has been observed in hippocampus and hypothalamus following brief or prolonged maternal deprivation during the first week of life (Cirulli, 2001). A similar effect was also reported to occur at postnatal day-ll in pups reared with hypercorticosteronemic mothers (Scaccianoce et al., 2001). These changes may represent a protective coping mechanism to limit the impact of a stressful situation, but could also interfere with the program of brain maturation and contribute to long-lasting changes in brain function as a consequence of adverse life events during development.
Brain-derived neurotrophic factor The regulation of another neurotrophin, BDNF, appears to be somewhat different from NGF and has been investigated by several researchers. Since BDNF is an important player in neuronal plasticity, it may represent an ideal mediator for short- and long-term changes taking place following exposure to stress. Single or repeated immobilization stress decreases BDNF expression in the hippocampus (Smith et al., 1995c; Vaidya et al., 1997; Butterweck et al., 2001; Roceri et al., 2002): the reduction of neurotrophin levels following immobilization is rapid, occurring as early as 1 h after the beginning of the stress, and mainly affects dentate gyrus cells, whereas the effects on CA3 and CA1 layers appears to be less pronounced and consistent (Butterweck et al., 2001). A decrease of hippocampal neurotrophin levels is observed following swim stress (Russo-Neustadt et al., 2001) and it has also been associated with a psychological stress, which consists in the reexposure to cues previously associated with footshock (Rasmusson et al., 2002). The magnitude of stressinduced downregulation of BDNF expression in the hippocampus is quite variable (and not reproduced by all authors) depending upon the type and duration of stress as well as the time elapsed between the end of stress and the sacrifice of the animals.
Corticosterone injection reduces BDNF mRNA levels in hippocampal dentate gyrus, suggesting the contribution of adrenal steroids in stress-induced regulation of BDNF mRNA levels (Smith et al., 1995c; Hansson et al., 2000; Schaaf et al., 2000). Despite the fact that adrenalectomy does not produce any consistent change in BDNF gene expression (Barbany and Persson, 1992; Smith et al., 1995c; Hansson et al., 2000), the downregulation of the neurotrophin following acute restraint stress was less pronounced in adrenalectomized rats, thus suggesting that adrenal hormones contribute, to a certain extent, to stress-induced changes in BDNF expression (Smith et al., 1995c). Interestingly it has been demonstrated that 5HT2A, but not 5HT2C or 5HT1A, receptor antagonists can effectively antagonize the reduction of hippocampal BDNF mRNA following stress, suggesting that an enhanced release of serotonin may play a role in these regulatory mechanisms (Vaidya et al., 1997). It remains to be established if other neurotransmitters such as dopamine and glutamate, which are known to affect BDNF expression and are released during stress, may participate in the modulation of the neurotrophin under adverse conditions. However it is difficult to reconcile an enhanced release of glutamate following stress (Moghaddam, 2002) with hippocampal BDNF downregulation, since it is known that glutamate increases, and not decreases, its expression (Zafra et al., 1990; Kokaia et al., 1993). Conversely, stimulation of dopamine D2 receptors reduces the mRNA levels for BDNF in the hippocampus (Fumagalli et al., 2001), although it is not well established if stress can effectively increase dopamine efflux in the hippocampal region, as it does in other brain regions such as the prefrontal cortex and nucleus accumbens (Finlay et al., 1995). Since stress is an important component of many neuropsychiatric disorders, a reduced production of BDNF, mainly within the dentate gyrus, can contribute to some of the defects that characterize stress-related disorders, such as depression and PTSD. Prolonged exposure to stress or glucocorticoids may determine structural changes within the hippocampus, characterized by atrophy of apical dendrites in CA3 pyramidal neurons, which is accompanied by specific cognitive deficits in spatial learning and memory (Magarinos et al., 1997;
671 McEwen, 2000a,b; Sapolsky, 2000). Such effect may originate from a loss of neuropil volume, an inhibition of the genesis of new neurons and glia or the loss of pre-existing neuronal or glial cells. Even though it is known that glutamate can play a role in these events (McEwen, 2000b), we may speculate that the dendritic atrophy, that is observed following stress, can be a consequence of reduced trophic supports provided by BDNF (and other trophic factors) in this structure. Although withdrawal of trophic factor support may not necessarily lead, per se, to cellular damage or neuronal cell death, it may render specific structures or cellular phenotypes more vulnerable to toxic or noxious stimuli. With this regard, it may be hypothesized that, under stressful situations, the vulnerability threshold of a specific cellular phenotype can be reduced because the protective "supply" of neurotrophin is diminished. Furthermore stress can alter brain function with impairment in learning and memory: it must be kept in mind that BDNF, beyond its important neurotrophic activity, exerts a relevant role in neuronal plasticity, regulates neurotransmitter release and has a role in memory and cognition (Schinder and Poo, 2000; Poo, 2001). Although the role of stress and corticosteroids on cognition depends upon the context of the learning task (de Kloet et al., 1999), the rapid reduction of BDNF m R N A levels following stress may prevent the initial encoding of contextual memory associated with a traumatic experience, thus representing a sort of defensive mechanism. However, a reduced expression of the neurotrophin after prolonged severe stress, may be one of the mechanisms through which stress and glucocorticoid hormones will impair brain function leading to cognitive deterioration and cellular atrophy (Magarinos et al., 1997; de Kloet et al., 1999; McEwen, 2000b). If this is the case pharmacological intervention aimed at reinstating the correct expression of BDNF may represent a valuable strategy to ameliorate functional defects associated with stress. Accordingly electroconvulsive seizure therapy and chronic antidepressant treatment blocked the downregulation of BDNF m R N A in the hippocampus in response to restraint stress (Nibuya et al., 1995) although, in another report, chronic exposure to imipramine failed to counteract the changes
occurring in hippocampal BDNF after acute or chronic immobilization (Butterweck et al., 2001). A further note is that stress may not directly alter the basal expression of a specific trophic factor, but it may interfere with its activity-dependent regulation. For example, BDNF expression is upregulated during learning and exercise, as well as in other experimental paradigms, and these mechanisms may be impaired following stress exposure. If this is the case, the alteration of BDNF activity can be "unmasked" during specific tasks that require the proper modulation of the neurotrophin expression. Another aspect that deserves attention is the anatomical specificity of stress-related changes in BDNF expression. Acute or repeated immobilization stress increases BDNF m R N A levels in the hypothalamus and pituitary (Smith et al., 1995b), suggesting that, similar to NGF, this neurotrophin may be important in brain regions that are primarily involved in the function of the HPA axis. This finding was confirmed by other investigators, who demonstrated not only a rapid upregulation of the neurotrophin but a differential modulation of its transcripts, which originates by specific 5' exons, in response to immobilization (Rage et al., 2002). The rat BDNF gene has a complex structure, with four 5' noncoding exons (exons I to IV) and one 3' exon (exon V) encoding the mature BDNF protein (Timmusk et al., 1993). Transcription of these exons is regulated by multiple promoters that yield ten different BDNF mRNAs: although each BDNF m R N A encodes an identical BDNF protein, these transcripts are differentially expressed and regulated throughout the brain (Timmusk et al., 1993; Lauterborn et al., 1998). This may affect RNA stability and trafficking, as well as translation, suggesting that a differential usage of each promoter may represent an important mechanism in the regulation of BDNF function. Following stress it was shown that the increase of exon Ill m R N A levels was rapid (within 15 min) but transient, whereas exon I expression was increased only after 3 h of immobilization (Rage et al., 2002). These changes are localized in the paraventricular and supraoptic nuclei suggesting a possible role of the neurotrophin in autocrine and paracrine regulation of arginin-vasopressin secretory process, as well as in the neuroplastic and functional changes of the hypothalamus following stress.
672 Reducing glucocorticoid feedback through adrenalectomy upregulates BDNF expression in the paraventricular nuclei of the hypothalamus, as well as in the anterior pituitary, an effect that was normalized by corticosterone replacement therapy (Smith et al., 1995b). Furthermore the elevation of hypothalamic BDNF levels after immobilization stress appears to be more pronounced in adrenalectomized rats. These data suggest that adrenal hormones exert an inhibitory role on BDNF expression in these regions reminiscent of their negative feedback on corticotrophin-releasing factor (CRF) in the paraventricular nuclei, but not in extrahypothalamic areas. The mechanism by which stress can increase the neurotrophin expression in the hypothalamus and pituitary gland has not been established. Glutamate is a likely player because it is released during stress and it is able to stimulate BDNF transcription (Zafra et al., 1991), although other mediators, such as IL-113 may also contribute to the observed effects. The role of BNDF in the hypothalamus is a matter of investigation. Apart from its ability to modulate dendritic branching and synaptic strength (Poo, 2001), BDNF may mediate the synthesis of specific neuropeptides (Carnahan and Nawa, 1995) and appears to be involved in the control of body weight and food consumption. Exogenous administration of the neurotrophin can determine a reduction in weight, whereas mice with a conditional knockout of the BDNF gene show an increase in body weight as well as in daily food intake, effects that can in part be mediated by elevated levels of leptin and insulin (Rios et al., 2001). Interestingly, weight loss is observed during prolonged stress, suggesting a possible association of this effect with the upregulation of hypothalamic BDNF expression. Another brain structure where BDNF expression is regulated by stress is the prefrontal-cingulate cortex, whose function appears to be altered in schizophrenia and depression. A single immobilization stress increases BDNF mRNA levels both in prefrontal and cingulate cortex (Molteni et al., 2001b), an effect that, similar to the hypothalamus, may be attributed to an enhanced release of dopamine and glutamate (Finlay et al., 1995; Moghaddam, 2002) rather than being related to glucocorticoids. Interestingly, chronic stress reduces the expression of the neurotrophin in prefrontal
cortex (Roceri et al., 2004), suggesting that adaptive changes taking place following chronic stress may recapitulate defects observed in psychiatric disorders, including reduced activity of dopamine and glutamate within the prefrontal cortex, which may ultimately contribute to BDNF downregulation. On this basis, although a rapid elevation of BDNF mRNA levels may represent a compensatory-protective mechanism, prolonged exposure to stress will have a negative impact on the neuroplastic capacity of prefrontal cortex function through a downregulation of the neurotrophin expression. Stress-induced changes in BDNF expression are attenuated in aged animals. It has been reported that aging per se decreases the hippocampal levels of the neurotrophin in rats as well as in monkeys (Smith and Cizza, 1996; Hayashi et al., 2001). Exposure of old rats to an immobilization stress determined a less pronounced reduction of hippocampal expression and failed to alter the levels of BDNF mRNA in the hypothalamus, suggesting that different molecular mechanisms may subserve the modulation of this neurotrophin in specific brain structures. Since the acute modulation of NGF under the same experimental conditions was not affected by aging, it may be inferred that the age-dependent impairment in these regulatory mechanisms may be specific for BDNF (Smith and Cizza, 1996). There is evidence that the stress-induced regulation of BDNF expression may represent a mechanism contributing to long-term changes in brain plasticity following stress exposure during development. It is known that stressful events occurring early in development may alter the normal program of brain maturation and produce long-lasting effects on brain function, which alter stress responsiveness and may increase the vulnerability to psychiatric disorders (Meaney, 2001). Accordingly, several animal models that can mimic such events have been developed in order to characterize the molecular changes contributing to long-term impairment in brain function (Lipska and Weinberger, 2000). A reduction of hippocampal BDNF mRNA and protein levels occurred in adult rats that were exposed to a single period of 24 maternal deprivation on postnatal day 9 (Roceri et al., 2002), suggesting that changes in neurotrophin expression may represent one of the mechanisms through which
673 stress during development produce long-lasting effects on brain function. Although the regulation of BDNF biosynthesis following stress exposure has been investigated in great detail, limited data are available on the changes produced by stress on BDNF signaling machinery. The expression of Trk-B, the high-affinity receptor for BDNF is reduced after an acute immobilization stress in the hypothalamus (Givalois et al., 2001), whereas the catalytic form of the receptor appears to be upregulated in the hippocampus following chronic stress (Nibuya et al., 1999). These changes may oppose those observed for BDNF suggesting the possibility that they may represent a compensatory mechanism to stress exposure.
Neurotrophin-3 The regulation of neurotrophin-3 following stress is opposite with respect to BDNF. In fact, its m R N A levels are upregulated in the hippocampus by chronic, but not acute, stress or following prolonged exposure to corticosterone (Barbany and Persson, 1992; Smith et al., 1995c). A similar upregulation of NT-3 expression has also been observed in the locus coeruleus (Smith et al., 1995a). Surgical removal of the adrenal glands reduces NT-3 m R N A levels in hippocampal dentate gyrus and within the CA2 pyramidal layer (Barbany and Persson, 1992; Hansson et al., 2000) and prevents stress-induced changes of the neurotrophin (Smith et al., 1995c), thus suggesting the important role exerted by circulating glucocorticoids in these mechanisms. In analogy to what has been observed with other trophic molecules, such as FGF-2 (see later), it may be hypothesized that the upregulation of NT-3 biosynthesis may represent a compensatory response aimed at preventing or limiting stress-induced damage in the hippocampus.
Fibroblast growth factors FGF-2 is the prototype member of a large family of neurotrophic molecules existing in different protein isoforms, which display selective subcellular localization thus implying different functional roles (Bikfalvi et al., 1997). FGF-2 binds to four related tyrosine
kinase receptors (FGFR1-4), which exist in different splice variants and, in many respects, appear similar to other growth factor receptors (Klint and ClaessonWelsh, 1999). FGF-2, a potent angiogenic factor, may stimulate hematopoiesis and play an important role in the differentiation and function of the central nervous system. Its m R N A and protein are found at relatively high concentrations in several regions of the embryonic, postnatal, and adult central nervous system, where it is mainly expressed in astroglial cells. FGF-2 can stimulate neonatal and adult brain neurogenesis (Palmer et al., 1999), plays an important role in regeneration after CNS injury and may participate in a cascade of events to facilitate neuronal repair and survival. Its neuroprotective activity has been shown over a wide range of neuronal phenotypes in vitro (Mattson et al., 1989) as well as in vivo (Anderson et al., 1988; Otto and Unsicker, 1990). The expression of FGF-2 in the brain is regulated by different neurotransmitter and hormonal pathways. In accordance with the possibility that its modulation may represent a rapid protective mechanism to maintain cell homeostasis and reduce neuronal damage under challenging or adverse situations, an acute immobilization stress determines an upregulation of FGF-2 in several brain regions of adult animals (Molteni et al., 2001a). Within some structures, such as the hippocampal formation, the effect is probably mediated by glucocorticoid hormones: in fact exogenous administration of adrenal steroids increases FGF-2 m R N A and protein levels (Riva et al., 1995b; Hansson et al., 2000), whereas adrenalectomy reduces its expression in hippocampus and frontal cortex (Riva et al., 1995a; Hansson et al., 2000). However we cannot rule out the possibility that neurotransmitters, such as dopamine, glutamate or norephinephrine, which are released upon stress exposure, can contribute to stress-related changes in FGF-2 expression. In fact, it has been previously shown that all these mediators can increase the biosynthesis of FGF-2 in specific brain structures (Follesa and Mocchetti, 1993; Riva et al., 1996; Roceri et al., 2001). The modulation of FGF-2 by stress is rapid and transient and, to some extent, resembles the modulation of FGF-2 described in other experimental paradigms (Riva et al., 1992; Riva et al., 1994).
674 Being that FGF-2 is neuroprotective (Anderson et al., 1988; Otto and Unsicker, 1990) and that its endogenous production may be relevant to preserve neuronal function under adverse situations (Rowntree and Kolb, 1997), it may be inferred that the prompt upregulation after stress exposure represents a possible strategy to preserve neuronal viability under challenging situations. It is interesting to notice that the expression of FGF-2 is induced by stress in dopamine producing regions (ventral tegmental area and substantia nigra) as well as in dopaminergic target regions, including striatum and prefrontal cortex, to underline a close relation between stress, dopamine and FGF-2. In this regard, we may speculate that the upregulation of the trophic factor may contribute to the sensitizing effects of stress and glucocorticoids in analogy to what has been previously described for amphetamine (Flores et al., 1998; Flores et al., 2000). Although the regulation of FGF-1 (acidic FGF), a close congener of FGF-2, has not been investigated following stress exposure, surgical removal of adrenal glands reduces its m R N A levels in frontal cortex and prevents its upregulation elicited by kainic acid (Riva et al., 1995a), suggesting a functional role of adrenal steroids also in the control of brain FGF-1 expression.
Conclusions The data accumulated over the last few years demonstrate that profound alterations in the expression profile of trophic molecules, mainly neurotrophins and fibroblast growth factors, can take place in response to stress. These changes can occur at different developmental stages and with a specific anatomical profile, thus suggesting that they depend upon different molecular mechanisms. Most of the studies have focussed on changes in m R N A and protein levels of different neurotrophic factors, but limited information is still available on their signaling pathways. In particular it will be crucial to assess if stress exposure may affect not only the biosynthesis of these proteins within selected brain regions, but could also regulate their subcellular distribution, local translation and receptor interaction. For example, in the case of BDNF, it will be important
to analyze different isoforms and investigate whether the changes in the transcription of 5' exons will interfere with the localization and targeting of the neurotrophin within specific cellular populations. Furthermore, changes in signaling pathways will ultimately influence post-receptor mechanisms involving the modulation of different intracellular cascades that, for example, might lead to changes in proteins involved in cellular survival and vulnerability (Manji et al., 2000). In summary, changes in the expression and function of neurotrophic factors following stress may represent a major component of the plastic changes set in motion within the CNS in order to cope with challenging situations. However repetitive stress or prolonged adverse situations may lead to a protracted reduction of neurotrophic factor production that could enhance cellular vulnerability. If this is indeed the case, pharmacological strategies aimed at reinstating the normal expression and function of neurotrophic molecules within selected brain regions might prove useful for the treatment of neuropsychiatric disorders where stress represent a major vulnerability factor.
References Alleva, E. and Santucci, D. (2001) Psychosocial vs. "physical" stress situations in rodents and humans: role of neurotrophins. Physiol. Behav., 73: 313-320. Aloe, L., Alleva, E., Bohm, A. and Levi-Montalcini, R. (1986) Aggressive behavior induces release of nerve growth factor from mouse salivary gland into the bloodstream. Proc. Natl. Acad. Sci. U S A, 83: 6184-6187. Aloe, L., Bracci-Laudiero, L., Alleva, E., Lambiase, A., Micera, A. and Tirassa, P. (1994) Emotional stress induced by parachute jumping enhances blood nerve growth factor levels and the distribution of nerve growth factor receptors in lymphocytes. Proc. Natl. Acad. Sci. U S A, 91: 10440-10444. Anderson, K.J., Dam, D., Lee, S. and Cotman, C.W. (1988) Basic fibroblast growth factor prevents death of lesioned cholinergic neurons in vivo. Nature, 332: 360-361. Barbany, G. and Persson, H. (1992) Regulation of Neurotrophin mRNA Expression in the Rat Brain by Glucocorticoids. Eur. J. Neurosci., 4: 396-403. Barbany, G. and Persson, H. (1993) Adrenalectomy attenuates kainic acid-elicited increases of messenger RNAs for neurotrophins and their receptors in the rat brain. Neuroscience, 54: 909-922.
675 Berninger, B., Garcia, D.E., Inagaki, N., Hahnel, C. and Lindholm, D. (1993) BDNF and NT-3 induce intracellular Ca2§ elevation in hippocampal neurones. Neuroreport, 4: 1303-1306. Bigi, S., Maestripieri, D., Aloe, L. and Alleva, E. (1992) NGF decreases isolation-induced aggressive behavior, while increasing adrenal volume, in adult male mice. Physiol. Behav., 51: 337-343. Bikfalvi, A., Klein, S., Pintucci, G. and Rifkin, D.B. (1997) Biological roles of fibroblast growth factor-2. Endocr. Rev., 18: 26-45. Black, I.B. (1999) Trophic regulation of synaptic plasticity. J. Neurobiol., 41: 108-118. Butterweck, V., Winterhoff, H. and Herkenham, M. (2001) St John's wort, hypericin, and imipramine: a comparative analysis of mRNA levels in brain areas involved in HPA axis control following short-term and long-term administration in normal and stressed rats. Mol. Psychiatry, 6: 547-564. Canossa, M., Griesbeck, O., Berninger, B., Campana, G., Kolbeck, R. and Thoenen, H. (1997) Neurotrophin release by neurotrophins: implications for activity-dependent neuronal plasticity. Proc. Natl. Acad. Sci. U S A, 94: 13279-13286. Carnahan, J. and Nawa, H. (1995) Regulation of neuropeptide expression in the brain by neurotrophins. Potential role in vivo. Mol. Neurobiol., 10: 135-149. Chao, M.V. and Bothwell, M. (2002) Neurotrophins: to cleave or not to cleave. Neuron, 33: 9-12. Cirulli, F. (2001) Role of environmental factors on brain development and nerve growth factor expression. Physiol. Behav., 73: 321-330. de Kloet, E.R., Oitzl, M.S. and Joels, M. (1999) Stress and cognition: are corticosteroids good or bad guys? Trends Neurosci., 22: 422-426. Duman, R.S., Malberg, J., Nakagawa, S. and D'Sa, C. (2000) Neuronal plasticity and survival in mood disorders. Biol. Psychiatry, 48: 732-739. Edwards, R.H., Selby, M.J., Garcia, P.D. and Rutter, W.J. (1988) Processing of the native nerve growth factor precursor to form biologically active nerve growth factor. J. Biol. Chem., 263: 6810-6815. Eide, F.F., Vining, E.R., Eide, B.L., Zang, K., Wang, X.Y. and Reichardt, L.F. (1996) Naturally occurring truncated trkB receptors have dominant inhibitory effects on brain-derived neurotrophic factor signaling. J. Neurosci., 16: 3123-3129. Finlay, J.M., Zigmond, M.J. and Abercrombie, E.D. (1995) Increased dopamine and norepinephrine release in medial prefrontal cortex induced by acute and chronic stress: effects of diazepam. Neuroscience, 64: 619-628. Flores, C., Rodaros, D. and Stewart, J. (1998) Long-lasting induction of astrocytic basic fibroblast growth factor by repeated injections of amphetamine: blockade by concurrent treatment with a glutamate antagonist. J. Neurosci., 18: 9547-9555.
Flores, C., Samaha, A.N. and Stewart, J. (2000) Requirement of endogenous basic fibroblast growth factor for sensitization to amphetamine. J. Neurosci., 20: RC55. Follesa, P. and Mocchetti, I. (1993) Regulation of basic fibroblast growth factor and nerve growth factor mRNA by beta-adrenergic receptor activation and adrenal steroids in rat central nervous system. Mol. Pharmacol., 43: 132-138. Fumagalli, F., Santero, R., Gennarelli, M., Giorgio, R. and Andrea Riva, M. (2001) Decreased hippocampal BDNF expression after acute systemic injection of quinpirole. Neuropharmacology, 40: 954-957. Givalois, L., Marmigere, F., Rage, F., Ixart, G., Arancibia, S. and Tapia-Arancibia, L. (2001) Immobilization stress rapidly and differentially modulates BDNF and TrkB mRNA expression in the pituitary gland of adult male rats. Neuroendocrinology, 74: 148-159. Gould, E. and Tanapat, P. (1999) Stress and hippocampal neurogenesis. Biol. Psychiatry, 46: 1472-1479. Hansson, A.C., Cintra, A., Belluardo, N., Sommer, W., Bhatnagar, M., Bader, M., Ganten, D. and Fuxe, K. (2000) Gluco- and mineralocorticoid receptor-mediated regulation of neurotrophic factor gene expression in the dorsal hippocampus and the neocortex of the rat. Eur. J. Neurosci., 12: 2918-2934. Hayashi, M., Mistunaga, F., Ohira, K. and Shimizu, K. (2001) Changes in BDNF-immunoreactive structures in the hippocampal formation of the aged macaque monkey. Brain Res., 918: 191-196. Hefti, F. and Weiner, W.J. (1986) Nerve growth factor and Alzheimer's disease. Ann. Neurol., 20: 275-281. Huang, E.J. and Reichardt, L.F. (2001) Neurotrophins: roles in neuronal development and function. Annu. Rev. Neurosci., 24: 677-736. Jedema, H.P. and Moghaddam, B. (1994) Glutamatergic control of dopamine release during stress in the rat prefrontal cortex. J. Neurochem., 63: 785-788. Jovanovic, J.N., Czernik, A.J., Fienberg, A.A., Greengard, P. and Sihra, T.S. (2000) Synapsins as mediators of BDNFenhanced neurotransmitter release. Nat. Neurosci., 3: 323-329. Kang, H.J. and Schuman, E.M. (1995) Neurotrophin-induced modulation of synaptic transmission in the adult hippocampus. J. Physiol. Paris, 89:11-22. Kaplan, D.R. and Miller, F.D. (2000) Neurotrophin signal transduction in the nervous system. Curr. Opin. Neurobiol., 10: 381-391. Kim, J.J. and Diamond, D.M. (2002) The stressed hippocampus, synaptic plasticity and lost memories. Nat. Rev. Neurosci., 3: 453-462. Klein, R., Conway, D., Parada, L.F. and Barbacid, M. (1990) The trkB tyrosine protein kinase gene codes for a second neurogenic receptor that lacks the catalytic kinase domain. Cell, 61: 647-656.
676 Klint, P. and Claesson-Welsh, L. (1999) Signal transduction by fibroblast growth factor receptors. Frontiers in Bioscience, 4: D 165-177. Knipper, M., da Penha Berzaghi, M., Blochl, A., Breer, H., Thoenen, H. and Lindholm, D. (1994) Positive feedback between acetylcholine and the neurotrophins nerve growth factor and brain-derived neurotrophic factor in the rat hippocampus. Eur. J. Neurosci., 6: 668-671. Kokaia, Z., Gido, G., Ringstedt, T., Bengzon, J., Kokaia, M., Siesjo, B.K., Persson, H. and Lindvall, O. (1993) Rapid increase of BDNF mRNA levels in cortical neurons following spreading depression: regulation by glutamatergic mechanisms independent of seizure activity. Brain Res. Mol. Brain Res., 19: 277-286. Korte, M., Carroll, P., Wolf, E., Brem, G., Thoenen, H. and Bonhoeffer, T. (1995) Hippocampal long-term potentiation is impaired in mice lacking brain-derived neurotrophic factor. Proc. Natl. Acad. Sci. U S A, 92: 8856-8860. Ladd, C.O., Huot, R.L., Thrivikraman, K.V., Nemeroff, C.B., Meaney, M.J. and Plotsky, P.M. (2000) Long-term behavioral and neuroendocrine adaptations to adverse early experience. Prog. Brain Res., 122: 81-103. Lakshmanan, J. (1986) Beta-nerve growth factor measurements in mouse serum. J. Neurochem., 46: 882-891. Lauterborn, J.C., Poulsen, F.R., Stinis, C.T., Isackson, P.J. and Gall, C.M. (1998) Transcript-specific effects of adrenalectomy on seizure-induced BDNF expression in rat hippocampus. Brain Res. Mol. Brain Res., 55: 81-91. Levi-Montalcini, R. (1987) The nerve growth factor 35 years later. Science, 237:1154-1162. Lewin, G.R. and Barde, Y.A. (1996) Physiology of the neurotrophins. Annu. Rev. Neurosci., 19: 289-317. Linden, D.J. and Connor, J.A. (1995) Long-term synaptic depression. Annu. Rev. Neurosci., 18: 319-357. Lipska, B.K. and Weinberger, D.R. (2000) To model a psychiatric disorder in animals: schizophrenia as a reality test. Neuropsychopharmacology, 23: 223-239. Lu, B. and Gottschalk, W. (2000) Modulation of hippocampal synaptic transmission and plasticity by neurotrophins. Prog. Brain Res., 128:231-241. Magarinos, A.M., Verdugo, J.M. and McEwen, B.S. (1997) Chronic stress alters synaptic terminal structure in hippocampus. Proc. Natl. Acad. Sci. USA, 94: 14002-14008. Manji, H.K., Moore, G.J., Rajkowska, G. and Chen, G. (2000) Neuroplasticity and cellular resilience in mood disorders. Mol. Psychiatry, 5: 578-593. Mattson, M.P., Murrain, M., Guthrie, P.B. and Kater, S.B. (1989) Fibroblast growth factor and glutamate: opposing roles in the generation and degeneration of hippocampal neuroarchitecture. J. Neurosci., 9: 3728-3740. McAllister, A.K. (1999) Subplate neurons: a missing link among neurotrophins, activity, and ocular dominance plasticity? Proc. Natl. Acad. Sci. U S A, 96: 13600-13602.
McEwen, B.S. (2000a) Effects of adverse experiences for brain structure and function. Biol. Psychiatry, 48: 721-731. McEwen, B.S. (2000b) The neurobiology of stress: from serendipity to clinical relevance. Brain Res., 886: 172-189. McEwen, B.S. and Sapolsky, R.M. (1995) Stress and cognitive function. Curr. Opin. Neurobiol., 5: 205-216. Meaney, M.J. (2001) Maternal care, gene expression, and the transmission of individual differences in stress reactivity across generations. Annu. Rev. Neurosci., 24: 1161-1192. Mocchetti, I., Spiga, G., Hayes, V.Y., Isackson, P.J. and Colangelo, A. (1996) Glucocorticoids differentially increase nerve growth factor and basic fibroblast growth factor expression in the rat brain. J. Neurosci., 16:2141-2148. Moghaddam, B. (2002) Stress activation of glutamate neurotransmission in the prefrontal cortex: implications for dopamine-associated psychiatric disorders. Biol. Psychiatry, 51: 775-787. Molteni, R., Fumagalli, F., Magnaghi, V., Roceri, M., Gennarelli, M., Racagni, G., Melcangi, R.C. and Riva, M.A. (2001a) Modulation of fibroblast growth factor-2 by stress and corticosteroids: from developmental events to adult brain plasticity. Brain Res. Brain Res. Rev., 37: 249-258. Molteni, R., Lipska, B.K., Weinberger, D.R., Racagni, G. and Riva, M.A. (2001b) Developmental and stress-related changes of neurotrophic factor gene expression in an animal model of schizophrenia. Mol. Psychiatry, 6: 285-292. Nibuya, M., Morinobu, S. and Duman, R.S. (1995) Regulation of BDNF and trkB mRNA in rat brain by chronic electroconvulsive seizure and antidepressant drug treatments. J. Neurosci., 15: 7539-7547. Nibuya, M., Takahashi, M., Russell, D.S. and Duman, R.S. (1999) Repeated stress increases catalytic TrkB mRNA in rat hippocampus. Neurosci. Lett., 267: 81-84. Ninkina, N., Adu, J., Fischer, A., Pinon, L.G., Buchman, V.L. and Davies, A.M. (1996) Expression and function of TrkB variants in developing sensory neurons. EMBO J., 15: 6385-6393. Otto, D. and Unsicker, K. (1990) Basic FGF reverses chemical and morphological deficits in the nigrostriatal system of MPTP-treated mice. J. Neurosci., 10: 1912-1921. Palmer, T.D., Markakis, E.A., Willhoite, A.R., Safar, F. and Gage, F.H. (1999) Fibroblast growth factor-2 activates a latent neurogenic program in neural stem cells from diverse regions of the adult CNS. J. Neurosci., 19: 8487-8497. Patapoutian, A. and Reichardt, L.F. (2001) Trk receptors: mediators of neurotrophin action. Curr. Opin. Neurobiol., 11: 272-280. Patterson, S.L., Abel, T., Deuel, T.A., Martin, K.C., Rose, J.C. and Kandel, E.R. (1996) Recombinant BDNF rescues deficits in basal synaptic transmission and hippocampal LTP in BDNF knockout mice. Neuron, 16: 1137-1145. Poo, M.M. (2001) Neurotrophins as synaptic modulators. Nature Rev. Neurosci., 2: 24-32.
677 Pozzo-Miller, L.D., Gottschalk, W., Zhang, L., McDermott, K., Du, J., Gopalakrishnan, R., Oho, C., Sheng, Z.H. and Lu, B. (1999) Impairments in high-frequency transmission, synaptic vesicle docking, and synaptic protein distribution in the hippocampus of BDNF knockout mice. J. Neurosci., 19: 4972-4983. Rage, F., Givalois, L., Marmigere, F., Tapia-Arancibia, L. and Arancibia, S. (2002) Immobilization stress rapidly modulates BDNF mRNA expression in the hypothalamus of adult male rats. Neuroscience, 112:309-318. Rasmusson, A.M., Shi, k. and Duman, R. (2002) Downregulation of BDNF mRNA in the hippocampal dentate gyrus after re-exposure to cues previously associated with footshock. Neuropsychopharmacology, 27: 133-142. Rios, M., Fan, G., Fekete, C., Kelly, J., Bates, B., Kuehn, R., Lechan, R.M. and Jaenisch, R. (2001) Conditional deletion of brain-derived neurotrophic factor in the postnatal brain leads to obesity and hyperactivity. Mol. Endocrinol., 15: 1748-1757. Riva, M.A., Donati, E., Tascedda, F., Zolli, M. and Racagni, G. (1994) Short- and long-term induction of basic fibroblast growth factor gene expression in rat central nervous system following kainate injection. Neuroscience, 59: 55-65. Riva, M.A., Fumagalli, F., Blom, J.M., Donati, E. and Racagni, G. (1995a) Adrenalectomy reduces FGF-1 and FGF-2 gene expression in specific rat brain regions and differently affects their induction by seizures. Mol. Brain Res., 34: 190-196. Riva, M.A., Fumagalli, F. and Racagni, G. (1995b) Opposite regulation of basic fibroblast growth factor and nerve growth factor gene expression in rat cortical astrocytes following dexamethasone treatment. J. Neurochem., 64: 2526-2533. Riva, M.A., Gale, K. and Mocchetti, I. (1992) Basic fibroblast growth factor mRNA increases in specific brain regions following convulsive seizures. Mol. Brain Res., 15:311-318. Riva, M.A., Molteni, R., Lovati, E., Fumagalli, F., Rusnati, M. and Racagni, G. (1996) Cyclic AMP-dependent regulation of fibroblast growth factor-2 messenger RNA levels in rat cortical astrocytes: comparison with fibroblast growth factor-1 and ciliary neurotrophic factor. Mol. Pharmacol., 49: 699-706. Roceri, M., Molteni, R., Fumagalli, F., Gennarelli, M., Racagni, G., Corsini, G.U., Maggio, R. and Riva, M.A. (2001) Stimulatory role of dopamine on FGF-2 expression in rat striatum. J. Neurochem., 76: 990-997. Roceri, M., W., H., Racagni, G., B.A., E. and Riva, M.A. (2002) Early maternal deprivation reduces the expression of BDNF and NMDA receptor subunits in rat hippocampus. Mol. Psychiatry, 7: 609-616. Roceri, M., Cirulli, F., Pessina, C., Peretto, P., Racagni, G. and Riva, M.A. (2004) Postnatal repeated maternal deprivation produces age dependent changes of BDNF expression in selected rat brain regions. Biological Psychiatry, 55: 708-714.
Rowntree, S. and Kolb, B. (1997) Blockade of basic fibroblast growth factor retards recovery from motor cortex injury in rats. Eur. J. Neurosci., 9: 2432-2441. Russo-Neustadt, A., Ha, T., Ramirez, R. and Kesslak, J.P. (2001) Physical activity-antidepressant treatment combination: impact on brain-derived neurotrophic factor and behavior in an animal model. Behav. Brain Res., 120: 87-95. Sapolsky, R.M. (2000) Glucocorticoids and hippocampal atrophy in neuropsychiatric disorders. Archives of General Psychiatry, 57: 925-935. Scaccianoce, S., Catalani, A., Lombardo, K., Consoli, C. and Angelucci, L. (2001) Maternal glucocorticoid hormone influences nerve growth factor expression in the developing rat brain. Neuroreport, 12: 2881-2884. Scaccianoce, S., Lombardo, K. and Angelucci, L. (2000) Nerve growth factor brain concentration and stress: changes depend on type of stressor and age. Int. J. Dev. Neurosci., 18: 469-479. Schaaf, M.J., De Kloet, E.R. and Vreugdenhil, E. (2000) Corticosterone effects on BDNF expression in the hippocampus. Implications for memory formation. Stress, 3: 201-208. Schinder, A.F., Berninger, B. and Poo, M. (2000) Postsynaptic target specificity of neurotrophin-induced presynaptic potentiation. Neuron, 25: 151-163. Schinder, A.F. and Poo, M.M. (2000) The neurotrophin hypothesis for synaptic plasticity. Trends Neurosci., 23: 639-645. Smith, M.A. and Cizza, G. (1996) Stress-induced changes in brain-derived neurotrophic factor expression are attenuated in aged Fischer 344/N rats. Neurobiol. Aging, 17: 859-864. Smith, M.A., Makino, S., Altemus, M., Michelson, D., Hong, S.K., Kvetnansky, R. and Post, R.M. (1995a) Stress and antidepressants differentially regulate neurotrophin 3 mRNA expression in the locus coeruleus. Proc. Natl. Acad. Sci. U S A, 92: 8788-8792. Smith, M.A., Makino, S., Kim, S.Y. and Kvetnansky, R. (1995b) Stress increases brain-derived neurotropic factor messenger ribonucleic acid in the hypothalamus and pituitary. Endocrinology, 136: 3743-3750. Smith, M.A., Makino, S., Kvetnansky, R. and Post, R.M. (1995c) Stress and glucocorticoids affect the expression of brain-derived neurotrophic factor and neurotrophin-3 mRNAs in the hippocampus. J. Neurosci., 15: 1768-1777. Sofroniew, M.V., Howe, C.L. and Mobley, W.C. (2001) Nerve growth factor signaling, neuroprotection, and neural repair. Annu. Rev. Neurosci., 24: 1217-1281. Spillantini, M.G., Aloe, L., Alleva, E., De Simone, R., Goedert, M. and Levi-Montalcini, R. (1989) Nerve growth factor mRNA and protein increase in hypothalamus in a mouse model of aggression. Proc. Natl. Acad. Sci. U S A, 86: 8555-8559. Thoenen, H. (1995) Neurotrophins and neuronal plasticity. Science, 270: 593-598.
678 Thoenen, H. (2000) Neurotrophins and activity-dependent plasticity. Prog. Brain Res., 128: 183-191. Thoenen, H., Bandtlow, C. and Heumann, R. (1987) The physiological function of nerve growth factor in the central nervous system: comparison with the periphery. Rev. Physiol. Biochem. Pharmacol., 109: 145-178. Thoenen, H., Bandtlow, C., Heumann, R., Lindholm, D., Meyer, M. and Rohrer, H. (1988) Nerve growth factor: cellular localization and regulation of synthesis. Cell Mol. Neurobiol., 8: 35-40. Timmusk, T., Palm, K., Metsis, M., Reintam, T., Paalme, V., Saarma, M. and Persson, H. (1993) Multiple promoters direct tissue-specific expression of the rat BDNF gene. Neuron, 10: 475-489. Tongiorgi, E., Righi, M. and Cattaneo, A. (1997) Activitydependent dendritic targeting of BDNF and TrkB mRNAs in hippocampal neurons. J. Neurosci., 17: 9492-9505. Ueyama, T., Kawai, Y., Nemoto, K., Sekimoto, M., Tone, S. and Senba, E. (1997) Immobilization stress reduced the
expression of neurotrophins and their receptors in the rat brain. Neurosci. Res., 28: 103-110. Vaidya, V.A., Marek, G.J., Aghajanian, G.K. and Duman, R.S. (1997) 5-HT2A receptor-mediated regulation of brainderived neurotrophic factor mRNA in the hippocampus and the neocortex. J. Neurosci., 17: 2785-2795. Zafra, F., Castren, E., Thoenen, H. and Lindholm, D. (1991) Interplay between glutamate and gamma-aminobutyric acid transmitter systems in the physiological regulation of brainderived neurotrophic factor and nerve growth factor synthesis in hippocampal neurons. Proc. Natl. Acad. Sci. U S A, 88: 10037-10041. Zafra, F., Hengerer, B., Leibrock, J., Thoenen, H. and Lindholm, D. (1990) Activity dependent regulation of BDNF and NGF mRNAs in the rat hippocampus is mediated by non-NMDA glutamate receptors. EMBO J., 9: 3545-3550.
T. Steckler, N.H. Kalin and J.M.H.M. Reul (Eds.) Handbook of Stress and the Brain, Vol. 15
ISBN 0-444-51173-3 Copyright 2005 Elsevier B.V. All rights reserved CHAPTER 5.3
Transcription factors as modulators of stress responsivity Ronald S. Duman*, David H. Adams and Birgitte B. Simen Departments of Psychiatry and Pharmacology, Laboratory of Molecular Psychiatry, Yale University School of Medicine, 34 Park Street, New Haven, CT 06508, USA
Abstract: Exposure to stress can lead to both adaptive and maladaptive changes that control neuronal function and behavior. The mechanisms underlying these adaptive changes include regulation of synaptic transmission, intracellular signal transduction, gene expression, and even structural alterations. Because acute and chronic exposure to stress can lead to long-term changes in neuronal function, special emphasis has been placed on regulation of transcription factors and patterns of gene expression that could underlie these changes. In this chapter, the basic mechanisms that regulate transcription factors and gene expression, including cis- and trans-acting factors, are discussed. The influence of stress on three major classes of transcription factors, activating transcription factor (ATF) (e.g., the cAMP response elementbinding protein or CREB), activator protein-1 (AP-1) (e.g., c-Fos and c-Jun), and nuclear factor kappa B (NF-~B) is examined. Characterization of the transcription factors and target genes underlying the actions of stress will provide critical information for understanding stress-related neurobiological disorders and ultimately better treatment interventions.
unique patterns of gene expression in different brain regions and even within a single neuron. These unique patterns of expression are involved in shaping the function of the brain and its ability to adapt and generate long-term and informed responses to subsequent stimuli. Exposure to short- or long-term stress produces significant and sometimes profound effects on neuronal function and behavior, and many of these effects occur at the level of gene transcription. This chapter will provide an overview of the fundamental concepts of gene transcription, and will then focus on several classes of transcription factors that have been extensively studied in the context of stress, anxiety, and depression. These include the cAMP response element-binding protein (CREB), the c-Fos/activator protein-1 (AP-1), and the NF~:B systems. This is not meant to be an exhaustive list of the transcription factors regulated by stress, but a more focused overview of a few significant factors that have been examined in this context. The glucocorticoid receptors represent another major class of transcription factors
Introduction
The identification and characterization of genes that control neuronal function and behavior have been the focus of intense research in recent years. The goal of this work is to understand the genes that control complex behavior and thereby identify gene mutations that contribute to neurobiological disorders. The completion of the human genome project, as well as the sequencing of the D N A of other species, has provided the coding information that is needed to study the molecular basis of neuronal function and behavior. However, D N A sequence alone does not tell us which genes are turned on and off in response to different endocrine, immune, environmental, and behavioral conditions. This information is critical because it represents one of the primary mechanisms by which the brain processes information and allows an organism to make appropriate adaptive responses to the same or related stimuli. The expression of genes in the brain is regulated by multiple internal and external stimuli that can induce discrete and 679
680 that are influenced by elevated levels of glucocorticoids that are activated by stress. Glucocorticoid receptor regulation of gene expression will be covered in another chapter.
DNA and gene transcription Gene expression is controlled by a complex interaction of transcription factors (referred to as transregulatory elements) with specific sequences of D N A in the promoter elements of genes (cis elements). The process of gene transcription and the expression of cassettes of genes under different conditions are of primary importance because these processes control all aspects of cellular/neuronal function (for reviews of gene transcription, see Armstrong and Montminy, 1993; Nestler et al., 2001). In 1953, Watson and Crick reported on the structure of DNA. They postulated that single strands of nucleotides, made up of adenine (A), thymine (T), guanine (G), and cytosine (C), are able to form a double helix by pairing of complementary strands (A with T and G with C). Double-stranded D N A has the characteristics required for duplication of genetic material during cell division and reproduction and for expression of genes that are necessary for cell survival and function. Gene transcription and expression are tightly controlled and most of the nuclear D N A is in an inactive state, tightly coiled around nucleosomes that are the major components of chromosomes. This form of D N A is inaccessible to transcription factors and must be uncoiled for initiation of gene transcription to occur. Relaxation of the nucleosomes, which are made up largely of histones, requires enzymatic processing and remodeling of the nucleosomes. The histone acetylases, one of the major classes of enzymes responsible for remodeling of nucleosomes, modify core DNA-binding proteins and allow for recruitment of transcription factors and R N A polymerase. This is referred to as the initiation phase of gene transcription, which is then followed by elongation and termination. Histone deacetylases return D N A to the coiled/bound nucleosome state and contribute to inactivation of gene transcription. Interestingly, histone deacetylases are inhibited by valproic acid, an anticonvulsant drug used for the
treatment of bipolar disorder (Phiel et al., 2001). Inhibition of histone deacetylase leads to increased expression of genes that are thought to contribute to the therapeutic action of valproic acid and other mood-stabilizing drugs, as well as its side effects. The initiation of gene transcription is controlled by interactions of transcription factors with specific D N A sequences in the gene promoter. The elements that determine where initiation occurs and control basal rates of transcription are referred to as core elements. The association of R N A polymerase to the core promoter, referred to as a T A T A box because of the high number of T and A nucleotides, is required for initiation (Fig. 1). Genes that do not contain a T A T A box have a poorly conserved element referred to as an initiator. R N A polymerase binds to the core promoter upon remodeling of a nucleosome and this step is required for transcriptional initiation. Type II R N A polymerase (pol II) is utilized for transcription of m R N A , while other forms are used for very large R N A (I) or for small nuclear R N A (snRNA) DNA ~
strandf
i'
)
~
~
~
CRE , ~
"
5'
transcription,./~~ activation i..i .....~
RNA
Fig. 1. Schematic model of cis- and trans-acting DNA elements involved in regulation of gene transcription. This model depicts a complex of transcriptional proteins associated with a TATA box or transcription initiation site. The TATA-binding protein (TBP) and RNA polymerase II (pol II), as well as several additional binding proteins, make up this complex. Transcription activation requires association with a promoter element, such as a cAMP response element (CRE) and the CRE-binding protein (CREB). Such elements are often located several hundreds of basepairs upstream from the TATA box but they can associate with the TATA complex by folding of the DNA strand upon itself. CREB binds to the CRE, but does not become fully active until it is phosphorylated (P). This allows for binding with the CREB-binding protein (CBP) that in turn couples to and activates the pol II transcription complex, resulting in the synthesis of RNA.
681 (I and III). The TATA-binding protein is responsible for binding of pol II as well as other transcription factors and cofactors and is therefore critical for initiation of basal transcription. Promoter elements located further upstream from the initiator help to recruit activator and/or repressor proteins and control higher rates of RNA synthesis and gene expression. The mRNA formed in the nucleus is transported to the cytoplasm, where it is translated to form cellular proteins. It is important to point out that posttranscriptional modifications of mRNA also play a critical role and are important for further regulation and fine tuning of specific patterns and types of genes expressed. For example, certain genes can encode multiple splice variants, each of which may have different regulatory and functional domains. Another example is RNA editing whereby a single nucleotide can be altered and result in an amino acid switch that changes the function of a protein. The stability and cellular localization of mRNA are additional mechanisms for regulation. The half-life of mRNA is controlled by sequences found in the 3' untranslated region (or 3' UTR) that influence stability and degradation. Sequences in the 3'UTR are also thought to act as signals for transport of mRNA to distal sites, such as axon and dendrite terminals, for local protein expression. The combination of these and other mechanisms provides many sites for fine tuning the translation and expression of proteins in a unique fashion and thereby control of neuronal function.
Transcription factor families Basal levels of gene transcription occur when RNA polymerase binds to the TATA box/initiation site, but efficient and higher rates of transcription require additional gene transcription factors. Transcription factors bind relatively close to the initiation site or bind to promoter elements that can be located several kilobases upstream of the initiation site (Nestler et al., 2001). Transcription factors are referred to as either promoters or enhancers depending on the distance from the initiation site, although functionally there is little difference because the distal enhancers can be brought into close
proximity to the TATA box/initiation site when the DNA loops upon itself (Fig. 1). In addition to a DNA-binding domain, transcription factors generally have activation and regulatory domains. Activation often requires an interaction with another subunit of the same or a different transcription factor for complete activation. These dimers may act in concert to enhance transcription, or a heterodimer may repress activity compared to that in response to a homodimer, or vice versa. The requirement for dimers that enhance or repress transcription is another mechanism for fine-tuning gene transcription. In this chapter we will discuss three major families of transcription factors that are regulated by stress and the immune system. These are the CREB-like transcription factors, the AP-1, and the NF~cB family. Each of these classes of transcription factors is regulated by a different mechanism. The primary means for activation of most CREB family members is by phosphorylation of specific amino acid residues in the regulatory domain. NFleB is also activated by phosphorylation, including phosphorylation of an NF~cB-binding protein that then releases NF~cB. Members of the AP-1 family are regulated primarily by induction of the total amount of the transcription factor protein, although these factors also have sites for regulation by phosphorylation. Another major class of transcription factor that plays a major role in the stress response is the glucocorticoid receptor, a member of the steroid hormone receptor superfamily. Steroid hormone receptors are cytoplasmic proteins that translocate to the nucleus upon binding to a specific class of steroid. Other members of this class include receptor/transcription factors for thyroid hormones, sex steroids (estrogen, progesterone, and testosterone), retinoic acid, and vitamin D.
Stress and the transcription factor CREB CREB and related transcription factors were initially studied and identified to contribute to the long-term adaptations that underlie learning and memory (Silva et al., 1998). However, CREB is expressed throughout the brain and is now known to be regulated by a variety of stimuli and in turn to regulate several classes of genes that influence many different aspects of CNS function. This includes acute and long-term
682 There are three different splice variants of C R E B found in the brain, ~, [3, and A. C R E B was originally identified as a transcriptional mediator of cAMPsignaling and c A M P - d e p e n d e n t protein kinase. However, C R E B is also phosphorylated and activated by other protein kinases, including Ca 2+dependent protein kinases and ribosomal $6 kinase (RSK) (Fig. 2). The phosphorylation of C R E B (pCREB) by multiple signal transduction pathways makes this transcription factor a point of convergence for rapid as well as long-term adaptive signaling pathways. This could play an i m p o r t a n t
responses to stress. This section will provide a description of the structural and functional aspects of C R E B and then will describe studies demonstrating the regulation and function of C R E B in stress and related conditions.
CREB structure and function C R E B belongs to the activating transcription factor family (ATF), and as the name of this family implies most members are activators of gene transcription.
Ca 2+
, '1'
....
DAG
cAMP Ras Raf MEK
IP3
/
Ca 2+
ERK RS
RNA
SRE ~
~RL
Fig. 2. Diagram of signal-transduction pathways that can activate CREB and that increase c-Fos gene expression. G protein receptorcoupled second messenger, neurotrophic factor, and Ca 2+ activated pathways activate protein kinases that subsequently regulate CREB and c-Fos gene expression. Regulation of cAMP levels is controlled by receptors that either stimulate or inhibit adenylyl cyclase (AC) via coupling to Gs or Gi, respectively. Activation of adenylyl cyclase and elevation of cAMP levels stimulates cAMP-dependent protein kinase (PKA), one of several protein kinases that can phosphorylate CREB at Ser133. Activation of phospholipase C (PLC) and the phosphotidyl inositol (PI) pathway occurs via receptor coupling with Gq. Activation of PLC leads to the formation of inositol trisphosphate (IP3) and diacylglycerol (DAG), which stimulates protein kinase C (PKC). Ca 2+ can be released from intracellular stores by IP3, or by influx through ion channels (e.g., voltage-dependent or glutamate-N-methyl D aspartate). Ca 2+, in conjunction with calmodulin, activates Ca2+/calmodulin-dependent protein kinase (CaMK). PKC and CaMK can also phosphorylate and activate CREB. In addition to these second messenger-dependent pathways, neurotrophic factor activation of Trk receptors and the Ras-RafMEK-ERK pathway can lead to regulation of gene expression. ERK can activate ribosomal $6 kinase (RSK), another CREB kinase. Phosphorylation of CREB leads to transcriptional activation of the c-Fos promoter which contains a CRE. In addition, ERK can directly activate c-Fos gene expression via phosphorylation of a serum response factor (SRF) which binds to a serum response element (SRE) in the promoter of the c-Fos gene.
683 role in the function of CREB in response to acute and chronic stress exposures. These and other pathways may be differentially regulated by stress in subsets of neurons and glia and result in a unique geneexpression signature. A number of technological approaches, from single-cell real-time polymerase chain reaction and genechip analysis, to double and triple labeling of proteins in cells, are being used to identify these unique signatures. CREB and related family members bind to a specific sequence of DNA, referred to as the cAMP response element (CRE) that is located in the promoter regions of genes. CREB binds with highest affinity to the consensus CRE, an eight basepair, palindromic sequence, T G A C G T C A . CREB also binds to C R E sequences that have one or two substitutions, although affinity of CREB for these elements is reduced. The binding of CREB to D N A requires the interaction of two molecules of CREB or related family members to form either homo- or heterodimers. Most dimers of A T F factors result in transcriptional activation, although there are a few examples of heterodimers that result in repression. The ability of CREB to form a dimer is dependent on the leucine zipper domain (Fig. 3). This region has a leucine repeat every seven amino acids and hydrophobic interactions of the leucine residues of two
transcription factors results in the formation of a "leucine zipper" that stabilizes the dimer. The leucine zipper domain is adjacent to a highly basic D N A binding domain. The name basic leucine zipper, or bZIP, refers to transcription factors that utilize these structural and functional motifs. H o m o - or heterodimers of CREB and related proteins are capable of binding to C R E sites, but transcriptional activation predominantly occurs when CREB is phosphorylated (see Figs. 1-3). There are several phosphorylation sites in the CREB molecule, but it is phosphorylation of a conserved amino acid residue, Ser 133, that is necessary for activation of gene transcription. Phosphorylation of Ser 133 is necessary for the interaction of CREB with the CREB-binding protein (CBP), which in turn interacts with the basal transcription complex at the TATA box/initiator of a gene and thereby results in transcriptional activation (Fig. 1). Another closely related subgroup of the A T F family is referred to as the C R E modulators or CREMs. The C R E M transcription factors include members that are regulated by either phosphorylation or expression. Most of the C R E M isoforms, like CREB, are phosphorylated and this leads to activation of gene transcription. However, there is also a transcriptional inhibitor, referred to as
activation domain
DNA binding domain CREB
i /
~iil~
.... ...... CREM
Fig. 3. Schematic structure of the CREB and CREM transcription factors. These models depict the primary functional domains of CREB and CREM. This includes the activation domain, also referred to as the P box or kinase-inducible domain, which contains a phosphorylation site for transcriptional activation. The amino terminus of the proteins contains the basic domain that binds to DNA and the leucine zipper domain that is responsible for dimerization of CREB and related proteins. Also shown at the bottom is the structure of ICER, or the inducible cAMP early response factor. ICER is generated by use of an alternative start site in the CREM gene that is activated by cAMP/Ca 2+ signaling and a CRE site. This results in the synthesis of a truncated form of the transcription factor that lacks the kinase-inducible domain. ICER is able to dimerize with CREB, CREM and related proteins, but cannot be activated by phosphorylation. ICER thereby serves as a negative-feedback transcription factor that shuts down CRE-mediated gene expression.
684 inducible cAMP early repressor (ICER). ICER is especially interesting because it is induced by activation of a CRE in an alternative promoter of the CREM gene. Initiation from this alternative site results in the formation of a truncated form of CREM that lacks the kinase-inducible domain (Fig. 3). This is an example of a transcription factor isoform that can dimerize with an activator like CREM or CREB, but because it cannot be phosphorylated it represses gene transcription. In this way ICER serves as a negative-feedback mechanism for counterregulation of CRE-mediated gene expression.
of the hypothalamus. Exposure to stress results in a rapid stimulation of the HPA axis and the release of existing CRF, as well as activation of CRF gene expression (Kovacs and Sawchenko, 1997). The CRF gene contains a CRE in the promoter region that could underlie its regulation by CREB and stress. Studies to directly examine the regulation of CRF in vivo by CREB have not been conducted. For example, the influence of expression of a dominant negative mutant of CREB in the PVN or null mutation of CREB on the expression of CRF in PVN could be examined to determine if CREB is necessary for the induction of CRF by stress.
Regulation of CREB by acute physical stress Regulation of CREB by psychological stress The potential role of CREB in responses to stress has been a topic of interest in recent years. These studies demonstrate that CREB is activated in several brain regions by exposure to acute stress, suggesting that this transcription factor could underlie early gene expression responses. The activation of CREB varies with the type of stress exposure (e.g., physical vs. psychological stress) and the brain regions examined. Acute exposure to a physical stressor, such as an electrified prod, ether, hyperosmolar solutions, or hypothermia, increases pCREB in the paraventricular nucleus (PVN) of the hypothalamus and the locus coeruleus (LC) (Borsok et al., 1994; Kovacs and Sawchenkno, 1997; Legradi et al., 1997; Bruijnzeel et al., 2001). The activation of CREB has been studied with regard to regulation of target genes in these regions. In LC, one of the suggested target genes of CREB is tyrosine hydroxylase, the ratelimiting enzyme for the synthesis of noradrenaline. Stress increases the expression of tyrosine hydroxylase in the LC and the promoter of this gene contains a CRE (Lewis et al., 1987). Other genes that could be regulated by CREB and stress include certain isoforms of adenylyl cyclase, which could contribute to an elevation of cAMP-CREB signaling (Chao et al., 2002). In the PVN, one of the key target genes that is regulated by stress and could be a target of CREB is corticotrophin-releasing factor (CRF). CRF is one of the key endocrine factors in the hypothalamicpituitary-adrenal (HPA) axis. This neuropeptide is expressed in the parvocellular neurons in the PVN
Another type of acute stress paradigm that has been examined is the forced swim test, which is suggested to be a model of psychological stress (Bilang-Bleuel et al., 2002). In this model, animals are exposed to swim stress for short periods of time (e.g., 5-15 min) and eventually become immobile. The forced swim test was originally designed as a model of antidepressant action as treatment with most classes of antidepressants decreases immobility time (Porsolt et al., 1977). Exposure to forced swim results in a biphasic induction of pCREB in the dentate gyrus of the hippocampus, many subregions of cerebral cortex, and amygdala, but not in the PVN, dorsal raphe, or LC. Levels of pCREB are dramatically increased between 15 and 30min and then return to basal levels, or below by 1 h (Bilang-Bleuel et al., 2002). Interestingly, there is a second surge in levels of pCREB by 6 h and this effect is sustained for at least 48 h. The biphasic time course has similarities with the early and late phases of long-term potentiation and induction of pCREB in this cellular model (CaMKII and then PKA mediated, respectively), raising the possibility that pCREB regulates genes that contribute to long-term changes in neuronal function and behavior. Levels of Fosimmunoreactivity are increased in many of the same regions, but are also increased in other regions, including PVN, LC, dorsal raphe, and bed nucleus of the stria terminalis (Bilang-Bleuel et al., 2002). The induction of pCREB in higher limbic forebrain structures in response to forced swim stress
685 has been discussed by Bilang-Bleuel and colleagues (2002) with regard to the psychological stress associated with this model. Forced swim stress also includes a physical component and activates deep subcortical structures as well. The higher limbic structures where pCREB is induced are involved in processing sensory information, as well as cognitive and emotional inputs compared to those regions where induction of pCREB is observed in response to physical stress (i.e., PVN and LC). This indicates that different neuronal circuits are activated depending on the type of stress exposure (psychological vs. physical stress) (Bilang-Bleuel et al., 2002). A similar pattern was observed for induction of mineralocorticoid receptors (Gesing et al., 2001). The induction of pCREB is observed in populations of neurons that represent the initial input subregions within these circuits (dentate gyrus in hippocampus; lateral nucleus in the amygdala). This suggests that modulation of pCREB may be involved in adjusting neuronal function at a proximal level and thereby affecting processing of information entering the circuit.
Nucleus accumbens CREB: an emotional gating switch The influence of forced swim stress, as well as other types of stress, on pCREB immunoreactivity in the nucleus accumbens has also been examined (Pliakas et al., 2001; Barrot et al., 2002). These studies demonstrate that forced swim, footshock, restraint, or social stress increase pCREB immunoreactivity in the nucleus accumbens shell and to a lesser extent in core, but not dorsal striatum. Repeated unpredictable stress also increases pCREB in the nucleus accumbens (Barrot et al., 2002). In addition, Barrot and colleagues have utilized a CRE transgenic reporter mouse to demonstrate that CRE-mediated gene expression is also increased under these conditions. Chronic morphine administration also increases pCREB- and CRE-mediated gene expression in NAc (Barrot et al., 2002). These studies demonstrate that the rewarding stimuli as well as stressful stimuli increase pCREB- and CRE-mediated gene expression in the nucleus accumbens shell. This has led to the hypothesis by Barrot and colleagues
that emotional stimuli, regardless of the valence, increase pCREB- and CRE-mediated gene expression in the nucleus accumbens. The possible role of CREB in the functional response to anxiogenic, aversive, and nociceptive stimuli has also been examined to directly test the hypothesis that CREB acts as an emotional gating sensor in the nucleus accumbens (Barrot et al., 2002). In general, when CREB in nucleus accumbens is increased the responses in behavioral tests of these endpoints are decreased: (1) viral expression of CREB in the nucleus accumbens reduces the preference for opiates or sucrose; (2) viral expression of CREB in the nucleus accumbens decreases anxietyrelated behavior (i.e., plus maze, open field) and expression of dominant negative CREB (mCREB) has the opposite effect; (3) studies of aversive and/or nociceptive responses demonstrate that viral expression of CREB reduces the sensitivity to naloxone in a conditioned aversion paradigm and mCREB has the opposite effect; (4) analysis of unconditioned responses demonstrate the expression of CREB in the nucleus accumbens increases the threshold footshock intensities required to elicit vocalization or jumping (increased threshold), while mCREB decreases threshold (i.e., animals vocalize and jump at lower intensity). Finally, expression of CREB in the nucleus accumbens decreases swimming/struggling in the forced swim test and decreases active avoidance in the learned helplessness paradigm, while mCREB expression has the opposite effects (Pliakas et al., 2001; Newton et al., 2002).
Influence of repeated stress on CREB The influence of chronic or repeated stress on activation of CREB has not been studied as extensively. Bruijnzeel and colleagues (2001) have investigated the influence of repeated stress on pCREB in the PVN to determine if CREB could contribute to the increased expression of CRF in response to repeated stress that has also been observed (Bruijnzeel et al., 1999). Prior exposure to footshock stress increases the induction of CRF upon challenge with another stress (Bruijnzeel et al., 2001). However, this paradigm does not lead to increased expression of pCREB upon the second challenge
686 stress. This suggests that the elevated induction of CRF is not due to increased activation of pCREB. However, one potential problem with the analysis is that only the total number of pCREB immunoreactive cells was determined. Although this is a typical analysis it is possible that there are different amounts of pCREB per cell and a small increase in cells that already express pCREB could be missed. It is also possible that other proteins that make up the transcriptional complex, such as the CREB-binding protein, are upregulated and more efficiently recruit pCREB. It is also possible that other transcription factors regulate the expression of CRF in PVN. Previous studies have demonstrated that prior footshock stress increases the induction of Fos-immunoreactivity in the PVN in response to a challenge stress (Rivest and Rivier, 1994; Bruijnzeel et al., 1999). Another study has examined the influence of repeated, chronic footshock stress (21days) on pCREB immunoreactivity (Trentani et al., 2002). The results of this study are interesting and demonstrate that in contrast to the acute stress paradigms, chronic stress decreases levels of pCREB in subregions of cerebral cortex (prefrontal, cingulate, and perirhinal) and paraventricular thalamic nucleus. There was no effect on pCREB in somatosensory cortex, dentate gyrus, or PVN in this report. A study of maternal isolation stress (1 h per day from D2 to D9) also demonstrates that levels of pCREB are decreased in several brain regions when examined at a later developmental time point (postnatal day 80) (Huang et al., 2002). These studies demonstrate that chronic stress can result in downregulation of pCREB in certain brain regions. It is interesting to speculate that activation of pCREB by acute stress may represent a normal adaptive response and that this effect is reversed with chronic exposure to stress in certain brain regions.
Role of CREB in conditioned responses Owing to the well-characterized role of CREB in learning and memory, the regulation and function of CREB in conditioned fear has been extensively examined. These studies have reported biphasic regulation of pCREB in parietal cortex, hippocampus, and amygdala. The early phase (0-30min)
correlates with the unconditioned stimulus (footshock stress), while the late phase (3-6 h) is associated with the conditioned response (freezing in response to context associated with footshock) (Stanciu et al., 2001). A recent study found a greater pCREB response in animals exposed to context and then footshock versus animals receiving footshock at the same time as context (Stanciu et al., 2001). This suggests a role for pCREB in memory consolidation, in agreement with regulation of hippocampal pCREB during the late phase of memory consolidation (Bernabeu et al., 1997). The time course of pCREB expression significantly varies depending on the type and duration of the stimuli. This suggests that the early phase induction of pCREB may be more closely related to the arousal and anxiety induced by the experimental conditions than the process related to memory consolidation. In contrast to pCREB, induction of Fos is observed between 60 and 90min after footshock, and this appears to be independent of the stimulus type or duration (see Stanciu et al., 2001). This suggests a highly constrained regulation of the Fos gene.
CREB in the etiology and treatment
of depression Mood disorders are often associated with stress, which can precipitate an episode of depression or can worsen an existing situation. Therefore, stress or stress-related behavioral paradigms are often used as models of depression and/or antidepressant actions. The regulation of CREB by acute and chronic stress suggests that this transcription factor could also be involved in depression. This possibility is supported by both basic and preclinical studies of CREB. Basic research studies first demonstrated a role for CREB in the actions of antidepressant treatments. These studies report that chronic antidepressant treatment increases CREB in the hippocampus and cerebral cortex of rodents (Fig. 4) (Frechilla et al., 1998; Nibuya et al., 1996; Thome et al., 2000). Upregulation of CREB expression and function is dependent on chronic antidepressant treatment, consistent with the time course for the therapeutic action of antidepressants. In addition, several different classes of antidepressants, including
687
Antidepressant treatment
andNE 'ui0 a5,H~T .:, br.a
Inhibit
5-HT
or
NE
,,~-
" .................. ~'...................................
-
PDE4 ~ inhibitor
Ca2+~endent ,,~,,
.:i.... cAMP
o
....
kinases
/
!ii ~::.....~
nucleus
..y
: 5-HT
:./i: ~
PKA f
/ ITrophic actions: altered ,i~:'~ synaptic plasticity and i' I neuronal morphology
CREB ~i
......
il
..
BDNF gene expression Fig. 4. Model depicting antidepressant regulation of the cAMP-CREB cascade and gene targets. Antidepressants block the reuptake or metabolism of NE, as well as 5-HT, and increase synaptic levels of these monoamines. Chronic antidepressant administration results in adaptations of the receptor-coupled signal transduction pathways and regulation of gene expression. One pathway that is regulated by antidepressant treatment is the cAMP-CREB cascade. Chronic antidepressant treatment leads to upregulation of cAMP-dependent protein kinase (PKA), increased levels of this kinase in nuclear fractions, and increased function and expression of the cAMP response element-binding protein (CREB). Inhibitors of phosphodiesterase type 1V (PDE4) increase levels of cAMP and are known to have antidepressant efficacy in behavioral models and in clinical trials. CREB can also be regulated by Ca2+-stimulated protein kinases, as well as ribosomal $6 kinase (see Fig. 2) and thereby acts as a common transcription factor target for multiple signal transduction pathways. Chronic antidepressant treatment and the cAMP-CREB cascade also increase the expression of specific gene targets in limbic brain structures, most notably brain-derived neurotrophic factor (BDNF) and its receptor TrkB. Upregulation of the cAMPCREB cascade and increased expression of BDNF/TrkB are thought to produce antidepressant effects, in part, by blocking or reversing the atrophy and decreased neurogenesis resulting from stress.
5-HT and noradrenaline-selective reuptake inhibitors increase C R E B , suggesting that this transcription factor is a c o m m o n d o w n s t r e a m target of antidepressants. The influence of antidepressant t r e a t m e n t on the function and expression of C R E B has been examined using several different approaches. Antidepressant t r e a t m e n t increases the expression of C R E B m R N A and immunoreactivity, indicating that the total
a m o u n t of C R E B protein is increased (Nibuya et al., 1996). Levels of C R E B binding to synthetic D N A containing a consensus C R E (i.e., determined in a gel mobility shift assay) and levels of p C R E B determined by i m m u n o h i s t o c h e m i s t r y are upregulated by antidepressant treatment, suggesting that the function of C R E B is increased (Nibuya et al., 1996; T h o m e et al., 2000). This possibility has been confirmed by studies d e m o n s t r a t i n g that antidepressant t r e a t m e n t
688 increases CRE-mediated gene transcription in a line of transgenic mice that expresses a CRE-regulated reporter gene, [3-galactosidase (Thome et al., 2000). These mice can be used to visualize CRE-mediated gene expression in vivo in response to various types of pharmacological or behavioral stimuli. The transcriptional activity of CREB can also be assessed by the expression of target genes that contain CRE sites. Two genes of interest that have been identified are brain-derived neurotrophic factor (BDNF) and its receptor TrkB. Chronic, but not acute antidepressant treatment increases the expression of BDNF and TrkB mRNA in the hippocampus and this effect is blocked in CREB null mutant mice (Nibuya et al., 1995; 1996; Conti et al., 2002). In contrast to antidepressant treatment, stress decreases the expression of BDNF in the hippocampus (Nibuya et al., 1999; Smith et al., 1995). Decreased expression of BDNF could contribute to the atrophy and loss of cells resulting from chronic stress exposure (see Duman et al., 2000). The exon III-specific promoter
signals
IgedUTd ~,~ ~.O~~nu~eu~ Ub Fig. 5. Model depicting NF•B signaling to the nucleus. Signals transduced from cell-surface receptors converge on I~cB kinase (IKK), which is activated by phosphorylation. IKK in turn phosphorylates IKB, causing it to become a substrate for an ubiquitin ligase. After ubiquitination, IKB is rapidly degraded to release the active NF~cB dimer (here shown as a p50/p65 heterodimer). The active NF~cB complex partitions preferentially to the nucleus where it activates transcription from
~cB-containing promotors.
of BDNF contains a Ca2+/CRE site, referred to as a CaRE that is responsible for the induction of BDNF expression in response to activation of the cAMPPKA cascade, as well as in response to neuronal depolarization and stimulation of CaZ+-calmodulin dependent protein kinase (Tao et al., 1998). The function of CREB in the actions of antidepressant treatment has also been examined using viral-mediated gene transfer and mutant mouse approaches. Viral-mediated expression of CREB in the hippocampus produces an antidepressant-like effect in the forced swim and learned helplessness paradigms (Chen et al., 2001). Preliminary studies demonstrate that viral expression of CREB in the amygdala also produces an antidepressant response in the learned helplessness model of depression (Wallace et al., 2002). However, as mentioned above, viral or transgenic expression of CREB in the nucleus accumbens results in a prodepressive phenotype in both the forced swim and learned helplessness models (Pliakas et al., 2001; Newton et al., 2002). These studies demonstrate that CREB influences behavior in models of depression, but that the effect depends on the brain region examined. This is not surprising because CREB is known to influence different target genes in these brain structures. In the hippocampus and amygdala, one of the targets of CREB is BDNF, which is capable of producing an antidepressant effect (Shirayama et al., 2002). In the nucleus accumbens, one of the key target genes of CREB is prodynorphin, which can produce aversive effects that could contribute to the prodepressive effects of CREB expression in this brain region (Carlezon et al., 1998; Pliakas et al., 2001; Newton et al., 2002). Clinical postmortem studies also demonstrate that CREB is altered in depressed patients. There is one report demonstrating that levels of CREB immunoreactivity are decreased in temporal cerebral cortex of patients not on antidepressant medication at the time of death (Dowlatshahi et al., 1998). In contrast, levels of CREB immunoreactivity are significantly increased in temporal cortex of patients receiving an antidepressant at the time of death. Further studies of the levels of CREB expression, as well as function, in postmortem brains of depressed patients must be conducted to determine if altered CREB is a marker of depression. These preliminary studies are consistent with the hypothesis that CREB may be
689 involved in the pathophysiology and treatment of depression.
Stress and the AP-1 family transcription factors AP-1 transcription factors represent another family of proteins that regulate gene expression. The primary mechanism for regulation of these proteins is via induction of gene expression. The AP-1 proteins bind to the AP-1 promoter element, which is a sequence of seven nucleotides and is similar to the consensus CRE sequence, TGACTCA. Although this sequence only differs from the CRE sequence by one nucleotide, it is sufficient to confer a relatively high degree of selective binding of the AP-1 transcription factors over the CREB-like factors. There are many genes in the brain that contain AP-1 elements, including those for neuropeptides, neurotransmitter synthetic enzymes, receptors, and neurotrophic factors.
AP-1 structure and function AP-1 transcription factors, like CREB and ATF family members, belong to the superfamily of basicleucine zipper DNA-binding proteins. AP-1 transcription factors form dimers via the leucine zipper domain and bind to DNA via the adjacent basic amino acid-rich domain. The two major families of AP-1 factors are the Fos and Jun transcription factors. Members of the Fos family include c-Fos, Fos-related antigen- 1 and -2 (FRA- 1 and FRA-2), FosB and the FosB splice variant ~FosB. The Jun family includes c-Jun, JunB, and JunD. A functional dimer is made up of one Fos and one Jun family member. Homodimers of Jun or Fos are not uncommon, but the DNA-binding affinity of these complexes is lower than for the Fos/Jun heterodimers. Under basal, unstimulated conditions cellular levels of most Fos and Jun transcription factors are low, and in some cells undetectable. Activation of many signal-transduction pathways increases the expression levels of Fos and Jun resulting in the formation of AP-1 complexes. JunD is one exception in that it is constitutively expressed. The rapid induction of most AP-1 transcription factors has led to their classification as immediate early genes (lEGs). The protypical lEG is c-Fos, which can be
induced rapidly, within minutes of the presentation of an extracellular stimulus. The ability of c-Fos to be turned on so rapidly is due to the presence of multiple response elements in the promoter, which includes three CREs. Activation of cAMP and/or Ca2+-dependent protein kinases and CREB thereby result in rapid induction of c-Fos expression (Fig. 2). Induction of c-Fos gene expression also occurs via additional signaling cascades, including the MAP kinase-ERK signaling pathway. Increased activity of this cascade can led to phosphorylation of CREB via activation of RSK. ERK signaling also results in activation of another transcription factor, Elk-l, which forms a complex with the serum response factor (SRF) that binds to serum response element (SRE) in the c-Fos promoter (Fig. 2). AP-1 transcription factors can also be regulated by phosphorylation, which in many cases functions as a negative-feedback mechanism to turn off AP-1mediated gene expression. AP-l-mediated transcription is further complicated by formation of heterodimers with other transcription factors such as p-CREB and nuclear steroid receptors. These cross-family dimers bind to different consensus sequences with differing affinities. Glucocorticoid receptors and Ap-1 proteins can antagonize one another both in vitro and in vivo (Karin and Chang, 2001). The mechanisms of this interference are not completely understood but may include direct protein-protein interactions as well as inhibition of upstream signaling molecules involved in the activation of AP-1 transcription. Jun N-terminal kinase (JNK) is stimulated by growth factors and cytokines and enhances AP-1 activity by induction of c-Fos and c-Jun transcription and phosphorylation of Jun proteins. Glucocorticoids inhibit JNK activity, so this is one possible mechanism of glucocorticoid-mediated inhibition of AP-1 activity.
Stress and regulation of los transcription factors A brief overview of the effects of stress on c-Fos and lEG expression will be presented in this section. Stress-induced alterations in the levels or activity of c-Fos and other lEGs are likely important for regulating both the adaptive and detrimental
690 responses to stress. Stress increases the expression of c-Fos and other AP-1 family members in both endocrine organs and various brain regions. However, it should be noted that further studies are needed to determine if these AP-1 transcription factors underlie the actions of stress on gene expression and function. This must be done using approaches similar to those described for CREB, particularly inducible transgenic and knockout mice combined to determine the role of c-Fos/AP-1 transcription factors in stressstimulated gene expression.
c-Fos
mapping
Although c-Fos and other IEGs ultimately function as transcription factors and regulate downstream target genes, c-Fos has most widely been used as a functional marker of activity in neurons and neuronal circuitries after a variety of stimuli. Expression of c-Fos and related IEG transcription factors is very rapid and robust and occurs in response to a variety of receptor-coupled signal-transduction pathways. Very low basal levels (i.e., unstimulated conditions) of c-Fos/IEG expression provides a high signal to noise ratio. This approach is not without limitations, which include a lack of expression in certain cell types and nonspecific induction. Expression of c-Fos has been used to determine the neuronal circuits underlying the neuroendocrine, autonomical, and behavioral responses induced by stress, c-Fos mapping indicates that acute stress challenges activate the HPA axis, specifically the CRF-containing parvocellular neurons of the hypothalamus. The induction of c-Fos in CRFcontaining neurons is intensity dependent and correlates with the increases in plasma corticosterone (Ericsson et al., 1994; Campeau and Watson, 1997). In addition to the hypothalamic PVN, acute stressors increase c-Fos mRNA and immunoreactivity in various brain regions. Stress can be classified as physical stress such as hemorrhage, ether, hyperosmolarity, and hyperthermia and psychological stress such as footshock, restraint, and immobilization as discussed above in relation to CREB activation. The neurocircuitry of both physical and psychological stressors as determined by c-Fos mapping generally includes the effector neurons in
the PVN, cingulate cortex, lateral septum, septohypothalamic nucleus, medial preoptic area, bed nucleus of stria terminalis, central amygdala, dorsal raphe, and locus coeruleus (Kovacs, 1998). In addition, psychological stressors such as swim or restraint stress activate neocortex, allocortex, hippocampus, nucleus accumbens, and medial amygdala brain regions involved in processing sensory, cognitive, and emotional input (Kovacs, 1998; Lopez et al., 1999; Bilang-Bleuel et al., 2002). However, there is some degree of specificity to the c-Fos response depending upon the stress that is administered. For instance, swim stress causes a relatively greater induction of c-Fos in the hippocampus than does restraint stress (Lopez et al., 1999). In addition, even when similar brain regions are activated by stress, the afferent inputs that mediate this activation are stressor specific.
Fos expression and chronic stress Repeated stimulation of the c-Fos gene eventually leads to a refractory state where further transcription is limited. This is likely due to counterregulatory mechanisms, including the induction of inhibitory transcription factors such as ICER, that block CRE-meditated gene expression. However, under conditions of repeated and long-term activation of the c-Fos gene the accumulation an alternative splice variant known as AFosB is observed. Unlike Fos, which degrades very rapidly (i.e., half-life of minutes to hours), AFosB is relatively stable and has a very long half-life of days to weeks (see Nestler et al., 2001). This different pattern of expression is thought to contribute to the long-term adaptations to repeated stimulation. Such differences in the temporal stability of AP-1 transcription factors, combined with different patterns of stimulation, provide mechanisms for discrete regulation of gene expression and cellular function. The activation of c-Fos following acute stress provides a map of the circuitry involved in the stress response. However, the pattern of c-Fos induction after repeated or chronic stress may reveal pathological processes. Repeated stress or chronic glucocorticoid administration attenuates the subsequent
691 induction of c-Fos, Fos B, and Jun B expression in the PVN by acute immoblization stress (Umemoto et al., 1997). Similarly, repeated restraint stress reduces c-Fos expression compared to acute restraint in the medial amygdala, hippocampus, septum, and brainstem. However, the degree of habituation of c-Fos and as well as other immediate early genes after repeated stress varies between brain regions and specific stressors (Chen and Hebert, 1995; Stamp and Herbert, 1999). The decreased activation of c-Fos after repeated stressors suggests that other members of the AP-1 family may mediate the effects of chronic stress. Sustained elevations of other members of the c-Fos family have been reported after chronic stimuli as discussed above. Protein products of the AFosB splice variant, termed chronic FRAs, are induced in a region-specific manner by a variety of chronic stimuli including electroconvulsive seizures, chronic drug of abuse administration, and lesions (Nestler et al., 1999). However, repeated immobilization stress does not alter the expression of the chronic FRA gene products of the FosB gene in the adrenal medulla (Nankova et al., 2000). In contrast, FRA-2 expression is increased by acute stress and to even greater levels by chronic stress in the adrenal medulla (Nankova et al., 2000). Other FRAs are also increased in the adrenal medulla, but these appear to be distinct from the chronic FRAs induced in the brain after ECS and the AFosB products. FRA-2 is induced in the PVN after capsaicin-induced stress (Honkaniemi et al., 1994). Additional studies are needed to completely characterize the effects of chronic stress on the large family of c-Fos transcription factors in brain regions that mediate the stress response. Inducible transgenic and knockout mice will be useful for linking the expression of c-Fos/AP-1 transcription factors with gene expression and behaviors produced by chronic stress.
c-Fos target genes As discussed above, c-Fos and other related family members ultimately function as transcription factors and regulate downstream target genes through interaction with an AP-1 promoter element to either stimulate or repress transcription. There are a
number of genes that contain the AP-1 consensus sequence within their promoter, including vasopressin, enkephalin, dynorphin, somatostatin, cholecystokinin, luteinizing hormone-releasing hormone, tyrosine hydroxylase (TH), and glutamic acid decarboxylase (GAD). However, direct evidence for gene regulation by AP-1 transcription factors is limited. In the adrenal medulla acute and chronic stressinduced increases in the catecholamine synthesis enzymes, TH, and dopamine [3-hydroxylase (DBH) are correlated with increased binding of AP-1 factors (Sabban and Kvetnansky, 2001). A single immobilization stress increases binding of c-Fos and c-Jun to the AP-1 transcription site and the binding is not further elevated with repeated stress (Nankova et al., 1994). Studies of c-Fos knockout mice suggest that c-Fos is necessary for chronic stress-induced DBH expression in the adrenals of female rats and in the brainstem, independent of gender (Serova et al., 1998). However, TH and phenyl-ethanolamine Nmethyltransferase (PNMT) levels are still increased by chronic stress in c-Fos knockout mice (Serova et al., 1998). This could result from the actions of other members of the c-Fos family that are driving expression of TH and DBH after chronic stress. FRA-2 activates the TH and DBH promoters in a cell-expression system in agreement with this possibility (Nankova et al., 2000). AP-1 transcription factors also play a role in regulating genes involved in cell survival (Shaulian and Karin, 2002). Depending upon the composition of the AP-1 proteins, the complex can either positively or negatively regulate cell proliferation. Fibroblasts derived from c-Fos or FosB single knockout mice proliferate normally, but fibroblasts from double c-Fos/FosB knockout mice have reduced proliferation. JunD and c-Jun knockout mice show similar deficits in cell proliferation. The induction of cyclin D1 is repressed in these knockout mice compared to wildtype. AP-1 proteins bind and activate the cyclin D1 promoter. Induction of cyclin D1 is critical for cell-cycle progression. Cyclin D1 binds to and activates G1 phase cyclin-dependent kinases (CDKs) that in turn activate and inhibit proteins that facilitate the transition from G1 phase to S phase of the cell cycle. These data suggest that the induction of cyclin D1 is one mechanism by which AP-1
692 proteins stimulate cell proliferation. However, not all AP-1 proteins promote proliferation. Studies with transgenic mice overexpressing JunB suggest that JunB antagonizes the effects of c-Jun and inhibits proliferation in a cell-type specific manner. Consistent with its ability to antagonize c-Jun, JunB inhibits the cyclin D1 promoter. There is also evidence that AP-1 proteins are involved in regulating genes that induce apoptosis (Shaulian and Karin, 2002). c-Fos is induced by kainic acid which induces apoptosis in the hippocampus through glutamate receptor stimulation. In addition, overexpression of c-Jun or c-Fos in various cell lines can be proapoptotic. Inhibition of c-Jun through the expression of a dominant negative mutant protects neurons from apoptosis induced by N G F withdrawal or chronic depolarization. One established AP-1 target gene that is proapoptotic is the Fas-ligand (FasL), as c-Jun has been shown to induce FasL. However, c-Jun is not proapoptotic in all cell or tissue types as demonstrated by c-Jun knockout mice from which some cell types undergo massive apoptosis. It is likely the balance between proapoptotic and antiapoptotic target genes that ultimately determines whether AP-1 activity leads to cell death or increases cell survival.
NF-KB-mediated gene transcription Nuclear factor kappa B (NF-~B) is a ubiquitous transcription factor activated by a variety of cellular stresses (Sen and Baltimore, 1986). Originally identified as an inducer of immunoglobulin ~c light-chain expression, it has since been implicated in protective responses to mutagenic factors, inflammation, infections, redox stress, radiation, and CNS injury as well as many homeostatic functions. In this section, we will give an overview of NF-KB's composition and regulation, with particular emphasis on its protective stress-responsive functions, both in the immune system and in the CNS.
Molecular compos#ion and activation of NF-lcB NF-KB is composed of hetero- and homodimers of five Rel family proteins, NF-~cB1 (p50), NF-~cB2 (p52), RelA (p65), RelB, and c-Rel, all of which can bind to DNA. The three latter subunits also function
as transcriptional activators, and one of these must therefore be present in an active NF-•B complex (Ghosh et al., 1998). The most common and therefore most intensely studied ("prototypical") NF-~cB is the p50-RelA heterodimer. Under basal conditions, most of the NF-KB in a cell exists in an inactive complex with an inhibitor, one of the IKB-family proteins (Baeuerle and Baltimore, 1988). When I~cB is phosphorylated by IKB kinase (IKK), it becomes a proteasomal substrate and is rapidly degraded, thereby releasing the active NF-~cB complex, which is now preferentially located in the nucleus. IKK consists of two kinase subunits, IKK~ and IKK[3, and a noncatalytic subunit IKK~, (Zandi, et al., 1997; Rothuort et al., 1998). NF-KB can also be activated through a noncanonical pathway, wherein p52 precursor in association with RelB is phosphorylated by NIK kinase in an IKKcz-dependent manner and cleaved to release the active p52-RelB dimer. This pathway is utilized during development of lymphoid organs and adaptive immunity (Senftleben et al., 2001). After activation, the nuclear NF-~cB complex binds to ~B promoter elements and increases expression of target genes (Sen and Baltimore, 1986). Conditions that lead to activation of NF-~cB are often linked to stress, be it an infection, tissue damage, or a toxic chemical compound that threatens the homeostasis of the organism. Thus, NF-~cB activation is driven by such diverse stressors as proinflammatory cytokines, acute-phase proteins, and cigarette smoke. NF-~cB also responds to DNA damage from irradiation, shear stress, and backup of unfolded proteins in the endoplasmic reticulum (Pahl, 1999). In response to the activation of NF-~cB, genes containing the KB promoter element are rapidly transcribed. Among these are many molecules central to the immune system, adhesion molecules, acutephase proteins, and a large variety of transcription factors (Pahl, 1999). In addition to relief of NF-~cB inhibition through the canonical IKK-IKB pathway, the activity of NF-~cB can be modulated by several factors. For instance, PKA-mediated phosphorylation of p65 renders NF-~cB competent to bind to p300/CBP, a cofactor that appears to be necessary not only for CREB-mediated transcription, but also for NF-KB activity (Zhong et al., 1998). ~PKC, casein kinase II and IKK~-mediated phosphorylation events enhance
693 the transcriptional activity of NF-~cB (Leitges et al., 2001). In addition, p300/CBP-dependent acetylation of RelA at one site enhances activity, and further acetylation increases NF-~cB's affinity for ~B sites (Chen et al., 2002).
Antiapoptotic effects of NF-IcB NF-KB protects against apoptosis by induction of a number of antiapoptotic target genes. Targeted deletion of RelA in mice causes lethality around embryonic days 15-16 due to liver apoptosis, and other cell types lacking NF-~cB are also more prone to apoptosis (Beg et al., 1995; Beg and Baltimore, 1996; Van Antwerp et al., 1996). Moreover, embryonic liver apoptosis phenotypes are observed in both IKK[3 and IKK7 knockouts (Li and Verma, 2002). Apoptosis is in many cases initiated by signaling through members of the tumor necrosis factor (TNF) family of receptors. Activation of TNF receptor I by the proinflammatory cytokine TNF-a initiates several signaling cascades including JNK and a proapoptotic pathway leading to activation of caspase-8, but the outcome is attenuated by the simultaneous activation of NF-~cB (Liu et al., 1996). By comparison, activation of another TNF receptor family member, Fas, which leads to similar proapoptotic events, does not efficiently induce NF-~cB and therefore causes full-blown apoptosis (Karin and Lin, 2002). Antiapoptotic genes transcribed upon NF-~B activation include lAPs (inhibitors of apoptosis), TRAF1 and 2 (TNF receptor-associated factors), GADD45[3, and c-FLIP (Karin and Lin, 2002). lAPs directly associate with caspases and prevent activation of initiator caspase 9 and inhibit effector caspases 3 and 7 (Deveraux and Reed, 1999). c-FLIP mimics caspases in its composition, but contains a catalytically inactive effector domain and appears to interfere with caspase-8 recruitment and activation (Krueger et al., 2001). Members of the Bcl-2 protein family are also expressed upon NF-~:B regulation, including A1, Bcl-XL, and Bcl-2 itself (all antiapoptotic), whereas the proapoptotic Bax protein is downregulated (Bentires-Alj et al., 2001; Karin and Lin et al., 2002). GADD4513 and XIAP (X-linked inhibitor of apoptosis) may protect against apoptosis by interfering with proapoptotic JNK signaling (De Smaele et al., 2001; Tang et al., 2001). Finally,
TRAF1 and 2 are critical components of the TNF receptor super family signaling pathway, and their induction by NF-~B may play a role in modulating the balance between pro- and antiapoptotic signals (Karin and Lin, 2002). Pulling in the opposite direction is the fact that many of the components of the signaling pathways leading to NF-~B activation are caspase targets (including NF-~cB itself), and cleavage of these attenuates the antiapoptotic outcome. Additionally, I~:B can be cleaved to render the molecule resistant to the phosphorylation step necessary for its degradation to release active NF-~cB (Karin and Lin, 2002). NF-~B may also in certain situations promote apoptosis, rather that fight it, but there are only a few examples of this. The most interesting may be that MEKl-rsk-dependent activation of NF-~cB by tumor supressor p53 is necessary for p53-induced apoptosis (Ryan et al., 2000). The generally cell-protective effect of NF-~cB activation is, not surprisingly, a double-edged sword with regard to oncogenesis. For instance, as many as 10% of lymphocyte neoplasias have, in addition to other abnormalities, mutations in their Rel or IKB sequences (Foo and Nolan, 1999). Several members of the NF-~B pathway induce transformation in vivo or in vitro when mutated or dysregulated, and many common oncogenes affect NF-~B activation. Additionally, activation of NF-~B in response to chemotherapy and radiation counteracts the ability of the treatment to induce apoptosis of the cancer cells (Baldwin, 2001). It is also of concern that NF-~B regulates the expression of adhesion molecules, some cell-cycle proteins, and COX-2, and therefore may be involved in proliferation, inhibition of differentiation, angiogenesis, and metastasis (Baldwin, 2001). Therefore, inhibition of NF-~B may be useful in treatment of certain cancers, and some experimental tumors are indeed responsive to such treatment. However, considering the sometimes opposing effects of NF-~cB actions, more studies are needed to determine the overall benefit of such treatment in vivo.
Role of NF-IcB in immune system activation The NF-~:B pathway is intimately involved in maturation and activation of most immune cells by
694 controlling the expression of a broad spectrum of immune system effectors, including MHC proteins, proinflammatory cytokines, chemokines, interferons, and adhesion molecules. Individual actions of the pathway effectors have been elucidated partly by transgenic techniques. For instance, c-Rel knockout mice have defects in immune cell activation leading to decreased production of cytokines and immunoglobulins (Kontgen et al., 1995). RelB knockouts show dendritic cell defects and die postnatally from T-celldependent multiorgan inflammation (Weih et al., 1996). Removal of p50 or p52 also causes immune defects, especially involving B cells (Li and Verma, 2002). During an immune response, NF-~B is activated rapidly to induce an inflammatory response. The major pathways of activation include signaling by proinflammatory cytokines, especially TNF-a and IL- 1 (interleukin- 1). In addition, the pattern recognition receptors called Toll-like receptors induce NF-KB in response to bacterial and viral products, such as lipopolysaccharide, dsRNA, and lipopeptides as part of the innate immune response. Antigenspecific T cells that are activated both through their T-cell receptors and costimulatory signals also induce NF-~cB. This in turn promotes production of IL-2, an essential component of a humoral immune response. NF-KB induction in other immune cells leads to massive secretion of more proinflammatory molecules, including IL-1, IL-6, IL-8, TNF-a, COX-2, matrix metalloproteinases, and inducible nitric oxide synthase (Li and Verma, 2002). Several of these molecules lead to further NF-KB activation, propagating an inflammatory response to protect the host against the infectious agent. However, the analogy of the double-edged sword also applies here, since many viruses have acquired ~B sites in their viral promotors. Thus, when the host's immune response to the virus activates NF-~:B to fight against the viral infection, transcription of viral proteins is enhanced. This may partly explain how certain viruses, including EBV and HIV-1, maintain chronic infections (Pahl, 1999). Several commonly used antiinflammatory drugs inhibit NF-~cB activation (in addition to their other effects). These include corticosteroids and nonsteroidal antiinflammatory drugs such as aspirin, at least some of which inhibit the phosphorylation of I~:B proteins. The antiinflammatory cytokines IL-10
and IL-13 suppress nuclear localization of NF-~:B and upregulate boB expression. Specific blockade of NF-~cB activation continues to be a focus for new antiinflammatory drug development (Epinat and Gilmore, 1999; Yamamoto and Gaynor, 2001).
Role of NF-lcB in CNS pathology and stress Neuronal NF-KB has important neuroprotective roles in response to CNS injury such as that following ischemia and seizures. It also appears to attenuate neuronal death in ALS, Alzheimer's, Parkinson's, and Huntington's diseases. In contrast, microglial NF-KB activation appears to promote neuronal degradation, and the end result of general NF-KB activation is therefore dependent on the balance of neuronal and glial responses (Mattson and Camandola, 2001). For example, data from studies with p50 knockout mice suggests that ischemic neuronal death is enhanced by NF-KB activation, whereas NF-KB protects neurons against seizurerelated excitotoxicity (Schneider et al., 1999; Yu et al., 1999). In addition to proinflammatory cytokines, potent CNS inducers of NF-~cB include bradykinin, glutamate, increases in intracellular Ca 2+, and reactive oxygen species, all of which are important mediators of CNS pathology (Mattson and Camandola, 2001). CNS injury-responsive NF-KB-induced molecules include the proinflammatory cytokines TNF-a and IL-6, 13APP ([3-amyloid protein precursor), Mn-SOD (manganese superoxide dismutase), calbindin, ICAM-1, GFAP, and NAIP-1 (neuronal apoptosis inhibitory protein-I), in addition to the antiapoptotic factors described above (Mattson and Camandola, 2001). ]3APP has neurotrophic properties and also further induces NF-~cB activation. Mn-SOD is a neuroprotective mitochondrial antioxidant molecule, and calbindin is involved in calcium-mediated neuronal signaling and cell death. ICAM-1 and GFAP mediate intracellular interactions and structural stability of glial cells, respectively, but the functional outcomes of their upregulation are unclear. In addition to its role in CNS injury, NF-KB is also involved in regulation of synaptic function. In this context, NF-~zB is activated in response to membrane depolarization, low-frequency stimulation,
695 and during long-term potentiation of synaptic transmission. In fact, pretreatment with ~cB decoy D N A (to "soak up" activated NF-~cB) in hippocampal slices abolishes the ability to induce long-term depression and significantly decreases the amplitude of long-term potentiation (Albensi and Mattson, 2000). The effects of NF-~cB activation in this phenomenon are not well understood, but induced genes may include N M D A and A M P A glutamate receptor subunits (Furukawa and Mattson, 1998). NF-~B activation has also been suggested to be involved in stress and some psychiatric diseases. Emotional distress in women scheduled for breast biopsies led to decreased levels of NF-~:B that returned to baseline when stress was relieved, providing a possible explanation for the phenomenon of stress-induced immunosuppression (Nagabhushan et al., 2001). Gene expression analysis in frontal cortex from patients with bipolar disorder showed a significant increase in NF-~cB transcription factor complex components, and increased levels of the same gene transcripts were found in some patients with schizophrenia and depression (Sun et al., 2001). However, it is not clear whether these increases reflect a protective response or an integral part of the pathology.
Conclusions Significant progress has been made in elucidating the molecular mechanisms that control gene transcription and genetic code of humans and other species. This work provides the tools necessary to study the genes that are differentially expressed in response to stress and other stimuli and that ultimately control neuronal function and behavior. This is an exciting and interesting time as we begin to unravel the fundamental molecular basis for complex behaviors and the genetic basis that underlie differences between individuals. The studies outlined in this chapter regarding a few classes of transcription factors and target genes provide a framework for future studies that will provide a complete analysis of the signal transduction and gene expression patterns that are critical to stress responses. Using well-defined approaches that clearly define the intensity and type of stress (e.g., physical vs. psychological stress) will
eventually elucidate the complex cellular responses that underlie adaptive changes as well as those that contribute to maladaptive alterations in response to stress.
Acknowledgments This work is supported by USPHS grants MH45481 and 2 PO1 MH25642, a Veterans Administration National Center Grant for PTSD, and by the Connecticut Mental Health Center.
References Albensi, B.C. and Mattson, M.P. (2000) Evidence for the involvement of TNF and NF-kappaB in hippocampal synaptic plasticity. Synapse, 35: 151-159. Armstrong, R. and Montminy, M.R. (1993) Transsynaptic control of gene expression. Ann. Rev. Neurosci., 16: 17-29. Baeuerle, P.A. and Baltimore, D. (1988) I kappa B: a specific inhibitor of the NF-kappa B transcription factor. Science, 242: 540-546. Baldwin, A.S. (2001) Control of oncogenesis and cancer therapy resistance by the transcription factor NF-kappaB. J. Clin. Invest., 107: 241-246. Barrot, M., Olivier, J.D.A., Perrotti, L.I., DiLeone, R.J., Berton, O., Eisch, A.J., Impey, S., Storm, D.R., Neve, R.L., Yin, J.C., Zachariou, V. and Nestler, E.J. (2002) CREB activity in the nucleus accumbens shell controls gating of behavioral responses to emotional stimuli. PNAS, 99: 11435-11440. Beg, A.A. and Baltimore, D. (1996) An essential role for NF-kappaB in preventing TNF-alpha-induced cell death. Science, 274: 782-784. Beg, A.A., Sha, W.C., Bronson, R.T., Ghosh, S. and Baltimore, D. (1995) Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-kappa B. Nature, 376: 167-170. Bentires-Alj, M., Dejardin, E., Viatour, P., Van Lint, C., Froesch, B., Reed, J.C., Merville, M.P. and Bours, V. (2001) Inhibition of the NF-kappa B transcription factor increases Bax expression in cancer cell lines. Oncogene, 20:2805-2813. Bernabeu, R., Bevilaqua, L., Ardenghi, P., Bromberg, E., Schmitz, P., Bianchin, M., Izquierdo, I. and Medina, J.H. (1997) Involvement of hippocampal cAMP/cAMP-dependent protein kinase signaling pathways in a late memory consolidation phase of aversively motivated learning in rats. PNAS, 94: 7041-7046. Bilang-Bleuel, A., Rech, J., De Carli, S., Holsboer, F. and Reul, J.M.H.M. (2002) Forced swimming evokes a biphasic response in CREB phosphorylation in extrahypothalamic
696 limbic and neocortical brain structures in the rat. Eur. J. Neurosci., 15: 1048-1060. Borsok, D., Konradi, C., Falkowski, O., Comb, M. and Hyman, S. (1994) Molecular mechanisms of stress-induced proenkephalin gene regulation: CREB interacts with the proenkephalin gene in the mouse hypothalamus and is phosphorylated in response to hyperosmolar stress. Mol. Endocrinol., 8: 240-248. Bruijnzeel, A., Stam, R., Compaan, J.C., Croiset, G., Akkermans, L.M., Olivier, B. and Wiegant, V.M. (1999) Long-term sensitization of Fos-responsivity in the rat central nervous system after a single stressful experience. Br. Res., 819: 15-22. Bruijnzeel, A., Stam, R., Compaan, J.C. and Wiegant, V.M. (2001) Stress-induced sensitization of CRH-ir but not P-CREB-ir responsivity in the rat central nervous system. Brain Res., 908: 187-196. Campeau, S. and Watson, S.J. (1997) Neuroendocrine and behavioral responses and brain pattern of c-fos induction associated with audiogenic stress. J. Neuroendocrinol., 9: 577-588. Carlezon, W.J., Thome, J., Olson, V.G., Lane-Ladd, S.B., Brodkin, E.S., Hiroi, N., Duman, R.S., Neve, R.L. and Nestler, E.J. (1998) Regulation of cocaine reward by CREB. Science, 282: 2272-2275. Chao, J., Ni, Y.G., Bolanos, C.A., Rahman, Z., DiLeone, R.J. and Nestler, E.J. (2002) Characterization of the mouse adenylyl cyclase type VIII gene promoter: regulation by cAMP and CREB. Eur. J. Neurosci., 16: 1284-1294. Chen, X. and Herbert, J. (1995) Regional changes in c-fos expression in the basal forebrain and brainstem during adaptation to repeated stress: correlations with cardiovascular, hypothermic and endocrine responses. Neurosci., 64: 675-685. Chen, A.-H., Shirayama, Y., Shin, K.-H., Neve, R.L. and Duman, R.S. (2001) Expression of the cAMP response element binding protein (CREB) in hippocampus produces antidepressant effect. Biol. Psychiat., 49: 753-762. Chen, L.F., Mu, Y. and Greene, W.C. (2002) Acetylation of RelA at discrete sites regulates distinct nuclear functions of NF-kappaB. EMBO J., 21: 6539-6548. Conti, A., Cryan, J.F., Dalvi, A., Lucki, L. and Blendy, J.A. (2002) CREB is essential for the upregulation of BDNF transcription, but not the behavioral or endocrine responses to antidepressant drugs. J. Neurosci., 22: 3262-3268. De Smaele, E., Zazzeroni, F., Papa, S., Nguyen, D.U., Jin, R., Jones, J., Cong, R. and Franzoso, G. (2001) Induction of gadd45beta by NF-kappaB downregulates pro-apoptotic JNK signalling. Nature, 414: 308-313. Deveraux, Q.L. and Reed, J.C. (1999) IAP family proteinssuppressors of apoptosis. Genes Dev., 13: 239-252. Dowlatshahi, D., MacQueen, G.M., Wang, J.F. and Young, L.T. (1998) Increased temporal cortex CREB concentrations and
antidepressant treatment in major depression. The Lancet, 352: 1754-1755. Duman, R.S., Malberg, J., Nakagawa, S. and D'Sa, C. (2000) Neuronal plasticity and survival in mood disorders. Biol. Psychiatry. 48: 732-739. Epinat, J.C. and Gilmore, T.D. (1999) Diverse agents act at multiple levels to inhibit the Rel/NF-kappaB signal transduction pathway. Oncogene, 18: 6896-6909. Ericsson, A., Kovacs, K.J. and Sawchenko, P.E. (1994) A functional anatomical analysis of central pathways subserving the effects of interleukin-1 on stress-related neuroendocrine neurons. J. Neurosci., 14: 897-913. Foo, S.Y. and Nolan, G.P. (1999) NF-kappaB to the rescue: RELs, apoptosis and cellular transformation. Trends Genet., 15: 229-235. Frechilla, D., Otano, A. and Del Rio, J. (1998) Effect of chronic antidepressant treatment on transcription factor binding activity in rat hippocampus and frontal cortex. Progr. Neuro. Psychopharm. Biol. Psychiatr., 22: 787-802. Furukawa, K. and Mattson, M.P. (1998) The transcription factor NF-kappaB mediates increases in calcium currents and decreases in NMDA- and AMPA/kainate-induced currents induced by tumor necrosis factor-alpha in hippocampal neurons. J. Neurochem., 70: 1876-1886. Gesing, A., Bilang-Bleuel, Droste A., Linthorst, S.K., Holsboer, F. and Reul, J.M.H.M. (2001) Psychological stress increased hippocampal mineralocorticoid receptor levels: involvement of corticotropin-releasing hormone. J. Neurosci., 21: 4822-4829. Ghosh, S., May, M.J. and Kopp, E.B. (1998) NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu. Rev. Immunol., 16: 225-260. Honkaniemi, J., Kononen, J., Kainu, T. and Pelto-Hiukko, M. (1994) Induction of multiple immediate early genes in rat hypothalamic paraventricular nucleus after stress. Br. Res. Mol. Br. Res., 25: 234-241. Huang, L., Holmes, G.L., Lai, M.C., Hung, P.L., Wang, C.L., Wang, T.J., Yang, C.H., Liou, C.W. and Yang, S.N. (2002) Maternal deprivation stress exacerbates cognitive deficits in immature rats with recurrent seizures. Epilepsia, 43: 1141-1148. Karin, M. and Chang, L. (2001) AP-l-glucocorticoid receptor crosstalk taken to a higher level. J. Endocrinol., 169: 447-451. Karin, M. and Lin, A. (2002) NF-kappaB at the crossroads of life and death. Nat. Immunol., 3: 221-227. Kontgen, F., Grumont, R.J., Strasser, A., Metcalf, D., Li, R., Tarlinton, D. and Gerondakis, S. (1995) Mice lacking the c-rel proto-oncogene exhibit defects in lymphocyte proliferation, humoral immunity, and interleukin-2 expression. Genes Dev., 9: 1965-1977. Kovacs, K. (1998) c-Fos as a transcription factor: a stressful (re) view from a functional map. Neurochem. Int., 33: 287-297.
697 Kovacs, K. and Sawchenko, P.E. (1997) Sequence of stressinduced alterations in indices of synaptic and transcriptional activation in parvocellular neurosecretory neurons. J. Neurosci., 16: 262-273. Krueger, A., Baumann, S., Krammer, P.H. and Kirchhoff, S. (2001) FLICE-inhibitory proteins: regulators of death receptor-mediated apoptosis. Mol. Cell. Biol., 21: 8247-8254. Legradi, G., Holzer, D., Kapcala, L.P. and Lechan, R.M. (1997) Glucocorticoids inhibit stress-induced phosphorylation of CREB in corticotropin-releasing hormone neurons of the hypothalamic paraventricular nucleus. Neuroendocrinology, 66:3550-3554. Leitges, M., Sanz, L., Martin, P., Duran, A., Braun, U., Garcia, J.F., Camacho, F., Diaz-Meco, M.T., Rennert, P.D. and Moscat, J. (2001) Targeted disruption of the zetaPKC gene results in the impairment of the NF-kappaB pathway. Mol. Cell., 8: 771-780. Lewis, E., Harrington, C.A. and Chikaraishi, D.M. (1987) Transcriptional regulation of the tyrosine hydroxylase gene by glucorticoid and cyclic AMP. PNAS, 84: 3550-3554. Li, Q. and Verma, I.M. (2002) NF-kappaB regulation in the immune system. Nat. Rev. Immunol., 2: 725-734. Liu, Z.G., Hsu, H., Goeddel, D.V. and Karin, M. (1996) Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-kappaB activation prevents cell death. Cell 87: 565-576. Lopez, J., Akil, H. and Watson, S.J. (1999) Neural circuits mediating stress. Biol. Psychiat., 45: 1461-1471. Mattson, M.P. and Camandola, S. (2001) NF-kappaB in neuronal plasticity and neurodegenerative disorders. J. Clin. Invest., 107: 247-254. Nagabhushan, M., Mathews, H.L. and Witek-Janusek, L. (2001) Aberrant nuclear expression of AP-1 and NFkappaB in lymphocytes of women stressed by the experience of breast biopsy. Brain Behav. Immun., 15: 78-84. Nankova, B., Kvetnansky, R., McMahon, A., Viskupic, E., Hiremagalur, B., Frankle, G., Fukuhara, K., Kopin, I.J. and Sabban, E.L. (1994) Induction of tyrosine hydroxylase gene expression by a nonneuronal nonpituitary-mediated mechanism in immobilization stress. PNAS, 91: 5937-5941. Nankova, B., Rivkin, M., Kelz, M., Nestler, E.J. and Sabban, E.L. (2000) Fos-related antigen 2: potential mediator of the transcriptional activation in rat adrenal medulla evoked by repeated immobilization stress. J. Neurosci., 20: 5647-5653. Nestler, E., Kelz, M.B. and Chen, J. (1999) DeltaFosB: a molecular mediator of long-term neural and behavioral plasticity. Brain Res., 835: 10-17. Nestler, E.J., Hyman, S.E. and Malenka, R.C. (2001) Signaling to the nucleus. In: Molecular Neuropharmacology (eds. E.J. Nestler, S.E. Hyman and R.C. Malenka). McGraw Hill, New York, NY, pp. 115-137. Newton, S., Thome, J., Wallace, T.L., Shirayama, Y., Schlesinger, L., Sakai, N., Chert, N., Neve, R., Nestler, E.J. and Duman, R.S. (2002) Inhibition of cAMP response
element-binding protein or dynorphin in the nucleus accumbens produces an antidepressant-like effect. J Neurosci., 24: 10883-10890. Nibuya, M., Morinobu, S. and Duman, R.S. (1995) Regulation of BDNF and trkB mRNA in rat brain by chronic electroconvulsive seizure and antidepressant drug treatments. J. Neurosci., 15: 7539-7547. Nibuya, M., Nestler, E.J. and Duman, R.S. (1996) Chronic antidepressant administration increases the expression of cAMP response element binding protein (CREB) in rat hippocampus. J. Neurosci., 16: 2365-2372. Nibuya, M., Takahashi, M., Russell, D.S. and Duman, R.S. (1999) Chronic stress increases catalytic TrkB mRNA in rat hippocampus. Neurosci. Letts., 267: 81-84. Pahl, H.L. (1999) Activators and target genes of Rel/NFkappaB transcription factors. Oncogene, 18: 6853-6866. Phiel, C., Zhang, F., Huang, E.Y., Guenther, M.G., Lazzar, M.A. and Klein, P.S. (2001) Histone deacetylase is a direct target of valproic acid, a potent anticonvulsant, mood stabilizer, and teratogen. J. Biol. Chem., 276: 3673436741. Pliakas, A., Carlson, R.R., Neve, R.L., Konradi, C., Nestler, E.J. and Carlezon, W.A. (2001) Altered responsiveness to cocaine and increased immobility in the forced swim test associated with elevated CREB expression in the nucleus accumbens. J. Neurosci., 21: 7397-7403. Porsolt, R., Le Pichon, M. and Jalfre, M. (1977) Depression: a new animal model sensitive to antidepressant treatments. Nature, 266: 730-732. Rivest, S. and Rivier, C. (1994) Stress and interleukin-1 beta-induced activation of c-fos, NGF1-B and CRF gene expression in the hypothalamic PVN: comparison between Sprague-Dawley, Fisher-344 and Lewis rats. J. Neuroendocrinol., 6: 101-117. Rothwarf, D.M., Zandi, E., Natoli, G. and Karin, M. (1998) IKK-gamma is an essential regulatory subunit of the IkappaB kinase complex. Nature 395: 297-300. Ryan, K.M., Ernst, M.K., Rice, N.R. and Vousden, K.H. (2000) Role of NF-kappaB in p53-mediated programmed cell death. Nature, 404: 892-897. Sabban, E. and Kvetnansky, R. (2001) Stress-triggered activation of gene expression in catecholaminergic systems: dynamics of transcriptional events. Trends Neurosci., 24: 91-98. Schneider, A., Martin-Villalba, A., Weih, F., Vogel, J., Wirth, T. and Schwaninger, M. (1999) NF-kappaB is activated and promotes cell death in focal cerebral ischemia. Nat. Med., 5: 554-559. Sen, R. and Baltimore, D. (1986) Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell, 46:705-716. Senftleben, U., Cao, Y., Xiao, G., Greten, F.R., Krahn, G., Bonizzi, G., Chen, Y., Hu, Y., Fong, A., Sun, S.C. and Karin, M. (2001) Activation by IKKalpha of a second,
698 evolutionary conserved, NF-kappa B signaling pathway. Science, 293: 1495-1499. Serova, L., Saez, E., Spiegelman, B.M. and Sabban, E.L. (1998) c-Fos deficiency inhibits induction of mRNA for some, but not all, neurotransmitter biosynthetic enzymes by immobilization stress. J. Neurochem., 70: 1935-1940. Shaulian, E. and Karin, M. (2002) AP-1 as a regulator of cell life and death. Nat. Cell. Biol., 4: E131-E136. Shirayama, Y., Chen, A.C.-H., Nakagawa, S., Russell, R.S. and Duman, R.S. (2002) Brain derived neurotrophic factor produces antidepressant effects in behavioral models of depression. J. Neurosci., 22: 3251-3261. Silva, A., Kogan, J.H., Frankland, P.W. and Kida, S. (1998) CREB and memory. Ann. Rev. Neurosci., 21: 127-148. Smith, M.A., Makino, S., Kvetnansky, R. and Post, R.M. (1995) Stress alters the express of brain-derived neurotrophic factor and neurotrophin-3 mRNAs in the hippocampus. J. Neurosci., 15: 1768-1777. Stamp, J. and Herbert, J. (1999) Multiple immediate-early gene expression during physiological and endocrine adaptation to repeated stress. Neurosci., 94: 1313-1322. Stanciu, M., Radulovic, J. and Spiess, J. (2001) Phosphorylated cAMP response element binding protein in the mouse brain after fear conditioning: relationship to Fos production. Mol. Brain Res., 94: 15-24. Sun, Y., Zhang, L., Johnston, N.L., Torrey, E.F. and Yolken, R.H. (2001) Serial analysis of gene expression in the frontal cortex of patients with bipolar disorder. Br. J. Psychiatry, Suppl 41: s137-s141. Tang, G., Minemoto, Y., Dibling, B., Purcell, N.H., Li, Z., Karin, M. and Lin, A. (2001) Inhibition of JNK activation through NF-kappaB target genes. Nature, 414: 313-317. Tao, X., Finkbeiner, S., Arnold, D.B., Shaywitz, A.J. and Greenberg, M.E. (1998) Ca2+ influx regulates BDNF transcription by a CREB family transcription factor-dependent mechanism. Neuron, 20: 709-726. Thome, J., Sakai, N., Shin, K.H., Steffen, C., Zhang, Y.-J., Impey, S., Storm, D.R. and Duman, R.S. (2000) cAMP response element-mediated gene transcription is upregulated
by chronic antidepressant treatment. J. Neurosci., 20: 4030-4036. Trentani, A., Kuipers, S.D., Ter Horst, G.J. and Den Boer, J.A. (2002) Selective chronic stress-induced in vivo ERK1/2 hyperphosphorylation in medial prefrontocortical dendrites: implications for stress-related cortical pathology? Eur. J. Neurosci., 15: 1681-1691. Umemoto, S., Kawai, Y., Ueyama, T. and Senba, E. (1997) Chronic glucocorticoid administration as well as repeated stress affects the subsequent acute immobilization stressinduced expression of immediate early genes but not that of NGFI-A. Neurosci., 80: 763-773. Van Antwerp, D.J., Martin, S.J., Kafri, T., Green, D.R. and Verma, I.M. (1996) Suppression of TNF-alpha-induced apoptosis by NF-kappaB. Science, 274: 787-789. Wallace, T., Stellitano, K.E., Neve, R.L. and Duman, R.S. (2002) Effects of CREB expression in the basolateral amygdala on behavioral models of depression and anxiety. Soc. for Neuros. Sci. Abstract. Weih, F., Durham, S.K., Barton, D.S., Sha, W.C., Baltimore, D. and Bravo, R. (1996) Both multiorgan inflammation and myeloid hyperplasia in RelB-deficient mice are T cell dependent. J. Immunol., 157: 3974-3979. Yamamoto, Y. and Gaynor, R.B. (2001) Therapeutic potential of inhibition of the NF-kappaB pathway in the treatment of inflammation and cancer. J. Clin. Invest., 107: 135-142. Yu, Z., Zhou, D., Bruce-Keller, A.J., Kindy, M.S. and Mattson, M.P. (1999) Lack of the p50 subunit of nuclear factor-kappaB increases the vulnerability of hippocampal neurons to excitotoxic injury. J. Neurosci., 19: 8856-8865. Zandi, E., Rothwarf, D.M., Delhase, M., Hayakawa, M. and Karin, M. (1997) The IkappaB kinase complex (IKK) contains two kinase subunits, IKKalpha and IKKbeta, necessary for IkappaB phosphorylation and NF-kappaB activation. Cell 91: 243-252. Zhong, H., Voll, R.E. and Ghosh, S. (1998) Phosphorylation of NF-kappa B p65 by PKA stimulates transcriptional activity by promoting a novel bivalent interaction with the coactivator CBP/p300. Mol. Cell, 1: 661-671.
T. Steckler, N.H. Kalin and J.M.H.M. Reul (Eds.) Handbook of Stress and the Brain, Vol. 15
ISBN 0-444-51173-3 Copyright 2005 Elsevier B.V. All rights reserved CHAPTER 5.4
Experience, structural plasticity and neurogenesis Jennifer D. Peters* and Elizabeth Gould Department of Psychology, Princeton University, Princeton, NJ 08544, USA
Abstract" The adult hippocampus is a structurally plastic region in which dendritic remodeling, synaptogenesis, neurogenesis, and cell death have all been reported to occur in the absence of damage or pathology. These ongoing structural changes are modulated by both hormones and experience, and may contribute to aspects of hippocampal function, such as learning and memory, response to novelty, and regulation of the hypothalamic-pituitary-adrenal (HPA) axis. In turn, environmental complexity, learning, stress, and aversive experience appear to shape the hippocampus by regulating its structural plasticity. This chapter focuses on structural changes in the adult hippocampus, including dendritic remodeling and neurogenesis, and how these processes are affected by hormones and experience throughout life.
Hormones and experience: hippocampal plasticity in the unstressed brain
The adult hippocampus exhibits a variety of dynamic structural phenomena previously believed to occur only during development. This structural plasticity appears to be modulated by both hormones and experience, suggesting that these changes may represent mechanisms that underlie hippocampal function. In this regard, the hippocampus has been linked to several functions that are affected by stress, including learning, response to novelty, and regulation of the hypothalamic-pituitary-adrenal (HPA) axis. The first section of this chapter examines hormones and experiences that appear to contribute to ongoing anabolic processes in the unstressed adult hippocampus. The second section focuses on how stress hormones and aversive experience affect these processes, and discusses some implications that this high-degree of plasticity may have for understanding how the adult brain normally functions and what goes wrong in cases of psychiatric illness.
The adult hippocampus has been shown to undergo significant structural change in the absence of damage or pathology. Hormones and experience appear to induce fluctuations in the number and complexity of synapses and dendritic spines, the production and survival of new neurons, and overall hippocampal volume throughout life.
Synaptic and dendritic remodeling Considerable transformations of hippocampal synapses and dendrites have been observed in response to changing behavioral states, hormonal levels, or environmental conditions. For example, during periods of torpor in the ground squirrel, mossy fiber synapses on CA3 pyramidal cells become smaller and show fewer postsynaptic densities and dendritic spine infoldings, but within two hours of arousal, their structures are completely restored (Popov and Bocharova, 1992). These rapid morphological changes parallel those seen in the apical dendrites of hippocampal pyramidal cells, which
*Corresponding author. Tel.: (609) 258-5625, (609) 258-4483; Fax: (609) 258-1113; E-mail:
[email protected] 699
700 appear much shorter, less-branched, and have fewer dendritic spines prior to arousal (Popov et al., 1992). Excitatory synapses on pyramidal cells in the CA1 region of female rodents are continuously formed and retracted in response to fluctuations in ovarian steroid levels via an NMDA receptormediated mechanism (McEwen et al., 2001). These estrous cycle oscillations in synapse number are coincident with changes in dendritic spine density on CA1 pyramidal neurons, such that during proestrus, rats have approximately 30% more spines than during late estrus (Woolley et al., 1990a). Environmental enrichment has long been known to produce pronounced structural changes in the brains of laboratory animals (Diamond et al., 1964). These paradigms usually give animals access to larger cages, different toys and opportunities for sensory stimulation and physical exercise, and a chance for increased social interaction and learning experiences. Exposure to laboratory enrichment early in life enhances the growth of granule and pyramidal cell dendrites in the developing rat hippocampus (Fiala et al., 1978). Other studies have noted increases in cell volumes and the number of dendritic branches in pyramidal cells, and increased dendrite length in the granule cells of the dentate gyrus in mice raised to adulthood in enriched environments. These effects appeared to be independent of physical activity, as mice with running wheels, but not other aspects of enrichment, did not show these changes (Faherty et al., 2003). Although the timescale and persistence of the effects of enrichment are not as well-characterized in the adult hippocampus as they are in other brain regions, enriched environment living for as little as 4 days increases spine number and length in the adult rat cortex (Wallace et al., 1992), and rats returned to isolated laboratory cages show persistent dendritic changes for several weeks following 30 days or more experience in enriched environments (Camel et al., 1986). One parameter that may potentially contribute to the environmental enrichment effect is learning, or the opportunity for the animals to engage in a greater variety of behaviors that involve the hippocampus. Some investigators have observed an increase in spine density on granule cells in the dentate gyrus of the hippocampus six hours after training on a passive
avoidance paradigm, an effect that persists for fewer than 3 days (O'Malley et al., 1998). A similar learning-associated effect was seen in granule cells after water maze training (O'Malley et al., 2000). An increase in basal spine density on the pyramidal cells of the CA1 region of the hippocampus was measured 24h post-training on either a trace eyeblink (hippocampus-dependent) or delay eyeblink (hippocampus-independent) conditioning task. No changes were observed in the dentate gyrus in this experiment. When given an NMDA receptor antagonist, animals neither acquired the conditioned responses nor showed increases in CA1 pyramidal spine density (Leuner et al., 2003). Taken together, these results suggest that learning alters dendritic spines, and it seems likely that these dendritic changes (which are generally accepted to indicate changes in the number of excitatory synapses as well) contribute to the learning process itself. Adult
neurogenesis
Although it was previously assumed that neurogenesis was restricted to early developmental periods, the adult brain of a variety of mammalian species, including humans, continues to generate significant numbers of neurons throughout life (Gould and Gross, 2002). One brain region where this phenomenon is particularly robust is the hippocampus. Approximately 9000 cells are produced each day in the dentate gyrus of the adult rat, the majority of which appear to differentiate into neurons (Cameron and McKay, 2001). Like the resident population of granule neurons, these adult-generated cells extend axons into the CA3 region (Stanfield and Trice, 1988; Hastings and Gould, 1999; Markakis and Gage, 1999) and form synapses (Kaplan and Hinds, 1977). Many of the new neurons die after several weeks (Cameron et al., 1993b; Biebl et al., 2000; Gould et al., 2001), but despite their transient existence, these cells appear to contribute to some aspects of hippocampal function, such as learning (Gould and Gross, 2002), reaction to novelty (Lemaire et al., 1999), and mood regulation (Santarelli et al., 2003). Adult neurogenesis, like dendritic remodeling, is modulated by hormones and the environment. Ovarian steroid manipulations demonstrate that transiently high levels of estrogen result in
701 increases in the rate of cell proliferation in the dentate gyrus. Ovariectomy diminishes the number of new neurons, whereas estrogen replacement restores neuron production to normal. A natural fluctuation in cell proliferation was also observed across the estrous cycle, such that female rats in proestrus produced more neurons than in other phases when estrogen levels are lower (Tanapat et al., 1999). Numerous studies suggest that environmental complexity can enhance the number of new neurons in the hippocampus over the number observed in control animals. The first study to report this examined black-capped chickadees and found that birds living in the wild had more new neurons than those living in captivity. A seasonal difference in neurogenesis was observed as well, such that during the season of maximal seed caching and retrieval, more new neurons were maintained as compared to other times of year (Barnea and Nottebohm, 1994). These findings suggested that activation of the hippocampus, by engagement in spatial navigation learning (or some other seasonal activity), enhanced the survival of new neurons. Another study showed more directly that food storage and retrieval stimulates hippocampal neurogenesis, since marsh tits that were allowed to forage for food showed considerable increases in neurogenesis over age-matched controls (Patel et al., 1997). Subsequent work demonstrated that living in a laboratory enriched environment setting also enhanced the survival of newly generated granule cells in the dentate gyrus of adult rats and mice (Kempermann et al., 1997; Nilsson et al., 1999). Some of this enrichment effect may also be due to increased motor activity, as mice given access to a running wheel have been shown to have increased neurogenesis over controls (see Brown et al., 2003). Another possibility is that various learning tasks involving the hippocampus increase the number of new cells either by upregulating cell proliferation or enhancing the survival of cells produced in adulthood. Support for this hypothesis comes from studies that show twice the number of adult-generated neurons in the dentate gyri of rats trained in hippocampus-dependent associative learning tasks over controls or rats trained in analogous tasks that do not require the hippocampus (Gould et al., 1999a). Subsequent research has indicated that these new neurons actually participate in the learning
process, since the toxin methylazoxymethanol acetate (MAM), which kills proliferating cells, not only blocked ongoing hippocampal neurogenesis but also the formation of trace memories. When rats were allowed to recover and neurogenesis resumed, the ability to acquire new trace memories returned (Shors et al., 2001b). It appears that only certain hippocampus-dependent learning tasks require ongoing neurogenesis, as MAM treatment does not disrupt all kinds of hippocampus-dependent learning (Shors et al., 2002). The continuous addition of new neurons with their immature (and thus unique) properties suggests that adult-generated neurons may play a crucial role in some forms of associative learning (Gould et al., 1999b).
Hippocampal volume Some evidence suggests that the overall volume of the hippocampus is related to some of its learning and memory functions. Vertebrate species with behavioral adaptations involving a need for extensive spatial navigation learning exhibit larger hippocampi than similar species without these adaptations. For example, birds and small mammals that cover wide territories in search of food or mates have significantly larger hippocampal volumes than other related species living in more restricted areas (see Sherry et al., 1992). Furthermore, avian species that presumably engage the hippocampus to distribute and relocate seeds in disparate locations show variations in the hippocampal formation across seasons, such that periods of intense caching and retrieval are correlated with increases in hippocampal volume (Smulders et al., 1995). These seasonal differences are not found in similar species of non-caching birds (Lee et al., 2001). Sex differences in hippocampal volume have also been reported in birds and mammals. These dimorphisms may be related to sex differences in hippocampus-dependent behavior. For example, female parasitic brooding cowbirds spend considerable amounts of time searching for and remembering the location of appropriate nests in which to lay eggs. They have larger hippocampi than male cowbirds who do not engage in such behaviors (Sherry et al., 1993). In addition, polygamous male voles who
702 traverse wide territories in search of mates have larger hippocampi than the more sedentary females (Jacobs et al., 1990). Differences in hippocampal volume have also been reported within same sex conspecifics, including in humans. Cab drivers in London exhibit larger posterior hippocampi, and smaller anterior hippocampi, than controls (Maguire et al., 2000). It is possible that an individual's hippocampal structure prior to experience might impose a predisposition to engage in certain behaviors or alter the likelihood of succeeding in activities that require certain types of learning, as becoming a cab driver inevitably involves extensive spatial navigation learning, but it may also be that the actual process of engaging in behaviors that require the hippocampus alters the size and shape of this brain region. Considerable evidence suggests that experience is, in fact, capable of modulating hippocampal structure, even in adulthood. Laboratory-based experimental manipulations in environment suggest that more complex experiences lead to gross changes in the hippocampus. When compared to control animals living in standard laboratory housing, animals living in a more complex environment had larger dentate gyri (Kempermann et al., 1997). While it is unclear whether enriched environment studies actually reflect the influence of experience on the hippocampus, as opposed to removing the effects of deprivation, these findings do suggest that conditions capable of activating the hippocampus alter its size. Changes in hippocampal volume might reflect any number of mechanisms, including alterations in total cell number, the length or complexity of dendritic arborizations, or the density of connections between cells. Since structural plasticity at each of these levels has been reported in the intact hippocampus of a variety of species under a variety of conditions that involve that brain region, it seems likely that the behaviors of the hippocampus in turn contribute to hippocampal function in a more general sense.
Stress hormones and aversive experience: hippocampal plasticity and psychopathology Owing to its high density of glucocorticoid receptors (Van Eekelen and De Kloet, 1992) and putative role
in regulating shut-off of the HPA axis (Feldman and Conforti, 1980), the hippocampus has been the focus of intensive investigation regarding the influence of stress on the brain. A number of studies have shown that aversive experiences and stress hormones mediate several aspects of structural plasticity in the adult hippocampus (Fig. 1).
Dendritic remodeling and cell survival Stress appears to affect hippocampal dendritic spines differently depending upon the sex of the animal, the cell type examined, and the duration and intensity of the stressor. Male rats grow more pyramidal cell spines 24 h following exposure to an acute stressor. This increase in spine density appears to correlate with stress-induced enhancements in hippocampusdependent learning. An opposite effect of stress is observed in female rats during diestrus; acute stress decreases the number of dendritic spines on CA1 pyramidal cells, a change that correlates with a stressinduced decrement in learning (Shors et al., 1998, 2001 a; Wood and Shors, 1998, Wood et al., 2001). Chronic, in contrast to acute, exposure to stress or stress hormones (glucocorticoids such as corticosterone), causes reversible dendritic atrophy in the apical dendrites of pyramidal cells in the CA3 region of the adult hippocampus. For instance, 21 days of 6-h-per-day restraint stress or 21 days of exogenous corticosterone treatment leads to significant atrophy of these dendrites (Woolley et al., 1990b; Watanabe et al., 1992c; Magarinos and McEwen, 1995a). Glucocorticoids are implicated in this change, since treatment with cyanoketone, an adrenal steroid synthesis blocker, prevents this stress-induced dendritic atrophy (Magarinos and McEwen, 1995b). Social stress in rats and tree shrews was found to have a similar effect on CA3 pyramidal cell dendrites (Magarinos et al., 1996; McKittrick et al., 2000). Additional components of the stress response, in concert with or downstream from glucocorticoids, likely contribute to dendritic remodeling. Stress is thought to increase the release of glutamate in the hippocampus, leading to the activation of N M D A receptors. Treatment with phenytoin, which blocks glutamate release, or blockade of N M D A receptors, preserved CA3 dendrites exposed to stress or stress
703
CA1 E
Fig. 1. Diverse actions of stress and adrenal steroids on the adult hippocampus. (A) Dendritic spine density increases on pyramidal cells of the CA1 region of male rats within 24h following acute stress. The opposite finding, a decrease in spine density, was demonstrated in female rats stressed during diestrus (Shors et al., 2001a). (B) Chronic exposure to stress or glucocorticoids causes apical dendritic atrophy in pyramidal cells of the CA3 region (Woolley et al., 1990b; Watanabe et al., 1992c; Magarinos et al., 1996; McKittrick et al., 2000). (C) Chronic exposure to extreme stress or high levels of glucocorticoids may result in CA3 pyramidal cell death (Uno et al., 1989; Sapolsky et al., 1990). (D) Basal levels of glucocorticoids prevent apoptosis in granule cells, but adrenalectomy results in an increase in pyknotic cells in the dentate gyrus (Gould et al., 1991b; Cameron and Gould, 1996). (E) Stressors or elevated glucocorticoids suppress the proliferation of granule cell precursors in the adult dentate gyrus (Gould et al., 1992, 1997, 1998; Cameron and Gould, 1994; Tanapat et al., 2001). hormones (Watanabe et al., 1992a; Magarinos and McEwen, 1995b). N M D A receptor activity is enhanced by serotonin ( R a h m a n n and Neumann, 1993), which is released in response to many stressors as well (see Chaouloff, 2000). Giving tianeptine, an atypical tricyclic antidepressant that reduces extracellular serotonin, serves to prevent stress- or corticosterone-induced atrophy of dendrites in the hippocampus (Watanabe et al., 1992b). Dendritic atrophy may ultimately result from glutamate excitotoxicity, or increased levels of intracellular calcium following activation of N M D A receptors. Excessive amounts of calcium can be harmful to cells by causing depolymerization or proteolysis of the cytoskeleton, allowing degradation of dendrites. Shrinkage of dendrites may serve a protective function, reducing the number of excitatory synapses and subsequent risk of excitotoxicity, or such a process might herald cell death. Chronic, long-term treatment of rodents and monkeys with high doses of glucocorticoids has been demonstrated to lead to degeneration even beyond dendritic atrophy in the hippocampus, as evidenced by irregularities in the CA3 and CA2 cell layers, shrinkage and condensation of the soma, and sometimes nuclear pyknosis in pyramidal neurons (Sapolsky et al., 1985, 1990). This pattern of damage was also observed in monkeys subjected to severe or
fatal stress during their lifetimes, further suggesting that stress can seriously compromise the hippocampus (Uno et al., 1989). As is postulated to be the case for dendritic atrophy, this extreme cell damage and eventual death is hypothesized to be mediated by the glucocorticoid receptor (GR) (Packan and Sapolsky, 1990) along with the accompanying excessive excitatory neurotransmission resulting in oxidative damage, compromised energy utilization, and possibly the induction of programmed cell death cascades associated with excess intracellular calcium (Reagan and McEwen, 1997; Sapolsky, 2000). Whether stress and glucocorticoids directly kill cells or simply lower their capacities to withstand injury (even bouts of hypoglycemia or ischemia that would be tolerated by healthy cells) remains debatable (see Lee et al., 2002). It is important to note, however, that cell death is not always observed when high levels of glucocorticoids are sustained over time, or even if electrophysiological or cognitive impairments are evident (Kerr et al., 1991; Bodnoff et al., 1995).
Adult
neurogenesis
Stress and glucocorticoids are potent modulators of hippocampal neurogenesis. Exogenous applications
704 of supraphysiological levels of corticosterone during development or later in life decreases the rate of proliferation of granule cell precursors in the rat (Gould et al., 199 la, 1992; Cameron and Gould, 1994; Gould, 1994). Alternatively adrenalectomy stimulates cell proliferation in the dentate gyrus during development and adulthood (Gould et al., 1992; Cameron and Gould, 1994). Still further evidence linking adrenal steroids to the modulation of hippocampal neurogenesis stems from the fact that conditions that elevate glucocorticoids, such as natural aging, have been shown to correlate with lower rates of granule cell production, and adrenalectomy in aged rats reverses this trend (Kuhn et al., 1996; Cameron and McKay, 1999). Stressful situations, which involve elevations in glucocorticoid levels and often increased glutamatergic transmission as well (Moghaddam et al., 1994), have been demonstrated to inhibit hippocampal neurogenesis in a variety of mammalian species. Acute exposure of rats to the odor of their natural predator, the fox, rapidly suppresses precursor proliferation, and ultimately the production of immature neurons, in the dentate gyrus. This effect involves adrenal steroids since normalizing corticosterone levels, by adrenalectomizing animals and replacing with low dose corticosterone in the drinking water, prevents the stress-induced decrease in cell proliferation (Tanapat et al., 2001). Tree shrews respond similarly to acute subordination stress, and chronic one-hour exposures to dominant animals for 28 days results not only in significant reductions in cell proliferation, but also decreased dentate gyrus volumes in the subordinates (Gould et al., 1997; Lucassen et al., 2001). A single brief exposure to an aggressive resident marmoset, an experience known to elevate cortisol levels, significantly suppresses cell proliferation in the dentate gyrus of intruder marmosets (Gould et al., 1998). These findings suggest that stress-induced inhibition of neurogenesis is a phenomenon that is common to many mammalian species. Since most progenitor cells in the dentate gyrus of adult animals do not appear to express mineralocorticoid or glucocorticoid receptors, adrenal steroids probably affect proliferation via an indirect mechanism (Cameron et al., 1993a). Recent evidence suggests that this mechanism involves NMDA
receptor activation, since blocking the activation of NMDA receptors prevents the stress-induced suppression of cell proliferation, whereas activating NMDA receptors prevents the adrenalectomyinduced increase in proliferation (Cameron et al., 1998). Independent of adrenal steroids, competitive and noncompetitive NMDA receptor antagonists enhance proliferation, as do lesions of the entorhinal cortex, which normally provides excitatory input to the dentate gyrus (Cameron et al., 1995). Experimentally induced seizures (which involve extreme levels of excitatory neurotransmission) have been shown to increase hippocampal neurogenesis (Parent et al., 1997; Nakagawa et al., 2000), which may seem to contradict the above findings. However, it is important to note that such seizures cause cell death, a condition that stimulates hippocampal neurogenesis (Gould and Tanapat, 1997; Kelsey et al., 2000). Although several studies have demonstrated a role for glucocorticoids in controlling the production of new neurons in the dentate gyrus both during development and in adulthood, many questions remain unanswered. First, is there a rebound of cell proliferation after glucocorticoid treatment (or stress) ceases? A recent report suggests that proliferation remains dampened for at least nine days following inescapable shock training (Malberg and Duman, 2003), but whether this suppression is common to different stressors and how long it may continue is unclear. Second, if cell proliferation does undergo a compensatory increase, it is uncertain whether such a rebound could suffice to make up for the deficits accumulated over long periods of intense stress. Third, and perhaps most importantly, the functional consequences of these stress-induced changes remain unknown. This issue is particularly difficult to address because stress and glucocorticoids are known to affect so many processes within the hippocampus that any measurable behavioral, cognitive, or affective changes are difficult to link to one cellular mechanism.
Hippocampal structure and psychiatric disorders In general, a wide array of psychiatric and endocrine disorders appear to result in reduced hippocampal volume in humans. Decreased hippocampal size has
705 been observed in all of the following conditions: Cushing's syndrome (hypercortisolemia) (Starkman et al., 1992), recurrent depression (Sheline et al., 1996), post-traumatic stress disorder (PTSD) (Bremner et al., 1995; Gurvits et al., 1996), and schizophrenia (Bogerts et al., 1993; Fukuzako et al., 1996). It is not yet clear whether these disorders cause the hippocampus to become smaller, or whether patients who are born with smaller hippocampi are then predisposed to develop these disorders. On the one hand, some studies of patients with major depression suggest that damage to the hippocampus accumulates over time, since patients experiencing their first episode have hippocampi of similar volume to agematched controls, whereas patients who have had several recurrences show decreases in volume that correlate with the amount of time spent depressed (Sheline et al., 1999; MacQueen et al., 2003). On the other hand, some recent evidence suggests that smaller hippocampi in PTSD are present prior to the development of the condition, however the degree of atrophy has also been related to the duration of trauma (Gurvits et al., 1996; Gilbertson et al., 2002). Since stress often precedes or appears to be linked to the development of psychiatric disorders, and since these conditions are often associated with hippocampus-related cognitive deficits, it is tempting to speculate that differences in hippocampal volume in these human conditions are the result of hormonal or experiential modulation of synapses, dendrites, and the production and survival of new neurons. Many investigators have shown that antidepressant treatment in animals reverses stress-induced changes in neurogenesis, mitigates reductions in hippocampal volume, and prevents dendritic atrophy (Czeh et al., 2001; D u m a n et al., 2001; Malberg and Duman, 2003). However, a stronger link between these animal studies and clinical reports will require the development of convincing animal models of these disorders and higher resolution neuroimaging. Furthermore, much work is needed to link these structural changes to symptoms associated with the specific psychopathology that accompanies different conditions (McEwen and Magarinos, 2001). In summary, the hippocampus appears to play a critical role in learning and memory, affect, and HPA axis regulation. In turn, enrichment and learning,
as well as stress and aversive experience, seem to modulate hippocampal structure and function. Ongoing remodeling of synapses and dendrites, continued cell birth and death, and gross volume fluctuations may be important for normal functioning. However, this plasticity may also make the hippocampus particularly vulnerable to structural abnormalities that can lead to pathology.
References Barnea, A. and Nottebohm, F. (1994) Seasonal recruitment of hippocampal neurons in adult free-ranging black-capped chickadees. Proc. Natl. Acad. Sci. USA, 91: 11217-11221. Biebl, M., Cooper, C.M., Winkler, J. and Kuhn, H.G. (2000) Analysis of neurogenesis and programmed cell death reveals a self-renewing capacity in the adult rat brain. Neurosci. Lett., 291: 17-20. Bodnoff, S.R., Humphreys, A.G., Lehman, J.C., Diamond, D.M., Rose, G.M. and Meaney, M.J. (1995) Enduring effects of chronic corticosterone treatment on spatial learning, synaptic plasticity, and hippocampal neuropathology in young and mid-aged rats. J. Neurosci., 15: 61-69. Bogerts, B., Lieberman, J.A., Ashtari, M., Bilder, R.M., Degreef, G., Lerner, G., Johns, C. and Masiar, S. (1993) Hippocampus-amygdala volumes and psychopathology in chronic schizophrenia. Biol. Psychiatry., 33: 236-246. Bremner, J.D., Randall, P., Scott, T.M., Bronen, R.A., Seibyl, J.P., Southwick, S.M., Delaney, R.C., McCarthy, G., Charney, D.S. and Innis, R.B. (1995) MRI-based measurement of hippocampal volume in patients with combat-related posttraumatic stress disorder. Am. J. Psychiatry, 152: 973-981. Brown, J., Cooper-Kuhn, C.M., Kempermann, G., Van Praag, H., Winkler, J., Gage, F.H. and Kuhn, H.G. (2003) Enriched environment and physical activity stimulate hippocampal but not olfactory bulb neurogenesis. Eur. J. Neurosci., 17(10): 2042-2046. Camel, J.E., Withers, G.S. and Greenough, W.T. (1986) Persistence of visual cortex dendritic alterations induced by postweaning exposure to a "superenriched" environment in rats. Behav. Neurosci., 100:810-813. Cameron, H.A. and Gould, E. (1994) Adult neurogenesis is regulated by adrenal steroids in the dentate gyrus. Neuroscience, 61: 203-209. Cameron, H.A. and Gould, E. (1996) Distinct populations of cells in the adult dentate gyrus undergo mitosis or apoptosis in response to adrenalectomy. J. Comp. Neurol., 369: 56-63. Cameron, H.A. and McKay, R.D. (1999) Restoring production of hippocampal neurons in old age. Nat. Neurosci., 2: 894-897.
706 Cameron, H.A. and McKay, R.D. (2001) Adult neurogenesis produces a large pool of new granule cells in the dentate gyrus. J. Comp. Neurol., 435: 406-417. Cameron, H.A., Woolley, C.S. and Gould, E. (1993a) Adrenal steroid receptor immunoreactivity in cells born in the adult rat dentate gyrus. Brain Res., 611: 342-346. Cameron, H.A., Woolley, C.S., McEwen, B.S. and Gould, E. (1993b) Differentiation of newly born neurons and glia in the dentate gyrus of the adult rat. Neuroscience, 56: 337-344. Cameron, H.A., McEwen, B.S. and Gould, E. (1995) Regulation of adult neurogenesis by excitatory input and NMDA receptor activation in the dentate gyrus. J. Neurosci., 15: 4687-4692. Cameron, H.A., Tanapat, P. and Gould, E. (1998) Adrenal steroids and N-methyl-D-aspartate receptor activation regulate neurogenesis in the dentate gyrus of adult rats through a common pathway. Neuroscience, 82: 349-354. Chaouloff, F. (2000) Serotonin, stress and corticoids. J. Psychopharmacol., 14: 139-151. Czeh, B., Michaelis, T., Watanabe, T., Frahm, J., de Biurrun, G., van Kampen, M., Bartolomucci, A. and Fuchs, E. (2001) Stress-induced changes in cerebral metabolites, hippocampal volume, and cell proliferation are prevented by antidepressant treatment with tianeptine. Proc. Natl. Acad. Sci. USA, 98(22): 12796-12801. Diamond, M.C., Drech, D. and Rosenzweig, M.R. (1964) The effects of an enriched environment on the histology of the rat cerebral cortex. J. Comp. Neurol., 123:111-120. Duman, R.S., Nakagawa, S. and Malberg, J. (2001) Regulation of adult neurogenesis by antidepressant treatment. Neuropsychopharmacology, 25(6): 836-844. Faherty, C.J., Kerley, D. and Smeyne, R.J. (2003) A GolgiCox morphological analysis of neuronal changes induced by environmental enrichment. Brain Res. Dev. Brain Res., 141(1-2): 55-61. Feldman, S. and Conforti, N. (1980) Participation of the dorsal hippocampus in the glucocorticoid feedback effect on adrenocortical activity. Neuroendocrinology, 30: 52-55. Fiala, B.A., Joyce, J.N. and Greenough, W.T. (1978) Environmental complexity modulates growth of granule cell dendrites in developing but not adult hippocampus of rats. Exp. Neurol., 59: 372-383. Fukuzako, H., Fukazako, T., Hashiguchi, T., Hokazono, Y., Takeuchi, K., Hirakawa, K., Ueyama, K., Takigawa, M., Kajiya, Y., Nakajo, M. and Fujimoto, T. (1996) Reduction in hippocampal formation volume is caused mainly by its shortening in chronic schizophrenia: assessment by MRI. Biol. Psychiatry, 39: 938-945. Gilbertson, M.W., Shenton, M.E., Ciszewski, A., Kasai, K., Lasko, N.B., Orr, S.P. and Pitman, R.K. (2002) Smaller hippocampal volume predicts pathologic vulnerability to psychological trauma. Nat. Neurosci., 5: 1242-1247.
Gould, E. (1994) The effects of adrenal steroids and excitatory input on neuronal birth and survival. Ann. N. Y. Acad. Sci., 743: 73-92; discussion 92-93. Gould, E. and Gross, C.G. (2002) Neurogenesis in adult mammals: some progress and problems. J. Neurosci., 22(3): 619-623. Gould, E. and Tanapat, P. (1997) Lesion-induced proliferation of neuronal progenitors in the dentate gyrus of the adult rat. Neuroscience, 80: 427-436. Gould, E., Woolley, C.S., Cameron, H.A., Daniels, D.C. and McEwen, B.S. (1991a) Adrenal steroids regulate postnatal development of the rat dentate gyrus: II. Effects of glucocorticoids and mineralocorticoids on cell birth. J. Comp. Neurol., 313: 486-493. Gould, E., Woolley, C.S. and McEwen, B.S. (1991b) Adrenal steroids regulate postnatal development of the rat dentate gyrus: I. Effects of glucocorticoids on cell death. J. Comp. Neurol., 313: 479-485. Gould, E., Cameron, H.A., Daniels, D.C., Woolley, C.S. and McEwen, B.S. (1992) Adrenal hormones suppress cell division in the adult rat dentate gyrus. J. Neurosci., 12: 3642-3650. Gould, E., McEwen, B.S., Tanapat, P., Galea, L.A. and Fuchs, E. (1997) Neurogenesis in the dentate gyrus of the adult tree shrew is regulated by psychosocial stress and NMDA receptor activation. J. Neurosci., 17: 2492-2498. Gould, E., Tanapat, P., McEwen, B.S., Flugge, G. and Fuchs, E. (1998) Proliferation of granule cell precursors in the dentate gyrus of adult monkeys is diminished by stress. Proc. Natl. Acad. Sci. USA, 95: 3168-3171. Gould, E., Beylin, A., Tanapat, P., Reeves, A. and Shors, T.J. (1999a) Learning enhances adult neurogenesis in the hippocampal formation. Nat. Neurosci., 2: 260-265. Gould, E., Tanapat, P., Hastings, N.B. and Shors, T.J. (1999b) Neurogenesis in adulthood: a possible role in learning. Trends Cogn. Sci., 3: 186-192. Gould, E., Vail, N., Wagers, M. and Gross, C.G. (2001) Adultgenerated hippocampal and neocortical neurons in macaques have a transient existence. Proc. Natl. Acad. Sci. USA, 95: 3168-3171. Gurvits, T.V., Shenton, M.E., Hokama, H., Ohta, H., Lasko, N.B., Gilbertson, M.W., Orr, S.P., Kikinis, R., Jolesz, F.A., McCarley, R.W. and Pitman, R.K. (1996) Magnetic resonance imaging study of hippocampal volume in chronic, combat-related posttraumatic stress disorder. Biol. Psychiatry, 40:1091-1099. Hastings, N.B. and Gould, E. (1999) Rapid extension of axons into the CA3 region by adult-generated granule cells. J. Comp. Neurol., 413: 146-154. Jacobs, L.F., Gaulin, S.J., Sherry, D.F. and Hoffman, G.E. (1990) Evolution of spatial cognition: sex-specific patterns of spatial behavior predict hippocampal size. Proc. Natl. Acad. Sci. USA, 87: 6349-6352.
707 Kaplan, M.S. and Hinds, J.W. (1977) Neurogenesis in the adult rat: electron microscopic analysis of light radioautographs. Science, 197: 1092-1094. Kelsey, J.E., Sanderson, K.L. and Frye, C.A. (2000) Perforant path stimulation in rats produces seizures, loss of hippocampal neurons, and a deficit in spatial mapping which are reduced by prior MK-801. Behav. Brain Res., 107: 59-69. Kempermann, G., Kuhn, H.G. and Gage, F.H. (1997) More hippocampal neurons in adult mice living in an enriched environment. Nature, 386: 493-495. Kerr, D.S., Campbell, L.W., Applegate, M.D., Brodish, A. and Landfield, P.W. (1991) Chronic stress-induced acceleration of electrophysiologic and morphometric biomarkers of hippocampal aging. J. Neurosci., 11: 1316-1324. Kuhn, H.G., Dickinson-Anson, H. and Gage, F.H. (1996) Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation. J. Neurosci., 16: 2027-2033. Lee, A.L., Ogle, W.O. and Sapolsky, R.M. (2002) Stress and depression: possible links to neuron death in the hippocampus. Bipolar. Disord., 4:117-128. Lee, D.W., Smith, G.T., Tramontin, A.D., Soma, K.K., Brenowitz, E.A. and Clayton, N.S. (2001) Hippocampal volume does not change seasonally in a non food-storing songbird. Neuroreport, 12: 1925-1928. Lemaire, V., Aurousseau, C., Le Moal, M. and Abrous, D.N. (1999) Behavioral trait of reactivity to novelty is related to hippocampal neurogenesis. Eur. J. Neurosci., 11(11): 4006-4014. Leuner, B., Falduto, J. and Shots, T.J. (2003) Associative memory formation increases the observation of dendritic spines in the hippocampus. J. Neurosci., 23(2): 659-665. Lucassen, P.J., Vollmann-Honsdorf, G.K., Gleisberg, M., Czeh, B., De Kloet, E.R. and Fuchs, E. (2001) Chronic psychosocial stress differentially affects apoptosis in hippocampal subregions and cortex of the adult tree shrew. Eur. J. Neurosci., 14: 161-166. MacQueen, G.M., Campbell, S., McEwen, B.S., Macdonald, K., Amano, S., Joffe, R.T., Nahmias, C. and Young, L.T. (2003) Course of illness, hippocampal function, and hippocampal volume in major depression. Proc. Natl. Acad. Sci. USA, 100:1387-1392. Magarinos, A.M. and McEwen, B.S. (1995a) Stress-induced atrophy of apical dendrites of hippocampal CA3c neurons: comparison of stressors. Neuroscience, 69: 83-88. Magarinos, A.M. and McEwen, B.S. (1995b) Stress-induced atrophy of apical dendrites of hippocampal CA3c neurons: involvement of glucocorticoid secretion and excitatory amino acid receptors. Neuroscience, 69: 89-98. Magarinos, A.M., McEwen, B.S., Flugge, G. and Fuchs, E. (1996) Chronic psychosocial stress causes apical dendritic atrophy of hippocampal CA3 pyramidal neurons in subordinate tree shrews. J. Neurosci., 16: 3534-3540.
Maguire, E.A., Gadian, D.G., Johnsrude, I.S., Good, C.D., Ashburner, J., Frackowiak, R.S. and Frith, C.D. (2000) Navigation-related structural change in the hippocampi of taxi drivers. Proc. Natl. Acad. Sci. USA, 97: 4398-4403. Malberg, J.E. and Duman, R.S. (2003) Cell proliferation in adult hippocampus is decreased by inescapable stress: reversal by fluoxetine treatment. Neuropsychopharmacology, 28(9): 1562-1571. Markakis, E.A. and Gage, F.H. (1999) Adult-generated neurons in the dentate gyrus send axonal projections to field CA3 and are surrounded by synaptic vesicles. J. Comp. Neurol., 406: 449-460. McEwen, B. and Magarinos, A.M. (2001) Stress and hippocampal plasticity: implications for the pathophysiology of affective disorders. Hum. Psychopharmacol., 16(S1): $7-S19. McEwen, B., Akama, K., Alves, S., Brake, W.G., Bulloch, K., Lee, S., Li, C., Yuen, G. and Milner, T.A. (2001) Tracking the estrogen receptor in neurons: implications for estrogeninduced synapse formation. Proc. Natl. Acad. Sci. USA, 98: 7093-7100. McKittrick, C.R., Magarinos, A.M., Blanchard, D.C., Blanchard, R.J., McEwen, B.S. and Sakai, R.R. (2000) Chronic social stress reduces dendritic arbors in CA3 of hippocampus and decreases binding to serotonin transporter sites. Synapse, 36: 85-94. Moghaddam, B., Bolinao, M.L., Stein-Behrens, B. and Sapolsky, R. (1994) Glucocorticoids mediate the stressinduced extracellular accumulation of glutamate. Brain Res., 655: 251-254. Nakagawa, E., Aimi, Y., Yasuhara, O., Tooyama, I., Shimada, M., McGeer, P.L. and Kimura, H. (2000) Enhancement of progenitor cell division in the dentate gyrus triggered by initial limbic seizures in rat models of epilepsy. Epilepsia, 41: 10-18. Nilsson, M., Perfilieva, E., Johansson, U., Orwar, O. and Eriksson, P.S. (1999) Enriched environment increases neurogenesis in the adult rat dentate gyrus and improves spatial memory. J. Neurobiol., 39: 569-578. O'Malley, A., O'Connell, C. and Regan, C.M. (1998) Ultrastructural analysis reveals avoidance conditioning to induce a transient increase in hippocampal dentate spine density in the 6 hour post-training period of consolidation. Neuroscience, 87(3): 607-613. O'Malley, A., O'Connell, C., Murphy, K.J. and Regan, C.M. (2000) Transient spine density increases in the mid-molecular layer of hippocampal dentate gyrus accompany consolidation of a spatial learning task in the rodent. Neuroscience, 99(2): 229-232. Packan, D.R. and Sapolsky, R.M. (1990) Glucocorticoid endangerment of the hippocampus: tissue, steroid and receptor specificity. Neuroendocrinology, 51: 613-618. Parent, J.M., Yu, T.W., Leibowitz, R.T., Geschwind, D.H., Sloviter, R.S. and Lowenstein, D.H. (1997) Dentate granule cell neurogenesis is increased by seizures and contributes to
708 aberrant network reorganization in the adult rat hippocampus. J. Neurosci., 17: 3727-3738. Patel, S.N., Clayton, N.S. and Krebs, J.R. (1997) Spatial learning induces neurogenesis in the avian brain. Behav. Brain Res., 89: 115-128. Popov, V.I. and Bocharova, L.S. (1992) Hibernation-induced structural changes in synaptic contacts between mossy fibres and hippocampal pyramidal neurons. Neuroscience, 48: 53-62. Popov, V.I., Bocharova, L.S. and Bragin, A.G. (I992) Repeated changes of dendritic morphology in the hippocampus of ground squirrels in the course of hibernation. Neuroscience, 48: 45-51. Rahmann, S. and Neumann, R.S. (1993) Activation of 5-HT2 receptors facilitates depolarization of neocortical neurons by N-methyl-D-aspartate. European Journal of Pharmacology, 231: 347-354. Reagan, L.P. and McEwen, B.S. (1997) Controversies surrounding glucocorticoid-mediated cell death in the hippocampus. J. Chem. Neuroanat., 13: 149-167. Santarelli, L., Saxe, M., Gross, C., Surget, A., Battaglia, F., Dulawa, S., Weisstaub, N., Lee, J., Duman, R., Arancio, O., Belzung, C. and Hen, R. (2003) Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants. Science, 301(5634): 805-809. Sapolsky, R.M. (2000) The possibility of neurotoxicity in the hippocampus in major depression: a primer on neuron death. Biol. Psychiatry, 48: 755-765. Sapolsky, R.M., Krey, L.C. and McEwen, B.S. (1985) Prolonged glucocorticoid exposure reduces hippocampal neuron number: implications for aging. J. Neurosci., 5: 1222-1227. Sapolsky, R.M., Uno, H., Rebert, C.S. and Finch, C.E. (1990) Hippocampal damage associated with prolonged glucocorticoid exposure in primates. J. Neurosci., 10: 2897-2902. Sheline, Y.I., Wang, P.W., Gado, M.H., Csernansky, J.G. and Vannier, M.W. (1996) Hippocampal atrophy in recurrent major depression. Proc. Natl. Acad. Sci. USA, 93: 3908-3913. Sheline, Y.I., Sanghavi, M., Mintun, M.A. and Gado, M.H. (1999) Depression duration but not age predicts hippocampal volume loss in medically healthy women with recurrent major depression. J. Neurosci., 19: 5034-5043. Sherry, D.F., Jacobs, L.F. and Gaulin, S.J. (1992) Spatial memory and adaptive specialization of the hippocampus. Trends Neurosci., 15: 298-303. Sherry, D.F., Forbes, M.R., Khurgel, M. and Ivy, G.O. (1993) Females have a larger hippocampus than males in the brood-parasitic brown-headed cowbird. Proc. Natl. Acad. Sci. USA, 90: 7839-7843. Shors, T.J., Lewczyk, C., Pacynski, M., Mathew, P.R. and Pickett, J. (1998) Stages of estrous mediate the stress-induced impairment of associative learning in the female rat. Neuroreport, 9: 419-423.
Shors, T.J., Chua, C. and Falduto, J. (2001a) Sex differences and opposite effects of stress on dendritic spine density in the male versus female hippocampus. J. Neurosci., 21: 6292-6297. Shors, T.J., Miesegaes, G., Beylin, A., Zhao, M., Rydel, T. and Gould, E. (2001b) Neurogenesis in the adult is involved in the formation of trace memories. Nature, 410: 372-376. Shors, T.J., Townsend, D.A., Zhao, M., Kozorovitskiy, Y. and Gould, E. (2002) Neurogenesis may relate to some but not all types of hippocampal-dependent learning. Hippocampus, 12: 578-584. Smulders, T.V., Sasson, A.D. and DeVoogd, T.J. (1995) Seasonal variation in hippocampal volume in a foodstoring bird, the black-capped chickadee. J. Neurobiol., 27: 15-25. Stanfield, B.B. and Trice, J.E. (1988) Evidence that granule cells generated in the dentate gyrus of adult rats extend axonal projections. Exp. Brain Res., 72: 399-406. Starkman, M.N., Gebarski, S.S., Berent, S. and Schteingart, D.E. (1992) Hippocampal formation volume, memory dysfunction, and cortisol levels in patients with Cushing's syndrome. Biol. Psychiatry, 32: 756-765. Tanapat, P., Hastings, N.B., Reeves, A.J. and Gould, E. (1999) Estrogen stimulates a transient increase in the number of new neurons in the dentate gyrus of the adult female rat. J. Neurosci., 19(14): 5792-5801. Tanapat, P., Hastings, N.B., Rydel, T.A., Galea, L.A. and Gould, E. (2001) Exposure to fox odor inhibits cell proliferation in the hippocampus of adult rats via an adrenal hormone-dependent mechanism. J. Comp. Neurol., 437: 496-504. Uno, H., Tarara, R., Else, J.G., Suleman, M.A. and Sapolsky, R.M. (1989) Hippocampal damage associated with prolonged and fatal stress in primates. J. Neurosci., 9: 1705-1711. Van Eekelen, J.A. and De Kloet, E.R. (1992) Co-localization of brain corticosteroid receptors in the rat hippocampus. Prog. Histochem. Cytochem., 26: 250-258. Wallace, C.S., Kilman, V.L., Withers, G.S. and Greenough, W.T. (1992) Increases in dendritic length in occipital cortex after 4 days of differential housing in weanling rats. Behav. Neural Biol., 58: 64-68. Watanabe, Y., Gould, E., Cameron, H.A., Daniels, D.C. and McEwen, B.S. (1992a) Phenytoin prevents stress- and corticosterone-induced atrophy of CA3 pyramidal neurons. Hippocampus, 2: 431-435. Watanabe, Y., Gould, E., Daniels, D.C., Cameron, H. and McEwen, B.S. (1992b) Tianeptine attenuates stressinduced morphological changes in the hippocampus. Eur. J. Pharmacol., 222:157-162. Watanabe, Y., Gould, E. and McEwen, B.S. (1992c) Stress induces atrophy of apical dendrites of hippocampal CA3 pyramidal neurons. Brain Res., 588: 341-345.
709 Wood, G.E. and Shors, T.J. (1998) Stress facilitates classical conditioning in males, but impairs classical conditioning in females through activational effects of ovarian hormones. Proc. Natl. Acad. Sci. USA, 95: 4066-4071. Wood, G.E., Beylin, A.V. and Shors, T.J. (2001) The contribution of adrenal and reproductive hormones to the opposing effects of stress on trace conditioning in males versus females. Behav. Neurosci., 115: 175-187.
Woolley, C.S., Gould, E., Frankfurt, M. and McEwen, B.S. (1990a) Naturally occurring fluctuation in dendritic spine density on adult hippocampal pyramidal neurons. J. Neurosci., 10: 4035-4039. Woolley, C.S., Gould, E. and McEwen, B.S. (1990b) Exposure to excess glucocorticoids alters dendritic morphology of adult hippocampal pyramidal neurons. Brain Res., 531: 225-231.
This Page Intentionally Left Blank
T. Steckler, N.H. Kalin and J.M.H.M. Reul (Eds.) Handbook of Stress and the Brain, Vol. 15
ISBN 0-444-51173-3 Copyright 2005 Elsevier B.V. All rights reserved CHAPTER 5.5
Adult neurogenesis in rodents and primates" functional implications Eberhard Fuchs* and Boldizsfir Cz6h Clinical Neurobiology Laboratory, German Primate Center, Kellnerweg 4, 37077, G6ttingen, Germany
Abstract: Within the last two decades, our view of the mature mammalian brain has been changed. It is far from being fixed and immutable, as a number of factors such as environmental stimulation, learning, growth factors, glucocorticoid, and sexual hormones, stress, aging, neurotransmitters such as glutamate and serotonin, and a number of drugs regulate the production of new neurons in the adult dentate gyrus. This newfound capacity has forced a new look at plasticity of the brain, an organ previously considered to have an anatomically stable structure. The presence of neuronal precursors in the adult mammalian brain suggests a new level of plasticity for brain regions, whereby neurons are constantly replaced. Moreover, it raises questions regarding the underlying mechanisms of the newly generated neurons, and how they influence the functioning of the differentiated adult brain. The evolutionary conservation of adult neurogenesis and the persistently high level of neuron production throughout adulthood suggest that this process is of fundamental biological importance. The functional implications of adult neurogenesis remain unknown, but several studies have suggested a link between the formation of new neurons in the dentate gyrus and learning.
of the future to change, if possible, this harsh decree." (Cajal, 1928). This dogma of neurobiology was initially challenged almost four decades ago. Using 3H-thymidine autoradiography, Altman and Das (1965) discovered the production of new granular neurons in the dentate gyrus and olfactory bulb of adult rat brains. Although only limited work followed this initial finding, it confirmed and further substantiated the neuronal character of the newly generated hippocampal cells by demonstrating that they receive synaptic input and extend axons into the mossy fiber pathway that projects to the CA3 subfield (Kaplan and Hinds, 1977; Stanfield and Trice, 1988). The next landmark was in the early 1980s, when a substantial neurogenesis in the vocal control nucleus of the adult canary brain was demonstrated (Goldman and Nottebohm, 1983) and a functional link between behavior, song learning, and the production of new neurons could be established (Alvarez-Buylla et al., 1988). Neuronal turnover in the high vocal control centers is thought
New neurons for the adult mammalian brain? The story of a controversy Unlike most body cells such as those in the skin, the gut or the blood, which are constantly renewed and easily regenerated, the brain, and in particular the mammalian brain, has always been regarded as a nonrenewable organ. Most neurons of the adult central nervous system are terminally differentiated. Sometimes the adult brain can compensate for damage by making new connections among surviving neurons. However, it cannot repair itself because it lacks the stem cells that are necessary for neuronal regeneration. This limitation and unclear evolutionary benefit were first described by Santiago Ram6n y Cajal, who stated: "In adult centers the nerve paths are something fixed, ended, immutable. Everything may die, nothing may be regenerated. It is for science *Corresponding author. Tel.: +49-551-3851 130; Fax: +49-551-3851 307; E-mail:
[email protected] 711
712 to play a role in the modification of perceptual memories or motor programs for song production in these animals (Goldman, 1998). Despite several attempts, neurogenesis could not be demonstrated in the brains of adult nonhuman primates such as rhesus monkeys, thereby leading to the assumption that neuronal replication could not be tolerated in primates - including h u m a n s - because it might interfere with learning and memory. In his initial study, Rakic (1985) investigated neurogenesis in adult rhesus monkeys using 3H-thymidine, examining major structures and subdivisions of the brain including the visual, motor, and association neocortex, hippocampus, and olfactory bulb. Rakic found "not a single heavily labeled cell with the morphological characteristics of a neuron in any brain in any adult animal" and concluded that "all neurons of the rhesus monkey brain are generated during prenatal and early postnatal life" (Rakic, 1985; Eckenhoff and Rakic, 1988). Furthermore, Rakic argued that "a stable population of neurons may be a biological necessity in an organism whose survival relies on learned behavior acquired over a long period of time." These reports had a profound influence on the development of the field. Presumably, these negative results formed the basis for researchers of the time to show little interest in neurogenesis in the adult mammalian brain. This view was clearly changed, with the field consequently reviving and "exploding" when the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) was introduced for labeling newborn neurons (Miller and Nowakowski, 1988). Using this new technique, it became clear that adult hippocampal neurogenesis in mammals is not restricted only to rodents but has been conserved throughout mammalian evolution. The formation of new granule neurons was demonstrated in tree shrews (Gould et al., 1997), animals considered to be phylogenetically located between insectivores and primates. This evidence was followed by initial reports of neurogenesis in the brains of marmoset monkeys (Gould et al., 1998), a small nonhuman primate from South America, and in the rhesus monkey, a typical representative of the nonhuman Old-world primates (Gould et al., 1999b; Kornack and Rakic, 1999).
Work with terminally ill patients has confirmed that humans also generate new neurons. The thymidine analog BrdU was injected into patients to monitor tumor cell proliferation. Some of these individuals subsequently died from their illness and small samples of hippocampus were evaluated for the presence of BrdU-labeled neurons. Since BrdU was systemically administered, all dividing cells would have been labeled and, indeed, newborn neurons were detected in the granule cell layer of all individuals (Eriksson et al., 1998). These data unequivocally show that neurogenesis is a common phenomenon across mammalian species.
The BrdU method Like 3H-thymidine, which was the label of choice in the initial experiments, BrdU is incorporated into a dividing progenitor cell during the S-phase. Fully differentiated neurons do not divide and cannot integrate the label. The BrdU immunolabel is not quantifiable in the same way as 3H-thymidine, which allows the counting of silver grains, thereby enabling objective criteria for strong or weak labeling. Depending on the survival time after injection, BrdU is a marker of proliferating cells (short survival time) and their progeny (longer survival time). BrdU labeling is a nonisotopic method and is visualized with immunocytochemical methods. Importantly, the BrdU technique has two major advantages. First, it allows stereological estimation of the total number of newborn cells and by this the effects of different treatments can be evaluated and compared. Second, combining BrdU labeling with other immunocytochemical labels permits the determination of the phenotypes of the new cells. Markers for immature neurons are class III B-tubulin (Tujl), doublecortin, turned-off-after-division 64KDa (TOAD-64), or polysialated neural cell adhesion molecule (PSANCAM). Markers for mature neurons are antibodies directed against neuron-specific nuclear protein (NeuN), microtubule-associated-protein 2 (MAP-2), or the calcium-binding protein, calbindin. Doublestaining with markers for nonneuronal cells can demonstrate a glial phenotype: glial fibrillary acidic protein (anti-GFAP) or anti-S-100B can visualize astrocytes, whereas oligodendrocytes can be labeled
713 by anti-O4 or anti-CNP (2',Y-cyclic nucleotide 3'-phosphodiesterase). It should be noted, however, that there is an ongoing debate about the accuracy of the BrdU technique (Gould and Gross, 2002; Hayes and Nowakowski, 2002). Some groups argue that application of an inappropriately high dose of BrdU may give false positive results (Rakic, 2002a).
Restriction of adult neurogenesis in the mammalian brain It has been reported that within vertebrates, at least for all bony fish investigated so far, there is an enormous potential for the production of new neurons in the adult brain. In gymnotiform fish, an average of 105 cells, corresponding to approximately 0.2% of the total population of the adult brain, are A
in the S-phase within any 2-h period (Zupanc, 1999). Comparing the massive neurogenesis observed throughout the life span of fish, amphibians and even birds with that observed in mammals, it is clear that the extent and the capacity for adult neurogenesis has diminished during the course of evolution. Furthermore, a local restriction is observed. In contrast to the widespread parenchymal neuronal migration by new neurons in the adult avian brain, there is a clear spatial restriction in the mammalian brain. Here, two regions of active proliferation generate neurons continuously throughout life, namely the subependymal zone of the lateral ventricle and the dentate gyrus of the hippocampal formation (see sections: "The dentate gyrus of the hippocampal formation" and "The subependymal zone," Fig. 1). In the rat dentate gyrus, approximately 9000 new cells are generated daily, i.e. 0.75% of the existing granule cell population or one new cell per 130 mature granule neurons each day (Cameron and B
lateral view
C
horizontal section
D OB
;C
sagittal section
frontal section
Fig. 1. Areas of neurogenesis in the adult mammalian brain. (A) Lateral view of a tree shrew brain displaying the localization of the hippocampus (HC). (B) Schematic drawing of horizontal section of the hippocampal formation, displaying the localization of neural progenitor cells within the germinative subgranular zone (sgz) adjacent to the granule cell layer (gcl) of the dentate gyrus (DG). Newly generated granule cells (gc) develop synapses on their cell bodies and dendrites extend axons and contact CA3 pyramidal cells (pc). (C) Schematic view of the ventricular subependymal zone (SZ), where multipotential self-renewing stem cells reside and spontaneously proliferate. Newly generated cells then migrate along the rostral migratory stream (RMS) toward the olfactory bulb (OB), into which they finally incorporate. (D) In the frontal section, the subependymal (or subventricular) zone appears adjacent to the wall of the lateral ventricle. NC: neocortex; CB: cerebellum; CC: corpus callosum; mf: mossy fibers.
714 McKay, 2001). This is a relatively small percentage when compared to the total number of granular neurons, which is estimated to be in the order of 106, depending on the strain and age of the animal and the counting method used (Amaral and Witter, 1989; West et al., 1991). Since only about 30% of the BrdU-positive cells survive permanently and differentiate into neurons (Kempermann et al., 2003), it was therefore estimated that approximately 6% of the total granule cell population is renewed within one month (Cameron and McKay, 2001). Because of their presumed ability to form new synapses rapidly, newly generated neurons might be responsible for a greater proportion of new connections than the resident neurons, thereby having a proportionately larger influence on the physiology and functioning of the hippocampal formation. Morphologically indistinguishable from their neighbors with synapses on cell bodies and dendrites (Markakis and Gage, 1999), adult-generated granule neurons are thought to be integrated into the neuronal network. However, it is not clear whether the new neurons receive the same inputs as the ones produced during development. Kornack and Rakic (1999) estimated that in the adult macaque hippocampus approximately 0.004% of the total granule cell population is generated per day, i.e. one new neuron per 24,000 existing granule neurons each day. In the adult human hippocampus, cells do proliferate, yielding 50-400 BrdU-positive cell/mm 3 within the germinative zone (Erikson et al., 1998). Another study examining cell formation in the human hippocampal formation of newborn infants came to a much more cautious conclusion, stating that "neurogenesis in the adult primate brain may possibly be very limited or absent" (Seress et al., 2001). In their study, Seress et al. (2001) investigated postmortem hippocampal tissue of human infants from the 24th gestational week until the end of the first postnatal year. To detect cell proliferation, they used an endogenous mitotic marker Ki-67, instead of the exogenous marker BrdU. The authors found that newly generated cells represent ~ 1% of the granule cell population in the newborn infant hippocampus, but they claimed that the majority (80%) of them had glial morphology, while a maximum of 20% of the labeled cells were granule cells. Thus, it appears that
in primates newborn neurons represent a much smaller fraction of the mature granule cells compared to rodents. Whether these newly generated cells in the human hippocampus become functional and, if so, their role remains unknown.
The dentate gyrus of the hippocampal formation Adult neurogenesis is found in the dentate gyrus of the hippocampal formation (Fig. 1A, B), a unique brain structure that contains a high degree of structural plasticity and is intimately involved in the processing and storage of new information (Suzuki and Eichenbaum, 2000). During development of the hippocampus, a secondary germinal zone, separate from the ventricle wall, is formed along the border between the hilus and the granular cell layer, i.e. the subgranular zone (Altman and Bayer, 1990). Developmental studies have revealed that in both rodents and primates, the majority of neurons in the entorhinal cortex, subicular complex, and Ammon's horn are generated before birth. The major difference in hippocampal development between primates and rodents is the formation of the dentate gyrus. In rodents, approximately 85% of the dentate granule cells are formed postnatally, whereas similar numbers of cells are formed already prenatally in primates (Bayer, 1980; Rakic and Nowakowski, 1981). In rhesus monkeys the formation of granule cells starts as early as week E38, but the majority of them are generated between E60-E120 (Rakic and Nowakowski, 1981). In humans, the granule cell layer first appears during the 13-14th gestational week, and similar to rhesus monkeys, the majority of the granule cells are formed throughout the second trimester (Humphrey, 1967; Seress et al., 2001). At term, about 50-60% of the adult number of granule cells are present in the dentate gyrus of the rhesus monkey (Keuker et al., 2003), and this estimate is approximately 70-85% in case of humans (L. Seress, personal communication). Neurogenesis in the dentate gyrus continues throughout life, but displays a steady decline from early postnatal days (Kuhn et al., 1996). Nevertheless, neurogenesis is still detectable into
715 very old age in both rodents and nonhuman primates (Kuhn et al., 1996; Gould, 1999b). Importantly, neural stem cells that exhibit longterm self-renewal and multipotentiality can be isolated from the adult ventricular subependyma, whereas the adult dentate gyrus does not contain resident stem cells. Instead, separate neuronal and glial progenitors with only limited self-renewal capacity are the source of newly generated dentate neurons throughout adulthood (Seaberg and van der Kooy, 2002). In the last few years, a theory has been developed that describes this population of cells (Gage, 1998, 2000). According to this theory, the cells are dividing asymmetrically resulting in daughter cells that enter the pathway leading to differentiation. The remaining cells are thought to form a sustaining pool of proliferating cells. From studies in rodents, it was estimated that about 50% of the BrdUpositive cells develop a neuronal phenotype, migrate into the granular cell layer, become morphologically indistinguishable from the other surrounding granule cells, and are capable of extending axonal projections along the mossy fiber tract to their natural target area, the hippocampal CA3 region (Cameron et al., 1993b; Markakis and Gage, 1999). About 15% of the BrdU-positive cells differentiate into glia cells. The remaining 35% do not show a clear neuronal or glial phenotype up to four weeks after cell division. Thus, the new neuron is the end product of a series of steps consisting of proliferation, survival, migration, differentiation, and establishment of functional connections with other neurons.
The subependymal zone The subependymal zone of the adult lateral ventricle (Fig. 1C, D) gives rise to new neurons and is seen as a residual proliferative zone left over from the embryonic neural tube. Multipotential, self-renewing stem cells in the adult subependymal zone are the source of newly generated cells that pass through the rostral migratory stream, complete their last divisions, and incorporate into the olfactory bulb where they differentiate into interneurons (Luskin, 1993; Lois and Alvarez-Buylla, 1994; Menezes et al.,
1995). Furthermore, Kornack and Rakic (2001a) provided evidence for the presence of spontaneous adult neurogenesis in the primate subependymal zone, which raises the possibility that an active subependymal zone/rostral migratory stream system is also present in humans. The migration into the olfactory bulb has been described as a tangential chain migration, which does not require radial glia as a stationary partner (Lois et al., 1996). It is important to note that two neuronal phenotypes are generated in the olfactory bulb, GABAergic granule cells, and dopaminergic periglomerular interneurons (McLean and Shipley, 1988; Betarbet et al., 1996). Moreover, retroviral fate mapping studies confirmed that multipotential neural stem cells in the ventricle wall also generate glial cells (Goldman, 1995; Morshead et al., 1998). Therefore, within specific regions of the adult brain, all signals are present for instructing stem cells to generate glia and neurons with specific neurotransmitter phenotypes.
Neurogenesis in forebrain areas In an unperturbed mammalian brain, neurogenesis is thought to be strictly limited to the dentate gyrus and the subependymal ventricle wall. Other areas of the brain contain an abundant population of proliferative precursors, but these cells generate only glia. The most common assumption is that neurogenic zones are defined by the location of the neural stem/ progenitor cells in the adult. However, this may not be true. Immature progenitors in white matter generate oligodendrocytes in vivo and in vitro (Wolswijk and Noble, 1992; Horner et al., 2000), but recent studies show that these "glial" progenitors can actually make neurons in culture if treated with the appropriate growth factors (Palmer et al., 1999; Kondo and Raft, 2000; Nunes et al., 2003). This implies that the local environment, not the distribution of cells, is the key factor in defining where neurons are made. A progenitor cell may "see" quite specific local environments depending on where it resides, and it would react appropriately to the local signals being produced by neighboring cells. In the dentate gyrus, the progenitor cell neighbors include other
716 precursors, glia, granule cell neurons and, surprisingly, vascular endothelium. Recent studies show that the neural progenitor cells in the hippocampal subgranular zone proliferate in small clusters and that these clusters are located around the periphery of small capillaries (Palmer et al., 2000). Dividing endothelia are found within the core of many proliferating clusters and this angiogenic microenvironment appears to be relatively unique to the hippocampal subgranular zone and the ventricular subependymal zone. Precursors in white matter do not associate with vessels and it is possible, though not yet shown, that this vascular environment provides some of the cues necessary for stem cells to generate neurons. There have been a few reports on spontaneous adult neurogenesis in neocortical structures. Kaplan (1981) reported on continuous neurogenesis in the three-month-old rat visual cortex. Recently, Gould and coworkers demonstrated that in adult nonhuman primates new neurons originating from the subependymal zone migrate through the white matter to the prefrontal, inferior temporal, and posterior parietal cortices, where they extend axons (Gould et al., 1999c, 2001). Furthermore, Bernier et al. (2002) reported on adult neurogenesis in the amygdala, piriform cortex, and adjoining inferior temporal cortex in squirrel monkeys and macaques. These findings invoked great skepticism in the scientific community (Nowakowski and Hayes, 2000; Rakic, 2002b). Clearly, because of the considerable conceptual and biomedical implications of this claim, it is essential to validate the reliability and robustness of this putative phenomenon. Accordingly, Kornack and Rakic (2001b) examined the proliferation and phenotypic differentiation of cells in the cerebrum of adult rhesus monkeys, but their findings do not substantiate the claim of neurogenesis in adult nonhuman primate neocortex. Further, similar negative findings were reported recently by Koketsu et al. (2003). Today there is an agreement that in nonhuman primates continuous adult neurogenesis takes place in the hippocampal dentate gyrus and in the subependymal zone, with cells from the latter region migrating to the olfactory bulb. However, additional studies are needed to clarify the extent of adult neurogenesis in other regions of the primate brain.
Neuromodulatory factors regulating adult hippocampal neurogenesis Steroid hormones
Glucocorticoid hormones, such as cortisol (in primates) and corticosterone (in rats) secreted by the adrenal cortex, have been shown to inhibit the production of new granule neurons by suppressing the proliferation of granule cell precursors. The suppressive action of glucocorticoids seems to have biological relevance. Conditions associated with hypercortisolism, such as stress and ageing, are also associated with reduced granular cell production in rodents, tree shrews, and nonhuman primates (Gould et al., 1997, 1998; Cameron and McKay, 1999). On the other hand, removal of circulating glucocorticoids by adrenalectomy results in a clear increase in neurogenesis in the dentate gyrus of young adult rats (Cameron and Gould, 1994). The mechanism by which glucocorticoids inhibit adult neurogenesis is unknown. Glucocorticoids may act in part via mineralocorticoid receptors (Gass et al., 2000; Fischer et al., 2002). However, newborn cells in the subgranular zone do not express detectable levels of glucocorticoid or mineralocorticoid receptors (Cameron et al., 1993a). In contrast to the suppressive effects of glucocorticoids on cell proliferation, the ovarian steroid estrogen has been shown to stimulate the proliferation of granule cell precursors in the dentate gyrus of adult female rats. This increase in the rate of cell proliferation occurs naturally across the rat estrous cycle, with maximal levels of cell production during proestrus, a time when estrogen levels are highest (Tanapat et al., 1999). This finding raises the interesting question regarding which mechanisms mediate the survival of newly generated cells in females as well as in males. Several studies suggest that glutamate receptors may mediate the effects of glucocorticoids on hippocampal proliferation. N-methyl-aspartate (NMDA) receptor blockade or entorhinal cortical lesions (which deprive the hippocampus of a major glutamatergic input) prevent glucocorticoidmediated decreases in adult neurogenesis (Cameron et al., 1995). NMDA receptor blockade also prevents adrenalectomy mediated increases in adult
717 neurogenesis (Cameron et al., 1998). Alternatively, stress- and glucocorticoid-induced changes in other steroids, such as estrogen or testosterone, or in neurotransmitter systems (see following section), could conceivably be involved, especially because plasma stress hormone levels do not always correlate with the rate of adult hippocampal neurogenesis (Czeh et al., 2001, 2002).
Neurotransmitters As indicated previously, another strong regulator of proliferation is the glutamatergic input to the granule layer of the dentate gyrus. NMDA receptor agonists and antagonists decrease or increase cell proliferation respectively (Cameron et al., 1995). Nitric oxide (NO) seems to be another negative regulator of cell proliferation in the hippocampal dentate gyrus (Packer et al., 2003). Recent evidence supports the view that other neurotransmitter systems influence the production of new granule neurons in the dentate gyrus. For example, serotonin may stimulate granule cell production (Brezun and Daszuta, 2000a,b), whereas depletion of serotonin reduces neurogenesis (Brezun and Daszuta, 1999). In line with the growing body of evidence suggesting that mood stabilizers and antidepressants exert neurotrophic effects (Chen et al., 2000; Malberg et al., 2000), recent reports showed that different classes of antidepressant drugs, as well as the NMDA receptor antagonist MK-801, may prevent stress-induced suppression of adult hippocampal cell proliferation (Gould et al., 1997; Czeh et al., 2001; Lee et al., 2001; van der Hart et al., 2002; Malberg and Duman, 2003). It should be noted that to date, receptors neither for steroid hormones nor for glutamate or other neurotransmitters have been found on hippocampal progenitor cells. It will be interesting in future studies to identify the types of receptors expressed by these progenitor cells; such receptors should provide clues as to the intercellular signals that are critical in regulating adult neurogenesis.
Growth factors Although the neuromodulatory signals discussed in the previous section trigger proliferation, the direct
mitogenic stimulus to the progenitor cells appears to be mediated via growth factors, such as epidermal growth factor (EGF). Dentate precursor cells are known to express EGF receptors (Okano et al., 1996) and direct infusion of the growth factor into the dentate gyrus stimulates proliferation (Tanapat and Gould, 1997). Chronic infusion of EGF into the ventricular system of adult rats triggered neurogenesis, predominantly in the subependymal zone, and was nearly ineffective in stimulating proliferation in the subgranular zone (Kuhn et al., 1997). Moreover, when using this route of administration, EGF induced a prominent phenotypic shift that led to more astrocytes and fewer neurons. Intracerebroventricular administration of the vascular endothelial growth factor (VEGF) stimulates neurogenesis both in the subependymal zone and in the subgranular zone of the dentate gyrus, where the VEGFR2/Flk-1 receptor was colocalized with the immature neurons (Jin et al., 2002). Via peripheral application, selective induction of neurogenesis has also been achieved using FGF-2 (Wagner et al., 1999) or IGF-1 (Aberg et al., 2000). It is presently unclear whether growth factors can pass directly through the blood-brain barrier or whether other mechanisms such as angiogenesis are triggered, which have a secondary positive effect on neurogenesis.
Systemic influences on neurogenesis
Strain, age, and environment Regulation of adult hippocampal neurogenesis has different regulatory levels, including cell proliferation, survival, and differentiation. Several studies have examined whether strain differences could possibly affect adult hippocampal neurogenesis, revealing that different aspects of adult hippocampal neurogenesis are differentially influenced by the genetic background (Kempermann et al., 1997a; Kempermann and Gage, 2002a,b). Another internal factor that has a strong effect on adult hippocampal neurogenesis is age. With increasing age, the proliferation rate and ratio of cells differentiating into neurons decreases, whereas the percentage of surviving newborn cells remains constant (Kuhn et al., 1996; Gould et al., 1999b).
718 Interestingly, reduction of corticosteroid levels in aged rats could restore the naturally low rate of cell proliferation (Cameron and McKay, 1999). This may suggest that the neuronal progenitor population in the dentate gyrus in fact remains stable during ageing, but the spontaneous rate of neurogenesis is suppressed by elevated levels of corticosteroids. External stimuli and environmental complexity enhance the survival of new neurons in the adult brain. This was first demonstrated in black-capped chickadees (Barnea and Nottebohm, 1994, 1996). Newly formed neurons were shown to survive longer in birds living in their natural environment compared to those animals living in captivity. Moreover, seasonal differences in the adult-generated hippocampal neurons correlated with seasonal changes in food storage and retrieval, behaviors that require spatial learning (Barnea and Nottebohm, 1994, 1996). This line of research has been extended to the mammals by studies that analyzed the stimulatory effect of the environment on adult dentate neurogenesis (Kempermann et al., 1997b; van Praag et al., 2000). "Enriched environment" paradigms, where female mice are placed into housing conditions that are more similar to their natural surroundings, have been shown to increase neurogenesis by stimulating a better survival of the newly generated cells. The "enriched" animals also showed improved motor skills and better performance in learning tasks. Most importantly, the stimulatory effect on neurogenesis occurred at all ages, including senescence, even when the animals were housed under enriched conditions for only a few weeks (Kempermann et al., 1998). Among the stimulatory factors within an enriched environment, voluntary physical activity appears to be a very strong activator of the proliferation of hippocampal progenitor cells (van Praag et al., 1999). The sole introduction of a running wheel into a standard laboratory home cage doubled hippocampal neurogenesis, suggesting that physiological parameters, such as blood flow, glucose uptake, and neovascularization, could be mediators of this effect. Today it is evident that the mammalian hippocampus does indeed show an activity-dependent regulation of adult neurogenesis. These findings imply that data related to the number, regulation, and longevity of newly generated cells must be
interpreted in light of the manner in which animals are housed. Relatively impoverished environments may also account for the detection of low numbers of new neurons in some regions, or the inability to find new neurons in some areas. Environmental complexity is not only related to structural enrichment. Exposing mice to an odorenriched environment can markedly increase the survival of newborn cells in the olfactory bulb (Rochefort et al., 2002). Furthermore, this effect is region specific, because enriched odor exposure does not influence hippocampal neurogenesis and the mice living in an odor-enriched environment display improved olfactory memory without a change in spatial learning performance (Rochefort et al., 2002). Another study using mutant mice demonstrated that deficits in the migration of olfactory-bulb neuron precursors result in an impairment of discrimination between odors, whereas general olfactory functions are unaltered (Gheusi et al., 2000).
Learning Probably the most convincing evidence for a functional role of newborn neurons comes from studies of songbirds. For instance, in canaries, song is specific to males, which modify their repertoire by listening to congeners, and by adding, dropping, or altering song syllables. Some studies have shown that rates of neuron turnover in the high-vocal center (HVC), a nucleus involved in song production, are highest at times of year when canaries modify their songs, whereas song stability is greatest when canaries breed and recruitment of new neurons is at its lowest (Kirn et al., 1994; Alvarez-Buylla and Kirn, 1997). It has been hypothesized that neuronal replacement in the HVC provides a cellular basis for song plasticity in adult canaries (Alvarez-Buylla et al., 1992; Kirn and Nottebohm, 1993). A similar correlation has been reported among foodstoring birds, which cache food and retrieve caches based on the spatial memory of their locations. In black-capped chickadees, recruitment of hippocampal neurons increases in the autumn at the peak of caching behavior (Barnea and Nottebohm, 1994, 1996). A study on mountain chickadees, however, could not replicate this correlation
719 (Pravosudov et al., 2002). Such results suggest that seasonal patterns of hippocampus-dependent learning (spatial memory for cache locations) might also correlate with differential patterns of neurogenesis, thereby affecting the number of neurons in the mammalian dentate gyrus. In rodents, Gould et al. (1999a) were the first to demonstrate that learning can increase the survival of the newly generated granule cells. In naive laboratory animals, the majority of the newborn neurons degenerate within two weeks of their production (Cameron et al., 1993b; Kempermann et al., 2003). It was demonstrated that if rats were trained to learn hippocampal-dependent tasks like place learning in a Morris water maze (Morris et al., 1982) or trace eyeblink conditioning (Solomon et al., 1986), then these learning experiences could significantly increase the survival of the newly generated granule cells (Gould et al., 1999a). This stimulating effect was attributed to learning, and not mere general experience, because exposure of animals to the same environment and conditions in the absence of learning had no effect on the number of new neurons. Furthermore, learning tasks that do not require the hippocampus, like delay eyeblink conditioning and cue learning in a Morris water maze (Schmaltz and Theios, 1972; Morris et al., 1982), did not alter the number of new neurons. These data clearly demonstrate that an enriched environment and certain types of learning are sufficient to enhance the number of new neurons in the dentate gyrus of adult rats and suggest the possibility that these new cells may play a role in learning. In a seminal study, Shors et al. (2001) used a toxin, the DNA-methylating agent methylazoxymethanol acetate (MAM), to block cell proliferation. MAM dramatically diminished the number of adult-generated cells in the dentate gyrus of rats, which resulted in a significant impairment of hippocampal dependent, but not hippocampal-independent forms of associative memory formation. In this study, adult rats were trained with delay or trace eyeblink conditioning. This task requires the animals to learn to associate two stimuli, a conditioned stimulus (white noise) with an unconditioned stimulus (shock to the eyelid), and as the animal learns, it blinks in response to the conditioned stimulus. During delay conditioning, the conditioned stimulus and
unconditioned stimulus overlap, and acquisition does not require an intact hippocampus (Schmaltz and Theios, 1972). During trace eyeblink conditioning, the animals have to associate the two stimuli separated temporally (during the trace interval), and acquisition of the trace conditioning requires intact hippocampus (Solomon et al., 1986; Weiss et al., 1999). Treating the rats with MAM for two weeks reduced the number of newly generated cells in the hippocampal dentate gyrus by 75-84%, yet the animals could rapidly acquire the hippocampalindependent task of delay conditioning, whereas their conditioned responses during trace conditioning were reduced by ~ 60% (Shors et al., 2001). Until now, this study is probably the most compelling evidence indicating the role of adult-generated neurons in mammalian learning. Contrasting somewhat to the findings of Shors et al. (2001), in a recent experiment conducted in our laboratory we could not demonstrate any learning impairment after stress-induced suppression of dentate cytogenesis in a hippocampal-dependent task (Bartolomucci et al., 2002). We exposed adult male tree shrews to five weeks of daily psychosocial stress and tested them repeatedly on a holeboard apparatus using two different learning tasks devised to evaluate hippocampal-dependent and hippocampal-independent cognitive function. We could show that despite the fact that stress significantly suppressed hippocampal neurogenesis, learning was enhanced in a hippocampal-dependent task in which animals had to learn the spatial distribution of hidden food rewards. Importantly, this stress-induced improvement of learning was found only for the number of reference memory errors (opening of an unbaited hole), whereas working memory performance, as measured by the number of repeated choices, was not affected by the stress. However, it should be emphasized that in the study by Shors et al. (2001), the disruption of dentate cell proliferation was much more pronounced than in our experiment and, more importantly, the memory tasks employed in the two experiments account for different memory types - namely, the accurate timing of a learned response, and the acquisition and retrieval of a complex spatial distribution. These conflicting results seemed to be resolved by a more recent study from the same group (Shors et al., 2002). As in their
720 previous study, they used again the antimitotic agent MAM, and tested whether reduction of cytogenesis affected learning and performance associated with different hippocampal-dependent tasks: spatial navigation learning in a Morris water maze, contextual fear conditioning, and trace fear conditioning. Reduction of new neurons in the adult hippocampus was associated with impaired performance in the trace fear conditioning paradigm but affected neither spatial navigation learning in the Morris water maze nor contextual fear conditioning. It is possible that spatial navigation learning can still occur with a very small percentage of new neurons. Another possibility is that only certain types of hippocampal-dependent learning require new cells, and spatial navigation learning is not one of those types. Nevertheless, a study using genetically different mouse strains did show a significant correlation between adult hippocampal neurogenesis and parameters describing the acquisition of the Morris water maze task (Kempermann and Gage, 2002a). A recent study used fractionated brain irradiation to block the formation of new neurons in the dentate gyrus (Madsen et al., 2003). Blockade of dentate neurogenesis induced significant impairment in a place recognition task using a T-maze, but lesioned animals performed equally to controls when tested in the Morris water maze (Madsen et al., 2003). Furthermore, several recent studies made an attempt to correlate hippocampal cell proliferation with water maze performance in aged rats, but majority of them failed to support a direct relationship of adult neurogenesis with spatial learning and memory capability in the Morris water maze (Merrill et al., 2003; Bizon and Gallagher, 2003), except one (Drapeau et al., 2003). Yet another hypothesis suggested that the functional role of these adult-generated granule cells does not relate to the acquisition of new memories. Instead, they may play a role in the periodic clearance of outdated hippocampal memory traces after cortical memory consolidation, thereby ensuring that the hippocampus is continuously available to process new memories (Feng et al., 2001). Apparently, the outcome of experiments investigating the role of adult hippocampal neurogenesis in learning is largely dependent on the approach, i.e. which specific learning paradigm is used for
evaluating hippocampal-dependent performance. The functional role of the hippocampus is broad (Suzuki and Clayton, 2000) and a great variety of learning tests can be used for assessment, each of which may be specific to a certain type of associative learning. For example, some groups put extra effort into their experimental paradigm by not using animals that were housed and tested in laboratory conditions; rather, they investigated wild animals living in their natural environment. A well-designed study on wild eastern gray squirrels (a long-lived mammal that scatter-hoards food) found negative results when testing whether seasonal variations in spatial memory processing (i.e. increased processing during caching season in the autumn) correlate with changes in neurogenesis and total granule cell number in the hippocampal dentate gyrus (Lavenex et al., 2000).
Acute and chronic
stressful experience
Collectively, the above-discussed observations demonstrate that cell proliferation in the dentate gyrus can be modulated by environmental signals and experience. However, environmental signals can also be detrimental to the functioning of neurogenesis. Stressful experiences are known to activate the hypothalamic-pituitary-adrenal (HPA) axis and increase levels of circulating adrenocortical steroid hormones, cortisol or corticosterone. There is compelling evidence demonstrating that both acute and chronic stressful experience can affect the production of new hippocampal granule cells by suppressing both the proliferation rate of precursor cells as well as the survival rate of the daughter cells. In collaboration with Elizabeth Gould, we were the first to demonstrate that acute psychosocial stress can dramatically suppress cell proliferation in the hippocampal dentate gyrus of adult tree shrews (Gould et al., 1997). In nonhuman primates, we found a similar effect of acute stressful experience (Gould et al., 1998) suggesting that these characteristics may be common to most mammalian species. In rats, a single exposure to a predator odor resulted in a marked reduction of cell proliferation (Tanapat et al., 2001). Chronic psychosocial stress seems to have a relatively mild suppressive effect on neurogenesis.
721 Subjecting tree shrews to five weeks of daily psychosocial stress resulted in a ~ 30% decrease of dentate cell proliferation (Czeh et al., 2001), and similar moderate effects were observed in rats (Czeh et al., 2002). Therefore, one may speculate that acute stress seems to cause a more robust inhibition of dentate cell proliferation, and proliferating cells may "habituate" after chronic stress and therefore are not as responsive to the inhibitory effects of stress hormones. However, once again the effect of stress on adult hippocampal neurogenesis may largely depend on the applied experimental stress paradigm. This could explain the findings of a recent study in which acute restraint stress of rats did not affect the proliferation rate of dentate precursor cells, whereas three weeks of daily restraint stress suppressed proliferation by ~ 2 0 % , and six weeks of chronic stress resulted in a substantial ~ 5 0 % reduction of neurogenesis, producing a significant decrement in the total number of granule cells by 13 % (Pham et al., 2003). It should be emphasized that comparisons across studies are often difficult, because of the different BrdU-labeling schedules. Importantly, stress exposure suppresses proliferation in the dentate gyrus, while proliferation in the subependymal zone is unaffected, indicating that the stress-induced decrease in cytogenesis is not due to a nonspecific effect, for example, decreased bioavailability of the marker molecule BrdU. Furthermore, there is compelling evidence that not only cell proliferation but also the survival of the newborn granule neurons are suppressed by stress (Czeh et al., 2002; Pham et al., 2003).
Long-term effect of prenatal stress It has become increasingly evident that the antecedents of many illnesses begin in fetal life, and further, that prenatal conditions can bias us toward either health or disease in the postpartum period. For example, several retrospective studies have confirmed that chronic maternal stress during pregnancy significantly increases the likelihood of disturbed physical and/or psychological development of the child (Jones and Tauscher, 1978; Meijer, 1985; Lou et al., 1994). An association has also been described between maternal stress and an increased probability
of schizophrenia (Huttunen and Niskanen, 1978; Myhrman et al., 1996; van Os and Selten, 1998) and depressive symptomatology (Watson et al., 1999; Brown et al., 2000) in prenatally stressed offspring. Studies investigating the long-term effect of prenatal stress on adult hippocampal neurogenesis are scarce. Lemaire et al. (2000) performed an extensive study on the effect of prenatal stress in rats. They restrained pregnant female rats during late pregnancy for 45 min three times a day, at the same time exposing them to bright light; adult dentate neurogenesis was later evaluated at different ages in the offspring. They reported that prenatal stress induced a life span reduction of hippocampal neurogenesis and decline in total granule cell numbers, accompanied by learning impairment in hippocampal-related spatial tasks (Lemaire et al., 2000). In a recent study that investigated whether prenatal stress can alter neural, hormonal, and behavioral status in nonhuman primates, pregnant rhesus monkeys were acutely stressed on a daily basis for 25% of their 24-week gestation using an acoustic startle protocol (Coe et al., 2003). At two-to-three years of age, hippocampal volume, cytogenesis in the dentate gyrus, and cortisol levels were evaluated in the offspring generated from stressed and control pregnancies. Prenatal stress, both early and late in pregnancy, resulted in reduced hippocampal volume and an inhibition of dentate gyrus neurogenesis. These changes were associated with higher cortisol levels, a more rapid escape from dexamethasone suppression, lower levels of exploration, and higher levels of motor behavior. These findings indicate that the prenatal environment can alter behavior, deregulate neuroendocrine systems, and affect the hippocampal structure of primates in a persistent manner. Moreover, these data strengthen pathophysiological hypotheses that propose an early neurodevelopmental origin for psychopathological vulnerability in adulthood.
Injury-induced adult neurogenesis Pathological conditions, such as ischemia or epileptic seizures, result in a marked increase of neurogenesis in the adult hippocampus. Enhanced dentate neurogenesis after experimental stroke may represent
722 a natural form of neural self-repair (Liu et al., 1998; Kee et al., 2001; Jin et al., 2001; Yagita et al., 2001), whereas seizures-induced neurogenesis may have either a reparative role or alternatively it can promote abnormal hyperexcitability (Parent et al., 1997, 1998; Scott et al., 1998; Blumcke et al., 2001; Parent, 2002). Ethanol exposure also seems to affect neurogenesis in the adult dentate gyrus, although so far the experimental results are somewhat conflicting. Some studies report decreased neurogenesis after ethanol exposure (Nixon and Crews, 2002; Herrera et al., 2003), whereas others demonstrate the opposite effect (Pawlak et al., 2002; Zharkovsky et al., 2003).
Functional role of adult-generated neurons The evolutionary conservation of adult neurogenesis in the mammalian brain suggests that it is of fundamental biological importance. Newly generated dentate granule cells become incorporated into the granule cell layer, attain the morphological and biochemical characteristics of neurons (Cameron et al., 1993b; Okano et al., 1993), develop synapses on their cell bodies, and dendrites (Kaplan and Hinds, 1977; Kaplan and Bell, 1984), extend axons into the CA3 region (Stanfield and Trice, 1988; Markakis and Gage, 1999), and generate action potentials (van Praag et al., 2002). They show distinct morphological and electrophysiological properties compared to mature granule cells (Liu et al., 2000), present a lower threshold for induction of long-term potentiation (LTP) and display robust LTP (Wang et al., 2000). Furthermore, the fact that continuous neurogenesis takes place in the hippocampal formation raises the possibility that newborn neurons could participate in learning. Indeed, a continually rejuvenating population of new neurons seems well suited for the proposed transient role of the hippocampal formation in information storage (Squire, 1992). The question of what the individual new neurons are used for is difficult to address experimentally. The major difficulty is that to date, no specific agent is available that can selectively block neurogenesis. Any of the currently available treatments that suppress adult neurogenesis may affect other brain regions or other processes in the same region. Probably, an elegant way to resolve this exciting question would be to
develop a mutant animal in which neurogenesis could be blocked in a specific region at particular times.
Acknowledgment We are grateful to J. Keuker for her help in the preparation of the figures.
References Aberg, M.A., Aberg, N.D., Hedbacker, H., Oscarsson, J. and Eriksson, P.S. (2000) Peripheral infusion of IGF-I selectively induces neurogenesis in the adult rat hippocampus. J. Neurosci., 20: 2896-2903. Altman, J. and Bayer, S.A. (1990) Migration and distribution of two populations of hippocampal granule cell precursors during the perinatal and postnatal periods. J. Comp. Neurol., 301: 365-381. Altman, J. and Das, G.D. (1965) Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. J. Comp. Neurol., 124: 319-335. Alvarez-Buylla, A. and Kirn, J.R. (1997) Birth, migration, incorporation, and death of vocal control neurons in adult songbirds. J. Neurobiol., 33: 585-601. Alvarez-Buylla, A., Theelen, M. and Nottebohm, F. (1988) Birth of projection neurons in the higher vocal center of the canary forebrain before, during, and after song learning. Proc. Natl. Acad. Sci. USA, 85: 8722-8726. Alvarez-Buylla, A., Ling, C.Y. and Nottebohm, F. (1992) High vocal center growth and its relation to neurogenesis, neuronal replacement and song acquisition in juvenile canaries. J. Neurobiol., 23: 396-406. Amaral, D.G. and Witter, M.P. (1989) The three-dimensional organization of the hippocampal formation: a review of anatomical data. Neuroscience, 31: 571-591. Barnea, A. and Nottebohm, F. (1994) Seasonal recruitment of hippocampal neurons in adult free-ranging black-capped chickadees. Proc. Natl. Acad. Sci. USA, 91: 11217-11221. Barnea, A. and Nottebohm, F. (1996) Recruitment and replacement of hippocampal neurons in young and adult chickadees: an addition to the theory of hippocampal learning. Proc. Natl. Acad. Sci. USA, 93: 714-718. Bartolomucci, A., de Biurrun, G., Czeh, B., van Kampen, M. and Fuchs, E. (2002) Selective enhancement of spatial learning under chronic psychosocial stress. Eur. J. Neurosci., 15:1863-1866. Bayer, S.A. (1980) Development of the hippocampal region in the rat. I. Neurogenesis examined with 3H-thymidine autoradiography. J. Comp. Neurol., 190: 87-114. Bernier, P.J., Bedard, A., Vinet, J., Levesque, M. and Parent, A. (2002) Newly generated neurons in the amygdala and
723 adjoining cortex of adult primates. Proc. Natl. Acad. Sci. USA, 99:11464-11469. Betarbet, R., Zigova, T., Bakay, R.A. and Luskin, M.B. (1996) Dopaminergic and GABAergic interneurons of the olfactory bulb are derived from the neonatal subventricular zone. Int. J. Dev. Neurosci., 14: 921-930. Bizon, J.L. and Gallagher, M. (2003) Production of new cells in the rat dentate gyrus over the lifespan: relation to cognitive decline. Eur. J. Neurosci., 18: 215-219. Blumcke, I., Schewe, J.C., Normann, S., Brustle, O., Schramm, J., Elger, C.E. and Wiestler, O.D. (2001) Increase of nestin-immunoreactive neural precursor cells in the dentate gyrus of pediatric patients with early-onset temporal lobe epilepsy. Hippocampus, 11:311-321. Brezun, J.M. and Daszuta, A. (1999) Depletion in serotonin decreases neurogenesis in the dentate gyrus and the subventricular zone of adult rats. Neuroscience, 89: 999-1002. Brezun, J.M. and Daszuta, A. (2000a) Serotonin may stimulate granule cell proliferation in the adult hippocampus, as observed in rats grafted with foetal raphe neurons. Eur. J. Neurosci., 12: 391-396. Brezun, J.M. and Daszuta, A. (2000b) Serotonergic reinnervation reverses lesion-induced decreases in PSA-NCAM labeling and proliferation of hippocampal cells in adult rats. Hippocampus, 10: 37-46. Brown, A.S., van Os, J., Driessens, C., Hoek, H.W. and Susser, E.S. (2000) Further evidence of relation between prenatal famine and major affective disorder. Am. J. Psychiatry, 157: 190-195. Cajal, R.S. (1928) Degeneration and regeneration of the nervous system. May, R.M. trans., Vol. II. Oxford University Press, London: Humphrey Milford, p. 750. Cameron, H.A. and Gould, E. (1994) Adult neurogenesis is regulated by adrenal steroids in the dentate gyrus. Neuroscience, 61: 203-209. Cameron, H.A. and McKay, R.D. (1999) Restoring production of hippocampal neurons in old age. Nat. Neurosci., 2: 894-897. Cameron, H.A. and McKay, R.D. (2001) Adult neurogenesis produces a large pool of new granule cells in the dentate gyrus. J. Comp. Neurol., 435: 406-417. Cameron, H.A., Woolley, C.S. and Gould, E. (1993a) Adrenal steroid receptor immunoreactivity in cells born in the adult rat dentate gyrus. Brain Res., 611: 342-346. Cameron, H.A., Woolley, C.S., McEwen, B.S. and Gould, E. (1993b) Differentiation of newly born neurons and glia in the dentate gyrus of the adult rat. Neuroscience, 56: 337-344. Cameron, H.A., McEwen, B.S. and Gould, E. (1995) Regulation of adult neurogenesis by excitatory input and NMDA receptor activation in the dentate gyrus. J. Neurosci., 15: 4687-4692. Cameron, H.A., Tanapat, P. and Gould, E. (1998) Adrenal steroids and N-methyl-D-aspartate receptor activation
regulate neurogenesis in the dentate gyrus of adult rats through a common pathway. Neuroscience, 82: 349-354. Chen, G., Rajkowska, G., Du, F., Seraji-Bozorgzad, N. and Manji, H.K. (2000) Enhancement of hippocampal neurogenesis by lithium. J. Neurochem., 75: 1729-1734. Coe, C.L., Kramer, M., Czeh, B., Gould, E., Reeves, A.J., Kirschbaum, C. and Fuchs, E. (2003) Prenatal stress diminishes neurogenesis in the dentate gyrus of juvenile rhesus monkeys. Biol. Psychiatry, 54: 1025-1034. Czeh, B., Michaelis, T., Watanabe, T., Frahm, J., de Biurrun, G., van Kampen, M., Bartolomucci, A. and Fuchs, E. (2001) Stress-induced changes in cerebral metabolites, hippocampal volume, and cell proliferation are prevented by antidepressant treatment with tianeptine. Proc. Natl. Acad. Sci. USA, 98: 12796-12801. Czeh, B., Welt, T., Fischer, A.K., Erhardt, A., Schmitt, W., Muller, M.B., Toschi, N., Fuchs, E. and Keck, M.E. (2002) Chronic psychosocial stress and concomitant repetitive transcranial magnetic stimulation: effects on stress hormone levels and adult hippocampal neurogenesis. Biol. Psychiatry, 52: 1057-1065. Drapeau, E., Mayo, W., Aurousseau, C., Le Moal, M., Piazza, P.V. and Abrous, D.N. (2003) Spatial memory performances of aged rats in the water maze predict levels of hippocampal neurogenesis. Proc. Natl. Acad. Sci. USA, 100: 14385-14390. Eckenhoff, M.F. and Rakic, P. (1988) Nature and fate of proliferative cells in the hippocampal dentate gyrus during the life span of the rhesus monkey. J. Neurosci., 8: 2729-2747. Eriksson, P.S., Perfilieva, E., Bjork-Eriksson, T., Alborn, A.M., Nordborg, C., Peterson, D.A. and Gage, F.H. (1998) Neurogenesis in the adult human hippocampus. Nat. Med., 4: 1313-1317. Feng, R., Rampon, C., Tang, Y.P., Shrom, D., Jin, J., Kyin, M., Sopher, B., Miller, M.W., Ware, C.B., Martin, G.M., Kim, S.H., Langdon, R.B., Sisodia, S.S. and Tsien, J.Z. (2001) Deficient neurogenesis in forebrain-specific presenilin-1 knockout mice is associated with reduced clearance of hippocampal memory traces. Neuron, 32:911-926. Fischer, A.K., von Rosenstiel, P., Fuchs, E., Goula, D., Almeida, O.F. and Czeh, B. (2002) The prototypic mineralocorticoid receptor agonist aldosterone influences neurogenesis in the dentate gyrus of the adrenalectomized rat. Brain Res., 947: 290-293. Gage, F.H. (1998) Stem cells of the central nervous system. Curr. Opin. Neurobiol., 8: 671-676. Gage, F.H. (2000) Mammalian neural stem cells. Science, 287: 1433-1438. Gass, P., Kretz, O., Wolfe, D.P., Berger, S., Tronche, F., Reichardt, H.M., Kellendonk, C., Lipp, H.P., Schmid, W. and Schutz, G. (2000) Genetic disruption of mineralocorticoid receptor leads to impaired neurogenesis and granule cell
724 degeneration in the hippocampus of adult mice. EMBO Rep., 1: 447-451. Gheusi, G., Cremer, H., McLean, H., Chazal, G., Vincent, J.D. and Lledo, P.M. (2000) Importance of newly generated neurons in the adult olfactory bulb for odor discrimination. Proc. Natl. Acad. Sci. USA, 97: 1823-1828. Goldman, J.E. (1995) Lineage, migration, and fate determination of postnatal subventricular zone cells in the mammalian CNS. J. Neurooncol., 24: 61-64. Goldman, S.A. (1998) Adult neurogenesis: from canaries to the clinic. J. Neurobiol., 36: 267-286. Goldman, S.A. and Nottebohm, F. (1983) Neuronal production, migration, and differentiation in a vocal control nucleus of the adult female canary brain. Proc. Natl. Acad. Sci. USA, 80: 2390-2394. Gould, E. and Gross, C.G. (2002) Neurogenesis in adult mammals: some progress and problems. J. Neurosci., 22: 619-623. Gould, E., McEwen, B.S., Tanapat, P., Galea, L.A. and Fuchs, E. (1997) Neurogenesis in the dentate gyrus of the adult tree shrew is regulated by psychosocial stress and NMDA receptor activation. J. Neurosci., 17: 2492-2498. Gould, E., Tanapat, P., McEwen, B.S., Flfigge, G. and Fuchs, E. (1998) Proliferation of granule cell precursors in the dentate gyrus of adult monkeys is diminished by stress. Proc. Natl. Acad. Sci. USA, 95: 3168-3171. Gould, E., Beylin, A., Tanapat, P., Reeves, A. and Shors, T.J. (1999a) Learning enhances adult neurogenesis in the hippocampal formation. Nat. Neurosci., 2: 260-265. Gould, E., Reeves, A.J., Fallah, M., Tanapat, P., Gross, C.G. and Fuchs, E. (1999b) Hippocampal neurogenesis in adult Old World primates. Proc. Natl. Acad. Sci. USA, 96: 5263-5267. Gould, E., Reeves, A.J., Graziano, M.S. and Gross, C.G. (1999c) Neurogenesis in the neocortex of adult primates. Science, 286: 548-552. Gould, E., Vail, N., Wagers, M. and Gross, C.G. (2001) Adultgenerated hippocampal and neocortical neurons in macaques have a transient existence. Proc. Natl. Acad. Sci. USA, 98: 10910-10917. Hayes, N.L. and Nowakowski, R.S. (2002) Dynamics of cell proliferation in the adult dentate gyrus of two inbred strains of mice. Brain Res. Dev. Brain Res., 134: 77-85. Herrera, D.G., Yague, A.G., Johnsen-Soriano, S., BoschMorell, F., Collado-Morente, L., Muriach, M., Romero, F.J. and Garcia-Verdugo, J.M. (2003) Selective impairment of hippocampal neurogenesis by chronic alcoholism: protective effects of an antioxidant. Proc. Natl. Acad. Sci. USA, 100: 7919-7924. Horner, P.J., Power, A.E., Kempermann, G., Kuhn, H.G., Palmer, T.D., Winkler, J., Thal, L.J. and Gage, F.H. (2000) Proliferation and differentiation of progenitor cells throughout the intact adult rat spinal cord. J. Neurosci., 20: 2218-2228.
Humphrey, T. (1967) The development of the human hippocampal fissure. J. Anat., 101: 655-676. Huttunen, M.O. and Niskanen, P. (1978) Prenatal loss of father and psychiatric disorders. Arch. Gen. Psychiatry, 35: 429-431. Jin, K., Minami, M., Lan, J.Q., Mao, X.O., Batteur, S., Simon, R.P. and Greenberg, D.A. (2001) Neurogenesis in dentate subgranular zone and rostral subventricular zone after focal cerebral ischemia in the rat. Proc. Natl. Acad. Sci. USA, 98: 4710-4715. Jin, K., Zhu, Y., Sun, Y., Mao, X.O., Xie, L. and Greenberg, D.A. (2002) Vascular endothelial growth factor (VEGF) stimulates neurogenesis in vitro and in vivo. Proc. Natl. Acad. Sci. USA, 99:11946-11950. Jones, F. and Tauscher, J. (1978) Residence under an airportlanding pattern as a factor in teratism. Arch. Environ. Health, 33: 10-12. Kaplan, M.S. (1981) Neurogenesis in the 3-month-old rat visual cortex. J. Comp. Neurol., 195: 323-338. Kaplan, M.S. and Bell, D.H. (1984) Mitotic neuroblasts in the 9-day-old and l 1-month-old rodent hippocampus. J. Neurosci., 4: 1429-1441. Kaplan, M.S. and Hinds, J.W. (1977) Neurogenesis in the adult rat: electron microscopic analysis of light radioautographs. Science, 197: 1092-1094. Kee, N.J., Preston, E. and Wojtowicz, J.M. (2001) Enhanced neurogenesis after transient global ischemia in the dentate gyrus of the rat. Exp. Brain Res., 136: 313-320. Kempermann, G. and Gage, F.H. (2002a) Genetic determinants of adult hippocampal neurogenesis correlate with acquisition, but not probe trial performance, in the water maze task. Eur. J. Neurosci., 16: 129-136. Kempermann, G. and Gage, F.H. (2002b) Genetic influence on phenotypic differentiation in adult hippocampal neurogenesis. Brain Res. Dev. Brain Res., 134: 1-12. Kempermann, G., Kuhn, H.G. and Gage, F.H. (1997a) Genetic influence on neurogenesis in the dentate gyrus of adult mice. Proc. Natl. Acad. Sci. USA, 94: 10409-10414. Kempermann, G., Kuhn, H.G. and Gage, F.H. (1997b) More hippocampal neurons in adult mice living in an enriched environment. Nature, 386: 493-495. Kempermann, G., Kuhn, H.G. and Gage, F.H. (1998) Experience-induced neurogenesis in the senescent dentate gyrus. J. Neurosci., 18: 3206-3212. Kempermann, G., Gast, D., Kronenberg, G., Yamaguchi, M. and Gage, F.H. (2003) Early determination and long-term persistence of adult-generated new neurons in the hippocampus of mice. Development, 130:391-399. Keuker, J.I., Luiten, P.G. and Fuchs, E. (2003) Preservation of hippocampal neuron numbers in aged rhesus monkeys. Neurobiol. Aging, 24: 157-165. Kirn, J.R. and Nottebohm, F. (1993) Direct evidence for loss and replacement of projection neurons in adult canary brain. J. Neurosci., 13: 1654-1663.
725 Kirn, J.R., O'Loughlin, B., Kasparian, S. and Nottebohm, F. (1994) Cell death and neuronal recruitment in the high vocal center of adult male canaries are temporally related to changes in song. Proc. Natl. Acad. Sci. USA, 91: 7844-7848. Koketsu, D., Mikami, A., Miyamoto, Y. and Hisatsune, T. (2003) Nonrenewal of neurons in the cerebral neocortex of adult macaque monkeys. J. Neurosci., 23: 937-942. Kondo, T. and Raft, M. (2000) Oligodendrocyte precursor cells reprogrammed to become multipotential CNS stem cells. Science, 289: 1754-1757. Kornack, D.R. and Rakic, P. (1999) Continuation of neurogenesis in the hippocampus of the adult macaque monkey. Proc. Natl. Acad. Sci. USA, 96: 5768-5773. Kornack, D.R. and Rakic, P. (2001a) The generation, migration, and differentiation of olfactory neurons in the adult primate brain. Proc. Natl. Acad. Sci. USA, 98: 4752-4757. Kornack, D.R. and Rakic, P. (2001 b) Cell proliferation without neurogenesis in adult primate neocortex. Science, 294: 2127-2130. Kuhn, H.G., Dickinson-Anson, H. and Gage, F.H. (1996) Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation. J. Neurosci., 16: 2027-2033. Kuhn, H.G., Winkler, J., Kempermann, G., Thal, L.J. and Gage, F.H. (1997) Epidermal growth factor and fibroblast growth factor-2 have different effects on neural progenitors in the adult rat brain. J. Neurosci., 17: 5820-5829. Lavenex, P., Steele, M.A. and Jacobs, L.F. (2000) The seasonal pattern of cell proliferation and neuron number in the dentate gyrus of wild adult eastern grey squirrels. Eur. J. Neurosci., 12: 643-648. Lee, H.J., Kim, J.W., Yim, S.V., Kim, M.J., Kim, S.A., Kim, Y.J., Kim, C.J. and Chung, J.H. (2001) Fluoxetine enhances cell proliferation and prevents apoptosis in dentate gyrus of maternally separated rats. Mol. Psychiatry, 6: 610, 725-728. Lemaire, V., Koehl, M., Le Moal, M. and Abrous, D.N. (2000) Prenatal stress produces learning deficits associated with an inhibition of neurogenesis in the hippocampus. Proc. Natl. Acad. Sci. USA, 97:11032-11037. Liu, J., Solway, K., Messing, R.O. and Sharp, F.R. (1998) Increased neurogenesis in the dentate gyrus after transient global ischemia in gerbils. J. Neurosci., 18: 7768-7778. Liu, X., Tilwalli, S., Ye, G., Lio, P.A., Pasternak, J.F. and Trommer, B.L. (2000) Morphologic and electrophysiologic maturation in developing dentate gyrus granule cells. Brain Res., 856: 202-212. Lois, C. and Alvarez-Buylla, A. (1994) Long-distance neuronal migration in the adult mammalian brain. Science, 264: 1145-1148. Lois, C., Garcia-Verdugo, J.M. and Alvarez-Buylla, A. (1996) Chain migration of neuronal precursors. Science, 271: 978-981.
Lou, H.C., Hansen, D., Nordentoft, M., Pryds, O., Jensen, F., Nim, J. and Hemmingsen, R. (1994) Prenatal stressors of human life affect fetal brain development. Dev. Med. Child Neurol., 36: 826-832. Luskin, M.B. (1993) Restricted proliferation and migration of postnatally generated neurons derived from the forebrain subventricular zone. Neuron, 11: 173-189. Madsen, T.M., Kristjansen, P.E., Bolwig, T.G. and Wortwein, G. (2003) Arrested neuronal proliferation and impaired hippocampal function following fractionated brain irradiation in the adult rat. Neuroscience, 119: 635-642. Malberg, J.E., Duman, R.S. (2003) Cell proliferation in adult hippocampus is decreased by inescapable stress: reversal by fluoxetine treatment. Neuropsychopharmacology, 28: 1562-1571. Malberg, J.E., Eisch, A.J., Nestler, E.J. and Duman, R.S. (2000) Chronic antidepressant treatment increases neurogenesis in adult rat hippocampus. J. Neurosci., 20: 9104-9110. Markakis, E.A. and Gage, F.H. (1999) Adult-generated neurons in the dentate gyrus send axonal projections to field CA3 and are surrounded by synaptic vesicles. J. Comp. Neurol., 406: 449-460. McLean, J.H. and Shipley, M.T. (1988) Postmitotic, postmigrational expression of tyrosine hydroxylase in olfactory bulb dopaminergic neurons. J. Neurosci., 8: 3658-3669. Meijer, A. (1985) Child psychiatric sequelae of maternal war stress. Acta Psychiatr. Scand., 72: 505-511. Menezes, J.R., Smith, C.M., Nelson, K.C. and Luskin, M.B. (1995) The division of neuronal progenitor cells during migration in the neonatal mammalian forebrain. Mol. Cell Neurosci., 6: 496-508. Merrill, D.A., Karim, R., Darraq, M., Chiba, A.A. and Tuszynski, M.H. (2003) Hippocampal cell genesis does not correlate with spatial learning ability in aged rats. J. Comp. Neurol., 459: 201-207. Miller, M.W. and Nowakowski, R.S. (1988) Use of bromodeoxyuridine-immunohistochemistry to examine the proliferation, migration and time of origin of cells in the central nervous system. Brain Res., 457: 44-52. Morris, R.G., Garrud, P., Rawlins, J.N. and O'Keefe, J. (1982) Place navigation impaired in rats with hippocampal lesions. Nature, 297:681-683. Morshead, C.M., Craig, C.G. and van der Kooy, D. (1998) In vivo clonal analyses reveal the properties of endogenous neural stem cell proliferation in the adult mammalian forebrain. Development, 125: 2251-2261. Myhrman, A., Rantakallio, P., Isohanni, M., Jones, P. and Partanen, U. (1996) Unwantedness of a pregnancy and schizophrenia in the child. Br. J. Psychiatry, 169: 637-640. Nixon, K. and Crews, F.T. (2002) Binge ethanol exposure decreases neurogenesis in adult rat hippocampus. J. Neurochem., 83: 1087-1093.
726 Nowakowski, R.S. and Hayes, N.L. (2000) New neurons: extraordinary evidence or extraordinary conclusion? Science, 288: 771. Nunes, M.C., Roy, N.S., Keyoung, H.M., Goodman, R.R., McKhann, G., Jiang, L., Kang, J., Nedergaard, M. and Goldman, S.A. (2003) Identification and isolation of multipotential neural progenitor cells from the subcortical white matter of the adult human brain. Nat. Med., 9: 439-447. Okano, H.J., Pfaff, D.W. and Gibbs, R.B. (1993) RB and Cdc2 expression in brain: correlations with 3H-thymidine incorporation and neurogenesis. J. Neurosci., 13: 2930-2938. Okano, H.J., Pfaff, D.W. and Gibbs, R.B. (1996) Expression of EGFR-, p75NGFR-, and PSTAIR (cdc2)-like immunoreactivity by proliferating cells in the adult rat hippocampal formation and forebrain. Dev. Neurosci., 18: 199-209. Packer, M.A., Stasiv, Y., Benraiss, A., Chmielnicki, E., Grinberg, A., Westphal, H., Goldman, S.A. and Enikolopov, G. (2003) Nitric oxide negatively regulates mammalian adult neurogenesis. Proc. Natl. Acad. Sci. USA, 100: 9566-9571. Palmer, T.D., Markakis, E.A., Willhoite, A.R., Safar, F. and Gage, F.H. (1999) Fibroblast growth factor-2 activates a latent neurogenic program in neural stem cells from diverse regions of the adult CNS. J. Neurosci., 19: 8487-8497. Palmer, T.D., Willhoite, A.R. and Gage, F.H. (2000) Vascular niche for adult hippocampal neurogenesis. J. Comp. Neurol., 425: 479-494. Parent, J.M. (2002) The role of seizure-induced neurogenesis in epileptogenesis and brain repair. Epilepsy Res., 50:179-189. Parent, J.M., Yu, T.W., Leibowitz, R.T., Geschwind, D.H., Sloviter, R.S. and Lowenstein, D.H. (1997) Dentate granule cell neurogenesis is increased by seizures and contributes to aberrant network reorganization in the adult rat hippocampus. J. Neurosci., 17: 3727-3738. Parent, J.M., Janumpalli, S., McNamara, J.O. and Lowenstein, D.H. (1998) Increased dentate granule cell neurogenesis following amygdala kindling in the adult rat. Neurosci. Lett., 247: 9-12. Pawlak, R., Skrzypiec, A., Sulkowski, S. and Buczko, W. (2002) Ethanol-induced neurotoxicity is counterbalanced by increased cell proliferation in mouse dentate gyrus. Neurosci. Lett., 327: 83-86. Pham, K., Nacher, J., Hof, P.R. and McEwen, B.S. (2003) Repeated restraint stress suppresses neurogenesis and induces biphasic PSA-NCAM expression in the adult rat dentate gyrus. Eur. J. Neurosci., 17: 879-886. Pravosudov, V.V., Lavenex, P. and Clayton, N.S. (2002) Changes in spatial memory mediated by experimental variation in food supply do not affect hippocampal anatomy in mountain chickadees (Poecile gambeli). J. Neurobiol., 51: 142-148. Rakic, P. (1985) Limits of neurogenesis in primates. Science, 227: 1054-1056.
Rakic, P. (2002a) Adult neurogenesis in mammals: an identity crisis. J. Neurosci., 22:614-618. Rakic, P. (2002b) Neurogenesis in adult primate neocortex: an evaluation of the evidence. Nat. Rev. Neurosci., 3: 65-71. Rakic, P. and Nowakowski, R.S. (1981) The time of origin of neurons in the hippocampal region of the rhesus monkey. J. Comp. Neurol., 196: 99-128. Rochefort, C., Gheusi, G., Vincent, J.D. and Lledo, P.M. (2002) Enriched odor exposure increases the number of newborn neurons in the adult olfactory bulb and improves odor memory. J. Neurosci., 22: 2679-2689. Schmaltz, L.W. and Theios, J. (1972) Acquisition and extinction of a classically conditioned response in hippocampectomized rabbits (Oryctolagus cuniculus). J. Comp. Physiol. Psychol., 79: 328-333. Scott, B.W., Wang, S., Burnham, W.M., De Boni, U. and Wojtowicz, J.M. (1998) Kindling-induced neurogenesis in the dentate gyrus of the rat. Neurosci. Lett., 248: 73-76. Seaberg, R.M. and van der Kooy, D. (2002) Adult rodent neurogenic regions: the ventricular subependyma contains neural stem cells, but the dentate gyrus contains restricted progenitors. J. Neurosci., 22: 1784-1793. Seress, L., Abraham, H., Tornoczky, T. and Kosztolanyi, G. (2001) Cell formation in the human hippocampal formation from mid-gestation to the late postnatal period. Neuroscience, 105: 831-843. Shors, T.J., Miesegaes, G., Beylin, A., Zhao, M., Rydel, T. and Gould, E. (2001) Neurogenesis in the adult is involved in the formation of trace memories. Nature, 410: 372-376. Shors, T.J., Townsend, D.A., Zhao, M., Kozorovitskiy, Y. and Gould, E. (2002) Neurogenesis may relate to some but not all types of hippocampal-dependent learning. Hippocampus, 12: 578-584. Solomon, P.R., Vander Schaaf, E.R., Thompson, R.F. and Weisz, D.J. (1986) Hippocampus and trace conditioning of the rabbit's classically conditioned nictitating membrane response. Behav. Neurosci., 100: 729-744. Squire, L.R. (1992) Memory and the hippocampus: a synthesis from findings with rats, monkeys, and humans. Psychol. Rev., 99: 195-231. Stanfield, B.B. and Trice, J.E (1988) Evidence that granule cells generated in the dentate gyrus of adult rats extend axonal projections. Exp. Brain Res., 72: 399-406. Suzuki, W.A. and Clayton, N.S. (2000) The hippocampus and memory: a comparative and ethological perspective. Curr. Opin. Neurobiol., 10: 768-773. Suzuki, W.A. and Eichenbaum, H. (2000) The neurophysiology of memory. Ann. N.Y. Acad. Sci., 911: 175-191. Tanapat, P. and Gould, E. (1997) EGF stimulates proliferation of granule cell precursors in the dentate gyrus of adult rats. Soc. Neurosci. Abstr., 23: 317. Tanapat, P., Hastings, N.B., Reeves, A.J. and Gould, E. (1999) Estrogen stimulates a transient increase in the number of new
727 neurons in the dentate gyrus of the adult female rat. J. Neurosci., 19: 5792-5801. Tanapat, P., Hastings, N.B., Rydel, T.A., Galea, L.A. and Gould, E. (2001) Exposure to fox odor inhibits cell proliferation in the hippocampus of adult rats via an adrenal hormone-dependent mechanism. J. Comp. Neurol., 437: 496-504. van der Hart, M.G., Czeh, B., de Biurrun, G., Michaelis, T., Watanabe, T., Natt, O., Frahm, J. and Fuchs, E. (2002) Substance P receptor antagonist and clomipramine prevent stress-induced alterations in cerebral metabolites, cytogenesis in the dentate gyrus and hippocampal volume. Mol. Psychiatry, 7: 933-941. van Os, J. and Selten, J.P. (1998) Prenatal exposure to maternal stress and subsequent schizophrenia. The May 1940 invasion of The Netherlands. Br. J. Psychiatry, 172: 324-326. van Praag, H., Kempermann, G. and Gage, F.H. (1999) Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nat. Neurosci., 2: 266-270. van Praag, H., Kempermann, G. and Gage, F.H. (2000) Neural consequences of environmental enrichment. Nat. Rev. Neurosci., 1: 191-198. van Praag, H., Schinder, A.F., Christie, B.R., Toni, N., Palmer, T.D. and Gage, F.H. (2002) Functional neurogenesis in the adult hippocampus. Nature, 415: 1030-1034. Wagner, J.P., Black, I.B. and DiCicco-Bloom, E. (1999) Stimulation of neonatal and adult brain neurogenesis by subcutaneous injection of basic fibroblast growth factor. J. Neurosci., 19:6006-6016.
Wang, S., Scott, B.W. and Wojtowicz, J.M. (2000) Heterogenous properties of dentate granule neurons in the adult rat. J. Neurobiol., 42: 248-257. Watson, J.B., Mednick, S.A., Huttunen, M. and Wang, X. (1999) Prenatal teratogens and the development of adult mental illness. Dev. Psychopathol., 11: 457-466. Weiss, C., Bouwmeester, H., Power, J.M. and Disterhoft, J.F. (1999) Hippocampal lesions prevent trace eyeblink conditioning in the freely moving rat. Behav. Brain Res., 99: 123-132. West, M.J., Slomianka, L. and Gundersen, H.J. (1991) Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator. Anat. Rec., 231: 482-497. Wolswijk, G. and Noble, M. (1992) Cooperation between PDGF and FGF converts slowly dividing O-2A adult progenitor cells to rapidly dividing cells with characteristics of O-2A perinatal progenitor cells. J. Cell Biol., 118: 889-900. Yagita, Y., Kitagawa, K., Ohtsuki, T., Takasawa, K., Miyata, T., Okano, H., Hori, M. and Matsumoto, M. (2001) Neurogenesis by progenitor cells in the ischemic adult rat hippocampus. Stroke, 32: 1890-1896. Zharkovsky, T., Kaasik, A., Jaako, K. and Zharkovsky, A. (2003) Neurodegeneration and production of the new cells in the dentate gyrus of juvenile rat hippocampus after a single administration of ethanol. Brain Res., 978: 115-123. Zupanc, G.K. (1999) Neurogenesis, cell death and regeneration in the adult gymnotiform brain. J. Exp. Biol., 202: 1435-1446.
This Page Intentionally Left Blank
T. Steckler, N.H. Kalin and J.M.H.M. Reul (Eds.) Handbook of Stress and the Brain, Vol. 15
ISSN 0-444-51173-3 Copyright 2005 Elsevier B.V. All rights reserved CHAPTER 5.6
Cellular and molecular analysis of stress-induced neurodegeneration methodological considerations J. Lu 1, Z. N6methy 1, J.M. Pego 2, J.J. Cerqueira 2, N.
Sousa 2
and O.F.X. Almeida 1'*
1Max_Planck Institute of Psychiatry, Kraepelinstrasse 2-10, D-80804 Munich, Germany :Life and Health Science Research Institute, Health Science School, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal
Abstract: Evidence that chronic hypercorticalism induces a broad spectrum of deleterious cellular effects in the brain has accumulated over the last two decades. These principal effects of hypercorticalism include neuronal atrophy, neuronal death and glial responses. Importantly, these changes, which may occur interdependently and/or concomitantly, lead to neurodegeneration. While there has been a significant expansion of the number of techniques available for examining effects of chronic stress in the brain, the cellular and molecular mechanisms underpinning stressinduced neurodegeneration are still only partially known. This article appraises the major current methodologies available for analyzing stress-induced neurodegeneration, and considers the advantages and limitations of each of these methods.
What do we understand by stress?
corticosteroid secretion are a crucial accompaniment of the chronic response to stress (Sapolsky et al., 2000). Briefly, in the event that adequate adaptive mechanisms cannot be recruited, chronic stress will result in a state of chronic hypercorticalism and, as a consequence, deleterious effects, including immune suppression and a variety of mental disturbances will emerge (Sapolsky et al., 2000).
Stress refers to the organism's attempt to mount an 'adaptive' (beneficial) response to aversive stimuli in order to maintain or restore homeostasis. Different sensory and motor systems are differentially activated depending on the quality and intensity of the stressful (aversive) stimulus, and the magnitude and duration of the response are influenced by the "context" of the stimulus (experience, mood, age, environmental factors) (Herman and Cullinan, 1997). Thus, extreme caution is necessary before generalizing about the effects of one particular "stress"; it is fair to say that much of the confusion existing in the field is a consequence of the false presumption that elevated corticosteroids mimic stress and/or one stressor is a representative of every stressor. Another important point to be noted is that prolonged elevations in
Forms of neurodegeneration Numerous studies have demonstrated that chronic hypercorticalism induces a broad spectrum of deleterious cellular effects in the brain, which can be conveniently categorized as neuronal atrophy, neuronal death, and glial responses. These changes, which may occur interdependently and/or concomitantly constitute neurodegeneration. The neurodegenerative changes associated with hypercorticalism are by far less-marked than the damage seen in the so-called
*Corresponding author: Tel.: 9 89 30622216; Fax: 9 89 306 22461; E-mail:
[email protected]. 729
730 neurodegenerative diseases (Parkinson's, Alzheimer's, etc.) and it would therefore, probably be more prudent to consider them as representative of the selective vulnerability of given brain regions. Most of the examples given in this review relate to the responses of hippocampal cells to stress (and pharmacological hypercorticalism). The hippocampus has been the most extensively studied brain region in this respect; its particular vulnerability to corticosteroids most probably reflects its high concentrations of corticosteroid (mineralocorticoid and glucocorticoid) receptors.
Neuronal
atrophy
An important notion to be kept in mind when referring to neuronal degeneration is that it does not necessarily imply the death of neurons. Indeed, most events perceived by a living organism (either positive or negative) are believed to modulate the structure of neuronal networks rather than lead to changes in neuronal number (Segal, 2002; Erickson et al., 2003). To evaluate dendritic arborizations one can use the Golgi technique, which selectively impregnates single neurons with silver chromate (Camillo Golgi, 1843-1924). This method has provided indispensable information about the way in which sets of neuronal elements contribute to the gross structure of the neuropil and tracts. Impregnations show up as black, purple or reddish-brown against a pale yellow background; it is essentially a stochastic technique, the exact chemical mechanism of which remains unclear. This approach allows impregnated neurons and boundaries in any region of interest to be traced and reconstructed from successive serial sections. Two-dimensional (2D) analysis can be performed from traces obtained using a drawing tube attached to a light microscope. This type of analysis does not require any sophisticated equipment and has been widely used in the past. However, it has one major disadvantage: converting a 3D probe into a 2D probe results in a loss of information on the suppressed dimension. To achieve 3D reconstructions, cell bodies, dendritic arborizations, and boundaries of the region of interest should be drawn (under 25-100 • oil immersion objectives) and plotted in 3D using a video computer system (e.g., Neurolucida
from MicroBrightField, Inc.). Three-dimensional models of neurons can be visualized using appropriate software. Three-dimensional reconstructions of neurons can be rotated around any of the x-, y-, and z-axes to allow the best visualization of the dendritic trees. Total dendritic lengths, number of segments/ bifurcations, Sholl analysis (which provides an estimate of dendritic densities, based on the number of intersections between concentric circles centered in cell soma and the dendritic segments) and spine densities are just some of the parameters this analysis provides. Importantly, the use of these techniques allowed the pioneers of neuroanatomy to recognize the organization of neuronal networks, and to eventually demonstrate the occurrence of remarkable alterations in dendritic trees and synaptic contacts following neuronal insults. Indeed, such knowledge existed long before the description of different forms of neuronal death. Several studies in the 1990s demonstrated that hypercorticalism (pharmacological or stress-induced) induces alterations in cytoplasm organelles (Miller et al., 1989) and, ultimately, atrophy of CA3 pyramidal cell dendrites in the hippocampal formation; (Watanabe et al., 1992; Magarinos and McEwen 1995a); subsequent work confirmed these results in this neuronal population but also observed similar alterations in all the other major subdivisions of the hippocampal formation (Sousa et al., 2000). Furthermore, the later studies noted a marked loss of synapes in at least one of the links of the intrinsic hippocampal circuitry (the mossy fiber-CA3 connection). It may therefore be concluded that elevated corticosteroids trigger structural responses within cytoplasmic organelles, dendrites, axons, and their synaptic contacts; importantly, such changes do not necessarily involve the irreversible loss of neurons (Sousa et al., 2000). Neuritic alterations of the type described above correlate with behavioral deficits and would appear to serve as the neuroanatomical basis of adaptive mechanisms underlying learning and memory (Erickson et al., 2003). The cellular basis of learning and memory has long been believed to include alterations in dendrites (mainly in spines) and in the number and structure of synapses (Cajal, 1893). The validity of this notion was explored in a number
731 of quantitative light and electron microscopic studies, which, in the majority of cases, showed that the richness of dendritic arborizations and the numerical density of synapses increases as a consequence of learning of novel behaviors. More recent studies have also shown that, despite numerical changes in dendritic spines and synapses, the cellular mechanisms of hippocampus-dependent associative learning include the remodeling of existing hippocampal synapses; these changes most likely reflect an involvement of signal transduction proteins and the transformation of silent postsynaptic synapses into active ones (Rusakov et al., 1997; Stewart et al., 2000). In light of these robust correlations between neuritic (dendritic spine and synapse) changes and cognitive performance, it seems warranted to conclude that perturbations of the former will result in impaired performance in hippocampus-dependent learning tasks. Most interestingly, although the neuritic atrophy and synaptic loss referred to above would be expected to provoke some degree of functional impairment, together with the fact that these paradigms are not necessarily associated with neuronal cell loss, it seems more than likely that neuronal reorganization (regrowth of dendrites and axons and establishment of new synapses) of damaged neuronal circuits is an important mechanism allowing recovery from insults (McEwen, 1999). The above proposition appears to be valid insofar that studies in rats have shown that, whereas no significant structural reorganization occurs during or immediately after the termination of elevated corticosteroid levels (by pharmacological means or after the imposition of stressors), significant reorganization does occur within one month of withdrawal from the damaging stimulus (Sousa et al., 2000). This so-called "reactive synaptogenesis" occurs throughout the hippocampal formation and is commensurate with restoration of spatial learning and memory to levels found in control animals. Thus, the more recent findings match well with older observations that hypercorticalism-induced cognitive impairment is a reversible phenomenon. Importantly, regeneration of dendritic, axonal, and synaptic elements does not seem to be compromised in conditions when profound neuronal loss has occurred, e.g., in the dentate granule cell layer
after adrenalectomy, a manipulation accompanied by marked collapse of the mossy fiber inputs to the CA3 pyramidal layer. Administration of low doses of corticosterone to adrenalectomized rats can, at least partially, restore the total dendritic length of granule cells and the volume and surface area of the mossy fiber terminals (Sousa et al., 1999a). In addition, substitution therapy with corticosterone results in complete recovery of the volume of the suprapyramidal bundle, number, and surface area of mossy fiber-CA3 synapses, and the surface area of dendritic excrescences (Sousa et al., 1999a). These observations on the fine structural adjustments fit with results of other work showing that behavioral functions impaired by adrenalectomy can be partially reinstated by the administration of corticosterone (McCormick et al., 1997). The evidence summarized above firmly indicates that alterations of the corticosteroid milieu can induce profound, but largely reversible, changes in the ultrastructural organization of the hippocampal formation; these bidirectional alterations, more than changes in neuron viability, may represent the neuroanatomical correlation of hippocampus-dependent learning and memory. Presently, there is no clear data available as to what neurochemical mechanisms might underlie the fine structural observations described above. However, N M D A and serotonin (5-HT) receptors appear to be key players since the administration of either N M D A antagonists or serotonin reuptake inhibitors have been shown to abrogate CA3 dendritic atrophy (Watanabe et al., 1992; Magarinos and McEwen, 1995b). Growth factors also seem to be likely mediators, as suggested by data showing that elevated corticosteroid levels (including those produced in response to stress) attenuate hippocampal brain-derived growth factor (BDNF) and nerve growth factor (NGF) levels (Smith et al., 1995; Hansson et al., 2000) and that adrenalectomy results in significant alterations in the levels of neurotrophin-3 and fibroblast growth factor2 (FGF-2) (Barbany and Persson, 1992; Hansson et al., 2000). Finally, it seems highly plausible that neurotrophins play a major role in the neuritic regrowth seen after recovery from exposure to high corticosteroid levels because the recovery phase is characterized by an increase in their synthesis (Smith et al., 1995).
732 Neuronal death Neuronal death can occur through one of the two basic mechanisms- necrosis or apoptosis (see Fig. 1 and for review Majno and Joris, 1995). Necrosis is the unexpected death of cells resulting from "external damage," usually mediated via destruction of the integrity of plasma membrane and/or the trophic support of the cell. Morphologically, there is lysis of the plasma membrane of the swollen necrotic cell, which leads to release of cytoplasmic components into the surrounding tissue spaces. Inflammatory cells, attracted by the necrotic debris, trigger tissue destruction. Necrosis of isolated cells can occur, although necrosis usually affects large clusters. Consequently, there is significant tissue inflammation (with subsequent repair and scarring), with permanent alteration of architecture and function. Since necrosis usually ensues from cytotoxins, the process is completed rapidly within seconds-to-minutes. Apoptosis differs from necrosis in that it involves the triggering of specific, sequentially occurring, events. Although the term apoptosis was originally coined to describe a specific morphological sequel, it is now known that apoptosis depends on activation of a genomic program; as such, the term apoptosis is
APOPTOSiS
A"
frequently used synonymously with the term programmed cell death (Fig. 2). It should be mentioned that most current methods for the detection of apoptosis can only detect late stages of the process, and that some programmed cell death may not involve the mechanisms of apoptosis (e.g., oncosisthe term oncosis (derived from onkos, meaning swelling) was proposed in 1910 by von Recklinghausen precisely to mean cell death with swelling; oncosis leads to necrosis with karyolysis and stands in contrast to apoptosis, which leads to necrosis with karyorhexis and cell shrinkage). In contrast to necrosis, apoptosis is a much slower process; depending on the initiating stimulus, apoptosis requires from a few hours to several days for its complete manifestation. Conceptually, this form of cell death is analogous to "suicide," inasmuch as death results from the activation of the dying cell's own death machinery. Apoptosis, first recognized by embryologists, has now come to be recognized as being important for maintaining tissue homeostasis and to constitute a major component of many pathological responses, including neurodegenerative diseases. It is important to note that the genetic program for apoptosis can be triggered by both intrinsic (e.g., during histogenesis) and extrinsic
J
Cell shrinkage, membrane blebbing ~ A n n e x i n
NECROSIS Cell swelling
V
+ Hoechst, Acridine orange EB,Pi
N uclear condensation
:~ P--
i
Loss of membrane integrity
/
Karyolysis
s
N uclear fragme ntation
~
Apoptotic bodies
(~)
41- TU NE L I
k
1
Fig. 1. Comparision of morphological changes in apoptosis and necrosis. Apoptosis, characterized by cell shrinkage, membrane blebbing, nuclear condensation, nuclear fragmentation, and apoptotic bodies developed in different stages of injury is shown on the left-side. As described in the main text, apoptotic cells can be identified in a variety of ways, some of which (annexin V-binding, Hoechst, acridine orange, and TUNEL staining) are indicated here. Note that the majority of methods are based primarily on changes in the properties of the cell membrane and nucleus. Necrosis, characterized by cell swelling, loss of membrane integrity, and karyolysis is shown on the right. Membrane-impermeable markers such as ethidium bromide (EB) and propidium iodide (PI) can be used to identify necrosis.
733 Stress/hypercorticalism Cytoplasm ic mem brane
.........
caspasess7ubst ~
......... .,,..
..... .,
"///
Downstream caipases activation Nuclear membrane DNA fragmentation, Chromatin condensation Fig. 2. Signal pathways in apoptosis. The mitochondrion as the integrator of apoptotic signals from stress or other factors such as nitric oxide (NO) or reactive oxygen species (ROS), can release cytochrome c and apoptosis-inducing factor (AIF); cytochrome c, together with Apafl, Caspase9, and ATP activate caspase3, which, in turn, activates downstream caspases for DNA cleavage. Note that Bcl2 from the mitochondrial membrane can prevent mitochondrial pore formation, which antagonizes cytochrome c activity; Bax can increase mitochondrial permeability. Separation of living/apoptotic cells by flow cytometry. For flow cytometric analysis, ethanolfixed cells are washed in phosphate-citrate buffer and stained with propidium iodide. As cells pass in front of a laser, they absorb, diffract, refract and reflect incident light, and emit fluorescence. The scattered light is focused by a lens into a photomultiplier, the emitted fluorescent signal is optically filtered through dichroic mirrors, and subsequently processed by wide bandpass filters selected to optimize the various fluorescent emissions; signals are detected by photomultiplier tubes, and based on fluorescence intensity profiles, living and apoptotic cells can be distinguished.
factors, including stressful stimuli and exogenous corticosteroids, although the intracellular signaling cascades and morphological changes are essentially the same in both situations. As already mentioned, apoptosis is now known to be important during embryogenesis/histogenesis but also in the course of normal tissue turnover. Of course, the mature brain is traditionally not regarded as an organ where cell and tissue turnover occurs, but with the increasing number of reports that, besides glial cells, neurons can also be generated in certain regions, the original concept does not seem to be strictly correct. Furthermore, it is being increasingly recognized that apoptosis makes a significant contribution to neural cell loss in pathological conditions, e.g., in neurodegenerative diseases such as Alzheimer's and Parkinson's disease (Honig and
Rosenberg, 2000; Friedlander, 2003). It is also pertinent to mention that a revisionist view with respect to the distinctive roles and mechanisms of necrosis and apoptosis has emerged since the late 1990s; according to these authors it is now accepted that virtually any insult just below the threshold to induce necrosis results in an apoptotic response (McConkey, 1998). The cellular response becomes relevant in this process in that, in contrast to the situation in necrosis, apoptosis involves active processes within the dying cell and does not merely depend on the insult itself. In contrast to most other cells, neurons have elaborate morphologies with complex neuritic arborizations that often extend long distances from the perikarya. It is in fact the richness of complex contacts between neurons that results in the establish-
734 ment of functional networks. With this concept in mind, it is not difficult to accept that neuronal degeneration does not necessarily imply neuronal death; neuronal atrophy and synaptic loss also represent forms of nervous tissue degeneration. It was recently shown that the biochemical cascades leading to apoptosis can be activated locally in synapses and dendrites (Mattson, 2000), indicating a much more complex role for apoptosis than previously envisaged, i.e., in synaptic loss and dendritic remodeling.
Glial response
Glia mediate neuroendocrine and neuroimmune functions that are altered in the face of a number of neuronal insults, including prolonged stress. The biological functions of glia involve changes in shape, interactions with neurons and other glia, and gene expression. Glia cells become activated in the presence of ongoing neurodegeneration and progress to produce what is termed "reactive gliosis" (Nichols, 1999; Liu and Hong, 2003). Since good markers to distinguish normal from reactive glia are not commonly available, most researchers currently depend on well-defined morphological criteria. In several neurodegenerative conditions, astrocytes exhibit hypertrophy and signs of metabolic activation, and astrocytic processes begin to entwine neurons. Microglia also become activated and subsets of these cells increase in number and may enter the phagocytic or reactive stage. Glial markers of brain aging and glial activation include glial fibrillary acidic protein (GFAP) and transforming growth factor (TGF)-[31, which are increased in astrocytes and microglia, respectively (Nichols, 1999). Interestingly, steroids (Laping et al., 1994), such as those produced in the adrenals (Melcangi et al., 1997), regulate the interactions between glia and neurons, and glial gene expression, including GFAP and TGF-[31. Despite the recognized relevance of the biological functions of glia, little is known about the effect of stress on hippocampal glial cells. Anecdotal evidence suggests an increase in glial cell number and signs of cytoplasmic transformation of astrocytes and microglia in areas of the brain implicated in stress-induced
disorders (namely the prefrontal cortex and the hippocampus) (Ramos-Remus et al., 2002). Based on these findings, it appears that the hippocampal glial response to chronic stress may be similar to that found in endangered or challenged hippocampal environments, such as in ischemia. A different line of evidence on the glial response to imbalances in the corticosteroid milieu has come from studies in surgically lesioned animals (Vijayan and Cotman, 1987). Animals with surgical entorhinal lesions concomitantly treated with hydrocortisone demonstrated more astrocytes and fewer nonastrocytes in the dentate outer molecular layer compared with untreated animals. Glia in the treated animals also showed a decrease in average optical density of cytoplasmic acid phosphatase staining. These findings suggest that hydrocortisone treatment prior to, and following, an entorhinal lesion accelerates lesioninduced migration of astrocytes to the outer molecular layer, and reduces the increase in microglial number resulting from the lesion. The observed effect on microglia may result from direct hormonal inhibition of local proliferation of microglia or from the well-known systemic anti-inflammatory action of glucocorticoids on monocytes, the putative precursors of brain microglia. In light of these findings it has been suggested that glucocorticoid hormones significantly alter the response of nonneuronal cells to neural tissue damage. Lending support to this view is the observation that adrenalectomized animals show induction of GFAP immunoreactivity, which occurs contemporaneously with neurodegeneration (Trejo et al., 1998). Although no variation in the total number of glial cells is found, signs of astroglial activation can be observed in the adrenalectomized group: astroglial cells change in size and shape, and their processes in the molecular layer, which normally show unipolarity become randomly organized (Sousa et al., 1997). Both effects are confined to the dentate gyrus and mossy fiber zone. The degeneration and astroglial reaction become more pronounced with increasing duration after adrenalectomy, and both can be prevented by placing animals on corticosterone replacement therapy. Results such as these illustrate the close relationship between the glial response and neuronal degeneration in the dentate gyrus following adrenalectomy, in terms of both, time and space (Sousa et al., 1997).
735
What are the neural targets of stressmediated degeneration? Corticosteroids are secreted distal to their brain targets but distribution maps of their receptors serve as reliable indicators of their sites of action. In a landmark study on the rat brain, Reul and de Kloet (1985) reported that radioactively labeled corticosterone binds with differing affinities to two distinct receptors, and that the hippocampus showed the highest signal retention for both receptors; subsequent cloning studies revealed significant homologies between the high-affinity and low-affinity central and peripheral corticosteroid receptors: mineralocorticoid (MR) and glucocorticoid (GR) receptors, respectively. In vitro studies showed that the high-affinity binding site in brain can also bind aldosterone; in practice however, the endogenous production of aldosterone only reaches concentrations sufficient to activate renal mineralocorticoid receptors (Funder, 1996); thus, cerebral MR show promiscuity in that, like GR, they bind corticosterone; however, since they have a ca. 10-fold greater affinity for corticosterone as compared to GR, MR are predominantly occupied during periods when corticosteroid levels are low, whereas GR only become occupied when corticosteroid secretion increases above a certain threshold (e.g., during stress or in pathological conditions). Further, the presence of two isoforms of the pre-receptor enzyme l lB-hydroxysteroid dehydrogenase, involved in the interconversion of corticosteroids to active and inactive forms, contribute to the selective access to intracellular receptors (Yau and Seckl, 2001). While GR are widely distributed throughout the brain, but are particularly concentrated in the hippocampus, hypothalamus, and lower brainstem, MR are almost exclusively confined to the hippocampus and other limbic structures such as the septum, central nucleus of the amygdala, the olfactory nucleus, and some hypothalamic nuclei (Van Eekelen et al., 1988; Ahima and Harlan, 1990; Ahima et al., 1991). Within the hippocampal formation, subfield-specific differences in MR and GR concentration profiles have been described: MR levels are high in CA1 pyramidal layer ~ granule cell layer (dentate gyrus) > CA3 pyramidal layer, and GR are concentrated in the CA1 ~ dentate gyrus >>
CA3 (Van Eekelen et al., 1988). The functional significance, if any, of these differential patterns of receptor distribution may be inferred from the known functions of the particular brain nuclei displaying high levels of MR and GR expression and/or ligand binding. At this juncture, it is important to point out that while the described patterns of MR and GR occurrence in the various hippocampal subdivisions may serve as eventual predictors of function, they do not necessarily reflect the receptor repertoire of individual cells in any region; further, it is still not known to what extent receptor composition (concentration of individual receptors or co-localization of MR and GR in the same cell) determines the fate of a particular cell (e.g., survival vs. death) in response to changes in the corticosteroid milieu.
Experimental paradigms for examining stress-mediated degeneration Designing models of stress implies a clear definition of the question under study; more specifically, if one wants to determine the effect of stress upon a specific region of the brain, several issues need to be considered. One of them is adaptation; if a single stressor is applied for a prolonged period, then the organism tends to adapt to that stressor and the stress response gets blunted. A second issue to consider is unpredictability; even when applying different stressors, care must be taken to avoid adaptation, e.g., by applying a battery of stressors at different clock times and in random order. A final point to consider is that stressors vary in quality; for example, physical and psychological stressors activate different regions of the brain, with the former depending on perception by brain stem centers as opposed to the latter, which depends on the activation of higher regions of the brain (in particular the limbic system). Obviously, comparisons between different experimental paradigms (and the results therefrom) must also take into account factors such as intensity and duration/ chronicity. A commonly used approach in evaluating the cellular effects of stress involves decomposition of the effectors of these actions, e.g., by mimicking the endocrine response to stress by administering high
736 doses of corticosteroids, a paradigm that does not exactly reproduce the physical, behavioral/emotional, and neurochemical manifestations of stress. Nevertheless, our current understanding of the actions mediated by the two corticosteroid receptors has largely benefited from the exploitation of the high selectivity of aldosterone (the prototypic MR agonist) and dexamethasone or RU28362 (prototypic GR agonists) as well as the antagonists spironolactone and RU28318 (for blocking MR effects) and RU38486 (for blocking GR effects). Further insights into the biological actions of MR and GR are now being gained from MR and GR gain- and loss-offunction mouse models (Muller et al., 2002). The use of such models has proved particularly useful in proving and understanding the importance and role of these receptors in stress-mediated neuronal damage, and neuronal disorders influenced by stress such as anxiety, depression, and dementia. While in vivo models are necessary for the evaluation of stress effects, in vitro models are indispensable for understanding the cellular and molecular mechanisms underlying those effects. The latter approach is particularly amenable to analysis at the molecular level, but the major caveat here is that in vitro observations do not necessarily apply to the whole organism whose ultimate response to the same stimulus reflects an integration of a plethora of adaptive and signaling pathways emanating from cells with diverse properties, e.g., the liver can substantially influence the response of the brain to endogenous and exogenous stress hormones. As a result, a neurotoxic stimulus in vitro might just happen to be protective or to have no effect in the living organism.
Use of stereology in analyzing neurodegeneration Another extremely relevant issue to consider when designing an experiment is the sensitivity and specificity of the methodological procedures employed to test the hypothesis. Obviously, the analysis of stressinduced neurodegeneration also follows this rule. A common first analytical approach is to make observations on histological sections. Histological sections define the normal appearance of tissue and organs, and detect natural or induced alterations in structure. Histological descriptions often include
terms such as "large," "small," "many," "few," "absent," or "present." Helpful as these terms are for the description of basic features, they are often open to subjectivity and, being qualitative, they do not allow statistical evaluation of the effects of a particular treatment, e.g., stress exposure. Quantitative data can take several forms, but all basically depend on counting cells in a section. One modern approach, superior to previous methods (Abercrombie, 1946; Weibel et al., 1966) is that of stereology, which is given detailed consideration below. Using stereology, one can obtain estimates of object volumes and derive numbers of objects from this data increasing the precision and relevance of data (Gundersen et al., 1988; West, 1999). The principle behind stereology is to recreate or estimate the properties of geometrical objects in space. Its application to tissue or organ sections allows relatively precise estimation of the geometrical properties of the objects in a given section. As space has three dimensions, objects within it have properties for each possible number of dimensions, and objects within a given space can be defined in terms of their volumes (3 dimensions), surfaces (2 dimensions), lengths (1 dimension) and numbers. Each of these properties can be estimated by stereological methods, usually a two-step procedure involving: sampling and subsequently measuring. A characteristic of many tissues and organs is that they contain a large number of the objects of interest, but too many to be measured individually. Producing a good sample is an essential step in stereological methods. Errors incurred during sampling can result in difficulties in obtaining meaningful stereological estimates later. Sampling usually starts before the investigator has any predictions as to the study outcome and even before the investigator has thought about applying stereological methods. To avoid later regrets, it is advisable to sample correctly from the very beginning; however, the researcher can be consoled by the fact that stereology-based sampling methods are compatible with all other types of analysis. One usually wants to make statements about a structure (e.g., the hippocampus) or a cellular population (neurons) by sampling only a part of the structure or population. If such statements are to be valid for the entire structure or population, the sample must be a representative one. Selecting
737 representative samples requires: (i) access to the entire structure or population; (ii) ability to recognize and/or define the entire structure or population; and (iii) that all parts of the structure or population contribute equally to the sample. These pre-requisites can be met by random sampling in one of two ways: (i) Random independent samples- This is the most obvious approach in which one selects an initial location at random. After measuring the objects of interest, subsequent locations for measurement are chosen independent of the first. When a sufficient number of locations has been sampled, the individual measurements are averaged. Despite providing reliable and reproducible estimates, this method is, however, not an efficient sampling procedure. (ii) Uniform random systematic (URS) s a m p l e s - In URS sampling, a random starting point is selected and samples are drawn at regular (or uniform systematic) intervals. Choosing a random starting point means that all areas to be analyzed have an equal chance to contribute to the final sample measure. By eliminating sample clustering, the URS sampling procedure, on average, yields a more accurate estimate than the random independent sampling approach, and is the recommended method of choice. In practice, sections are selected using the URS sampling procedure at the time of tissue sectioning. If, for example, every fifth section is collected, the only requirement is to assure compliance with the need to randomly select the initial section in the series. The next step in stereology is 'measuring' which involves relatively easy, routine work depending on identification of the object of interest and based on a simple set of rules. Curiously, the volume, surface, length, and number of objects are such basic parameters that it may be surprising to realize that methods for their accurate measurement only became available in the 1980s and did not enter widespread use until the 1990s. Stereological tools have now virtually replaced the earlier error-prone methods, which all suffered from the assumption that histological sections are two-dimensional images from which three-dimensional measurements were nevertheless attempted. The traditional methods involved certain well-grounded assumptions about the "missing dimension" in two-dimensional images. Inherently, the proximity to the true values achieved
using such assumption-based methods, depended largely on how good the assumptions were. In modern stereology, design-based methods have replaced assumption-based ones. These newer approaches involve measurements on a series of sections which in fact do have three dimensions. Therefore, information about the third dimension is based on fact, rather than assumption; obtaining precise measurements then depends on one other f a c t o r - the availability of good probes to apply to the sample. The selection of the adequate probe ultimately determines the precision of the estimation (West, 1999).
Estimation of volumes Estimating volumes using points is conceptually the easiest of all stereological methods and was first described by the Italian mathematician Bonaventura Cavalieri (1598-1647). The "Cavalieri Principle" holds that, if one places a grid of regularly spaced points over an object of interest, the measured surface area will be a function of the number of points falling within it. To calculate the volume of an object (in our case, section), one simply has to multiply the average areas of different sections by the thickness of each section. The point-counting principle can, theoretically, also be applied to very small objects like cells, but this would require very thin sections in order to reduce error and the ability to identify the object in consecutive sections. Other approaches, like the n u c l e a t o r - in which a point associated with a small particle (e.g., a nucleolus within a cell) is identified and from which rays are extended until the intersection of particle's boundaries to allow the estimation of its profile area and, subsequently, the absolute volume of the p a r t i c l e have been developed for this purpose (Gundersen et al., 1988).
Estimation of total cell numbers Measuring neuronal loss has preoccupied many neuroscientists interested in the effects of stress and glucocorticoids on the brain, in particular, the hippocampus. Such information can be generated by
738 simply counting the number of cells in a given section and comparing the values obtained with those for sections from an anatomically matched area in control (e.g., nonstressed) subjects. This procedure will yield an estimate of cell number per unit area (NA); to date, this is probably the most widely-used method to count neurons. The precision (relevance) of such an estimate relies entirely on how similar the sections being compared are. A serious (but common) error of such estimates is the "reference trap," which refers to how variation in the volume of reference can affect the final result. This can be illustrated by considering the fact that because the NA of granule cells in the hippocampus in two different sections from different experimental groups is similar, it does not necessarily follow that the total number of cells in each section is the same; this is because the volume (derived from the third dimension, which is not taken into account in deriving the NA value) of the dentate granule cell layer might differ significantly between individual sections and subjects. Design-based methods (e.g., estimating neurons using volumes within a probe) help avoid the introduction of such biases (West, 1999). Essentially, estimating numbers within a volume is just the corollary of estimating volumes with points. One takes two adjacent sections that are thinner than the diameter of the object (e.g., nuclei) to be counted; the objects visible in the second, but not first, section are counted. Then, the number of objects in the volume represented by the two sections will, on average, correspond to the number of objects counted in the second. This approach is called the (physical) dissector because the counting principle is based on a comparison of two sections. Application of this technique provides the numerical density (Nv) of objects (e.g., neurons) within a region of interest. Now, if the total volume of the structure (e.g., hippocampus) is known, the total number of objects (e.g., neurons) can be derived from the product of Nv and the total volume. The optical fractionator is another means for obtaining the total number of objects in a given 3D structure. It is based on the combination of systematic sampling (which yields an estimate of the fraction of the tissue- fractionator) and the dissector in thick optical sections that intrinsically have three dimensions.
Detection of cell death
The application of stereological methods to histological sections (e.g., stained with Nissl, Giemsa) can certainly provide information of neuronal loss based on the comparison of total number of surviving neurons between experimental groups (West et al., 1991; Sousa et al., 1999b). However, this approach can also be applied in combination with markers of neuronal death to directly determine the number of dying cells at a particular time-point. Several specific staining methods for detecting neurodegeneration have been developed but their use has not yet been generalized. The earliest markers of neuronal degeneration were based on silver-impregnation methods that provide unspecific indications of degeneration in neuronal soma and neurites. More recently, the use of two anionic fluorescein derivatives have proved very useful for the simple and definitive localization of neuronal degeneration in brain tissue sections. Initial work on the first generation fluorochrome, Fluoro-Jade, demonstrated the utility of this compound for the detection of neuronal degeneration induced by a variety of well-characterized neurotoxicants, including kainic acid, 3-nitropropionic acid, isoniazid, ibogaine, domoic acid, and high doses ofdizocilpine maleate (MK-801) (Schmued et al., 1997). After validation, the tracer was used to reveal previously unreported sites of neuronal degeneration associated with other neurotoxicants. Preliminary findings with a second-generation fluorescein derivative, Fluoro-Jade B, suggest that this tracer is a specific and selective marker for the identification of neurons undergoing degeneration (both apoptotic and necrotic) (Eyupoglu et al., 2003); Fluoro-Jade B also provides improved staining and can stain the distal portion as well as the proximal portion of the dissected axon (the so-called anterograde and retrograde degeneration after axotomy). Furthermore, FluoroJade tracers can be combined with other histologic methods, including immunofluoresence that can help in discriminating different types of neurodegeneration to obtain information on the neurochemical identity of the affected cells (Schmued and Hopkins, 2000); recent preliminary findings on a number of specialized applications of Fluoro-Jade include the detection of apoptosis, amyloid plaques, astrocytes, and dead cells in tissue culture.
739 An early observation concerning apoptosis was that cells entering apoptosis showed dramatic and characteristic changes in nuclear shape and organization (Fig. 1) (see for review Kerr et al., 1972; Wyllie, 1980; Ucker, 1991). It is still probably correct to say that the characteristic change in nuclear morphology is the most accurate indicator of the involvement of apoptosis in the death of a cell. This is true even in light of the apparently paradoxical observation that nuclear fragmentation per se is not essential for apoptosis; enucleated cells can still undergo other changes characteristic of apoptosis. This unequivocally demonstrates that the effectors of the apoptotic machinery are located in the cytoplasm. However, under normal conditions, changes in nuclear morphology remain an early and relatively unequivocal hallmark of apoptosis, with such changes occurring at an early point in the series of morphological events, usually soon after the onset of surface blebbing. Apoptosis is an ATP (energy)-dependent process (Reed and Green, 2002). Since ATP levels fall to a point where the cell can no longer perform basic metabolic functions, the cell will die. Apoptotic cells exhibit significant reductions in their ATP levels, which can serve as an early marker of cell death. Depletion of energy pools is, however, not specific to apoptosis. Either exposure to toxic agents (secondary necrosis) or metabolic damage (primary necrosis) can also induce drops in ATP levels, albeit rapid ones (Leist et al., 1999), followed by necrotic cell death. The change in both ATP and ADP levels (ADP/ATP ratio) has been used to differentiate apoptosis from necrosis (Bradbury et al., 2000). In contrast, cell proliferation and growth arrest can both be recognized by increased levels of ATP and decreased levels of ADP. Determination of the ADP/ATP ratio offers highly consistent results and with excellent correlation to other markers of apoptosis (e.g., TUNEL-based techniques and caspase assays) (Bradbury et al., 2000). c-Jun N-terminal kinase (JNK) is one of the main MAP kinase groups identified in mammals. Recent evidence suggests that activation of JNK plays an important role in neuronal apoptosis and other physiological and pathological processes (Ham et al., 2000). For measuring JNK activity easily in a large number of samples, one can use an assay that utilizes
an N-terminal c-Jun fusion protein bead to selectively "pull down" JNK from cell lysate; c-Jun phosphorylation is then measured using a phospho-c-Junspecific antibody. Alternatively, one might analyze JNK-specific activity by determining the phosphorylation of c-Jun by Western blotting using a phospho-c-Jun-specific antibody. Given the involvement of JNK in signaling pathways, which may not be directly related to apoptosis, care needs to be exercised in interpreting results obtained with such methods. One of the first questions to resolve whenever searching for neurodegeneration, whether necrotic or apoptotic, is the ability to distinguish if the cells undergoing degeneration are neurons or glial cells. For this, immunohistochemistry is the most convenient and commonly used approach. Using specific antibodies for each cell population, one can easily identify the lineage of dying cells. Numerous neural cell type-specific (neurons, astroglia, oligodendrocytes, etc.) markers are currently available. For example, one may use antiGFAP to label astrocytes, antidoublecortin to identify neuroblasts (stem cells), antiNeuN to mark mature, differentiated neurons, or antiTuJ1 to study fibers. As mentioned already, apoptosis is a genetically programmed phenomenon. A complex network of genes (Steller, 1995; Lossi and Merighi, 2003), in particular encoding members of the Bcl-2 family of proteins, play a central role in the regulation of apoptosis. Here, we focus on Bcl-2 family members as these have received most attention in the context of this article. The Bcl-2 family of proteins comprises death-inducer (proapoptotic) molecules such as Bax and Bcl-xs and death-repressor (antiapoptotic) molecules such as Bcl-2 and Bcl-xL. These various proteins, which can homo- or heterodimerize with each other, are activated by physiological or injurious stimuli, and appear to operate upstream of events leading to the final execution phase of the apoptotic process; the latter results from the activation of cysteine proteases, the caspases. Caspases convey the apoptotic signal in a proteolytic cascade, with caspases cleaving and activating other caspases that then degrade other cellular targets that lead to cell death (Friedlander, 2003). Caspase activation can directly initiate the permeability transition of the mitochondrial
740 membrane, resulting in the release of several mitochondrial proteins (see Fig. 2 for a simplified scheme). The large number of products developed to study caspases and their substrates is indirect testimony to their importance; because of space constraints, the authors here only review a few of these. Caspase-3 is a key protease that becomes activated during the early stages of apoptosis. Synthesized as an inactive proenzyme, the activated form cleaves and activates other caspases, in addition to cleaving specific targets in the cytoplasm and nucleus (e.g., DNA and nuclear membrane fragmentation). Once activated, caspase-3 serves as a marker for cells undergoing apoptosis. Several biotin- or FITC-tagged anti-active caspase 3 antibodies are available, facilitating their routine use (Gown and Willingham, 2002). Because caspase activity is likely to be the most specific indicator of the apoptotic process, the assay of caspase activity through the detection of specific cleavage products in target proteins represents a theoretically valid approach for measuring apoptosis. Recently, antibodies to the caspase-generated cleavage products of cytokeratin 18 have appeared on the market, with several studies demonstrating their utility, especially in cell culture, but probably also in fixed tissue sections (Leers et al., 1999). However, cytokeratin 18 is expressed only in certain cell types and this antibody is not broadly applicable to all cell types. The use of antibodies specific for more generally distributed cleaved substrates of caspases, such as the cleaved form of caspase 3, would have more general applicability (Srinivasan et al., 1998). Owing to their cell-permeable nature, a new line of cell-permeable fluorogenic caspase substrates enables the visualization of intracellular protease activities by standard fluorescence microscopy or multiparameter flow cytometry (see below) in living cells. The substrates, designed for caspase-1, caspase-6, caspase-8 (the caspase-3 processing enzyme), and caspase-9, detect early events in the apoptotic pathway before DNA degradation has started (Davis et al., 1998; Komoriya et al., 2000). Recently, these caspase substrates have been used to demonstrate that the pattern of caspase activation is not only dependent on the apoptosis-inducing agent employed, but also on the cell type (Komoriya et al., 2000). Events occurring downstream of caspase-3 activation include cleavage of poly(ADP-ribose)
polymerase (PARP), an enzyme implicated in DNA damage and repair mechanisms. Cleavage of PARP from the native 116kDa to 85kDa is considered a hallmark of apoptosis (Sallmann et al., 1997). The availability of FITC-tagged anti-PARP antibodies therefore, provide another useful marker of apoptosis. In healthy cells, cytochrome c is located in the space between the inner and outer mitochondrial membranes. An apoptotic stimulus triggers the release of cytochrome c from the mitochondria into the cytosol where it binds to Apaf-1. The cytochrome c/Apaf-1 complex activates caspase-9, which then activates caspase-3 and other downstream caspases. Cytochrome c released from the mitochondria into the cytosol can be detected by Western blotting using antibodies directed against cytochrome c. The procedure is simple, straightforward, and provides an effective means for detecting cytochrome c translocation from mitochondria into cytosol during apoptosis (Jemmerson et al., 1999). As already alluded to, Bcl-2 family proteins form complexes, these complexes can enter the mitochondrial membrane where they regulate the release of cytochrome c and other proteins. When Bax, for example, localizes to the mitochondrial membrane, it acts to increase mitochondrial permeability, induces the release of cytochrome c and other mitochondrial proteins, leading to apoptosis ultimately. In contrast, Bcl-2 and Bcl-XL prevent mitochondrial pore formation and therefore, block apoptosis. Antibodies (applications include immunocytochemistry, Western blotting) and gene probes (for Northern blotting, in situ hybridization histochemistry, and polymerase chain reaction analysis) are now available for measuring most key members of the Bcl-2 family in a variety of species, including humans, rats, and mice. Such studies have shown that Bcl-2 levels in the brain decline rapidly after birth, except for those areas displaying postnatal neurogenesis such as the dentate gyrus of the hippocampus. Further, numerous studies have shown that Bcl-2 expression can be induced in the adult brain, including the hippocampus, upon experience of various noxious stimuli. Unlike that of Bcl-2, the expression of Bcl-XL occurs in neurons from early development through to senescence. The proapoptotic protein Bax is expressed through all life stages whereas the smaller proapoptotic splice
741 variant of Bcl-2, Bcl-Xs is only barely detectable in the mature brain. To date, there is no evidence that corticosteroids, which represent the endocrine response to stress, can directly regulate or interact with any members of the bcl-2 gene family. Rather, corticosteroids appear to influence the pro- and antiapoptotic gene expression and activity by interacting with p53, a ubiquitously distributed tumor suppressor protein, which has been shown to induce and repress the transcription of bax and bcl-2; glucocorticoids were recently shown to enhance the transactivation potential of p53 (Crochemore et al., 2002). Although measurements of gene or protein expression of Bcl-2 family members might be reasonably expected to correlate with apoptosis, recent studies have shown that absolute levels of these molecules do not reflect the actual viability of neurons in situ. Rather, the ratio of expression of pro-apoptotic (e.g., Bax) to antiapoptotic (e.g., Bcl-2) molecules factor has proven to be the factor determining neuronal survival (Almeida et al., 2000) insofar that this derivative correlates with the incidence of apoptosis measured by histochemical techniques such as T U N E L (see below). Disruption of the mitochondrial transmembrane potential is one of the earliest events after apoptosis induction. Normally, cellular energy generated by mitochondrial respiration accumulates in the transmembrane space as an electron gradient called the mitochondrial transmembrane potential A~m. Disruption of the mitochondrial transmembrane potential occurs following the onset of apoptosis. Using a fluorescent lipophilic cation as a mitochondrial activity marker, one can measure differences in the fluorescence displayed by healthy cells versus apoptotic cells: in healthy cells, the dye accumulates and aggregates in the mitochondria, producing a bright red fluorescence, while in apoptotic cells the fluorescence cation cannot aggregate in the mitochondria because of the altered transmembrane potential, thus remaining in its monomeric (green fluorescent) form within the cytoplasm. These fluorescent signals are analyzed by flow cytometry using the FITC channel for green monomers and the propidium iodide (PI) channel for red aggregates. Additionally, apoptotic and healthy cells can be viewed simultaneously by fluorescence microscopy using a wide-band pass filter. Some kits combine
detection of disrupted mitochondrial transmembrane potential with changes in the composition of the plasma membrane. Flow cytometry can be summarized as a method for measuring physical and biochemical features of cell components on a cell-by-cell basis, primarily by optical means. Fluorescent dyes or fluorophoreconjugated antibodies are used to report the quantities of specific cellular components, density of cellular markers and r e c e p t o r s - or even activation state of various enzymes. Put simpler, flow cytometers are highly sophisticated fluorescence microscopes, where fixed or living cells are not attached to a well-defined surface, but rather travel one by one, by continuous flow of a stream of the suspension past a sensor. Each cell scatters some of the excitation laser light, and the labeled cells emit fluorescent signals from the dye. These two parameters are sensed by photodetectors, data are collected, and processed by a computer. The term 'FACS' is Becton-Dickinson's registered trademark and is an acronym for "FluorescenceActivated Cell Sorter." FACS is therefore, a machine that can rapidly separate cells in a suspension, based on the size and color of their fluorescence. (Note: not all flow cytometers are necessarily able to separate cells into different vials, but all can analyze the distribution of cell size and/or physical or biochemical cellular properties). These particular features of flow cytometric methods allow the identification and quantification of apoptotic cells as well as possible mechanisms of cell death. The main flow cytometric approaches that can be used to identify apoptotic cells may be summarized as follows: (1) Apoptosis-associated changes in cell size and granularity can be detected by analysis of laser light scattered by the cell. (2) Using annexin V in combination with propidium iodide (PI), it is possible to differentiate between healthy, early apoptotic, and necrotic cells on the basis of the distribution of plasma membrane phospholipids as well as changes in membrane integrity. (3) Fluorochromes like Rhodamine 123 (Rhod123) or 3,3'-dihexiloxadicarbocyanine (DiDOC6) reveal decreases in the mitochondrial transmembrane potential (A~m) that occurs early during apoptosis. (4) Apoptotic cells can be recognized by their fractional DNA content, or by the presence of DNA strand breaks using fluorochrome-labeled nucleotides attached to the 3'-OH
742 termini in a reaction catalyzed by exogenous terminal deoxynucleotidyl transferase (TdT). As regards the identification of putative mechanisms of cell death, after labeling with primary and fluorescent secondary antibodies, one can detect and measure: (a) cellular levels of death-related proteins (members of Bcl-2 family, proto-oncogenes like c-myc and ras, tumorsuppressor genes such as p53, etc.) or (b) study particular cell functions, such as mitochondrial metabolism, in the context of cell sensitivity to apoptosis. The main virtue of flow cytometry lies in the possibility of multiparametric, correlated analysis of a multitude of cell attributes and markers, thus addressing problems of cellular heterogeneity. Flow cytometry also provides more effective data acquisition as compared to fluorescence microscopy (which has similar capabilities, but in which the sample size is limited up to a few hundred cells. Flow cytometry can easily measure 10,000-100,000 cells per sample!). There are, however, certain difficulties associated with this technique, which have to be taken into consideration when using flow cytometry in general. With respect to cell death detection, a major problem is that the single parameter on which the identification of apoptotic or necrotic cells relies on, may be absent, when apoptosis is atypical. Moreover, in the case of nonfixed, living cells, the dissociation procedure may damage the plasma membrane, resulting in PI (a widely used cell viability dye) to enter and label the cells as if they were dead. Clumping of cells may also pose technical difficulties. Since high-speed FACS machines use high pressure to achieve rapid acquisition rate, limitations such as cell type and viability must also be considered. Externalization of phosphatidylserine (PS) and phosphatidylethanolamine are hallmarks of changes in the cell surface during apoptosis. Annexin V binds to PS with strong avidity and can be used as a marker of PS externalization using either microscopy or flow cytometry (when fluorescent-labeled annexin V is applied). Importantly, annexin V-binding cannot be applied to tissue sections or adherent cells, and when flow cytometric analysis is used on cell suspensions. PS are phospholipids only present on the cytoplasmic face of the plasma membrane and other internal membranes, and it remains unclear as to why certain
subpopulations of cells (< 30%) entering apoptosis externalize PS at a very early stage of the process, just after the fragmentation of the nucleus begins. However, since inhibition of caspase activity blocks PS externalization, a role for caspases is indicated. It is important to keep in mind that, during necrosis or the terminal lytic steps of apoptosis, PS that are actually localized on the inner face of the membrane might be accessed by annexin V, giving rise to false positives. When combined with a vital dye such as the red fluorescent DNA-binding compound PI, FITCannexin V labeling can be used to distinguish necrotic from apoptotic cells; this is because PI does not penetrate live cell membranes or cells in the early phases of apoptosis but only cells that have lost membrane integrity as a result of necrosis or very late apoptosis. The permeability of the plasma membrane is substantially different in necrotic and apoptotic cells, a fact that can be taken advantage of in the distinction between these forms of cell death. Thus, it is possible to distinguish between stages of apoptosis mainly on the basis of the plasma membrane permeability changes. Large-molecular weight DNA-binding dyes, such as PI or the homodimer of ethidium bromide (EB), cannot enter intact cells because of their large size and, without permeabilization treatments, do not label apoptotic cells until the final stage of cell lysis. On the other hand, in ethanolfixed cells, which have been subsequently washed in phosphate-citrate buffer, extraction of the lowmolecular weight DNA from apoptotic cells takes place, and apoptotic cells appear to the left of the normal G1 peak after PI staining (Fig. 3). This is a fast, simple, but not very specific, method for detecting apoptosis, and has the disadvantage that apoptosis of cells in late S phase or from G2 may be missed. Smaller dyes that can attach to DNA (such as DAPI, Hoechst 33342 or 33258), are furthermore able to enter, and differentially label apoptotic and healthy cells based on the condensation and subsequent fragmentation of the chromatin, which occurs early during apoptosis. Using flow cytometry, for instance, one can distinguish between healthy, apoptotic and dead cells by the simultaneous use of the blue-fluorescent Hoechst 33342 dye (which stains the condensed chromatin of apoptotic cells more brightly than that of normal cells) and PI, which
743
iiiiiiiiiiiiiiiiiiiii:~ii~i~iiii~iii~ii~i~.
!iiii!iii!!!ii!i!i!il
Low
Scatter and high ~g, le
!~!!!i!~!!!!!!!, ~ !,!,!
//I~ce~
iiiiiiiiiiii!iiiiiiiiiiii!ili!iii~i~Ii~i~i~i~i~i~i~
ii'!i!i!ii!il~ilil!il~ilil
i~i~i~i~i~i~i~i~ii~i~i~i~iiii~i~i~i~i~i~i~iii~iii~i~i~i~~ri~i~i~i~i~i~i~i~l
~!.~.i.i.~.i.!ii!iil
~ii!iii~iiii!iiiii!!ii!iii!iiii!i!ii!ii!i!i!i!i!i!i!i!i!iii!~!i!!!~
~i~i~!~i~!~i~
................................................................................... ..............................................................................
iiiiiiiiiiiiiiiiiii!ii
i•!!i•!i!•!i!i•!ii•!i!i!•!i!!!i!ii!•!i!i!i!i!iii!ii!i!i!iii!ii!i!i!i!i!i!i!ii!ii!iiiii!iii!iii
i!iiiiii!iiiii!!ii!i! ........ '
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . :~::~:~:~:~:~:~:~:~:~:~:~:~:~:~:~:~:~:~:~:i:~:~:~:~:~:~:~:~:~:~:~:~:~:~:~:~:~:~:~:~:~:~:'
iiiiiiiiiii .....................
iiiiii!iiiii!iiiiii!i~
i~i~i~!ii~iiii!i~ii~i~ii~ii~i~i~i~ii~ii~i!~i~i~i~iii~ii~[ii~i~ii~i~i~!~
iiiiiiiiii!
i~i~i!i~i~i~i~i~i~i!i~ii~i~ii~i~i~i!ii~i~i~i~i~iii~ii~i~i~i~i~!ii~i~i!i!i~i~
~,~,~,~,~,~,~,~,~,
!iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii
iiiiii!i
Coil ection lenses
Dichroic mirrors
Laser(s)
i
Cell ~
t:I~otomultiplier tubes
_+
Fig. 3. Separation of living/apoptotic cells by flow cytometry. For flow cytometric analysis, ethanol-fixed cells are washed in phosphate-citrate buffer and stained with propidium iodide. As cells pass in front of a laser, they absorb, diffract, refract, and reflect incident light, and emit fluorescence. The scattered light is focused by a lens into a photomultiplier, the emitted fluorescent signal is optically filtered through dichroic mirrors, and subsequently processed by wide-band pass filters selected to optimize the various fluorescent emissions; signals are detected by photomultiplier tubes, and based on fluorescence intensity profiles, living and apoptotic cells can be distinguished.
labels dead cells (Pollack and Ciancio, 1991). Acridine orange (AO) (another cell-permeant nucleic acid-binding dye that emits green fluorescence when bound to double-stranded DNA, and red fluorescence when bound to single-stranded DNA or RNA) is another useful probe for identifying apoptotic cells, because its metachromatic fluorescence is sensitive to DNA conformation. Careful combination of fluorescent dyes, furthermore, allows even more accurate determination of different stages of the apoptotic process: for example, 7-aminoactinomycin D (7-AAD) can be used alone or in combination with Hoechst 33342 to separate populations of live, early apoptotic, and late apoptotic, cells
(Schmid et al., 1994). In mixed cell populations, however, identification of cell types is necessary, and has also to be taken into consideration. For instance, the combination of acridine orange and ethidium bromide (AO/EB) is useful to accurately differentiate between healthy, early apoptotic, late apoptotic, and necrotic cells (Liegler et al., 1995), but cannot be used for phenotypic analyses due to the broad emission spectrum of AO and EB. Certain techniques, like DNA strand-break labeling by terminal deoxynucleotidyl transferase (TdT) can overcome this problem, but are technically very demanding. TdT adds biotinylated or digoxigenin-labeled nucleotides to the strand-breaks in the DNA of apoptotic
744 c e l l s - apoptotic cells therefore, can be detected by using fluorochrome-labeled streptavidin conjugates or fluorochrome-labeled anti-digoxygenin antibodies in flow cytometric analysis. By combining this procedure with phenotypic markers tagged to other dyes, it is even possible to obtain cell cycle profiles in cells of a given phenotype (Li et al., 1996). As mentioned earlier, changes in plasma membrane permeability are signs of late phases of cell lysis. Changes in mitochondrial membrane permeability, however, occur much earlier during apoptosis, and are considered to be a distinctive feature of early programmed cell death. The mitochondrial permeability transition (MPT) is intimately linked to the opening of a "megachannel," the permeability transition pore (PTP). Ionic equilibration through the PTP results in disruption of the mitochondrial transmembrane potential (A~m), uncoupling of the respiratory chain, and release of cytochrome c into the cytoplasm. Of all these features, changes in the mitochondrial permeability can be relatively easily followed by application of fluorescent dyes (e.g., DiOC6), while the subsequent ionic and electrical fluctuations can be investigated by patch-clamp techniques or certain fluorophores. While certain drugs, like the green-fluorescent calcein (which is produced from the nonfluorescent calcein-AM form within the cell itself) are used to indicate PTP opening, and, subsequently, the taking up of the dye into the mitochondrial matrix, others (like JC-1, JC-9, or DiOC6) do not just simply accumulate in the mitochondria, but also indicate changes in Aq/m in single-cell imaging or flow cytometric assays. Other dyes, like MitoTracker | Red CMXRos can be fixed by aldehyde-based fixatives and can thus be used for other subsequent analytical procedures such as immunocytochemistry, DNA end-labeling, in situ hybridization, or counterstaining. Ionic concentrations in the mitochondria can be monitored using patch-clamp techniques or fluorescent dyes like the CaZ+-sensor Rhod-2. Loss of DNA integrity is characteristic of apoptosis (Collins et al., 1997). When DNA extracted from apoptotic cells is analyzed using gel electrophoresis, a characteristic internucleosomal "ladder" of DNA fragments (typically, 180-200 bp in length) is revealed (Compton and Cidlowski, 1986; Walker
et al., 1999); larger DNA fragments have also been seen at earlier stages in apoptotic cell cultures. Although these electrophoretic methods are commonly used in apoptosis detection, the results they provide can present interpretational difficulties. Also, these methods cannot be easily applied, requiring extraction of DNA from large numbers of cells undergoing apoptosis in a relatively synchronous way; however, such synchrony is not always present, especially in tissues (Collins et al., 1997), and as noted above, apoptosis is a relatively rare event, occurring in only a subset of cells within a given structure, thus raising problems of sensitivity. A widely used method that has contributed much to our knowledge of stress- and corticosteroidinduced apoptosis in the brain is also based on the detection of DNA strand-breaks. This approach detects 3'-OH ends of single-stranded DNA after the addition of labeled nucleotides to the open ends of DNA in a procedure known as in situ end-labeling (ISEL). The latter may be achieved using either E. coli polymerase (or its Klenow fragment) in a method called in situ nick-translation (ISNT), or terminal transferase in a method referred to as terminal deoxynucleotidyltransferase-mediated dUTP nickend labeling (TUNEL) (Modak and Bollum, 1972; Gavrieli et al., 1992; Jin et al., 1999). These methods allow the cytochemical demonstration of free DNA strand openings. TUNEL staining is now widely used for the detection of apoptotic cells in tissue sections and cells in culture. Despite its apparent simplicity, unless used optimally, this technique may lack sensitivity and, worse, specificity. For example, TUNEL can reportedly label both apoptotic and necrotic cells, and potentially, proliferating cells also, although these problems are less-frequently encountered in tissue sections than when cultured cells are stained. Moreover, as already noted in the main text, apoptotic cells can be easily recognized on the basis of their unambiguous morphological characteristics. With regard to mitotic cells, it deserves mentioning that although chromatin condensation at telophase may mimic apoptosis, the greatest analogy between mitotic and apoptotic aspects occurs in abortive mitosis, a form of cell division that leads to active cell death (sometimes named "mitotic catastrophe").
745 The major problems associated with the TUNEL technique, especially in tissues, can be summarized as follows: (i) without pretreatment, TUNEL sensitivity is poor and can lead to false negatives; (ii) established pretreatments (proteinase K, microwaves) can easily result in labeling of morphologically normal nuclei; and (iii) the method depends on good fixation and can prove problematic when large tissue blocks are used (outside-inside gradients of penetration of fixative). Other considerations include: (i) the DNA breaks, which are targeted by TUNEL, are less accessible than intact DNA; (ii) besides apoptosis, DNA recombination, replication, repair or compaction-relaxation during mitosis, tissue electrocoagulation, autolysis, fixation, paraffin embedding, cutting, and pretreatments with H202, detergent, proteinase K, and microwaves can all result in DNA breaks; and (iii) DNA compaction (a hallmark of apoptosis) and protein cross-linking and precipitation induced by fixation can mask the 3-OH recessed ends. Despite the above caveats, TUNEL is still regarded as a reliable marker of the DNA fragmentation, which typically occurs in apoptosis. The key to distinguishing between apoptotic and nonapoptotic DNA is the cautious use of "break disclosure" reagents(detergents, proteases, microwaves). Extensive tests have led some authors to propose that optimal staining results from qualitative adaptations of
Table 1. Dyes commonly used for quantifying apoptosis by flow cytometry Marker dye
MW
Absorption Emission max. max.
DAPI 350.25 358 461 PI 668.4 535 617 DiOC6 572.73 484 501 Rhod 123 380.83 507 529 JC-1 652.23 514 529 Annexin V Depends on fluorescent co~ugate conjugates AO 301.82 500 526 7-AAD 1270 546 647 MitoTracker 531.52 578 599 Red| CMXRos Rhod-2, AM 1123.96 550 571 Hoechst 33342, 33258 623.96 352 461
retrieval techniques rather than retrieval reinforcement; for example, quite different pHs are necessary to obtain specific labeling in formalin- versus Bouin-fixed tissues. When fixation is controlled (e.g., homogeneous and light) as is usually the case in prospective studies, proteinase K alone may be sufficient for all cross-linking aldehyde fixatives (paraformaldehyde, formalin, B5). Proteinase K and microwave treatment may be necessary when tissues are fixed for too long and/or in precipitating solutions (Bouin's). Nonspecific (background) staining can also present a problem, even when optimal pretreatments are applied. This can be overcome by optimizing the detection system, e.g., dilution of the enzyme-coupled antibody, choice of enzyme, careful monitoring of color development. Absence of standardization of color reaction implies suboptimal quantification of those cells which might otherwise show morphological signs of apoptosis. Also to be remembered is that all labeled cells, irrespective of intensity of labeling, should to be counted as long as they show morphological features of apoptosis. Another method for detecting these single-strand ends is the use of monoclonal antibody reactive with single-stranded DNA (Naruse et al., 1994; Frankfurt et al., 1996). Since preservation/fixation procedures can have dramatic effects on the detection of singlestranded DNA (Labat-Moleur et al., 1998; Tateyama et al., 1998), careful consideration must be given to this issue and optimized for each cell type or tissue. The investigator should also keep in mind that in cases of overfixation, for example, open DNA strands will be inaccessible to assay reagents (Nakamura et al., 1997). This can be overcome by introducing protease treatments prior to ISNT or TUNEL procedures. Proteases must be used c a u t i o u s l y protease treatments can mimic the actions of endogenous caspases, thus leading to artefactual DNA strand-breaks. Here, it is also important to note that, depending on permeabilization and fixation protocols, some methods detect so-called preapoptotic nuclei in which strand breaks are detected in the absence of apoptosis-like changes in the morphology of the nucleus. Alternatively, positively labeled strand-breaks may not correlate with nuclear fragmentation in individual cells, or DNA strand-breaks may only become detectable
746 at relatively late stages of the apoptotic process (Collins et al., 1997). Recently, a number of authors have indicated reservations about the use of the TUNEL and ISNT assays for detecting apoptosis. It has become apparent that single-stranded DNA ends are not necessarily specific for apoptosis since they may also occur in necrotic cells (Kockx et al., 1998; Mizoguchi et al., 1998). Therefore, although these methods have been, and remain, very useful (their major advantage being that they can be applied directly to intact tissue sections, thus providing good anatomical resolution), the results they yield must be treated with extreme care; for example, in our studies (e.g. Hassan et al., 1996), we only consider TUNEL-positive cells as apoptotic if they simultaneously display the typical morphological characteristics of apoptotic cells; positively stained cells, which have a clearly defined nucleus and cell body are excluded, as are cell fragments and endothelial cells; further stringency is added by ensuring that the person performing the cell counts is unaware of the treatments.
Concluding remarks The main objective of this article was to provide a brief overview of the methodologies available to study the cellular and molecular basis of stressinduced neurodegeneration. While our coverage is by no means exhaustive, we aimed to review each of the major approaches in current use, both in brain tissue and cell culture, and to discuss each of the methods in terms of their advantages and inherent drawbacks; it should become obvious to the reader that no single method can be considered to be definitive by itself, and investigators are encouraged to confirm results obtained one method with that from an alternative technique whenever feasible, in order to avoid from misinterpretation of results. We also attempted to discuss certain important aspects of experimental design in the hope that the use of standardized procedures will contribute to our increased understanding of stress-induced neuronal damage.
List of abbreviations 5-HT
serotonin
7-ADD AIF AO APAF-1 BDNF CA DAPI DiDOC6 EB FACS FGF-2 FITC GFAP GR ISEL ISNT JC-1
7-aminoactinomycin D apoptosis-inducing factor acridine orange apoptotic protease activating factor 1 brain-derived nerve factor field of hippocampus 4'-6-diamidino-2-phenylindole 3,3'-dihexiloxa-dicarbocyanine ethidium bromide fluorescence-activated cell sorter fibroblast growth factor fluorescein isothiocyanate glial fibrillary acidic protein glucocorticoid receptor in situ end-labeling in situ nick-translation 5,5', 6,6'-tetra-chloro- 1,1 ',3,3'-tetraethyl benzimidazolyl-carbocyanine iodide JNK c-Jun N-terminal kinase MK-801 dizocilpine maleate MPT mitochondrial permeability transition MR mineralocorticoid receptor number per unit area NA NeuN neuronal-specific nuclear protein NGF nerve growth factor NMDA N-methyl-D-aspartic acid NO nitric oxide number per unit volume Nv PARP poly(ADP-ribose) polymerase PI propidium iodide PS phosphatidylserine permeability transition pore PTP rhodamine Rhod reactive oxygen species ROS terminal deoxynucleotidyl transferase TdT transforming growth factor TGF neuron-specific class III beta-tubulin TuJ1 TUNEL Terminal deoxynucleotidyl transferasemediated dUTP nick end-labeling uniform random systematic URS mitochondrial transmembrane potential AqJ m
Acknowledgements This article was written under the auspices of the German-Portuguese cooperation - Gabinete de Relaq6es Internacionais da Ci6ncia e do Ensino
747 S u p e r i o r ( G R I C E S ) and the G e r m a n A c a d e m i c E x c h a n g e Service ( D A A D ) . Zs. N 6 m e t h y was supp o r t e d by M a r i e Curie I n d i v d u a l F e l l o w s h i p f r o m the European Commission (QLK6-CT-2001-51072). M a n y of the m e t h o d s described here were established in the a u t h o r s ' l a b o r a t o r i e s by p a s t and present colleagues w h o are duly t h a n k e d .
References Abercrombie, M. (1946) Estimation of nuclear population from microtome sections. Anat. Rec., 94: 239-247. Ahima, R.S. and Harlan, R.E. (1990) Charting of type II glucocorticoid receptor-like immunoreactivity in the rat central nervous system. Neuroscience, 39: 579-604. Ahima, R., Krozowski, Z. and Harlan, R. (1991) Type I corticosteroid receptor-like immunoreactivity in the rat CNS: distribution and regulation by corticosteroids. J. Comp. Neurol., 313: 522-538. Almeida, O.F.X., Conde, G.L., Crochemore, C., Demeneix, B.A., Fischer, D., Hassan, A.H., Meyer, M., Holsboer, F. and Michaelidis, T.M. (2000) Subtle shifts in the ratio between pro- and antiapoptotic molecules after activation of corticosteroid receptors decide neuronal fate. FASEB J., 14: 779-790. Barbany, G. and Persson, H. (1992) Regulation of Neurotrophin mRNA expression in the rat brain by glucocorticoids. Eur. J. Neurosci., 4: 396-403. Bradbury, D.A., Simmons, T.D., Slater, K.J. and Crouch, S.P. (2000) Measurement of the ADP:ATP ratio in human leukaemic cell lines can be used as an indicator of cell viability, necrosis and apoptosis. J. Immunol. Methods, 240: 79-92. Cajal, R. (1893) Neue darstellung vom histologischen bau des centralnervensystem. Arch. Anat. Physiol., 319: 428. Collins, J.A., Schandi, C.A., Young, K.K., Vesely, J. and Willingham, M.C. (1997) Major DNA fragmentation is a late event in apoptosis. J. Histochem. Cytochem., 45: 923-934. Compton, M.M. and Cidlowski, J.A. (1986) Rapid in vivo effects of glucocorticoids on the integrity of rat lymphocyte genomic deoxyribonucleic acid. Endocrinology, 118: 38-45. Crochemore, C., Michaelidis, T.M., Fischer, D., Loeffler, J.P. and Almeida, O.F.X. (2002) Enhancement of p53 activity and inhibition of neural cell proliferation by glucocorticoid receptor activation. FASEB J., 16: 761-770. Davis, R.E., Mysore, V., Browning, J.C., Hsieh, J.C., Lu, Q.A. and Katsikis, P.D. (1998) In situ staining for poly(ADPribose) polymerase activity using an NAD analogue. J. Histochem. Cytochem., 46: 1279-1289. Erickson, K., Drevets, W. and Schulkin, J. (2003) Glucocorticoid regulation of diverse cognitive functions in
normal and pathological emotional states. Neurosci. Biobehav. Rev., 27: 233-246. Eyupoglu, I.Y., Savaskan, N.E., Brauer, A.U., Nitsch, R. and Heimrich, B. (2003) Identification of neuronal cell death in a model of degeneration in the hippocampus. Brain Res. Protoc., 11: 1-8. Frankfurt, O.S., Robb, J.A., Sugarbaker, E.V. and Villa, L. (1996) Monoclonal antibody to single-stranded DNA is a specific and sensitive cellular marker of apoptosis. Exp. Cell Res., 226: 387-397. Friedlander, R.M. (2003) Apoptosis and caspases in neurodegenerative diseases. New Engl. J. Med., 348: 1365-1375. Funder, J.W. (1996) Mineralocorticoid receptors in the central nervous system. J. Steroid Biochem. Mol. Biol., 56: 179-183. Gavrieli, Y., Sherman, Y. and Ben-Sasson, S.A. (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol., 119: 493-501. Gown, A.M. and Willingham, M.C. (2002) Improved detection of apoptotic cells in archival paraffin sections: immunohistochemistry using antibodies to cleaved caspase 3. J. Histochem. Cytochem., 50: 449-454. Gundersen, H.J.G., Bagger, P., Bendtsen, T.F., Evans, S.M., Korbo, L., Marcussen, N., Moller, A., Nielsen, K., Nyengaard, J.R., Pakkenberg, B., Sorensen, F.B., Vesterby, A. and West, M.J. (1988) The new stereological tools: disector, fractionator, nucleator and point sampled intercepts and their use in pathological research and diagnosis. APMIS, 96: 379-394. Ham, J., Eilers, A., Whitfield, J., Neame, S.J. and Shah, B. (2000) c-Jun and the transcriptional control of neuronal apoptosis. Biochem. Pharmacol., 60:1015-1021. Hansson, A.C., Cintra, A., Belluardo, N., Sommer, W., Bhatnagar, M., Bader, M., Ganten, D. and Fuxe, K. (2000) Gluco- and mineralocorticoid receptor-mediated regulation of neurotrophic factor gene expression in the dorsal hippocampus and the neocortex of the rat. Eur. J. Neurosci., 12:2918-2934. Hassan, A.H., von Rosenstiel, P., Patchev, V.K., Holsboer, F. and Almeida, O.F.X. (1996) Exacerbation of apoptosis in the dentate gyrus of the aged rat by dexamethasone and the protective role of corticosterone. Exp. Neurol., 140: 43-52. Herman, J.P. and Cullinan, W.E. (1997) Neurocircuitry of stress: central control of the hypothalamo-pituitary-adrenocortical axis. Trends Neurosci., 20: 78-84. Honig, L.S. and Rosenberg, R.N. (2000) Apoptosis and neurologic disease. Am. J. Med., 108: 317-330. Jemmerson, R., Liu, J., Hausauer, D., Lain, K.P., M ondino, A. and Nelson, R.D. (1999) A conformational change in cytochrome c of apoptotic and necrotic cells is detected by monoclonal antibody binding and mimicked by association of the native antigen with synthetic phospholipid vesicles. Biochemistry, 38: 3599-3609.
748 Jin, K., Chen, J., Nagayama, T., Chen, M., Sinclair, J., Graham, S.H. and Simon, R.P. (1999) In situ detection of neuronal DNA strand breaks using the Klenow fragment of DNA polymerase I reveals different mechanisms of neuron death after global cerebral ischemia. J. Neurochem., 72: 1204-1214. Kerr, J.F., Wyllie, A.H. and Currie, A.R. (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer, 26: 239-257. Kockx, M.M., Muhring, J., Knaapen, M.W. and de Meyer, G.R. (1998) RNA synthesis and splicing interferes with DNA in situ end labeling techniques used to detect apoptosis. Am. J. Pathol., 152: 885-888. Komoriya, A., Packard, B.Z., Brown, M.J., Wu, M.L. and Henkart, P.A. (2000) Assessment of caspase activities in intact apoptotic thymocytes using cell-permeable fluorogenic caspase substrates. J. Exp. Med., 191: 1819-1828. Labat-Moleur, F., Guillermet, C., Lorimier, P., Robert, C., Lantuejoul, S., Brambilla, E. and Negoescu, A. (1998) TUNEL apoptotic cell detection in tissue sections: critical evaluation and improvement critical evaluation and improvement. J. Histochem. Cytochem., 46: 327-334. Laping, N.J., Teter, B., Nichols, N.R., Rozovsky, I. and Finch, C.E. (1994) Glial fibrillary acidic protein: regulation by hormones, cytokines, and growth factors. Brain Pathol., 4: 259-75. Leers, M.P., Kolgen, W., Bjorklund, V., Bergman, T., Tribbick, G., Persson, B., Bjorklund, P., Ramaekers, F.C., Bjorklund, B., Nap, M., Jornvall, H. and Schutte, B. (1999) Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis. J. Pathol., 187: 567-572. Leist, M., Single, B., Naumann, H., Fava, E., Simon, B., Kuhnle, S. and Nicotera, P. (1999) Inhibition of mitochondrial ATP generation by nitric oxide switches apoptosis to necrosis. Exp. Cell Res., 249: 396-403. Li, X., Melamed, M.R. and Darzynkiewicz, Z. (1996) Detection of apoptosis and DNA replication by differential labeling of DNA strand breaks with fluorochromes of different color. Exp. Cell Res., 222: 28-37. Liegler, T.J., Hyun, W., Yen, T.S.B. and Stittes, D.P. (1995) Detection and quantification of live, apoptotic, and necrotic human peripheral lymphocytes by single-laser flow cytometry. Clin. Diagnostic Lab. Immunol., 2: 369-376. Liu, B. and Hong, J.S. (2003) Role of microglia in inflammation-mediated neurodegenerative diseases: mechanisms and strategies for therapeutic intervention. J. Pharmacol. Exp. Ther., 304: 1-7. Lossi, L. and Merighi, A. (2003) In vivo cellular and molecular mechanisms of neuronal apoptosis in the mammalian CNS. Prog. Neurobiol., 69: 287-312. Magarinos, A.M. and McEwen, B.S. (1995a) Stress-induced atrophy of apical dendrites of hippocampal CA3c neurons: comparison of stressors. Neuroscience, 69: 83-88.
Magarinos, A.M. and McEwen, B.S. (1995b) Stress-induced atrophy of apical dendrites of hippocampal CA3c neurons: involvement of glucocorticoid secretion and excitatory amino acid receptors. Neuroscience, 69: 89-98. Majno, G. and Joris, I. (1995) Apoptosis, oncosis, and necrosis. An overview of cell death. Am. J. Pathol., 146: 3-15. Mattson, M.P. (2000) Apoptotic and anti-apoptotic synaptic signaling mechanisms. Brain Pathol., 10:300-312. McConkey, D.J. (1998) Biochemical determinants of apoptosis and necrosis. Toxicol. Lett., 99: 157-168. McCormick, C.M., McNamara, M., Mukhopadhyay, S. and Kelsey, J.E. (1997) Acute corticosterone replacement reinstates performance on spatial and nonspatial memory tasks 3 months after adrenalectomy despite degeneration in the dentate gyrus. Behav. Neurosci., 111: 518-531. McEwen, B.S. (1999) Stress and the aging hippocampus. Front. Neuroendocrinol., 20: 49-70. Melcangi, R.C., Magnaghi, V., Cavarretta, I., Riva, M.A. and Martini, L. (1997) Corticosteroid effects on gene expression of myelin basic protein in oligodendrocytes and of glial fibrillary acidic protein in type 1 astrocytes. J. Neuroendocrinol., 9: 729-733. Miller, M.M., Antecka, E. and Sapolsky, R. (1989) Short term effects of glucocorticoids upon hippocampal ultrastructure. Exp. Brain Res., 77: 309-314. Mizoguchi, M., Manabe, M., Kawamura, Y., Kondo, Y., Ishidoh, K., Kominami, E., Watanabe, K., Asaga, H., Senshu, T. and Ogawa, H. (1998) Deimination of 70-kD nuclear protein during epidermal apoptotic events in vitro. J. Histochem. Cytochem., 46: 1303-1309. Modak, S.P. and Bollum, F.J. (1972) Detection and measurement of single-strand breaks in nuclear DNA in fixed lens sections. Exp. Cell Res., 75: 307-313. Muller, M., Holsboer, F. and Keck, M.E. (2002) Genetic modification of corticosteroid receptor signalling: novel insights into pathophysiology and treatment strategies of human affective disorders. Neuropeptides, 36:117-131. Nakamura, M., Yagi, H., Ishii, T., Kayaba, S., Soga, H., Gotoh, T., Ohtsu, S., Ogata, M. and Itoh, T. (1997) DNA fragmentation is not the primary event in glucocorticoidinduced thymocyte death in vivo. Eur. J. Immunol., 27: 999-1004. Naruse, I., Keino, H. and Kawarada, Y. (1994) Antibody against single-stranded DNA detects both programmed cell death and drug-induced apoptosis. Histochemistry, 101: 73-78. Nichols, N.R. (1999) Glial responses to steroids as markers of brain aging. J. Neurobiol., 40: 585-601. Pollack, A. and Ciancio, G. (1991) Cell-cycle phase-specific analysis of cells viability using Hoechst 33342 and propidium iodide after ethanol preservation. In: Darzynkiewicz, Z. and Crissman, H.A. (Eds.), Flow Cytometry. Academic Press, San Diego, pp. 19-24.
749 Ramos-Remus, C., Gonzalez-Castaneda, R.E., Gonzalez-Perez, O., Luquin, S. and Garcia-Estrada, J. (2002) Prednisone induces cognitive dysfunction, neuronal degeneration, and reactive gliosis in rats. J. Investig. Med., 50: 458-464. Reed, J.C. and Green, D.R. (2002) Remodeling for demolition: changes in mitochondrial ultrastructure during apoptosis. Mol. Cell, 9: 1-3. Reul, J.M. and de Kloet. E.R. (1985) Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation. Endocrinology, 117:2505-2511. Rusakov, D.A., Davies, H.A., Harrison, E., Diana, G., Richter-Levin, G., Bliss, T.V. and Stewart, M.G. (1997) Ultrastructural synaptic correlates of spatial learning in rat hippocampus. Neuroscience, 80: 69-77. Sallmann, F.R., Bourassa, S., Saint-Cyr, J. and Poirier, G.G. (1997) Characterization of antibodies specific for the caspase cleavage site on poly(ADP-ribose) polymerase: specific detection of apoptotic fragments and mapping of the necrotic fragments of poly(ADP-ribose) polymerase. Biochem. Cell Biol., 75: 451-456. Sapolsky, R.M., Romero, L.M. and Munck, A.U. (2000) How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocr. Rev., 21 : 55-89. Schmid, I., Uittenbogaart, C.H. and Giorgi, J.V. (1994) Sensitive method for measuring apoptosis and cell surface phenotype in human thymocytes by flow cytometry. Cytometry, 15: 12-20. Schmued, L.C. and Hopkins, K.J. (2000) Fluoro-Jade B: a high affinity fluorescent marker for the localization of neuronal degeneration. Brain Res., 874: 123-130. Schmued, L.C., Albertson, C. and Slikker Jr., W. (1997) Fluoro-Jade: a novel fluorochrome for the sensitive and reliable histochemical localization of neuronal degeneration. Brain Res., 751: 37-46. Segal, M. (2002) Changing views of Cajal's neuron: the case of the dendritic spine. Prog. Brain Res., 136: 101-107. Smith, M.A., Makino, S., Kvetnansky, R. and Post, R.M. (1995) Stress and glucocorticoids affect the expression of brain-derived neurotrophic factor and neurotrophin-3 mRNAs in the hippocampus. J. Neurosci., 15: 1768-1777. Sousa, N., Madeira, M.D. and Paula-Barbosa, M.M. (1997) Structural alterations of the hippocampal formation of adrenalectomized rats: an unbiased stereological study. J. Neurocytol., 26: 423-438. Sousa, N., Madeira, M.D. and Paula-Barbosa, M.M. (1999a) Corticosterone replacement restores normal morphological features to the hippocampal dendrites, axons and synapses of adrenalectomized rats. J. Neurocytol., 28: 541-558. Sousa, N., Paula-Barbosa, M.M. and Almeida, O.F.X. (1999b) Ligand and subfield specificity of corticoid-induced neuronal loss in the rat hippocampal formation. Neuroscience, 89: 1079-1087. Sousa, N., Lukoyanov, N.V., Madeira, M.D., Almeida, O.F.X. and Paula-Barbosa, M.M. (2000) Reorganization of the
morphology of hippocampal neurites and synapses after stress-induced damage correlates with behavioral improvement. Neuroscience, 97: 253-266. Srinivasan, A., Roth, K.A., Sayers, R.O., Shindler, K.S., Wong, A.M., Fritz, L.C. and Tomaselli, K.J. (1998) In situ immunodetection of activated caspase-3 in apoptotic neurons in the developing nervous system. Cell Death Differ., 5: 1004-1016. Steller, H. (1995) Mechanisms and genes of cellular suicide. Science, 267: 1445-1449. Stewart, M.G., Harrison, E., Rusakov, D.A., Richter-Levin, G. and Maroun, M. (2000) Re-structuring of synapses 24 hours after induction of long-term potentiation in the dentate gyrus of the rat hippocampus in vivo. Neuroscience, 100: 221-227. Tateyama, H., Tada, T., Hattori, H., Murase, T., Li, W.X. and Eimoto, T. (1998) Effects of prefixation and fixation times on apoptosis detection by in situ end-labeling of fragmented DNA. Arch. Pathol. Lab. Med., 122: 252-255. Trejo, J.L., Rua, C., Cuchillo, I. and Machin, C. (1998) Calbindin-D28k- and astroglial protein-immunoreactivities, and ultrastructural differentiation in the prenatal rat cerebral cortex and hippocampus are affected by maternal adrenalectomy. Dev. Brain Res., 108: 161-177. Ucker, D.S. (1991) Death by suicide: one way to go in mammalian cellular development? New Biol., 3: 103-109. Van Eekelen, J.A., Jiang, W., De Kloet, E.R. and Bohn, M.C. (1988) Distribution of the mineralocorticoid and the glucocorticoid receptor mRNAs in the rat hippocampus. J. Neurosci. Res., 21: 88-94. Vijayan, V.K. and Cotman, C.W. (1987) Hydrocortisone administration alters glial reaction to entorhinal lesion in the rat dentate gyrus. Exp. Neurol., 96: 307-320. Walker, P.R., Leblanc, J., Smith, B., Pandey, S. and Sikorska, M. (1999) Detection of DNA fragmentation and endonucleases in apoptosis. Methods, 17: 329-338. Watanabe, Y., Gould, E., Cameron, H.A., Daniels, D.C. and McEwen, B.S. (1992) Phenytoin prevents stress- and corticosterone-induced atrophy of CA3 pyramidal neurons. Hippocampus, 2: 431-435. Weibel, E.R., Kistler, G.S. and Scherle, W.F. (1966) Practical stereological methods for morphometric cytology. J. Cell Biol., 30: 23-38. West, M.J. (1999) Stereological methods for estimating the total number of neurons and synapses: issues of precision and bias. Trends Neurosci., 22:51-61. West, M.J., Slomianka, L. and Gundersen, H.J.G. (1991) Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator. Anat. Rec., 231: 482-497. Wyllie, A.H. (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature, 284: 555-556. Yau, J.L. and Seckl, J.R. (2001) llbeta-hydroxysteroid dehydrogenase type I in the brain; thickening the glucocorticoid soup. Mol. Psychiatry, 6: 611-614.
This Page Intentionally Left Blank
T. Steckler, N.H. Kalin and J.M.H.M. Reul (Eds.) Handbook of Stress and the Brain, Vol. 15
ISBN 0-444-51173-3 Copyright 2005 Elsevier B.V. All rights reserved CHAPTER 5.7
Enhancing resilience to stress" the role of signaling cascades Pei-Xiong Yuan*, Rulun Zhou, Neda Farzad, Todd D. Gould, Neil A. Gray, Jing Du and Husseini K. Manji Laboratory of Molecular Pathophysiology, National Institute of Mental Health, Building 49, Room B1EE16, 49 Convent Drive, Bethesda, MD 20892-4405, USA
Abstract: A central role of the brain involves both the perception and response to stressful stimuli. The mechanisms by which the brain responds to stress are of critical importance to the appropriate function of an organism. In this regard cellular resilience in the brain and responses at the neuronal level to stress have become an intriguing area of research. The results of stress in the brain appear to include atrophy of hippocampal neurons, other morphometric and structural brain changes, a decrease in neurotrophic support, and changes in behavior in preclinical models. The hypothalamicpituitary-adrenal (HPA) axis appears to play a critical role in mediating these effects. Increasing recent data implicate a critical role for glucocorticoids, and corticotrophic hormone-releasing factor (CRF), in long-term effects of early-life stress on hippocampal integrity and function. Clinical evidence is consistent with the preclinical evidence including structural and morphometric brain changes, and the finding that a significant percentage of patients with mood disorders display some form of HPA axis activation. Stress is a critical factor in the development of some psychiatric disorders. Some antidepressants, electroconvulsive shock therapy (ECT), and mood stabilizers (lithium) appear to modulate glucocorticoid receptor number and/or function, components of the HPA axis, and neurotrophic pathways and molecules in preclinical models. The possibility arises that regulation of these factors may be a principle component to the susceptibility to develop and the treatment of psychiatric disorders. We also discuss the role that epigenetic factors, perhaps mediated by stress, may have on behavior, and response to future stressors.
et al., 2000), for example, novelty stress and social defeat; physiological stress can be defined as disturbing an individual's internal milieu, leading to activation of regulatory mechanisms that serve to restore homeostasis (Kollack-Walker et al., 2000), for example, starvation, noise, cold exposure, or hemorrhage. Coping with stressful stimuli is often described as a conscious cognitive effort. However, it may also be physiological in nature, whereby the body activates a series of counteractive measures in response to a stressor. For example, changes in gene expression, neurotransmitter or receptor levels, or synaptic plasticity may all be adaptive responses to stress. Whether psychological or physiological, the brain plays a key role in perceiving, adapting and responding to stress. There is a vast array of neuronal
Introduction After many decades of stress research, beginning with the pioneering work of Hans Selye (Selye, 1956), the term "stress" is still defined today in a variety of ways. However, the term is generally accepted to include a disruptive force, whether good or bad, which affects the homeostatic balance of an organism. Stress may result from a disparate array of psychological or physiological stressors, or disruptive stimuli. Psychological stress can be defined as involving a reaction to an aversive stimulus in an individual's external environment (Kollack-Walker *Corresponding author. Tel.: + 1(301) 496 9802; Fax: + 1(301) 480-0123; E-mail:
[email protected] 751
752 cellular adaptive mechanisms that are of central importance in understanding the brain's response to stressful stimuli. Failure of these adaptive mechanisms may antedate illness; for example, much evidence suggest stress as an antecedent of psychiatric disorders (Heim and Nemeroff, 2001). For example, in clinical populations environmental events (for example, early childhood stressors) correlate with the development of psychiatric disorders in adults (Heim and Nemeroff, 2001). Particularly strong is the epidemiological evidence in regard to the development of depression and anxiety disorders (Heim and Nemeroff, 2001). Indeed, accumulated data suggests that various stressors such as physical abuse, sexual abuse, parental loss, and even prenatal stress are all correlated with the development of severe mood and anxiety disorders in adulthood (Heim and Nemeroff, 2001). Furthermore, the clinical presentation of psychiatric disorders is often associated with acute life events or ongoing stress in adulthood (Heim and Nemeroff, 2001). In total, these data lead to a hypothesis whereby early life stressors lead to a state of enhanced vulnerability, and that adult mood and anxiety disorders present when later life events have effects on the vulnerable brain of these individuals (Heim and Nemeroff, 2001). Evidence implicates the hypothalamic-pituitaryadrenal (HPA) axis (corticotrophic hormonereleasing factor (CRF) and glucocorticoids in particular) in long-term effects of early-life stress on hippocampal integrity and function. Most wellstudied is the hippocampus, where high levels of glucocorticoid receptors are thought to mediate stress effects including cellular morphological changes, and changes in regional circuits and gross behavioral differences. A growing body of data is specifically implicating glutamatergic neurotransmission in stress-induced hippocampal atrophy and death (McEwen, 1999; Sapolsky, 2000b). As we discuss, the abundant data for the critical roles of CRF and glucocorticoids are noteworthy in regard to the pathophysiology of mood disorders. In addition to directly causing neuronal atrophy, stress and glucocorticoids also appear to reduce cellular resilience, thereby making certain neurons more vulnerable to other insults, such as ischemia, hypoglycemia, and excitatory amino acid toxicity (Sapolsky, 2000a). The long-term effects of stress on
behavior may be mediated by epigenetic gene regulatory factors. This chapter focuses primarily on the adaptive neuronal resilience to stress and the mechanisms underlying this phenomenon. However, as we discuss at the end of the chapter, these data and their implications may have relevance for the treatment of severe psychiatric disorders. Thus, recent efforts to understand the cellular and molecular actions of mood stabilizers and antidepressants have focused efforts on understanding what enzymes, signaling pathways, and gene expression profiles are altered in the brain (Duman, 1998; Duman et al., 2000; Gould and Manji, 2002). This evidence suggests that the downstream targets of both antidepressants and mood stabilizers are critical intracellular signaling pathways (Gould et al., 2002b); many of the same pathways are those involved in modulation of stress and the stress response. Accumulating evidence suggests that these medications may target pathways involved in mediating cellular resilience and neuroplasticity within the brain (Duman et al., 2000; Manji et al., 2000b). It is possible that the effects of these medications are to counteract- at least in p a r t - the deleterious effects of stress that antedate the development of psychiatric illnesses.
Stress modulates neural plasticity "Neuroplasticity" subsumes diverse processes of vital importance by which the brain perceives, adapts and responds to a variety of internal and external stimuli. The manifestations of neuroplasticity in the adult central nervous system (CNS) have been characterized as including alterations of dendritic function, synaptic remodeling, long-term potentiation (LTP), axonal sprouting, neurite extension, synaptogenesis, and even neurogenesis (see (Mesulam, 1999) for an excellent overview). The adult brain is more plastic than previously believed. The alterations in cellular morphology resulting from various stressors have been the focus of considerable recent research (D'Sa and Duman, 2002) (Fig. 1). Remodeling of synaptic contacts and dendrites in the hypothalamus coinciding with the onset of lactation (Michaloudi et al., 1997; Stern and Armstrong, 1998) and branching of dendrites of cerebral-cortical neurons in an enriched environment
753
Stress
0,ucoco,,co,0
~ Glutamate ~
Unknown
Ca2+ Hyperactivationof Ca2+-dependent enzymes
I
Oxygenfree radicals
~1, Glucos!transporter and glucoseuptake
~II, BDNF
nerg,iapac, . I .,ro,h;c up,oO
Atrophy, Endangerment, and Death of Neurons Fig. 1. Cellular mechanisms by which stress and mood disorders may bring about impairments of structural plasticity. This figure depicts the multiple mechanisms by which stress and potentially affective episodes may attenuate cellular resiliency, thereby resulting in atrophy, death, and endangerment of hippocampal neurons. The primary mechanisms appear to be: (i) excessive NMDA and nonNMDA glutamatergic throughput; (ii) downregulation of cell surface glucose transporters, which are involved in bringing glucose into the cell. Reduced levels of glucose transporters thus reduce the neurons' energy reservoir, making them susceptible to energy failure when faced with excessive demands; (iii) reduction in the levels of BDNF, which is essential for the neuron's normal trophic support and synaptic plasticity. The well-documented reduction in glial cells may contribute to impairments of neuronal structural plasticity by reducing the neuron's energy supply and reduced glialmediated clearing of excessive synaptic glutamate. NMDA, N-methyl-D-aspartate glutamate receptor; GR, glucocorticoid receptor; BDNF, brain-derived neurotrophic factor. Modified and reproduced, with permission (Manji and Duman, 2001). and after training (Withers and Greenough, 1989), are just two examples of adaptive plasticity. Most studies of adaptive plasticity of neurons in response to stress, as well as hormones of the HPA axis, have focused on the hippocampus. This is due, in part, to the well-defined and easily studied neuronal
populations of the limbic brain regions, including the dentate gyrus granule cell layer, and the CA1 and CA3 pyramidal cell layers. These cell layers and their connections (mossy fiber pathway and Schaffer collateral) have also been used as cellular models of learning and memory (i.e., LTP). Another major reason that the hippocampus has been the focus of stress research is that the highest levels of glucocorticoid receptors (GR) are expressed in this brain region (Lopez et al., 1998). However, it is clear that stress and glucocorticoids also influence the survival and plasticity of neurons in other brain regions (e.g., prefrontal cortex, vide infra) that have not yet been studied in the same detail as the hippocampus. One of the most consistent effects of stress on cellular morphology is dendritic remodeling of hippocampal neurons (for reviews see (McEwen, 1999; Sapolsky, 2000a). The remodeling of dendrites is observed profoundly in the CA3 pyramidal neurons as atrophy-decreased number and length of the apical dendritic branches. The stress-induced atrophy of CA3 neurons occurs after two to three weeks of exposure to restraint stress or more long-term social stress, and has been observed in rodents and tree shrews (McEwen, 1999; Sapolsky, 2000a). Although the effects of chronic stress in the CA3 tend to be the greatest, subtle structural changes are also found in the CA1 and dentate gyrus after a one-month multiple stress paradigm (Sousa et al., 2000). Additionally, profound changes in the morphology of the mossy fiber terminals and significant loss of synapses were also observed. These alterations were partially reversible following rehabilitation from stress. Moreover, these fine structural changes also occur upon exposure to high levels of glucocorticoids, and were accompanied by impairments in spatial learning and memory. The latter were undetectable following rehabilitation, suggesting that activation of the HPA axis likely plays a major role in mediating the stress-induced neural plasticity and hippocampaldependent learning and memory (Sapolsky, 1996, 2000a; McEwen, 1999). The hippocampus has a very high concentration of glutamate and expresses both mineralcorticoid receptor (MR) and glucocorticoid receptor (GR), though G R may be relatively scarce in the hippocampus of primates (Patel et al., 2000; Sanchez et al., 2000), and more abundant in cortical regions. MR activation in the hippocampus (CA1)
754 is associated with reduced calcium currents, whereas activation of GR causes increased calcium currents and enhanced responses to excitatory amino acids. Very high levels of GR activation markedly increases calcium currents and leads to increased N-methyl-Daspartate (NMDA) receptor throughput that could predispose to neurotoxicity. Indeed, as we discuss in greater detail below, a growing body of data has implicated glutamatergic neurotransmission in stressinduced hippocampal atrophy and death ((McEwen, 1999); see Fig. 1). These observations are noteworthy with respect to the pathophysiology of mood disorders, since a significant percentage of patients with mood disorders display some form of HPA axis activation, and the subtypes of depression most frequently associated with HPA activation are those most likely to be associated with hippocampal volume reductions (Sapolsky, 2000a). A significant percentage of patients with Cushing's disease, in which pituitary gland adenomas result in cortisol hypersecretion, are also known to manifest prominent depressive symptoms, as well as hippocampal atrophy. Additionally, some patients with Cushing's disease show a reduction in hippocampal volume that correlates inversely with plasma cortisol concentrations; following corrective surgical treatment, enlargement of hippocampal volume in proportion to the treatment-associated decrement in urinary free-cortisol concentrations is observed (Starkman et al., 1999; Simmons et al., 2001). The HPA axis hyperactivity in mood disorder patients is most commonly measured by increased cortisol levels in plasma (especially at the circadian nadir), urine and CSF, increased cortisol response to adrenocorticotropin hormone (ACTH), blunted ACTH response to CRF challenge, enlarged pituitary and adrenal glands, and downregulation at postmortem examination of frontal cortical CRF. Reduced corticosteroid receptor feedback is implicated in this process by challenge studies with dexamethasone and dexamethasone plus CRF in bipolar and unipolar subjects (McQuade and Young, 2000; Reul and Holsboer, 2002). Hippocampal changes and HPA axis overactivation is also a finding in anxiety disorders where a number of clinical studies suggest that patients with post-traumatic stress disorder (PTSD) have a smaller hippocampal volume than matched
control subjects (Bremner et al., 1995, 1997; Gurvits et al., 1996; Stein et al., 1997; Charney and Bremner, 1999). To date, no quantitative neuroimaging studies have been performed in other anxiety disorders such as panic disorder, phobic disorder, or generalized anxiety disorder- but a single study does suggest the presence of abnormalities in the temporal lobe in panic disorder (Ontiveros et al., 1989; Charney and Bremner, 1999). Also, some studies suggest that there is an increase in cortisol release in response to stress in PTSD and panic disorder. Furthermore, it is generally consistent that there is a chronic increase of CRF in patients with anxiety. A cautionary note in the interpretation of the clinical studies is suggested by the results of the recent longitudinal studies undertaken to investigate the effects of earlylife stress and inherited variation in monkey hippocampal volumes (Lyons et al., 2001). In these studies, paternal half-siblings raised apart from one another by different mothers in the absence of fathers were randomized to one of three postnatal conditions that disrupted diverse aspects of early maternal care. These researchers found that all paternal halfsiblings, with small adult hippocampal volumes, responded to the removal of mothers after weaning with initially larger relative increases in cortisol levels (Lyons et al., 2001). Plasma cortisol levels 3 and 7 days later, and measures of cortisol-negative feedback in adulthood were not, however, correlated with hippocampal size. Thus, these studies suggest that small hippocampi also reflect an inherited characteristic of the brain, and highlight the need for caution in attribution of causality in the cross-sectional human morphometric studies of the hippocampus. A recent study by Gilbertson et al. also supports the hypothesis that smaller hippocampal volume is associated with susceptibility to stress (Gilbertson et al., 2002). The brains of monozygotic twin pairs, in which one twin experienced combat in Vietnam and the other did not, were imaged by MRI. As reported in prior studies, veterans who had developed PTSD displayed reduced hippocampal volume in comparison to those who did not. However, it was also observed that the combat-naive cotwins of the PTSD-sufferers also had reduced hippocampal volume, in comparison to cotwins of veterans who never developed PTSD. Likewise, the hippocampal volume of the combat-naive cotwins was inversely correlated
755 with the severity of PTSD symptoms in their veteran counterparts. These data suggest that a genetic contributor to PTSD susceptibility is associated with reduced hippocampal volume (and/or a propensity to sustain PTSD-unrelated hippocampal shrinkage). Although not as extensively studied as the hippocampus, recent research has also demonstrated histopathological changes in rat prefrontal cortex after corticosterone administration (Wellman, 2001). Thus, using a Golgi-Cox procedure, Wellman (2001) investigated pyramidal neurons in layer II-III of medial prefrontal cortex, and quantified dendritic morphology in three dimensions. This study demonstrated a significant redistribution of apical dendrites in corticosterone-treated animals, with the amount of dendritic material proximal to the soma being increased and distal dendritic material being decreased. These findings suggest that stress may produce a significant reorganization of the apical dendritic arbor from medial prefrontal cortex in rats. Most recently, Lyons (2002) demonstrated that four years after a brief stressor (intermittent postnatal separations from maternal availability) young adult squirrel monkeys showed significantly larger right ventral medial prefrontal volumes. Neither overall brain volumes nor left prefrontal measures were altered, suggesting selective (rather than nonspecific) effects. An intriguing observation of this study was that, similar to their hippocampal findings (vide supra), these investigators found a strong heritability of the right ventral medial prefrontal volume. Thus, in this study, certain fathers produced offspring with large right ventral medial prefrontal volumes, whereas others produced offspring with small right ventral medial prefrontal volumes (Lyons, 2002). Since the paternal half-siblings were raised apart by different mothers in the absence of fathers, the phenotypic similarities in right ventral medial prefrontal volume likely represent a major genetic contribution, effects which were not seen for other prefrontal regions (Lyons, 2002).
Effects of stress, glucocorticoids, and psychotrophic medications on hippocampal neurogenesis The demonstration that neurogenesis occurs in the adult human brain has reinvigorated research into
the cellular mechanisms by which the birth of new neurons is regulated in the mammalian brain (Eriksson et al., 1998). The localization of pluripotent progenitor cells and neurogenesis occurs in restricted brain regions. The greatest density of new cell birth is observed in the subventricular zone and the subgranular layer of the hippocampus. Cells born in the subventricular zone migrate largely to the olfactory bulb and those in the subgranular zone into the granule cell layer. The newly generated neurons send out axons and appear to make connections with surrounding neurons, indicating that they are capable of integrating into the appropriate neuronal circuitry in hippocampus and cerebral cortex. Neurogenesis in the hippocampus is increased by enriched environment, exercise, and hippocampaldependent learning (Kempermann et al., 1997; van Praag et al., 1999; Gould et al., 2000). Upregulation of neurogenesis in response to these behavioral stimuli and the localization of this process to hippocampus has led to the proposal that new cell birth is involved in learning and memory (Gould et al., 2000). Chronic, but not acute, antidepressant treatment also increases the neurogenesis of dentate gyrus granule cells (Jacobs et al., 2000; Manev et al., 2001; D'Sa and Duman, 2002). These studies demonstrate that chronic administration of different classes of antidepressant treatment, including noradrenaline (NA), selective serotonin reuptake inhibitors (SSRIs), and electroconvulsive seizure, increases the proliferation and survival of new neurons. Lithium also increases neurogenesis in the dentate gyrus (Chen et al., 2000b). In contrast, increased neurogenesis is not observed in response to chronic administration of nonantidepressant psychotropic drugs. Studies demonstrating that neurogenesis is increased by conditions that stimulate neuronal activity (e.g., enriched environment, learning, exercise) suggest that this process is also positively regulated by, and may even be dependent on, neuronal plasticity (Kempermann, 2002). It is clear that the enhancement of hippocampal neurogenesis by antidepressants serves to highlight the degree to which these effective treatments are capable of regulating long-term neuroplastic events in the brain. At this point, it is not completely clear precisely what the clinical significance of enhancement of adult hippocampal neurogenesis
756 by antidepressants truly represents. In view of the opposite effects of stress and antidepressants on hippocampal neurogenesis, it is quite plausible that alterations in hippocampal neurogenesis are fundamental to the clinical syndrome of depression (Jacobs et al., 2000; Manev et al., 2001; D'Sa and Duman, 2002; Kempermann, 2002). However, as Kempermann (2002) has clearly articulated, much more research is required in order to adequately link changes in adult hippocampal neurogenesis to the pathophysiology and treatment of depression. Recent studies have shown that decreased neurogenesis occurs in response to both acute and chronic stress (see (Gould et al., 2000)). Removal of adrenal steroids (i.e., adrenalectomy) increases neurogenesis and treatment with high levels of glucocorticoids reproduces the downregulation of neurogenesis that occurs in response to stress. Aging also influences the rate of neurogenesis. Although neurogenesis continues into late life, the rate is significantly reduced (Cameron and McKay, 1999). The decreased rate of cell birth could result from upregulation of the HPA axis and higher levels of adrenal steroids that occur in later life. Lowering glucocorticoid levels in aged animals restores neurogenesis to levels observed in younger animals, indicating that the population of progenitor cells remains stable but that it is inhibited by glucocorticoids (Cameron and McKay, 1999). Interestingly, studies in glucocorticoid receptor knockout mice showed significant alterations in hippocampal neurogenesis (Gass et al., 2000). A reduction of granule cell neurogenesis (up to a 65 % of control levels) was found in M R - / - mice, whereas G R - / - mice did not show neurogenic disruption, eventually relating the MR receptor in the pathogenesis of hippocampal changes observed in chronic stress and affective disorders (Gass et al., 2000). These preclinical observations raise the interesting possibility that CRF and GR antagonists, currently being developed for the treatment of mood and anxiety disorders, may have particular utility in the treatment of elderly depressed patients. Also, of potential relevance (noting the effect of hormonal fluctuations on mood disorders) for our understanding of the neurobiology and treatment of mood disorders, ovariectomy decreases the proliferation of new cells in the hippocampus, effects which
are reversed by estrogen replacement. The rate of neurogenesis fluctuates over the course of the estrus cycle in rodents, and the total rate of cell birth is higher in female rodents relative to males. In addition to potentially playing a role in the beneficial cognitive effects of estrogen, the regulation of neurogenesis by this gonadal steroid may also provide important clues about certain sexually dimorphic characteristics of mood disorders.
Mechanisms underlying stress-induced morphometric changes Glutamate, calcium, and apoptosis Microdialysis studies have shown that stress increases extracellular levels of glutamate in hippocampus, and NMDA glutamate receptor antagonists attenuate stress-induced atrophy of CA3 pyramidal neurons (McEwen, 1999; Sapolsky, 2000b). Although a variety of methodological issues remain to be fully resolved, the preponderence of the evidence to date suggests that the atrophy, and possibly death, of CA3 pyramidal neurons arises, at least in part, from increased glutamate neurotransmission (McEwen, 1999; Sapolsky, 2000b). It should be noted, however, that although NMDA antagonists block stressinduced hippocampal atrophy, there have not been any studies demonstrating that they are able to block the cell death induced by severe stress. This suggests that the mechanisms underlying atrophy and death may lie on a continuum, with severe (or prolonged) stresses "recruiting" additional pathogenic pathways in addition to enhanced NMDA-mediated neurotransmission. As discussed, stress increases extracellular levels of glutamate and sustained activation of NMDA, as well as nonNMDA ionotropic receptors could result in high intracellular levels of calcium. Overactivation of the glutamate ionotropic receptors is known to contribute to the neurotoxic effects of a variety of insults, including repeated seizures and ischemia (Fig. 1). Neurotoxicity follows as a response to overactivation of calcium-dependent enzymes and the generation of oxygen-free radicals. Stress or glucocorticoid exposure also compromises the metabolic capacity of neurons, thereby increasing the vulnerability to other types of neuronal insults.
757
Hypothalamic-pituitary-adrenal (HPA ) axis Activation of the HPA axis appears to play a critical role in mediating these effects, since stress-induced neuronal atrophy is prevented by adrenalectomy, and duplicated by exposure to high concentrations of glucocorticoids (reviewed in Sapolsky, 1996, 2000a; McEwen, 1999). Increasing recent data also suggests a critical role for CRF in long-term effects of earlylife stress on hippocampal integrity and function. Thus, the administration of CRF to the brains of immature rats has been demonstrated to reduce memory function throughout life; these deficits are associated with progressive loss of hippocampal CA3 neurons and chronic upregulation of hippocampal CRF expression, effects that do not require the presence of stress levels of glucocorticoids (Brunson et al., 2001). The CRF1 receptor, which binds CRF with higher affinity than CRF2 receptor, plays a major role in regulating adrenocorticotropin hormone (ACTH) release, and has been implicated in animal models of anxiety. Indeed, the central administration of CRF1 antisense oligodeoxynucleotides has been demonstrated to have anxiolytic effects against both CRF and psychological stressors. Although CRF2 receptors appear to act in an antagonistic manner, i.e., CRF~ activates and CRF2 attenuates the stress response, its precise role is still being characterized (reviewed in (Reul and Holsboer, 2002). Interestingly, pretreatment with a CRF antagonist also attenuates the stress-induced increases in MR levels in hippocampus, neocortex, frontal cortex, and amygdala (Gesing et al., 2001). Rats that underwent a stressor also showed increased ACTH and cortisol levels following the administration of an MR antagonist, suggesting that the upregulation of MR in the stressed group is associated with increased inhibitory tone of the HPA axis. Together, the abundant data for the critical roles of CRF and glucocorticoids are noteworthy with respect to the pathophysiology of mood disorders, since a significant percentage of patients with mood disorders display some form of HPA axis activation, and the subtypes of depression most frequently associated with HPA activation are those most likely to be associated with hippocampal volume reductions (Sapolsky, 2000a). A significant percentage of
patients with Cushing's disease, in which pituitary gland adenomas result in cortisol hypersecretion, are also known to manifest prominent depressive symptoms, as well as hippocampal atrophy. Additionally, some patients with Cushing's disease show a reduction in hippocampal volume that correlates inversely with plasma cortisol concentrations; following corrective surgical treatment, enlargement of hippocampal volume in proportion to the treatmentassociated decrement in urinary free-cortisol concentrations is observed (Starkman et al., 1999; Simmons et al., 2001). The HPA axis hyperactivity in mood disorder patients is generally manifest by increased cortisol levels in plasma (especially at the circadian nadir), urine, and CSF, increased cortisol response to ACTH, blunted ACTH response to CRF challenge, enlarged pituitary and adrenal glands, and downregulation at postmortem of frontal cortical CRF. Reduced corticosteroid receptor feedback is implicated in this process by challenge studies with dexamethasone and dexamethasone plus CRF in bipolar and unipolar subjects (McQuade and Young, 2000; Reul and Holsboer, 2002). Evidence in humans suggests that decreased corticosteroid receptor number (postmortem reduction of GR messenger ribonucleic acid (mRNA) (Webster et al., 1999)) may be present in the hippocampus of individuals with bipolar and unipolar disorder, and some antidepressants (tricyclics), electroconvulsive shock therapy (ECT), and mood stabilizers (lithium) may modulate GR number and/or function (reviewed in (Holsboer, 2000). Furthermore, transgenic mice with reduced GR have HPA and cognitive disturbance that may parallel depression in humans and that normalizes with antidepressant exposure (Steckler et al., 2001). Finally, antisense oligonucleotides targeted to GR reduced immobility on the forced swim test (suggesting an antidepressant-like effect), as does the antiglucocorticoid drug mifepristone (RU-486) (Korte et al., 1996). In addition to directly causing neuronal atrophy, stress and glucocorticoids also appear to reduce cellular resilience, thereby making certain neurons more vulnerable to other insults, such as ischemia, hypoglycemia, and excitatory amino acid toxicity (Sapolsky, 2000a). Thus, recurrent stress (and presumably recurrent mood disorders episodes, which
758 are often associated with hypercortisolemia) may lower the threshold for cellular death/atrophy in response to a variety of physiological (e.g., aging) and pathological (e.g., ischemia) events. The potential functional significance of these effects is supported by the demonstration that overexpression of the glucose transporter blocks the neurotoxic effects of neuronal insults (Fig. 1) (Sapolsky, 2000a,b; Manji and Duman, 2001b). Such processes may conceivably also play a role in the relationship between mood disorders and cerebrovascular events, considering that individuals who develop their first depressive episode in late-life have an increased likelihood of showing magnetic resonance imaging (MRI) evidence of cerebrovascular disease (Kumar et al., 1997; Murray and Lopez, 1997; Steffens and Krishnan, 1998; Steffens et al., 1999; Chemerinski and Robinson, 2000; Drevets, 2000). The precise mechanisms by which glucocorticoids exert these deleterious effects remain to be fully elucidated, but likely involve the inhibition of glucose transport (thereby diminishing capability of energy production and augmenting susceptibility to hypoglycemic conditions), and the aberrant, excessive facilitation of glutamatergic signaling (Sapolsky, 2000a). The reduction in the resilience of discrete brain regions including hippocampus and potentially prefrontal cortex, may also reflect the propensity for various stressors to decrease the expression of brain-derived neurotrophic factor (BDNF) in this region (Smith et al., 1995; Nibuya et al., 1999). The mechanisms underlying the downregulation of BNDF by stress have not been fully elucidated. However, as we discuss in a later section recent evidence suggests that mood stabilizers and antidepressants activate neurotrophic signaling pathways, and in particular have effects on BDNF-mediated signaling.
Neurotrophic signaling cascades: a focus on brain-derived neurotrophic factor(BDNF) Neurotrophins are a family of regulatory factors that mediate the differentiation and survival of neurons, as well as the modulation of synaptic transmission and synaptic plasticity (Patapoutian and Reichardt, 2001; Poo, 2001). The neurotrophin family now
include- among others- nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin (NT)-3, NT-4/5 and NT-6 (Patapoutian and Reichardt, 2001). These various proteins are closely related in terms of sequence homology and receptor specificity. They bind to and activate specific receptor tyrosine kinases belonging to the Trk family of receptors, including TrkA, TrkB, TrkC and a pan-neurotrophin receptor P75 (Patapoutian and Reichardt, 2001; Poo, 2001). Additionally, there are two isoforms of TrkB receptors: the full length TrkB and the truncated form of TrkB, which does not contain the intracellular tyrosine kinase domain (Fryer et al., 1996). The truncated form of TrkB can thus function as a dominant-negative inhibitor for the TrkB receptor tyrosine kinase, thereby providing another mechanism to regulate BDNF signaling in the CNS (Gonzalez et al., 1999). Neurotrophins can be secreted constitutively or transiently, and often in an activity-dependent manner. Recent observations support a model wherein neurotrophins are secreted from the dendrite and act retrogradely at presynaptic terminals where they act to induce long-lasting modifications (Poo, 2001). Within the neurotrophin family, BDNF is a potent physiological survival factor, which has also been implicated in a variety of pathophysiological conditions. The cellular actions of BDNF are mediated through two types of receptors: a high-affinity tyrosine receptor kinase (TrkB) and a low-affinity pan-neurotrophin receptor (p75). TrkB is preferentially activated by BDNF and NT4/5, and appears to mediate most of the cellular responses to these neurotrophins. BDNF and other neurotrophic factors are necessary for the survival and function of neurons (Mamounas et al., 1995), implying that a sustained reduction of these factors could affect neuronal viability. As discussed already, BDNF is best known for its long-term neurotrophic and neuroprotective effects, which may be very important for its putative role in the pathophysiology and treatment of mood disorders, and its putative role in the effects of stress (vide infra). In this context, it is noteworthy that although endogenous neurotrophic factors have traditionally been viewed as increasing cell survival by providing necessary trophic support, it is now clear that their survival-promoting effects are mediated in large part by an inhibition of cell death
759
cascades (Riccio et al., 1999). I n c r e a s i n g evidence suggests t h a t n e u r o t r o p h i c factors inhibit cell d e a t h cascades by activating the mitogen-activated p r o t e i n ( M A P ) kinase signaling p a t h w a y a n d the p h o s p h o t i d y l i n o s i t o l - 3 kinase ( P I - 3 K ) / A k t p a t h w a y (Fig. 2).
Signaling through the mitogen-activated protein (MAP) kinase cascade Shc (a p r o t e i n t h a t recognizes specific p h o s p h o t y r o sine residues on receptors) r e c r u i t m e n t a n d phosp h o r y l a t i o n results in r e c r u i t m e n t to the m e m b r a n e
BD~
~ ~ ~ ~ :~
~
T,.,1,. ~
i ~ ~ ~Ri!~:J~-~' ~ :~~ ~ ~ ~ .; .ii, .;~i5~ ~ .~ ~ it~
~~............. ~"~ii :~ ~-::'~I~- I~'~~:::i~i .............~~~. '~............... . ~ ~' ............... ~:::~:'
....
"
~
..... ........... .........
~~ C*~ ~~'........... :~:~~......... ~i,~:~iii~i~if~iii~
~ ~'~~ ~ ~
~ ;~ ::~i!~
............ ".......... ~' ............. "................ ~ ~""~' ................... ~:~-~... ~ ....
......
,
N~r~
~
....
#
..... .......................... /
"'-...
Fig. 2. Neurotrophins and the ERK MAP minase signaling cascade. Cell survival is dependent on neurotrophic factors, such as brainderived neurotrophic factor (BDNF) and nerve growth factor (NGF), and the expression of these factors can be induced by synaptic activity. The influence of neurotrophic factors on cell survival is mediated by activation of the mitogen-activated protein (MAP) kinase cascade. Activation of neurotrophic factor receptors, also referred to as Trks, results in activation of the MAP kinase cascade via several intermediate steps, including phosphorylation of the adaptor protein SHC and recruitment of the guanine nucleotide exchange factor son of sevenless (Sos). This results in activation of the small guanosine triphosphate- binding protein Ras, which leads to activation of a cascade of serine/threonine kinases. This includes Raf, MAP kinase (MEK), and MAP kinase also referred to as extracellular response kinase, or Erk). One target of the MAP kinase cascade is Rsk, which influences cell survival in at least two ways. Rsk phosphorylates and inactivates the proapoptotic factor BAD. Rsk also phosphorylates cyclic adenosine monophosphate response element-binding protein (CREB) and thereby increases the expression of the antiapoptotic factor bcl-2. These mechanisms underlie many of the long-term effects of neurotrophins, including neurite outgrowth, cytoskeletal remodeling, and cell survival. It is now clear, however, that BDNF also exerts many acute synaptic effects, including regulating the release of a number of neurotransmitters.
760 of a complex of the adaptor Grb-2 and the Ras exchange factor, son of sevenless (SOS), thereby stimulating transient activation of Ras. Ras, in turn, activates PI3K, the p38 MAPK/MAPK-activating protein kinase 2 pathway and the c-Raf/ERK pathway. Among the targets of ERK are the ribosomal $6 kinases (RSKs). Both RSK and MAPK-activating protein kinase 2 phosphorylate CRE-binding protein (CREB) and other transcription factors. Recent studies have demonstrated that the activation of the MAP kinase pathway can inhibit apoptosis by inducing the phosphorylation of BAD (Bcl-xl/Bcl-2 Associated Death promoter), and increasing the expression of the antiapoptotic protein Bcl-2, the latter effect likely involves the cAMP (cyclic adenosine monophosphate) response element binding protein (CREB) (Riccio et al., 1997; Bonni et al., 1999). Phosphorylation of BAD occurs via activation of Rsk. Rsk phosphorylates BAD and thereby promotes its inactivation. Activation of Rsk also mediates the actions of the MAP kinase cascade and neurotrophic factors on the expression of Bcl-2. Rsk can phosphorylate CREB, leading to induction of Bcl-2 gene expression (Fig. 2). MAP kinases are abundantly present in the brain, and in recent years have been postulated to play a major role in a variety of long-term CNS functions, both in the developing and mature CNS (Suzuki et al., 1995; Matsubara et al., 1996; Kornhauser and Greenberg, 1997; Fukunaga and Miyamoto, 1998; Robinson et al., 1998). With respect to their actions in the mature CNS, MAP kinases have been implicated in mediating neurochemical processes associated with long-term facilitation (Martin et al., 1997), long-term potentiation (English and Sweatt, 1996, 1997), associative learning (Atkins et al., 1998), one-trial and multi-trial classical conditioning (Crow et al., 1998), long-term spatial memory (Blum et al., 1999), and have also been postulated to integrate information from multiple, infrequent bursts of synaptic activity (Murphy et al., 1994). Important for the present discussion, MAP kinase pathways have recently been demonstrated to regulate the responses to environmental stimuli and stressors in rodents (Xu et al., 1997), and to couple protein kinase A (PKA) and protein kinase C (PKC) to CREB protein phosphorylation in area CA1 of hippocampus (Roberson et al., 1996, 1999).
These recent studies suggest the possibility of a broad role for the MAPK cascade in regulating gene expression in long-term forms of synaptic plasticity (Roberson et al., 1999). Overall, the data suggests that MAP kinases play important physiological roles in the mature CNS, and furthermore, may represent important targets for the actions of CNS-active agents (Nestler, 1998; Yuan et al., 1998). One important mechanism by which the MAP kinase signaling cascade inhibits cell death is by increasing the expression of the antiapoptotic protein Bcl-2 (Bonni et al., 1999; Finkbeiner, 2000). Bcl-2 is now recognized as a major neuroprotective protein, since Bcl-2 overexpression protects neurons against diverse insults including ischemia, MPTP (1-methyl-4phenyl-l,2,3,6-tetrahydropyridine), 13-amyloid, free radicals, excessive glutamate, and growth factor deprivation (Manji et al., 200 l c). Accumulating data suggests that not only is Bcl-2 neuroprotective, but that it also exerts neurotrophic effects and promotes neurite sprouting, neurite outgrowth, and axonal regeneration (Oh et al., 1996; Chen et al., 1997; Chen and Tonegawa, 1998; Chierzi et al., 1999; Holm et al., 2001). Moreover, a recent study demonstrated that severe stress exacerbates stroke outcome by suppressing Bcl-2 expression (DeVries et al., 2001). In this study, the stressed mice expressed ~ 70% less Bcl-2 mRNA than unstressed mice after ischemia. Furthermore, stress greatly exacerbated infarct in control mice but not in transgenic mice that constitutively express increased neuronal Bcl-2. Finally, high corticosterone concentrations were significantly correlated with larger infarcts in wild-type mice but not in Bcl-2 overexpressing transgenic mice. Thus, enhanced Bcl-2 expression appears to be capable of offsetting the potentially deleterious consequences of stressinduced neuronal endangerment, and suggests that pharmacologically induced upregulation of Bcl-2 may have considerable utility in the treatment of a variety of disorders associated with endogenous or acquired impairments of cellular resilience (vide infra). Overall, it is clear that the neurotrophic factor/MAP kinase/ Bcl-2 signaling cascade plays a critical role in cell survival in the CNS, and that there is a fine balance maintained between the levels and activities of cell survival and cell death factors; modest changes in this signaling cascade or in the levels of the Bcl-2 family of proteins (potentially due to genetic, illness,
761 or insult-related factors) may therefore profoundly affect cellular viability. Recently, it was reported that chronic stress (21 days footshock) induced a pronounced and persistent extracellular response kinase 1/2 (ERK1/2) hyperphosphorylation in dendrites of the higher prefrontocortical layers, while phospho-CREB was reduced in several cortical regions including frontal cortex (Trentani et al., 2002). Since CREB is phosphorylated and activated by phospho-ERK1/2 directly, this reduction indicate that chronic stress could downregulate CREB phosphorylation indirectly, and subsequently downregulate the transcription of some genes such as Bcl-2 and BDNF. As mentioned previously, severe stressors decrease the expression of Bcl-2 (DeVries et al., 2001)and BDNF (Smith et al., 1995; Nibuya et al., 1999) in brain regions, but the mechanisms that mediate this effect still remain unclear. An intriguing possibility holds that this effect may be attributable to dysregulation of the B D N F - E R K - C R E B coordination. This disruption of the coordination may be a key mechanism by which prolonged stress induces atrophy of selective subpopulations of vulnerable neurons and/or distal dendrites. Conceivably, the precise kinetics of ERK and CREB activation ultimately dictate whether the activated kinases participate in a cell survival or death-promoting pathway.
Phosphotidylinositol-3 kinase (PI3K-Akt) pathway: a major pathway mediating neuronal survival The PI3K-Akt pathway is also particularly important for mediating neuronal survival under a wide variety of circumstances. Trk receptors can activate PI3K through at least two distinct pathways, the relative importance of which differs between neuronal subpopulations. In many neurons, Ras-dependent activation of PI3K is the most important pathway through which neurotrophins promote cell survival. In some cells, however, PI3K can also be activated through three adaptor proteins, Shc, Grb-2, and Gab-1. Binding to phosphorylated tyrosine 490 of Shc results in recruitment of Grb-2. Phosphorylated Grb-2 provides a docking site for Gab-l, which in turn is bound by PI3K (Brunet et al., 2001).
PI3 kinase directly regulates certain cytoplasmic apoptotic pathways. Akt has been proposed to act both prior to the release of cytochrome-c by proapoptotic Bcl-2 family members, and subsequent to the release of cytochrome-c, by regulating components of the apoptosome. Akt phosphorylates the proapoptotic Bcl-2 family member BAD, thereby inhibiting BAD's proapoptotic functions (Datta et al., 1997). Akt may also promote survival in an indirect fashion by regulating another major signaling enzyme-glycogen synthase kinase 3 (GSK-3) (Woodgett, 2001). Elevated GSK-3 has been shown to promote apoptosis in cultured neurons (Bijur et al., 2000). Furthermore, neurotrophin withdrawal increases, whereas phosphorylation by Akt decreases GSK-3 activity (Hetman et al., 2000). Moreover, a series of studies indicates that Akt controls a major class of transcriptional f a c t o r s - the Forkhead box transcription factor, class O (FOXO) subfamily of Forkhead transcriptional regulators (FKHR, FKHRL1, and AFX). Several groups have independently shown that Akt directly phosphorylates FOXOs and inhibits their ability to induce the death genes (Brunet et al., 1999; Dijkers et al., 2000). Finally, activation of Akt also results in phosphorylation of nuclear factor-~cB (NF-KB). Transcription activated by NF-~cB has been shown recently to promote neuronal survival (Maggirwar et al., 1998). Thus, PI3K acting through Akt may promote survival by variety of mechanisms; precisely which of these mechanisms is operative in the actions of neurotrophins, and under what circumstances is the focus of extensive current research (Fig. 2).
Epigenetic gene regulatory factors likely regulate how organisms adapt to and respond to stress: regulation of neurotrophic factors may be important The discussion in the chapter have centered upon the fact that the stress response, mood disorders, and other behaviors, while traditionally being conceptualized as neurochemical disorders, are now being thought of in a more dynamic sense, wherein changes in the underlying pathophysiology of these processes are being reconceptualized as being due primarily to changes in synaptic plasticity and cellular resilience.
762 In the next section of this chapter we discuss the emerging evidence suggesting that antidepressant medications and mood stabilizers (valproic acid and lithium) upregulate BDNF (Duman et al., 2000) and other proteins with neurotrophic/neuroprotective functions in the brain (Manji and Chen, 2002). Furthermore, there exists a great deal of evidenceboth clinical and preclinical- showing that activating these pathways has functional effects (increased neurogenesis, neuroprotection, etc.). As discussed, it is well-established that acute stress -or the stress hormone corticosterone- can regulate the expression of neurotrophic molecules in rodents. Specifically, a number of studies have shown that acute stress and/or administration of exogenous corticosterone administration to rodents decreases the mRNA and protein levels of BDNF in the hippocampus (Smith et al., 1995). In addition to the broadly replicated BDNF results, studies have looked at other neurotrophic molecules, with variable results dependent upon the experimental conditions. Thus, both stress and thymoleptic medications regulate neurotrophic factors, and their regulation of these molecules appears to be opposite to one another. These diametric changes in the regulation of neurotrophic molecules give rise to the notion that regulation of these factors may be a principal component to the susceptibility and treatment of psychiatric disorders (Manji and Duman, 2001). In addition to the well-documented acute effects of stress, increasing e v i d e n c e - both preclinical and clinical- suggests that stress can have behavioral, biochemical, and cellular effects far temporally removed from the initial stressful event. These d a t a - by correlation and a s s o c i a t i o n - suggest major importance of these pathways in both the pathophysiology and treatment of mood disorders (Duman et al., 2000). In this brief section we discuss, some of the ways - in particular epigenetic m e c h a n i s m s - by which stress may have lasting effects on behavior, and response to future stressors. S t r e s s - and the individual response to s t r e s s appears to be a causal factor in the development of psychiatric diseases. In clinical populations environmental events (for example, early childhood stressors) correlate with the development of psychiatric disorders in adults (Heim and Nemeroff, 2001). Particularly strong is the epidemiological evidence in
regard to the development of depression and anxiety disorders (Heim and Nemeroff, 2001). Indeed, accumulated data suggests that various stressors such as physical abuse, sexual abuse, parental loss, and even prenatal stress are all correlated with the development of severe mood and anxiety disorders in adulthood (Heim and Nemeroff, 2001). Furthermore, the clinical presentation of psychiatric disorders is often associated with acute life events or ongoing stress in adulthood (Heim and Nemeroff, 2001). In toto, these data lead to a hypothesis whereby earlylife stressors lead to a state of enhanced vulnerability, and that adult mood and anxiety disorders present when later-life events have effects on the vulnerable brain of these individuals (Heim and Nemeroff, 2001). As witnessed by multiple studies of discordant monozygotic t w i n s - in addition to the aforementioned studies - where one has the disorder and the other does n o t - nonmendelian mechanisms must be operative to control behavior in genetically identical populations (Gottesman et al., 1982). A critical question thus becomes what are the mechanism by which early-life events regulate long-term changes in behavior and sustained differences in gene expression. Genetic vulnerability factors undoubtedly play a critical role, but these abundant d a t a - from multiple studies of discordant monozygotic twins - have shown that nongenetic (and more specifically nonmendelian genetic factors) are also critical mediators for the phenotypic expression of psychiatric diseases. Similarly, preclinical studies utilizing animals have found that early-life stress produces long-lasting and sometimes profound biochemical and behavioral changes that persist for a lifetime. While there are undoubtedly mendelian genetic contributions (both susceptibility and protective), which effect the impact of neonatal stressors on brain development, it is noteworthy that recent studies have also demonstrated nongenomic transmission across generations of not only maternal behavior, but also stress responses in rodents (Francis et al., 1999). Additional studies utilizing genetically identical inbred animals have identified specific environmental events, which can result in permanent alterations in behavior, gene expression, and subsequent responses to stimuli such as stress (Heim and Nemeroff, 2001; Sanchez et al., 2001). Specifically, a growing body of data has demonstrated that neonatal stress can have a major
763 impact on brain development, in particular by bringing about persistent changes in CRF-containing neurons, the HPA axis, the serotonergic system, the noradrenergic system, and the sympathetic nervous system (Graham et al., 1999). One well-studied model relies upon a maternal separation paradigm, wherein neonatal rodents are separated from their mother for defined periods. Behavioral, gene expression, and hormone production, changes have been noted to result from this paradigm, suggesting that there are epigenetic mechanisms that regulate these differential responses (Meaney, 2001). An additional line of studies by Meaney and colleagues has even documented that the degree of parental care that a newborn rat pup receives has lasting effects. This group has accumulated a large amount of data based on identifying litters in which pups were analyzed based upon the degree of maternal licking and grooming, followed by separation of groups receiving a standard deviation above and below the mean. Specific findings in these animals include lowered glucocorticoid receptor levels, increased HPA activity, and differential reactivity to stress (Ladd et al., 2000; Meaney, 2001; Weaver et al., 2001).
W h a t are the mechanisms by which early-life stress - both in humans and animals - leads to a state o f enhanced vulnerability?
The evidence discussed so far describe the clinical and preclinical evidence that nongenetic factors may play a role in behavioral and biochemical responses in the brain. In most areas of the brain neurons are not replaced. Thus, permanent and semipermanent modifications to deoxyribonucleic acid (DNA) that occur in early life, which affect gene transcription, could have downstream effects that persist for a lifetime- thus being temporally distant from the initial event. Mechanisms of gene regulation that are not determined solely by DNA base pair sequence are termed epigenetic and likely play a role in the formation of cellular memory and the modulation of neural circuitry in a manner that alters lifetime cellular and behavioral responses in an organism. Mendelian (DNA base pair) genetics involve inheritance patterns of nucleotide (cytosine, guanine, thymine, and adenine) inheritance. This is determined
almost exclusively by the gametes from the parents (except for spontaneous mutations, trinucleotide repeats, etc.). Whereas epigenetics (or the epigenome) refers to factors such as cytosine methylation and the affinity of DNA regions to nucleosomes (made up of histones). This type of inheritance can dramatically influence gene expression. The true extent of the dynamic mechanisms responsible is unknown, and is an active area of research. However, it is known to involve the interplay of transcription factors interacting with covalent DNA modifications, such as cytosine methylation, and the accessibility of DNA that is regulated by histone acetylation (Petronis, 2001; Geiman and Robertson, 2002). These epigenetic mechanisms are likely involved in modulating how previous experience may regulate subsequent behavioral responses (Meaney, 2001). Clearly, environmental factors play a role, but much of this may be primarily through epigenetic modifications. Thus, epigenetic interactions are likely prominent in gene expression, and formation of cellular memory. Indeed, work by Meaney and colleagues (with their grooming and licking model of parental care discussed above) has recently begun to document that some of the resultant changes - in particular GR regulation- may be mediated by epigenetic gene regulatory events (Weaver et al., 2002). The regulation of GR levels appears to be under the control of differential methylation of regions of the promoter, and the degree of parental care appears to regulate the methylation of this r e g i o n - and thus long-term gene expression (Weaver et al., 2002). A potential interesting avenue of research may be the role that early-life stress plays in regulating changes in neurotrophic signaling cascades. Epigenetic factors may play a role in regulating these cascades thereby contributing to a long-term state of reduced cellular resilience, and ultimately to phenotypic expression of disease. Future experiments will help to further delineate the effect of stress on neurotrophic molecules, the regulation of BDNF by stress, and provide a cellular model for studying these effects. They will additionally lay groundwork for potential future experiments addressing specific mechanisms of epigenetic gene regulation (such as cytosine methylation and histone acetylation) with specific relevance for studying gene-environment interactions in the pathophysiology of mood disorders, and suggest
764 mechanisms by which behavior can be regulated by epigenetic "marks," which are stable and permanent for the life of the organism.
Evidence that mood stabilizing and antidepressant medications regulate intracellular signaling pathways that exert critical neurotrophic] neuroprotective effects Noting the effects of stress on the brain, brain function, and behavior in preclinical models it is notable that the brains of patients with mood disorders show macroscopic and microscopic alterations that suggest impairments in neuroplasticity and cellular resilience. It is beyond the scope of this chapter to review these findings in depth; however, the evidence derives from postmortem and in vivo imaging. Studies utilizing neuroimaging report a decrease in frontal and temporal gray matter and an increase in ventricular size in patients with mood disorders (Drevets et al., 1997, 1999). Many studies also report a decrease in the size of some neuronal structures, including the hippocampus and portions of the basal ganglia (Drevets et al., 1999; Rajkowska, 2002). Functional imaging additionally reveals multiple abnormalities of regional cerebral blood flow and glucose metabolism in limbic and prefrontal cortical structures in mood disorders (Drevets et al., 1999; Drevets, 2000; Manji et al. 2001 a). Recent evidence using stereology techniques has identified decreased size and/or density of neurons, and decreased number and density of glial cells in portions of the anterior cingulate cortex, orbital frontal cortex, and dorsal lateral prefrontal cortex, as well changes in the hippocampus and amygdala, in patients with mood disorders (Rajkowska, 2000, 2002). Thus, both neuroimaging and postmortem findings suggest that the pathology of mood disorders may involve structural, as well as functional, changes in the brain. While there is not total reproducibility among studies, differences likely represent variations of experimental design, and as would be expected in heterogenous disorders such as mood disorders, in patient populations. Thus, research is required in order to understand if subtypes of depression, or mood disorders, are associated with any particular abnormality (Lenox et al., 2002).
As discussed more extensively earlier, endogenous neurotrophic factors are necessary for survival and functioning of neurons (Mamounas et al., 1995). They increase cell survival by providing necessary trophic support for growth, but also by exerting inhibitory effects on cell death cascades (Riccio et al., 1999). Evidence suggests that mood stabilizers and antidepressants may regulate these pathways. Antidepressant treatment in rats increases CREB phosphorylation and CREB-mediated gene expression in mouse limbic brain regions (Thome et al., 2000). Different classes of chronic antidepressant treatments, including NA and SSRIs and electroconvulsive seizure, upregulates CREB and BDNF expression, indicating that the CREB cascade and BDNF are common post-receptor targets of these therapeutic agents (Nibuya et al., 1995, 1996); furthermore the increase was only seen with chronic use, thus corresponding to the onset of action of these medications. More evidence that relates upregulation of these pathways and antidepressant utilization comes from antidepressant-like performance in behavioral models (Duman et al., 1999). Thus, it was observed that CREB overexpression in the dentate gyrus or BDNF injection results in an antidepressant-like effect in the learned-helplessness paradigm and the forced swim test model of antidepressant efficacy in rats (Siuciak et al., 1997; Chen et al., 2001; Shirayama et al., 2002). Chronic antidepressant treatment also results in an increase in the number of newly formed neurons (i.e., neurogenesis) in the hippocampus of rats. Another series of studies involving mood stabilizing medications lithium and valproic acid have demonstrated that these medications activate the ERK MAP kinase cascade (Yuan et al., 2001; Gould et al., 2002a). Chronic lithium or valproic acid robustly increases the levels of activated ERK in the frontal cortex and hippocampus of rats (G. Chen and H. K. Manji, unpublished observations). As described earlier, this pathway regulates the expression of the neuroprotective protein Bcl-2, and chronic treatment of rats with either lithium or valproic acid produces a twofold increase of Bcl-2 levels in the frontal cortex (Chen et al., 1999). Furthermore, chronic lithium treatment increases Bcl-2 levels in the mouse hippocampus (Chen et al., 2000a), and in cerebellar granule cells in culture
765 (Chen and Chuang, 1999). Valproic acid also increases Bcl-2 levels in human cells of neuronal origin (Yuan et al., 2001). It has also been observed that lithium is neuroprotective in animal models of ischemia, Huntington's disease, promotes neurogenesis in the hippocampus of rats, and is neuroprotective in many cell culture models (Manji et al., 1999; Chuang et al., 2002). Valproic acid also exerts neuroprotective actions in a number of cellular models including glutamate toxicity, [3-amyloid toxicity, and following exposure to other toxins (Bruno et al., 1995; Mark et al., 1995; Manji et al., 2000a; Hashimoto et al.,
2002). Thus, regulation of neurotrophic pathways is seen with both classes of medications (i.e., antidepressants and mood stabilizers) in limbic and frontal brain regions (Duman et al., 2000; Manji et al., 2000b). However, it is likely that therapeutic responses are generated by changes in specific cell types, and in neural pathways specific to each disorder. Proof from clinical studies are required to validate whether these actions are clinically relevant for the treatment of mood disorders. Based upon the above evidence longitudinal clinical studies were undertaken to address whether lithium produces effects consistent with changes in neuroplasticity. It was found using proton magnetic resonance spectroscopy (MRS) that chronic (4 week) lithium administration at therapeutic doses increases N-acetyl-aspartate (NAA, generally considered a marker of neuronal viability) concentration in the human brain in vivo (Moore et al., 2000a). These findings provide intriguing indirect support for the contention that: similar to the findings observed in the rodent brain and in human neuronal cells in culture, chronic lithium increases neuronal viability/ function in the human brain. As a follow up of the NAA findings, brain tissue volumes were examined using high-resolution three-dimensional MRI and validated quantitative brain tissue segmentation methodology to identify and quantify the various components by volume, including total brain white and gray matter content. This study revealed that 4 weeks of lithium at therapeutic doses significantly increases total gray matter content in the human brain of patients with bipolar disorder (Moore et al., 2000b). No significant changes were observed in
white matter volume or regional cerebral water content, thereby providing strong evidence that the observed increases in gray matter content are likely due to neurotrophic effects. Conclusions
Despite the collective logic of the adaptive mechanisms and impaired cellular resilience mentioned in this chapter, there are heterogeneous arrays of processes, involving intra- and intercellular actions, adaptations in neurons, glia, vasculature and peripheral organs. This chapter only represents a partial review of the cellular adaptive responses during stress. By understanding both the adaptations of the nervous system and the limits it faces in defending itself, efficacious clinical therapies may well emerge. Most importantly, impairments of structural plasticity and cellular resilience have also been implicated in the preclinic and clinic studies of mood disorders. Furthermore, antidepressants and mood stabilizers exert major effects on signaling pathways, which regulate neuroplasticity and cell survival (see review by (Manji et al., 2000c)). All of these findings have generated considerable excitement among the clinical neuroscience community, and are reshaping views about the neurobiological underpinnings of these stress-related disorders (Manji et al., 2000d, 2001b; D'Sa and Duman 2002; Nestler et al., 2002; Young, 2002). Abbreviations
NMDA CRF HPA GR ECT mRNA BDNF MAPK (PI-3K) CNS LTP MR ACTH
N-methyl-D-aspartate corticotrophic hormone-releasing factor; also called corticotrophin-releasing factor hypothalamic-pituitary-adrenal glucocorticoid receptor electroconvulsive shock therapy messenger ribonucleic acid brain-derived neurotrophic factor mitogen-activated protein kinase phosphotidylinositol-3 kinase central nervous system long-term potentiation mineralocorticoid receptor adrenocorticotrophic hormone
766 NGF NT Trk P75 PKA PKC SOS RSK CREB cAMP BAD Bcl-xl Bcl-2 MPTP GSK FOX FKHR AFX NF-KB GABA AMPA mEPSC mlPSC IR ION MEK ERK MRS NAA PTSD SSRI NA
nerve growth factor neurotrophin tyrosine kinase pan-neurotrophin receptor protein kinase A protein kinase C son of sevenless ribosomal $6 kinases cAMP response element binding protein cyclic adenosine monophosphate Bcl-xl/Bcl-2 Associated Death promoter B-cell lymphoma/leukemia-xl B-cell lymphoma/leukemia-2 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine glycogen synthase kinase Forkhead box transcription factor Forkhead transcriptional regulators? ALL1 fused gene from chromosome X nuclear factor ~:B gamma-aminobutyric acid alpha-amino- 3- hydro xy- 5-methylis o xazole-4-propionate miniature excitatory postsynaptic currents miniature inhibitory postsynaptic currents immunoreactivity isthmo-optic nucleus mitogen-activated/ERK-activating kinase extracellular response kinase magnetic resonance spectroscopy N-acetyl-aspartate post-traumatic stress disorder selective serotonin reuptake inhibitor noradrenaline
Acknowledgments We would like to acknowledge the support of the Intramural Research Program of the National Institute of Mental Heath and the Stanley Medical Research Institute. Due to space limitations we often cited review papers and apologize to those authors whose original data was not included.
References Atkins, C.M., Selcher, J.C., Petraitis, J.J., Trzaskos, J.M. and Sweatt, J.D. (1998) The MAPK cascade is required for mammalian associative learning. Nat. Neurosci., 1: 602-609. Bijur, G.N., De Sarno, P. and Jope, R.S. (2000) Glycogen synthase kinase-3beta facilitates staurosporine- and heat shock-induced apoptosis. Protection by lithium. J. Biol. Chem., 275: 7583-7590. Blum, S., Moore, A.N., Adams. F. and Dash, P.K. (1999) A mitogen-activated protein kinase cascade in the CA1/CA2 subfield of the dorsal hippocampus is essential for long-term spatial memory. J. Neurosci., 19: 3535-3544. Bonni, A., Brunet, A., West, A.E., Datta, S.R., Takasu, M.A. and Greenberg, M.E. (1999) Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms. Science, 286: 1358-1362. Bremner, J.D., Randall, P., Scott, T.M., et al. (1995) MRIbased measurement of hippocampal volume in patients with combat-related posttraumatic stress disorder. Am. J. Psychiatry, 152: 973-981. Bremner, J.D., Randall, P., Vermetten, E., et al. (1997) Magnetic resonance imaging-based measurement of hippocampal volume in posttraumatic stress disorder related to childhood physical and sexual abuse- a preliminary report. Biol. Psychiatry, 41: 23-32. Brunet, A., Bonni, A., Zigmond, M.J., et al. (1999) Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell, 96: 857-868. Brunet, A., Datta, S.R. and Greenberg, M.E. (2001) Transcription-dependent and -independent control of neuronal survival by the PI3K-Akt signaling pathway. Curr. Opin. Neurobiol., 11: 297-305. Bruno, V., Sortino, M.A., Scapagnini, U., Nicoletti, F. and Canonico, P.L. (1995) Antidegenerative effects of Mg(2+)valproate in cultured cerebellar neurons. Funct. Neurol., 10: 121-130. Brunson, K.L., Eghbal-Ahmadi, M., Bender, R., Chen, Y. and Baram, T.Z. (2001) Long-term, progressive hippocampal cell loss and dysfunction induced by early-life administration of corticotropin-releasing hormone reproduce the effects of early-life stress. Proc. Natl. Acad. Sci. USA, 98: 8856-8861. Cameron, H.A. and McKay, R.D. (1999) Restoring production of hippocampal neurons in old age. Nat. Neurosci., 2: 894-897. Charney, D.S. and Bremner, J.D. (1999) The neurobiology of anxiety disorders. In: Bunney, B.S. (Ed.), Neurobiology of Mental Illness. Oxford University Press, New York, pp. 494-517. Chemerinski, E. and Robinson, R.G. (2000) The neuropsychiatry of stroke. Psychosomatics, 41: 5-14. Chen, A.C., Shirayama, Y., Shin, K.H., Neve, R.L. and Duman, R.S. (2001) Expression of the cAMP response
767 element binding protein (CREB) in hippocampus produces an antidepressant effect. Biol. Psychiatry, 49: 753-762. Chen, D.F. and Tonegawa, S. (1998) Why do mature CNS neurons of mammals fail to re-establish connections following injury- functions of bcl-2. Cell Death Differ., 5:816-822. Chen, D.F., Schneider, G.E., Martinou, J.C. and Tonegawa, S. (1997) Bcl-2 promotes regeneration of severed axons in mammalian CNS. Nature, 385: 434-439. Chen, G., Zeng, W.Z., Yuan, P.X., et al. (1999) The moodstabilizing agents lithium and valproate robustly increase the levels of the neuroprotective protein bcl-2 in the CNS. J. Neurochem., 72: 879-882. Chen, G., Rajkowska, G., Du, F., Seraji-Bozorgzad, N. and Manji, H.K. (2000a) Enhancement of hippocampal neurogenesis by lithium. J. Neurochem., 75: 1729-1734. Chen, G., Masana, M.I. and Manji, H.K. (2000b) Lithium regulates PKC-mediated intracellular cross-talk and gene expression in the CNS in vivo. Bipolar Disorder 2000 Sep; 2(3 Pt 2)217-236. Review. Chen, R.W. and Chuang, D.M. (1999) Long term lithium treatment suppresses p53 and Bax expression but increases Bcl-2 expression. A prominent role in neuroprotection against excitotoxicity. J. Biol. Chem., 274: 6039-6042. Chierzi, S., Strettoi, E., Cenni, M.C. and Maffei, L. (1999) Optic nerve crush: axonal responses in wild-type and bcl-2 transgenic mice. J. Neurosci., 19: 8367-8376. Chuang, D.M., Chen, R., Chalecka-Franaszek, E., et al. (2002) Neuroprotective effects of lithium in cultured cells and animal model of diseases. Bipolar Disorders, 4: 129-136. Crow, T., Xue-Bian, J.J., Siddiqi, V., Kang, Y. and Neary, J.T. (1998) Phosphorylation of mitogen-activated protein kinase by one-trial and multi-trial classical conditioning. J. Neurosci., 18: 3480-3487. Datta, S.R., Dudek, H., Tao, X., et al. (1997) Akt phosphorylation of BAD couples survival signals to the cell - intrinsic death machinery. Cell, 91:231-241. DeVries, A.C., Joh, H.D., Bernard, O., et al. (2001) Social stress exacerbates stroke outcome by suppressing Bcl-2 expression. Proc. Natl. Acad. Sci. USA, 98: 11824-11828. Dijkers, P.F., Medema, R.H., Lammers, J.W., Koenderman, L. and Coffer, P.J. (2000) Expression of the pro-apoptotic Bcl-2 family member Bim is regulated by the forkhead transcription factor FKHR-L1. Curr. Biol., 10: 1201-1204. Drevets, W.C. (2000) Neuroimaging studies of mood disorders. Biol. Psychiatry, 48: 813-829. Drevets, W.C., Price, J.L., Simpson Jr., J.R., et al. (1997) Subgenual prefrontal cortex abnormalities in mood disorders. Nature, 386: 824-827. Drevets, W.C., Gadde, K. and Krishnan, R. (1999) Neuroimaging studies of depression. In: Bunney, B.S. (Ed.), Neurobiology of Mental Illness. Oxford University Press, New York, NY, pp 394-418. D'Sa, C. and Duman, R. (2002) Antidepressants and neuroplasticity. Bipolar Disorder, 4: 183.
Duman, R.S. (1998) Novel therapeutic approaches beyond the serotonin receptor. Biol. Psychiatry, 44: 324-335. Duman, R.S., Malberg, J. and Thome, J. (1999) Neural plasticity to stress and antidepressant treatment. Biol. Psychiatry, 46: 1181-1191. Duman, R.S., Malberg, J., Nakagawa, S. and D'Sa, C. (2000) Neuronal plasticity and survival in mood disorders. Biol. Psychiatry, 48: 732-739. English, J.D. and Sweatt, J.D. (1996) Activation of p42 mitogen-activated protein kinase in hippocampal long term potentiation. J. Biol. Chem., 271: 24329-24332. English, J.D. and Sweatt, J.D. (1997) A requirement for the mitogen-activated protein kinase cascade in hippocampal long term potentiation. J. Biol. Chem., 272: 19103-19106. Eriksson, P.S., Perfilieva, E., Bjork-Eriksson, T., et al. (1998) Neurogenesis in the adult human hippocampus. Nat. Med., 4: 1313-1317. Finkbeiner, S. (2000) CREB couples neurotrophin signals to survival messages. Neuron, 25:11-14. Francis, D., Diorio, J., Liu, D. and Meaney, M.J. (1999) Nongenomic transmission across generations of maternal behavior and stress responses in the rat. Science, 286: 1155-1158. Fryer, R.H., Kaplan, D.R., Feinstein, S.C., Radeke, M.J., Grayson, D.R. and Kromer, L.F. (1996) Developmental and mature expression of full-length and truncated TrkB receptors in the rat forebrain. J. Comp. Neurol., 374: 21-40. Fukunaga, K. and Miyamoto, E. (1998) Role of MAP kinase in neurons. Mol. Neurobiol., 16: 79-95. Gass, P., Kretz, O., Wolfer, D.P., et al. (2000) Genetic disruption of mineralocorticoid receptor leads to impaired neurogenesis and granule cell degeneration in the hippocampus of adult mice. EMBO Rep., 1: 447-451. Geiman, T.M. and Robertson, K.D. (2002) Chromatin remodeling, histone modifications, and DNA methylationhow does it all fit together? J. Cell. Biochem., 87:117-125. Gesing, A., Bilang-Bleuel, A., Droste, S.K., Linthorst, A.C., Holsboer, F. and Reul, J.M. (2001) Psychological stress increases hippocampal mineralocorticoid receptor levels: involvement of corticotropin-releasing hormone. J. Neurosci., 21: 4822-4829. Gilbertson, M.W., Shenton, M.E., Ciszewski, A., et al. (2002) Smaller hippocampal volume predicts pathologic vulnerability to psychological trauma. Nat. Neurosci., 5: 1242-1247. Gonzalez, M., Ruggiero, F.P., Chang, Q., et al. (1999) Disruption of Trkb-mediated signaling induces disassembly of postsynaptic receptor clusters at neuromuscular junctions. Neuron, 24: 567-583. Gottesman, I.I., Shields, J. and Hanson, D.R. (1982) Schizophrenia, the epigenetic puzzle. Cambridge University Press, Cambridge, New York. Gould, E., Tanapat, P., Rydel, T. and Hastings, N. (2000) Regulation of hippocampal neurogenesis in adulthood. Biol. Psychiatry, 48: 715-720.
768 Gould, T., Chen, G. and Manji, H. (2002a) Mood stabilizer psychopharmacology. Clinical Neuroscience Research, 2: 193-212. Gould, T., Du, J., Yuan, P., Chen, G. and Manji, H. (2002b) Evidence suggesting activation of the Wnt signaling pathway in rat brain after lithium and valproic acid administration. Biol. Psychiatry, 51: 39S. Gould, T.D. and Manji, H.K. (2002) The Wnt signaling pathway in bipolar disorder. Neuroscientist, 8. Graham, Y.P., Heim, C., Goodman, S.H., Miller, A.H. and Nemeroff, C.B. (1999) The effects of neonatal stress on brain development: implications for psychopathology. Dev. Psychopathol., 11: 545-565. Gurvits, T.V., Shenton, M.E., Hokama, H., et al. (1996) Magnetic resonance imaging study of hippocampal volume in chronic, combat-related posttraumatic stress disorder. Biol. Psychiatry, 40:1091-1099. Hashimoto, R., Hough, C., Nakazawa, T., Yamamoto, T. and Chuang, D.M. (2002) Lithium protection against glutamate excitotoxicity in rat cerebral cortical neurons: involvement of NMDA receptor inhibition possibly by decreasing NR2B tyrosine phosphorylation. J. Neurochem., 80: 589-597. Heim, C. and Nemeroff, C.B. (2001) The role of childhood trauma in the neurobiology of mood and anxiety disorders: preclinical and clinical studies. Biol. Psychiatry, 49: 1023-1039. Hetman, M., Cavanaugh, J.E., Kimelman, D. and Xia, Z. (2000) Role of glycogen synthase kinase-3beta in neuronal apoptosis induced by trophic withdrawal. J. Neurosci., 20: 2567-2574. Holm, K.H., Cicchetti, F., Bjorklund, L., et al. (2001) Enhanced axonal growth from fetal human bcl-2 transgenic mouse dopamine neurons transplanted to the adult rat striatum. Neuroscience, 104: 397-405. Holsboer, F. (2000) The corticosteroid receptor hypothesis of depression. Neuropsychopharmacology, 23: 477-501. Jacobs, B.L., Praag, H. and Gage, F.H. (2000) Adult brain neurogenesis and psychiatry: a novel theory of depression. Mol. Psychiatry, 5: 262-269. Kempermann, G. (2002) Regulation of adult hippocampal neurogenesis - implications for novel theories of major depression. Bipolar Disord., 4: 17-33. Kempermann, G., Kuhn, H.G. and Gage, F.H. (1997) More hippocampal neurons in adult mice living in an enriched environment. Nature, 386: 493-495. Kollack-Walker, S., Day, H.E.W. and Akil, H. (2000) Central stress neurocircuits. In: Encyclopedia of Stress. Ed. G. Fink, Academic Press, San Diego. Kornhauser, J.M. and Greenberg, M.E. (1997) A kinase to remember: dual roles for MAP kinase in long-term memory. Neuron, 18: 839-842. Korte, S.M., De Kloet, E.R., Buwalda, B., Bouman, S.D. and Bohus, B. (1996) Antisense to the glucocorticoid receptor in
hippocampal dentate gyrus reduces immobility in forced swim test. Eur. J. Pharmacol., 301: 19-25. Kumar, A., Miller, D., Ewbank, D., et al. (1997) Quantitative anatomic measures and comorbid medical illness in late-life major depression. Am. J. Geriatr. Psychiatry, 5: 15-25. Ladd, C.O., Huot, R.L., Thrivikraman, K.V., Nemeroff, C.B., Meaney, M.J. and Plotsky, P.M. (2000) Long-term behavioral and neuroendocrine adaptations to adverse early experience. Prog. Brain Res., 122: 81-103. Lenox, R.H., Gould, T.D. and Manji, H.K. (2002) Endophenotype in bipolar disorder. Am. J. Med. Genet., 114: 391-406. Lopez, J.F., Chalmers, D.T., Little, K.Y. and Watson, S.J. (1998) A.E. Bennett Research Award. Regulation of serotoninlA, glucocorticoid, and mineralocorticoid receptor in rat and human hippocampus: implications for the neurobiology of depression. Biol. Psychiatry, 43: 547-573. Lyons, D.M. (2002) Stress, depression, and inherited variation in primate hippocampal and prefrontal brain development. Psychopharmacology Bulletin, 36: 27-43. Lyons, D.M., Yang, C., Sawyer-Glover, A.M., Moseley. M.E. and Schatzberg, A.F. (2001) Early life stress and inherited variation in monkey hippocampal volumes. Arch. Gen. Psychiatry, 58:1145-1151. Maggirwar, S.B., Sarmiere, P.D., Dewhurst, S. and Freeman, R.S. (1998) Nerve growth factor-dependent activation of NFkappaB contributes to survival of sympathetic neurons. J. Neurosci., 18: 10356-10365. Mamounas, L.A., Blue, M.E., Siuciak, J.A. and Altar, C.A. (1995) Brain-derived neurotrophic factor promotes the survival and sprouting of serotonergic axons in rat brain. J. Neurosci., 15: 7929-7939. Manev, H., Uz, T., Smalheiser, N.R. and Manev, R. (2001) Antidepressants alter cell proliferation in the adult brain in vivo and in neural cultures in vitro. Eur. J. Pharmacol., 411: 67-70. Manji, H.K. and Chen, G. (2002) PKC, MAP kinases and the bcl-2 family of proteins as long-term targets for mood stabilizers. Mol. Psychiatry, 7(Suppl. 1): $46-56. Manji, H. and Duman, R. (2001a) Impairments of neuroplasticity and cellular resilience in severe mood disorder: implications for the development of novel therapeutics. Psychopharmacology Bulletin, 35: 5-49. Manji, H.K., Moore, G.J. and Chen, G. (1999) Lithium at 50: have the neuroprotective effects of this unique cation been overlooked? Biol. Psychiatry, 46: 929-940. Manji, H.K., Moore, G.J. and Chen, G. (2000a) Clinical and preclinical evidence for the neurotrophic effects of mood stabilizers: implications for the pathophysiology and treatment of manic-depressive illness. Biol. Psychiatry, 48: 740-754. Manji, H.K., Moore, G.J., Rajkowska, G. and Chen, G. (2000b) Neuroplasticity and cellular resilience in mood disorders. Mol. Psychiatry, 5: 578-593.
769 Manji, H.K., Moore, G.J. and Chen, G. (2000d) Lithium upregulates the cytoprotective protein Bcl-2 in the CNS in vivo: a role for neurotrophic and neuroprotective effects in manic depressive illness. J. Clin. Psychiatry, 61: 82-96. Manji, H.K., Moore, G.J. and Chen, G. (2001a) Bipolar disorder: leads from the molecular and cellular mechanisms of action of mood stabilizers. Br. J. Psychiatry, 178: s107119. Manji, H.K., Drevets, W.C. and Charney, D.S. (2001b) The cellular neurobiology of depression. Nat. Med., 7: 541-547. Manji, H.K., Moore, G.J. and Chen, G. (2001c) Bipolar disorder: leads from the molecular and cellular mechanisms of action of mood stabilizers. Br. J. Psychiatry, Suppl 41: s107-119. Mark, R.J., Ashford, J.W., Goodman, Y. and Mattson, M.P. (1995) Anticonvulsants attenuate amyloid beta-peptide neurotoxicity, Ca2+ deregulation, and cytoskeletal pathology. Neurobiol. Aging, 16:187-198. Martin, K.C., Michael, D., Rose, J.C., et al. (1997) MAP kinase translocates into the nucleus of the presynaptic cell and is required for long-term facilitation in Aplysia. Neuron, 18: 899-912. Matsubara, M., Kusubata, M,. Ishiguro, K., Uchida, T., Titani, K. and Taniguchi, H. (1996) Site-specific phosphorylation of synapsin I by mitogen-activated protein kinase and Cdk5 and its effects on physiological functions. J. Biol. Chem., 271: 21108-21113. McEwen, B.S. (1999) Stress and hippocampal plasticity. Annu. Rev. Neurosci., 22: 105-122. McQuade, R. and Young, A.H. (2000) Future therapeutic targets in mood disorders: the glucocorticoid receptor. Br. J. Psychiatry, 177: 390-395. Meaney, M.J. (2001) Maternal care, gene expression, and the transmission of individual differences in stress reactivity across generations. Annu. Rev. Neurosci., 24:1161-92. Mesulam, M.M. (1999) Neuroplasticity failure in Alzheimer's disease: bridging the gap between plaques and tangles. Neuron, 24: 521-529. Michaloudi, H.C., el Majdoubi, M., Poulain, D.A., Papadopoulos, G.C. and Theodosis, D.T. (1997) The noradrenergic innervation of identified hypothalamic magnocellular somata and its contribution to lactationinduced synaptic plasticity. J. Neuroendocrinol., 9: 17-23. Moore, G.J., Bebchuk, J.M., Hasanat, K., et al. (2000a) Lithium increases N-acetyl-aspartate in the human brain: in vivo evidence in support of bcl-2's neurotrophic effects? Biol. Psychiatry, 48: 1-8. Moore, G.J., Bebchuk, J.M., Wilds, I.B., Chen, G., Manji, H.K. and Menji, H.K. (2000b) Lithium-induced increase in human brain grey matter. Lancet, 356: 1241-1242. Murphy, T.H., Blatter, L.A., Bhat, R.V., Fiore, R.S., Wier, W.G. and Baraban, J.M. (1994) Differential regulation of calcium/calmodulin-dependent protein kinase II and p42
MAP kinase activity by synaptic transmission. J. Neurosci., 14: 1320-1331. Murray, C.J. and Lopez, A.D. (1997) Global mortality, disability, and the contribution of risk factors: global burden of disease study. Lancet, 349: 1436-1442. Nestler, E.J. (1998) Antidepressant treatments in the 21st century. Biol. Psychiatry, 44: 526-533. Nestler, E.J., Barrot, M., DiLeone, R.J., Eisch, A.J., Gold, S.J. and Monteggia, L.M. (2002) Neurobiology of depression. Neuron, 34:13-25. Nibuya, M., Morinobu, S. and Duman, R.S. (1995) Regulation of BDNF and trkB mRNA in rat brain by chronic electroconvulsive seizure and antidepressant drug treatments. J. Neurosci., 15: 7539-7547. Nibuya, M., Nestler, E.J. and Duman, R.S. (1996) Chronic antidepressant administration increases the expression of cAMP response element binding protein (CREB) in rat hippocampus. J. Neurosci., 16: 2365-2372. Nibuya, M., Takahashi, M., Russell, D.S. and Duman, R.S. (1999) Repeated stress increases catalytic TrkB mRNA in rat hippocampus. Neurosci. Lett., 267: 81-84. Oh, Y.J., Swarzenski, B.C. and O'Malley, K.L. (1996) Overexpression of Bcl-2 in a murine dopaminergic neuronal cell line leads to neurite outgrowth. Neurosci. Lett., 202: 161-164. Ontiveros, A., Fontaine, R., Breton, G., Elie, R., Fontaine, S. and Dery, R. (1989) Correlation of severity of panic disorder and neuroanatomical changes on magnetic resonance imaging. J. Neuropsychiatry Clin. Neurosci., 1: 404-408. Patapoutian, A. and Reichardt, L.F. (2001) Trk receptors: mediators of neurotrophin action. Curr. Opin. Neurobiol., 11: 272-280. Patel, P.D., Lopez, J.F., Lyons, D.M., Burke, S., Wallace, M. and Schatzberg, A.F. (2000) Glucocorticoid and mineralocorticoid receptor mRNA expression in squirrel monkey brain. J. Psychiatr. Res., 34: 383-392. Petronis, A. (2001) Human morbid genetics revisited: relevance of epigenetics. Trends Genet., 17: 142-146. Poo, M.M. (2001) Neurotrophins as synaptic modulators. Nat. Rev. Neurosci., 2: 24-32. Rajkowska, G. (2000) Postmortem studies in mood disorders indicate altered numbers of neurons and glial cells. Biol. Psychiatry, 48: 766-777. Rajkowska, G. (2002) Cell pathology in bipolar disorder. Bipolar Disord., 4: 105-116. Reul, J.M. and Holsboer, F. (2002) Corticotropin-releasing factor receptors 1 and 2 in anxiety and depression. Curr. Opin. Pharmacol., 2: 23-33. Riccio, A., Pierchala, B.A., Ciarallo, C.L. and Ginty, D.D. (1997) An NGF-TrkA-mediated retrograde signal to transcription factor CREB in sympathetic neurons. Science, 277:1097-1100. Riccio, A., Ahn, S., Davenport, C.M., Blendy, J.A. and Ginty, D.D. (1999) Mediation by a CREB family transcription
770 factor of NGF-dependent survival of sympathetic neurons. Science, 286: 2358-2361. Roberson, E.D., English, J.D. and Sweatt, J.D. (1996) A biochemist's view of long-term potentiation. Learn Mem., 3: 1-24. Roberson, E.D., English, J.D., Adams, J.P., Selcher, J.C., Kondratick, C. and Sweatt, J.D. (1999) The mitogenactivated protein kinase cascade couples PKA and PKC to cAMP response element binding protein phosphorylation in area CA1 of hippocampus. J. Neurosci., 19: 43374348. Robinson, M.J., Stippec, S.A., Goldsmith, E., White, M.A. and Cobb, M.H. (1998) A constitutively active and nuclear form of the MAP kinase ERK2 is sufficient for neurite outgrowth and cell transformation. Curr. Biol., 8:1141-1150. Sanchez, M.M., Young, L.J., Plotsky, P.M. and Insel, T.R. (2000) Distribution of corticosteroid receptors in the rhesus brain: relative absence of glucocorticoid receptors in the hippocampal formation. J. Neurosci., 20: 4657-4668. Sanchez, M.M., Ladd, C.O. and Plotsky, P.M. (2001) Early adverse experience as a developmental risk factor for later psychopathology: evidence from rodent and primate models. Dev. Psychopathol., 13: 419-449. Sapolsky, R.M. (1996) Stress, glucocorticoids, and damage to the nervous system: the current state of confusion. Stress, 1: 1-19. Sapolsky, R.M. (2000a) Glucocorticoids and hippocampal atrophy in neuropsychiatric disorders. Arch. Gen. Psychiatry, 57: 925-935. Sapolsky, R.M. (2000b) The possibility of neurotoxicity in the hippocampus in major depression: a primer on neuron death. Biol. Psychiatry, 48: 755-765. Selye, H. (1956) The Stress of Life. McGraw-Hill, New York. Shirayama, Y., Chen, A.C., Nakagawa, S., Russell, D.S. and Duman, R.S. (2002) Brain-derived neurotrophic factor produces antidepressant effects in behavioral models of depression. J. Neurosci., 22: 3251-3261. Simmons, N.E., Alden, T.D., Thorner, M.O. and Laws Jr., E.R. (2001) Serum cortisol response to transsphenoidal surgery for Cushing disease. J. Neurosurg., 95: 1-8. Siuciak, J.A., Lewis, D.R., Wiegand, S.J. and Lindsay, R.M. (1997) Antidepressant-like effect of brain-derived neurotrophic factor (BDNF). Pharmacol. Biochem. Behav., 56: 131-137. Smith, M.A., Makino, S., Kvetnansky, R. and Post, R.M. (1995) Stress and glucocorticoids affect the expression of brain-derived neurotrophic factor and neurotrophin-3 mRNAs in the hippocampus. J. Neurosci., 15: 1768-1777. Sousa, N., Lukoyanov, N.V., Madeira, M.D., Almeida, O.F. and Paula-Barbosa, M.M. (2000) Reorganization of the morphology of hippocampal neurites and synapses after stress-induced damage correlates with behavioral improvement. Neuroscience, 97: 253-266. Starkman, M.N., Giordani, B., Gebarski, S.S., Berent, S., Schork, M.A. and Schteingart, D.E. (1999) Decrease in
cortisol reverses human hippocampal atrophy following treatment of Cushing's disease. Biol. Psychiatry, 46: 1595-1602. Steckler, T., Rammes, G., Sauvage, M., van Gaalen, M.M., Weis, C., Zieglgansberger, W. and Holsboer, F. (2001) Effects of the monoamine oxidase A inhibitor moclobemide on hippocampal plasticity in GR-impaired transgenic mice. J. Psychiat. Res., 35(1): 29-42. Steffens, D.C. and Krishnan, K.R. (1998) Structural neuroimaging and mood disorders: recent findings, implications for classification, and future directions. Biol. Psychiatry, 43: 705-712. Steffens, D.C., Helms, M.J., Krishnan, K.R. and Burke, G.L. (1999) Cerebrovascular disease and depression symptoms in the cardiovascular health study. Stroke, 30:2159-2166. Stein, M.B., Koverola, C., Hanna, C., Torchia, M.G. and McClarty, B. (1997) Hippocampal volume in women victimized by childhood sexual abuse. Psychol. Med., 27: 951-959. Stern, J.E. and Armstrong, W.E. (1998) Reorganization of the dendritic trees of oxytocin and vasopressin neurons of the rat supraoptic nucleus during lactation. J. Neurosci., 18: 841-853. Suzuki, T., Okumura-Noji, K. and Nishida, E. (1995) ERK2type mitogen-activated protein kinase (MAPK) and its substrates in postsynaptic density fractions from the rat brain. Neurosci. Res., 22:277-285 Thome, J., Sakai, N., Shin, K., et al. (2000) cAMP response element-mediated gene transcription is upregulated by chronic antidepressant treatment. J. Neurosci., 20: 4030-4036. Trentani, A., Kuipers, S.D., Ter Horst, G.J. and Den Boer, J.A. (2002) Selective chronic stress-induced in vivo ERK1/2 hyperphosphorylation in medial prefrontocortical dendrites: implications for stress-related cortical pathology? Eur. J. Neurosci., 15(10): 1681-1691. van Praag, H., Kempermann, G. and Gage, F.H. (1999) Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nat. Neurosci., 2: 266-270. Weaver, I.C., La Plante, P., Weaver, S., et al. (2001) Early environmental regulation of hippocampal glucocorticoid receptor gene expression: characterization of intracellular mediators and potential genomic target sites. Mol. Cell. Endocrinol., 185: 205-218. Weaver, I.C.G., Cervoni, N., Szyf, M. and Meaney, M.J. (2002) Maternal behavior in infancy regulates methylation of hippocampal glucocorticoid receptor promoter. Abstract Soc. Neuroscience Annual Meeting, Vol. abstract number 866.9. Webster, O'Grady, Orthmann, et al. (1999) Decreased glucocorticoid receptor mRNA levels in individuals with depression, bipolar disorder and schizophrenia. Schizophrenia Research, 41:111.
771 Wellman, C.L. (2001) Dendritic reorganization in pyramidal neurons in medial prefrontal cortex after chronic corticosterone administration. J. Neurobiol., 49: 245-253. Withers, G.S. and Greenough, W.T. (1989) Reach training selectively alters dendritic branching in subpopulations of layer II-III pyramids in rat motor-somatosensory forelimb cortex. Neuropsychologia, 27: 61-69. Woodgett, J.R. (2001) Judging a protein by more than its name: gsk-3. Sci STKE, 2001: RE12. Xu, Q., Fawcett, T.W., Gorospe, M., Guyton, K.Z., Liu, Y. and Holbrook, N.J. (1997) Induction of mitogen-activated
protein kinase phosphatase-1 during acute hypertension. Hypertension, 30:106-111. Young, L.T. (2002) Neuroprotective effects of antidepressant and mood stabilizing drugs. J. Psychiatry Neurosci., 27: 8-9. Yuan, D., Komatsu, K., Tani, H., Cui, Z. and Kano, Y. (1998) Pharmacological properties of traditional medicines. XXIV. Classification of antiasthmatics based on constitutional predispositions. Biol. Pharm. Bull., 21:1169-1173. Yuan, P.X., Huang, L.D., Jiang, Y.M., Gutkind, J.S., Manji, H.K. and Chen, G. (2001) The mood stabilizer valproic acid activates mitogen-activated protein kinases and promotes neurite growth. J. Biol. Chem., 276:31674-31683.
This Page Intentionally Left Blank
SECTION 6
The Stressed Brain
This Page Intentionally Left Blank
T. Steckler, N.H. Kalin and J.M.H.M. Reul (Eds.) Handbook of Stress and the Brain, Vol. 15
ISBN 0-444-51173-3 Copyright 2005 Elsevier B.V. All rights reserved CHAPTER 6.1
Psychological and physiological stressors Krisztina J. Kovfics*, Ildik6 H. Mikl6s and Balfizs Bali Laboratory of Molecular Neuroendocrinology, Institute of Experimental Medicine, Hungarian Academy of Science, Szigony u. 43, Budapest H-1083, Hungary
Abstract: Stressors posed by the external and internal environment are divided into two main categories: physiological and psychological. These categories are not mutually exclusive and their classification is based on the differences in perception and registration of relevant stimuli and the afferent pathways that mediate and/or may modulate stress responses. This chapter summarizes and compares afferent mechanisms, activated circuits, as well as transcriptional, hormonal, and autonomic responses to physiological and psychological challenges. While both type of stressors initiate complex adaptive responses physiological stressors directly target homeostatic parameters, transduced via viscerosensory pathways, psychological stressors recruit various somatosensory and nociceptive afferentations, the information is processed through complex cortical and limbic circuits to include cognitive, learned, and emotional components. Functional anatomical mapping strategies that are based on the induction of immediate-early genes (i.e. c-fos) identified corticotropin-releasing factor (CRF)-synthesizing parvocellular neurons as a common target of both type of stressors. At the cellular level, double imaging technique revealed that even parvocellular neurosecretory neurons may respond differentially to categorically distinct paradigms. Significant differences have also been found at various medullary (nucleus of the solitary tract), subcortical, and limbic areas (amygdala and bed nucleus of stria terminalis) that involved in mediation of relevant information to the stress-related hypophysiotropic neurons. In addition, the chapter contains a brief summary on laboratory stressors used on human subjects.
presence of predators or changes in the individual's real or anticipated state in a social context. The concept of allostasis, introduced by Sterling and Eyer (1988), emphasizes the change ofsetpoints. McEwen broadened this concept and defined allostasis as a process maintaining stability through change and promoting adaptation and coping (McEwen and Stellar, 1993; McEwen, 1998a,b, 2000). At the time of birth of the stress concept, stressors have been regarded as what are now referred to as physiological stressors. Stressful stimuli provoke centrally mediated coordinated responses including neuroendocrine [i.e. activation of the hypothalamo-pituitary-adrenocortical (HPA) axis], autonomic (i.e. activation of the sympatho-adrenal system), and behavioral (i.e. flight or fight) changes. However, it became clear, that psychological challenges we, human beings, are experiencing every day are among the most powerful stimuli to induce these responses.
Introduction
Stressors have been defined by Selye as exogenous or endogenous challenges that threaten homeostasis (Selye, 1936; Selye, 1955). Indeed, vital parameters such as serum osmolarity and pH, extracellular sodium concentration, blood glucose levels, oxygen tension and core temperature should be kept in a narrow range around a f i x e d setpoint under tight homeostatic control. In addition to stressors that directly affect these physiological variables and require immediate corrections, there are challenges posed by the external or internal environment that initiate centrally mediated responses to serve adaptation. These situations involve failures to satisfy internal drives, aversive environmental stimuli, *Corresponding author Tel.: +36-1-210-9952; Fax: +36-1-210-9423; E-mail:
[email protected] 775
776
Stressor categories Stress paradigms used in the experimental stress research can be divided into two main categories: 1. Physiological stressors often referred to as homeostatic, systemic or physical stressors. These challenges target physiological parameters, their effect is mediated through well-defined internal or external receptor systems, the afferentation include viscerosensory pathways, activate subcortical autonomic circuits and directly affect stress-related motoneurons without significant cortical contribution. Examples include osmotic, metabolic, cardiovascular and immune stressors. Blood-borne signals [plasma osmolarity, immune mediators and certain hormones (i.e. angiotensin II and insulin)] are registered at the sensory circumventricular organs that lack the blood brain barrier (Katsuura et al., 1990; McKinley et al., 2003) and project to the paraventricular nucleus (PVN) (Kovacs and Sawchenko, 1993; Ericsson et al., 1994; Sawchenko et al., 2000). Blood pressure and volumerelated information is mediated through viscerosensory vagal and glossopharingeal inputs to the nucleus of the solitary tract (NTS) (Chan and Sawchenko, 1994; Chan and Sawchenko, 1998; Dampney and Horiuchi, 2003). Glucose-sensitive vagal inputs also reach NTS neurons that give rise ascending catecholaminergic pathways to the stress-related neurosecretory cells of the hypothalamus (Adachi et al., 1984; Nagase et al., 1993). Temperature(Scammell et al., 1993) and glucose (Williams et al., 2001) sensitive neurons were identified in the hypothalamus that may also provide direct inputs to the neurosecretory motoneurons (Fig. 1A). Thus, homeostatic challenges launch relative simple neuroendocrine reflexes by direct activation of the parvocellular CRF secreting neurons in the paraventricular nucleus to initiate the hormonal stress cascade. In addition to neuroendocrine responses, autonomic efferent projections to preganglionic cells groups in the medulla and spinal cord became also activated to induce adaptive cardiovascular, respiratory and other vegetative responses and include the activation of the sympatoadrenal system as well (Fig. 1B). 2. Psychological stressors are also called neurogenic, emotional, or processive stimuli. Registered
and initiated by complex somatosensory and nociceptive mechanisms, include less-specific exteroceptive and/or somatic inputs, processed through higher order brain circuits and involve learned, emotional, and cognitive components. Most often used emotional stressors are exposure to novel environment, restraint, immobilization, or footshock. The mechanisms that initiate a psychological stress response are less easily specified compared to physiological stress category. These probably involve spinal and trigeminal somatosensory and nociceptive pathways (spinothalamic-, trigeminothalamic-, medial leminiscus pathway, spinoreticulothalamic-, spinohypothalamic-, spinoreticular pathways). Scarce relevant viscerosensory information travel through the vagus and glossopharingeal nerves to the NTS (for review, see Sawchenko et al., 2000 and Pacak and Palkovits, 2001). Some of these pathways give collaterals to the hypothalamic effector neurons, but a great deal of relevant activational inputs is relayed at extrahypothalamic sites. Inputs from the extrahypothalamic areas do not reach directly the stressrelated motorneurons, their effect is integrated at the local GABA and glutamatergic interneuronal population in the periparaventricular region (Boudaba et al., 1996; Boudaba et al., 1997; Herman et al., 2002). Psychological stressors result in c-fos induction at cortical- (prefrontal) and subcortical limbic areas (septum, basolateral, and medial amygdala and bed nucleus of stria terminalis), midline thalamic nuclei, periaqueductal gray, locus coeruleus and catecholaminergic neurons in NTS and ventrolateral medulla (Cullinan et al., 1995; Li and Sawchenko, 1998; Dayas et al., 2001). Although these medullary cell groups are acknowledged as the major source of ascending aminergic pathways that stimulate CRF-secreting neurons in the PVN, their activation in response to psychogen stressors such as intermittent footshock is rather downstream to the recruitment of parvocellular stress-related neurons (Li et al., 1996; Sawchenko et al., 2000). Like physiological stressors, psychological and emotional stimuli also activate parvocellular neurosecretory motoneurons in the PVN and result in upregulation of neuropeptide gene expression in these cells (Ma et al., 1997; Viau and Sawchenko, 2002). Visceral responses to psychological stressors also include cardiovascular, respiratory, gastrointestinal
777
(A)[
CORTEX LIMBICCORTEX ,,
HYPOTHALAMUS[PVH-parvo)
Y T T T I.Tst I I Pi. I IcG I t NOCICEPTIV
VISCERAL BLOOD BORNE
SOMATOSENSORY
(B)[
I'GL ! t VISUAL
AUDITORY
CORTEX [
LIMBIC CORTEX
IA,..,to~./ oorvoI'V'~176176 I
~,
M~,~iol I.
l 1'
PITUITARY
t 1
PREGANGLIONIC NEURONS
ACTH
IML IDvc
vP
OXY ADRENAL [ Cortex I Medulla 1
ENDOCRINE
AUTONOMIC
BEHAVIORAL
Fig. 1. Schematic summary of afferent (A) and efferent (B) stress pathways. Abbreviations (A)- CG: central gray; CVOs: circumventricular organs; IGL: intergeniculate leaflet; MPL: medial paralemniscal nucleus; NTS: nucleus of the solitary tract; PPN: pedunculopontine nucleus; PVH-parvo: medial dorsal parvocellular subdivision of the hypothalamic paraventricular nucleus. Abbreviations (B)- AP: anterior pituitary; BNST: bed nucleus of stria terminalis; DVC: dorsal vagal complex; IML: intermediolateral cell column of the spinal cord; OXY:oxytocin; PP: posterior pituitary; VP: vasopressin.
and t h e r m o r e g u l a t o r y changes and are stressorspecific. Efferent pathways originate from cortical, limbic, hypothalamic and brainstem areas to activate m o t o r and a u t o n o m i c responses (Fig. 1B).
These stressor categories however, are not mutually exclusive. Experimentally used stressors as well as those posed by the environment should be viewed in their complexity. Immobilization or
778 footshock, for example, may cause pain that initiate well-defined stress reflex responses. Ether inhalation includes physiological stress components such as hypoxia and respiratory distress as well as emotional challenges such as restraint, aversive smell and loss of consciousness. Immune stimuli is often accompanied by hypotension and/or initiate sickness behavior (Berkenbosch et al., 1989). Stress responses can be conditioned and cross-sensitized among these categories (Goldstein et al., 1996; Morrow et al., 1999). In addition to stressors used in experimental biology, there are behaviorally more relevant, naturalistic stressors such as predator exposure or social stressors such as isolation, agonistic encounter (defeat) or changes in social hierarchy. Acute or chronic exposure of rats to predators (cats, ferrets) or their odors (synthetic fox fecal odor, trimethylthiazoline, TMT) evoke significant activation of the HPA axis (Blanchard et al., 1998; Morrow et al., 2000; Figueiredo et al., 2003). In most of these studies there is no direct contact between the subjects, indicating that visual, olfactory and auditory cues associated with the presence of the predator are able to start innate response programs, including the stress response. Brain regions, recruited by predator exposure correspond fairly well with those activated by emotional stressor (restraint), however, it is interesting to note that cat-exposed rats do not express c-fos m R N A in their PVNs in spite of the increase of CRF m R N A levels (Figueiredo et al., 2003). Agonistic encounter in males results in differential c-fos activation patterns in dominant and subordinate hamsters (Kollack-Walker et al., 1997) or in the resident and intruder rats (Halasz et al., 2002; Martinez et al., 2002). Fighting itself activates c-fos expression in the medial amygdala in both partners, however the number of areas showing increased neuronal activation is much higher in subordinates than in dominant males (Kollack-Walker et al., 1997; Martinez et al., 2002). Repeated exposure to social defeat results in a selective pattern of habituation of immediate-early gene expression in stress-releated pathways, however, there are brain areas showing persistent activation (Kollack-Walker et al., 1999). We refer to other chapters of this book for detailed neurobiological analysis of social stress effects.
Stressor characteristics Intensity of the stressors can be controlled experimentally in some cases: different doses of bacterial lipopolysaccharide (LPS) or cytokines (Ericsson et al., 1994) as immune stressors, different doses of insulin to induce hypoglycemia, amount of blood withdrawn during hemorrhage (Pacak and Palkovits, 2001), different intensity of noise to induce acoustic stress (Campeau and Watson, 1997) etc. Most stressors can also be categorized according to the duration of the challenge or the frequency of exposure to stressful situations. Acute stress means single exposure to a single challenge. In case of some stressors the duration of the challenge can be experimentally manipulated (footshock, ether, restraint, immobilization, swim, cold), in other cases, acute stress responses can only be initiated (hyperosmotic challenge, insulin-induced hypoglycemia, formaline injection). Repeated stress (may also be referred to as chronic intermittent stress) covers a situation when single exposure to one acute stressor is repeated once or couple of times daily for a prolonged time. Although most acute stressors can be repeated, emotional (restraint, immobilization) or immune (bacterial LPS or cytokine exposure) stressors are more often repeated than homeostatic challenges (hyperosmotic challenge, hypoglycemia etc.)
Chronic repeated variable stress paradigm (Chappell et al., 1986; Willner et al., 1992; Herman et al., 1995; Stout et al., 2002) includes different stressors from different classes, randomly distributed and repeated over a long time period. The term chronic stress refers to continuos exposure to a stressor. Certain experimentally induced certain disease states, such as arthritis (Sternberg et al., 1989; Harbuz et al., 1992), experimental allergic encephalomyelitis (EAE) (Harbuz et al., 1997), streptozotocin-induced diabetes (Akana et al., 1996; Chan et al., 2002), drug abuse (Sarnyai et al., 2001), as well as continuos social conflict or subordinate status (Albeck et al., 1997) are among the examples.
779 Adrenalectomy can also be regarded as a chronic stressor because of the metabolic imbalance caused by the lack of glucocorticoid hormones (Laugero et al., 2001).
Activity-dependent modifications of the stress responses During repeated or chronic exposure to homo- or heterotypic stressors, two general types of activitydependent changes can occur in the stress-related neuronal circuitry. Both involve changes in response produced by previous inputs: in other words the present response depends on the history of preceding inputs that can be regarded as a special form of memory. The first is a gradual decrement in response elicited by repeated or chronic application of the identical stressor referred to as habituation. The second is a progressive increase of the response: the phenomenon of sensitization or facilitation. In general, habituation occurs in response to nondamaging stimuli, which may enable an animal to remain in a relatively safe environment without eliciting a response constantly to the same innocuous stimulus. Sensitization has also an adaptive value to detect or to react to any stimulus earlier or in a more exaggerated way having been exposed to a harmful stimulus. Habituation Rats habituate their hormonal and transcriptional responses to repeated exposure to the same type (homotypic) of stressors. Habituation refers to the decrement in HPA activity that occurs with repeated exposure to the same or homotypic stressor. Basal measures of HPA activity remain unchanged or slightly elevated (Aguilera, 1994; Ma et al., 1997), however, even a few exposures to a stressor result in decreased or attenuated response to the last challenge of the same kind. Plasma CORT and hypothalamic CRF hetronuclear (hn)RNA responses are decreasing with increasing frequency of exposure to stress (Ma and Lightman, 1998). However, parvocellular AVP hnRNA levels seems to be facilitated by frequent exposure to restraint (Ma and Lightman, 1998).
In line with these changes, chronic intermittent stress differentially affects CRF and AVP stores and neuropeptide release from the median eminence (De Goeij et al., 1992a,b). Repeated hypoglycemia resulted in a decrease of CRF and an increase of AVP content and a shift in peptide release towards AVP (De Goeij et al., 1992). Neural or humoral mechanisms underlying stress habituation remains to be fully characterized. Theoretically, decreased drives or increased inhibitory mechanisms posed on the stress-related hypothalamic motorneurons could be involved. Recent findings implicate reduced afferent drive to the hypothalamic PVN in habituation. PVNprojecting cell groups that display attenuated activation to repeated stimuli included central autonomic (nucleus of the solitary tract, ventrolateral medulla, parabrachial nucleus) and limbic forebrain structures (septum, amygdala/bed nucleus of stria terminalis, prefrontal cortex) (Gosselink, 2002). However, no changes of c-fos expression are detected in hypothalamic areas involved in local inhibitory control of PVN. Involvement of stressor-specific "upstream" mechanisms in habituation is further supported by the increased responsibility of the axis to heterotypic stimuli that are mediated through distinct afferent inputs (Fernandes et al., 2002). In addition to the neural mechanisms, impaired negative feedback control of hypothalamic neurosecretory neurons that occur upon repeated exposure to stress may also be involved in habituation. Decreased negative feedback favors the synthesis and release of parvocellular AVP that may be responsible for exaggerated response to heterotypic challenges, which gain access to releasable neuropeptide pool through pathways that bypass the habituated ones. Decreased sensitivity of the glucocorticoid feedback, probably due to decreased number of glucocorticoid receptors and their interaction with transcription factors induced by CRF and AVP, is critical for the maintenance of ACTH responses in the presence of elevated plasma glucocorticoid levels during chronic stress (Aguilera, 1994; Herman et al., 1995). Stress-induced changes in hippocampal mineralocorticoid receptors (MR) might also contribute to altered regulation of HPA activity. Recent studies revealed CRF-dependent increase of MR in the hippocampus after psychologic stressors, which was associated with an increased MR-mediated tonic
780 inhibition of HPA axis (Gesing et al., 2001). In line with these studies Cole et al. suggest that mineralocorticoid receptors may also play an important role in constraining the HPA axis response to homotypic stressors in habituated rats (Cole et al.,
2000). Posterior division of the paraventricular nucleus of the thalamus (pPVTh) has been also implicated in habituation: lesion of this structure prevented habituation to repeated restraint without altering HPA responses to the first challenge (Bhatnagar et al., 2002). It should be noted however, that other reports did not support this hypothesis and showed no effect of PVTh or BNST on habituation to restraint (Fernandes et al., 2002).
~/m/ng A single exposure to one stressor induces delayed and long-lasting hyperresponsiveness in all indices of HPA axis activity to subsequent to homotypic and heterotypic stressors (Schmidt et al., 1995; Schmidt et al., 1996; Schmidt et al., 2001). Increased production, storage, and secretion of AVP from CRF-synthesizing hypophyseotropic neurons play a dominant role in this phenomenon (Aubry et al., 1999). It should be emphasized, however, that acute, repeated, chronic and primed stress-response categories are operational only in a certain experimental time domain and do not take into account the stress or allostatic load "history" of the subjects.
Facilitation, sensitization, cross-sensitization Prior exposure to repeated stressors results in habituation to the same stressor, however the responsiveness to heterotypic stressors is maintained or even facilitated (Scribner et al., 1993; Ma and Lightman, 1998). Rats exposed to repeated restraint and challenged with hypertonic saline show elevated CORT levels compared to naive animals. CRF transcription is induced normally in response to hypertonic saline injection in repeatedly restrained animals, while parvocellular AVP hnRNA levels rise more rapidly and to higher levels than in controls (Ma and Aguilera, 1999). Facilitated response to a novel challenge in rats habituated to another type of stressor may implicate differential effect of chronic/repeated stressor on synthesis and secretion of ACTH secretagogues. Increased synthesis of AVP in the parvocellular neurons and its accumulation in the external zone of the median eminence over that of CRF provide the neuropeptide basis for potentiation of ACTH release in response to a subsequent, heterotypic stressor (Schmidt et al., 1996; Schmidt et al., 2001). The posterior division of the paraventricular nucleus of the thalamus (pPVTh) seems to play an essential role in facilitation, pPVTh has been shown to inhibit the enhanced or facilitated HPA responses to novel, heterotypic restraint in previously chronically cold stressed rats (Bhatnagar et al., 2002). Sensitization occurs even after a single exposure to a stressor, which rather referred to as priming.
Strain and individual differences All aspects of the stress reactivity to various stressful challenges including neuroendocrine, autonomic, and behavioral responses show significant individual differences (Kabbaj and Akil, 2001) and depend on the strain (Harbuz, 1994; Dhabhar, 1997), pre- and postnatal experiences, and maternal care (Levine et al., 1991; Meaney et al., 1993; Reul et al., 1994; Abraham and Kovacs, 2000). We refer to Part II: Chapters 1.1; 1.5 and 1.6 of this book for details. Selye's concept of stress emphasized the nonspecific nature of the response to wide range of "nocuous agents" (i.e. stressors) and identified the stress triad (adrenal enlargement, gastric ulceration, and thymocolymphatic involution) as the hallmarks of the stress reaction. Although the stereotypic activation of stress-effector mechanisms has been confirmed even at molecular level, with the advent of the technical achievements in the analysis, data are accumulating supporting the heterogeneity of the responses, including differences in afferent and efferent pathways recruited during stress responses, plasma catecholamine profiles, and neurochemical measures.
Functional anatomical mapping of stress-activated circuits Adaptation of the functional activity mapping strategy that is based on the inducibility of different
781 immediate-early genes provided a useful tool in stress research to identify stress-related cells and extended circuits activated in response to various stressors. This approach confirmed the activation of CRFsecreting parvocellular neurosecretory neurons that initiate the neuroendocrine stress cascade, but also revealed the heterogeneity of afferent pathways and efferent mechanisms in response to different challenges.
Immediate-early gene markers of stress-induced neuronal activation Stereotypic inducibility of c-fos proto-oncogene rendered this immediate-early gene (lEG) to be the most widely used functional anatomical mapping tool to identify cells and extended circuitries that became activated in response to various stressful stimuli (Greenberg and Ziff, 1984; Ceccatelli et al., 1989; Morgan and Curran, 1991). In addition to c-fos, nerve growth factor-induced protein B (NGFI-B) and Fos-related antigens (FRAs) are also frequently used markers of neuronal activation in stress research (Chan et al., 1993; Hoffman et al., 1993; Kovacs, 1998).
9 Relation of lEG induction with release from tonic inhibition is not completely known, 9 Nuclear localization of the protein product does not allow to reveal connectivity and morphology of activated neurons.
Timing of immediate-early gene (lEG) induction following acute challenges Acute physiological, psychological, and immune challenges induce transient expression of lEG in the hypothalamic PVN (Honkaniemi et al., 1994; Hughes and Dragunow, 1995). Generally, c-fos mRNA cannot be detected under stress-free conditions, it's transcription is rapidly upregulated showing maximum 30 min after challenge, is diminished by 1 hour and is not detectable by 2-3h poststress (Ericsson et al., 1994; Cullinan et al., 1995). c-Fos protein is also undetectable under basal conditions, first c-Fos immunoreactive cell nuclei are revealed 15-30min after rapid stress, show maximum at 90-180min and decline thereafter (Morgan and Curran, 1991; Giovannelli et al., 1992; Kovacs and Sawchenko, 1996).
Markers of cellular activation in chronic situations
Pros of lEG-based mapping strategy 9 Baseline expression of lEGs is low, 9 Induced stereotypically in response to wide range of extracellular stimuli, 9 Phenotype of activated neurons can easily be identified, 9 Number of activated profiles can be quantitatively analyzed, 9 Differential induction of mRNA and protein product of IEGs allows identification of profiles responsive to two different challenges (Chaudhuri, 1997; Chaudhuri et al., 1997; Kovacs et al., 2001).
One of the significant drawbacks of c-fos-based activational mapping strategy is the transient induction of the marker, which does not allow detection of activated profiles under chronic situations (Hoffman et al., 1993). There are however, lEG markers, such as FRAs (Fos-related antigens, Fral and Fra2) that are induced in response to acute stimuli and, due to their long half-life, gradually accumulate in response to chronic and/or repeated stimuli (Sharp et al., 1991). Fos-related antigens but not c-fos are detected in the parvocellular neurosecretory neurons following adrenalectomy (Jacobson et al., 1990; Brown and Sawchenko, 1997).
Cons of lEG-based mapping strategy 9 Induction is transient, timing of the analysis is important, 9 May not equally label all activated neurons in a given circuit, 9 May not identify neurons that gain net inhibitory input after challenge,
Neuronal activation in the paraventricular hypophyseotropic neurons Most of acute stressful stimuli result in an induction of c-fos or NGFI-B in the dorsal medial part of the parvocellular subdivision of the PVN (mpPVN),
782
where hypophyseotropic CRF-secreting neurons are concentrated (Ceccatelli et al., 1989; Chan et al., 1993). However, there is a significant heterogeneity of IEG induction patterns seen in other functional domains of the PVN (see Fig. 2). Stressors that selectively activate CRF-synthesizing neurons in the PVN include novel environment (Handa et al., 1993), saline injection (Sharp et al., 1991), restraint (Ceccatelli et al., 1989; Abraham and Kovacs, 2000),
mpv
ether (Kovacs and Sawchenko, 1996), footshock (Rivest and Rivier, 1994), swim ( D u n c a n et al., 1993;
Cullinan et al., 1995), and IL-113 (Ericsson et al., 1994). The level of recruitment of m p P V N C R F cell
population following restraint, swim, hemorrhage and IL-113 seems to be submaximal, as c-Fos positive CRF cells account only for one-third of hypophyseotropic CRF cells in this subdivision (Dayas et al., 2001).
~
~
..
.
"-.
'~.:
">
-
" )%
.,"
,
9
9
.......
,
.
,
,i
9 "
Control ,o
.
.
.
.
Etfi~
,
,..,
.
.
,Nyp~lyc~,nia ~
_.
.
"-:-.,=?(:~ ,a\ % "
}
"\;
2"
..:." .,.
.
.: " .
I'PS
.
.""
',
'Hyoertonie
salt ~(.. .
, ,,:
.,%.