Peptide Receptors, Part II (Handbook of Chemical Neuroanatomy)

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PEPTIDE RECEPTORS PART II

This Page Intentionally Left Blank

H A N D B O O K OF CHEMICAL NEUROANATOMY Series Editors" A. Bj6rklund and T. H6kfelt

Volume 20

PEPTIDE RECEPTORS PART II Editors:

R. QUIRION Department of Psychiatry, Douglas Hospital Research Centre, 6875 Lasalle Boulevard, Montreal, QC H4H 1R3, Canada

A. BJORKLUND Department of Physiological Sciences, Wallenberg Neuroscience Center, Biomedical Center All, 22184 Lund, Sweden o,

T. HOKFELT Department of Neuroscience, Retzius Laboratory B3:4, Karolinska Institutet, Retzius v~ig 8, SE 17177 Stockholm, Sweden

2003

ELSEVIER A m s t e r d a m - B o s t o n - L o n d o n - New Y o r k - O x f o r d - Paris San Diego - San Francisco - S i n g a p o r e - Sydney - Tokyo

ELSEVIER SCIENCE B.V. Sara Burgerhartstraat 25 EO. Box 211, 1000 AE Amsterdam, The Netherlands 92003 Elsevier Science B.V. All rights reserved. This work is protected under copyright by Elsevier Science, and the following terms and conditions apply to its use:

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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 drugs dosages should be made. First edition 2003 Library of Congress Cataloging in Publication Data A catalog record from the Library of Congress has been applied for. British Library Cataloguingin Publication Data Peptide receptors Part 2 editors, R. Q u i r i o n , A. B j o r k l u n d , T. ( H a n d b o o k of c h e m i c a l n e u r o a n a t o m y ; v. 20) l.Neuropeptides - Receptors I . Q u i r i o n , Remi, 1955- I I . B j o r k l u n d , A n d e r s , I I I . H o k f e l t , T. (Tomas) 573.8'6465

ISBN: ISBN: ISSN:

Hokfelt.

-

1945-

0-444-50540-7 (volume) 0-444-90340-2 (series) 0924-8196 (series)

O 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 C. ABBADIE (p. 1) Laboratory of Molecular Neuropharmacology Memorial Sloan-Kettering Cancer Center 1275 York Avenue New York, NY 10021 USA S. AHMAD (p. 195) AstraZeneca R&D Montreal 7171 Frederick-Banting Ville St-Laurent Montreal, QC H4S 1Z9 Canada H. AKIL (p. 103) Mental Health Research Institute 206 Zena Pitcher Place Ann Arbor, MI 48109-0720 USA A. BEAUDET (p. 323) Department of Neurology and Neurosurgery Montreal Neurological Institute 3801 University Street Montreal, QC H3A 2B4 Canada J.K. CHAMBERS (p. 31) Vascular Biology Research SC1-H32-L3 SmithKline & Beecham R&D Pharmaceuticals NFSP-N Third Avenue Harlow, Essex CM19 5AW UK J.E. CLUDERAY (p. 31) Neuroscience Research, Neurophysiology and Imaging Research H 17/1130H04 SmithKline & Beecham R&D Pharmaceuticals NFSP-N Third Avenue Harlow, Essex CM 19 5AW UK

G. HERVIEU (p. 31 and 245) Department of Neuroscience SmithKline Beecham Pharmaceuticals Third Avenue Harlow, Essex CM19 5AW UK C. HOFFERT (p. 195) AstraZeneca R&D Montreal 7171 Frederick-Banting Ville St-Laurent Montreal, QC H4S 1Z9 Canada D. HUBATSCH (p. 195) AstraZeneca R&D Montreal 7171 Frederick-Banting Ville St-Laurent Montreal, QC H4S 1Z9 Canada L. MAULON (p. 31) Institute de Pharmacologie Mol6culaire et Cellulaire UMR 6097 CNRS 660 route des Lucioles Sophia-Antipolis, 06560 Valbonne France E MENNICKEN (p. 195) AstraZeneca R&D Montreal 7171 Frederick-Banting Ville St-Laurent Montreal, QC H4S 1Z9 Canada J.-L. NAHON (p. 31) Institut de Pharmacologie Mol6culaire et Cellulaire UMR 6097 CNRS 660 route des Lucioles Sophia-Antipolis, 06560 Valbonne France

C.R. NEAL, JR. (p. 103) Mental Health Research Institute and Department of Pediatrics 205 Zina Pitcher Place Ann Arbor, MI 48109-0720 USA

P. SARRET (p. 323) Department of Neurology and Neurosurgery Montreal Neurological Institute McGill University Montreal, QC H3A 2B4 Canada

D. O'DONNELL (p. 195) AstraZeneca R&D Montreal 7171 Frederick-Banting Ville St-Laurent Montreal, QC H4S 1Z9 Canada

R WALKER (p. 195) AstraZeneca R&D Montreal 7171 Frederick-Banting Ville St-Laurent Montreal, QC H4S lZ9 Canada

G.W. PASTERNAK (p. 1) Department of Neurology Memorial Sloan-Kettering Cancer Center 1275 York Avenue New York, NY 10021 USA

S.J. WATSON, JR. (p. 103) Department of Psychiatry Mental Health Research Institute 205 Zina Pitcher Place Ann Arbor, MI 48109-0720 USA

M. PELLETIER (p. 195) AstraZeneca R&D Montreal 7171 Frederick-Banting Ville St-Laurent Montreal, QC H4S 1Z9 Canada

S. WILSON (p. 31) Vascular Biology Research SC1-H32-L3 SmithKline & Beecham R&D Pharmaceuticals NFSP-N Third Avenue Harlow, Essex CM19 5AW UK

F. PRESSE (p. 31) Institut de Pharmacologie Mol6culaire et Cellulaire UMR 6097 CNRS 660 route des Luciles Sophia-Antipolis, 06560 Valbonne France T. SAKURAI (p. 245) Institute of Basic Medical Sciences University of Tsukuba Tsukuba, Ibaraki 305-8575 Japan

vi

M. YANAGISAWA (p. 245) Howard Hughes Medical Institute University of Texas Southwestern Medical Center at Dallas Dallas, TX 75253-9050 USA

Preface Peptide Receptors Part I was published in 2000 (as volume 16 of the Handbook of Chemical Neuroanatomy series). It summarized current knowledge on the discrete anatomical distribution of ten families of neuropeptide receptors expressed in the mammalian CNS. Part II is its natural complement with chapters coveting six additional families of neuropeptide receptors for ligands ranging from well known peptides such as the opioids and neurotensin to recently isolated ones like the orexins. As in the case of Part I, this volume integrates photomontages and maps of quantitative receptor autoradiography, in situ hybridization histochemistry and immunocytochemistry. Data derived from transgenic and knock-out animals are also summarized, helping to decipher the possible physiological and pathophysiological role(s) of a given peptide family. Some chapters also review current knowledge on the profile of internalization of the neuropeptidereceptor complex, an area of intense research activities that should help to better understand mechanisms involved in desensitization and tachyphylaxis. We hope that this volume will provide enjoyable reading and a useful source of information. Finally, we wish to express our warmest thanks to an outstanding group of contributors and to the editorial staff at Elsevier for the invaluable help in making this volume possible. REMI QUIRION ANDERS BJORKLUND TOMAS HOKFELT Montreal, Lund and Stockholm, May 2002

vii


Contents

List of Contributors P refa c e

I.

OPIOID RECEPTORS - C . ABBADIE AND G.W. PASTERNAK 1. 2. 3.

4.

5. 6. 7. 8. II.

vii

Introduction Opioids Opioid receptor subtypes 3.1. IX Opioid receptors 3.2. ~ Opioid receptors 3.3. ~: Opioid receptors Distribution of opioid receptors in the rat brain 4.1. Autoradiographic localization of opioid receptors 4.1.1. IX Opioid receptors 4.1.2. ~ Opioid receptors 4.1.3. ~: Opioid receptors 4.2. Opioid receptor mRNAs 4.2.1. IX Opioid receptors 4.2.2. ~ Opioid receptors 4.2.3. K Opioid receptors 4.3. Immunohistochemical distribution of the opioid receptors 4.3.1. Ix Opioid receptors 4.3.2. ~ Opioid receptors 4.3.3. ~: Opioid receptors 4.4. Ultrastructural localization of the opioid receptors Conclusions Abbreviations Acknowledgements References

THE MELANIN-CONCENTRATING H O R M O N E - G.J. HERVIEU, L. MAULON-FERAILLE, J.K. CHAMBERS, J.E. CLUDERAY, S. WILSON, F. PRESSE AND J.-L. NAHON A survey of the melanin-concentrating system: seminal background studies and pharmaceutical interest 1.1. MCH has a concerted set of actions in the fish 1.2. MCH also exists in mammals 1.3. MCH as a 'gut-brain' peptide 1.4. "A [mammalian] peptide still in search of functions"

1 1

4 5 5 7 8 8 8 9 9 12 12 15 16 17 17 19 19 21 22 22 24 24

31

31 31 32 33 34 ix

.

.

1.5. MCH regulates food intake in rats 1.6. SLC-1 and another orphan GPCR are paralogue receptors for MCH 1.7. The MCH system appears as a complex evolutionary model The pro-MCH gene, regulation of expression and precursor processing 2.1. Structure, chromosomal mapping and evolution of the pro-MCH gene and linked genes 2.2. Regulation of prepro-MCH gene expression 2.3. Peptide characterisation and precursor processing Features of the MCH system in the rat CNS 3.1. A striking hypothalamic localisation of the MCH immunoreactive cell bodies 3.2. Features of the MCH innervation within the mammalian brain 3.3. Colocalisation data 3.3.1. Neurochemical colocalisation 3.3.2. Functional colocalisation 3.4. Neurochemical environment and survival of MCH neurones 3.4.1. Neurochemical environment 3.4.2. Survival of MCH neurones in culture 3.5. Physiological secretion of MCH 3.6. Peripheral plasmatic and central MCH 3.7. Degradation of MCH by peptidases Central effects of MCH 4.1. MCH and the regulation of the HPA 4.2. MCH and reproductive functions 4.3. A role for MCH in regulating water balance 4.4. MCH and the control of feeding behaviour The MCH receptors 5.1. Bioassays available for melanotropins 5.2. MCH-binding sites 5.3. Molecular cloning, chromosomal localisation, and phylogeny 5.3.1. SLC-1 5.3.2. MCH2 5.4. Signalling 5.4.1. SLC-1 5.4.2. MCH2 5.5. Pharmacology 5.5.1. SLC-1 5.5.2. MCH2 5.6. Ligand-receptor structure-activity relationships 5.7. Central and peripheral distribution of the MCH receptor SLC-1 in the mammals 5.7.1. Overall distribution of SLC-1 mRNA and protein in the rodents 5.7.2. Quantitative RT-PCR (Taqman analysis) of SLC-1 gene expression in rat CNS and PNS 5.7.3. Immunochemical studies 5.7.4. Peripheral and central distribution studies of SLC-1 regional gene expression sites in the human

35 35 35 36 36 37 39 39 41 42 43 43 44 44 44 44 45 45 45 46 46 47 47 48 50 50 51 51 51 52 54 54 55 55 55 56 56 57 57 57 59 74

5.7.5. Autoradiographic ligand studies Central and peripheral distribution of the MCH receptor MCH2 in the mammals 5.9. Neurofunctional analysis Conclusion Abbreviations Acknowledgements References

76

5.8.

,

7. 8. 9. III.

NEUROANATOMICAL STUDIES OF THE OPIOID RECEPTOR-LIKE-1 RECEPTOR AND ITS ENDOGENOUS NEUROPEPTIDE ORPHANIN FQ (NOCICEPTIN) -C.R. NEAL JR., H. AKIL AND S.J. WATSON JR. .

2.

,

4. 5.

Introduction General characteristics 2.1. Kinetics and pharmacology 2.2. Cellular neurophysiological effects Biological effects of OFQ binding at the ORL1 receptor Anatomical studies of the orphanin peptide-receptor system In situ hybridization and immunohistochemistry studies 5.1. Methods 5.1.1. Animals 5.1.2. Tissue preparation 5.1.3. Preproorphanin and ORL1 cRNA probes 5.1.4. OFQ antibody production 5.1.5. Immunohistochemistry 5.1.6. In situ hybridization 5.1.7. Immunohistochemistry and in situ hybridization controls 5.2. Control results 5.2.1. Immunocytochemistry controls 5.2.2. In situ hybridization controls 5.3. Distribution of OFQ and the ORL 1 receptor in the rat forebrain 5.3.1. Cortex 5.3.2. Ventral forebrain 5.3.3. Septum 5.3.4. Basal ganglia 5.3.5. Basal telencephalon 5.3.6. Hypothalamus 5.3.7. Amygdala 5.3.8. Hippocampal formation and related structures 5.3.9. Thalamus 5.4. Distribution of OFQ and the ORL1 receptor in the rat brainstem and spinal cord 5.4.1. Mesencephalon 5.4.2. Cerebellum 5.4.3. Metencephalon 5.4.4. Myelencephalon 5.4.5. Spinal cord

76 78 85 86 92 92

103 103 104 104 105 105 106 107 107 107 108 108 108 108 109 109 109 109 110 110 111 129 130 131 131 132 134 135 136 137 137 139 140 141 142 xi

6.

7.

xii

Anatomical studies using 125I-[14Tyr]OFQ binding and agonist stimulated [35S]GTPyS receptor autoradiography 6.1. Methods 6.1.1. Animals 6.1.2. Tissue preparation 6.1.3. Peptide synthesis and iodination 6.1.4. Receptor autoradiography 6.1.5. Agonist-stimulated GTPyS receptor autoradiography 6.1.6. 125I-[14Tyr]OFQand agonist-stimulated [35S]GTPyS autoradiography controls 6.2. Control results 6.2.1. 125I-[14Tyr]OFQautoradiography controls 6.2.2. Agonist-stimulated [35S]GTPyS autoradiography controls 6.3. Pharmacological characterization of receptor binding 6.4. Distribution of OFQ binding in the rat forebrain 6.4.1. Cortex 6.4.2. Ventral forebrain 6.4.3. Septum 6.4.4. Basal ganglia 6.4.5. Basal telencephalon 6.4.6. Hypothalamus 6.4.7. Amygdala 6.4.8. Hippocampal formation and related structures 6.4.9. Thalamus 6.5. Distribution of OFQ binding in the rat brainstem and spinal cord 6.5.1. Mesencephalon 6.5.2. Cerebellum 6.5.3. Metencephalon 6.5.4. Myelencephalon 6.5.5. Spinal cord 6.6. Distribution of OFQ-stimulated GTPyS binding in the rat CNS Ontogeny studies 7.1. Methods 7.1.1. Animals 7.1.2. Rat developmental tissue preparation 7.1.3. Human developmental brain tissue procurement 7.1.4. Preproorphanin and ORL 1 cRNA probes 7.1.5. In situ hybridization 7.1.6. In situ hybridization controls 7.2. Expression of OFQ in the developing rat brain 7.2.1. E12-E22 7.2.2. P7-adult 7.3. Expression of ORL1 in the developing rat brain 7.3.1. E12-E22 7.3.2. P7-adult 7.4. OFQ and ORL 1 mRNA expression of in the developing human brain 7.4.1. OFQ 7.4.2. ORL1

144 144 144 144 144 144 145 145 146 146 146 147 147 148 148 148 148 149 149 149 150 150 150 150 151 151 152 152 153 155 155 155 155 155 156 156 156 156 156 158 159 159 161 161 161 164

8.

IV.

Physiological implications of OFQ and the ORL1 receptor 8.1. Comparisons with endogenous opioid systems 8.1.1. Proopiomelanocortin and the ~ receptor 8.1.2. Prodynorphin and the K receptor 8.1.3. Proenkephalin and the 3 receptor 8.2. Functional considerations of orphanin FQ and ORL1 circuitry 8.2.1. The limbic hypothalamic-pituitary-adrenal (L-HPA) axis 8.2.2. Learning and memory 8.2.3. Motor systems 8.2.4. Reinforcement and reward 8.2.5. Sexual behavior 8.2.6. Pain perception 8.2.7. Autonomic and physiologic functions 8.2.8. Special sensory systems 9. Concluding remarks 10. Abbreviations 11. Acknowledgements 12. References

165 165 166 166 167 168 168 169 169 170 171 172 173 173 174 174 184 184

LOCALIZATION OF GALANIN RECEPTOR SUBTYPES IN THE RAT CNS - D. O'DONNELL, E MENNICKEN, C. HOFFERT, D. HUBATSCH, M. PELLETIER, P. WALKER AND S. AHMAD

195

1. 2.

3.

4.

Introduction Galanin 2.1. Historical perspective 2.2. Distribution 2.3. Biological roles 2.3.1. Feeding 2.3.2. Cognition and memory 2.3.3. Sensory transmission/nociception 2.4. Therapeutic implications 2.5. Galanin antagonists 2.6. Genetic manipulations of galanin expression 2.7. Galanin-related peptides 2.7.1. Galanin message-associated peptide (GMAP) 2.7.2. Galanin-like peptide (GALP) Galanin receptor subtypes 3.1. Characterization of GALRs 3.2. Cloning of GALR subtypes 3.2.1. GALR1 3.2.2. GALR2 3.2.3. GALR3 3.3. The elusive galanin fragment receptor 3.4. Galanin-like receptors Localization of galanin receptors in the rat CNS 4.1. Distribution of 125I-galanin-binding sites in the rat CNS 4.1.1. Telencephalon

195 195 195 196 197 197 198 199 199 200 201 201 201 202 203 203 204 204 205 205 206 207 208 208 215 xiii

V.

4.1.2. Diencephalon 4.1.3. Mesencephalon 4.1.4. Rhombencephalon 4.1.5. Spinal cord 4.2. Distribution of GALR1 mRNA in the rat CNS 4.2.1. Telencephalon 4.2.2. Diencephalon 4.2.3. Mesencephalon 4.2.4. Rhombencephalon 4.2.5. Spinal cord 4.3. Distribution of GALR2 mRNA in the rat CNS 4.3.1. Telencephalon 4.3.2. Diencephalon 4.3.3. Mesencephalon 4.3.4. Rhombencephalon 4.3.5. Spinal cord 4.4. Distribution of GALR3 mRNA in the rat CNS 4.4.1. Telencephalon 4.4.2. Diencephalon 4.4.3. Mesencephalon 4.4.4. Rhombencephalon 4.4.5. Spinal cord 5. Expression of GALRs by glial cells 6. Localization of galanin receptors in the circumventricular organs of the rat 7. Localization of galanin receptors in dorsal root ganglia of the rat 7.1. Binding sites 7.2. Expression of different receptor subtypes 8. Concluding remarks 9. Abbreviations 10. Acknowledgements 11. References

216 216 216 216 217 217 217 219 219 219 221 221 223 223 223 225 225 227 227 227 227 228 228 229 229 230 231 231 233 235 236

OREXIN RECEPTORS M. YANAGIS AWA

245

.

xiv

-

T. SAKURAI, G. HERVIEU AND

Introduction 1.1. Discovery and identification of orexins/hypocretins 1.2. Structures of orexin-A and -B Biology of orexins 2.1. Prepro-orexin gene, structure and regulation of expression 2.2. Features of orexin system in mammals 2.2.1. Striking hypothalamic localization of orexin-containing neurons 2.2.2. Features of orexin innervation within mammalian brain 2.2.3. Neuroanatomical colocalization with other factors 2.2.4. Neuronal and humoral input to orexin neurons 2.3. Central effects of orexins in mammals 2.3.1. Feeding behavior

245 245 245 247 247 247 247 249 249 249 250 250

3.

4.

5. 6. 7. 8. 9.

10. 11. 12. 13. VI.

2.3.2. Behavioral studies 2.3.3. Water intake 2.3.4. Regulation of vigilance state and sleep process Orexin receptors 3.1. Structures 3.2. Chromosomal localization 3.3. Pharmacology 3.4. Signaling 3.5. Ligand-receptor structure-activity relationships Distribution of orexin receptor mRNA and protein in mammalian central nervous system 4.1. Overall distribution of orexin receptor mRNA in rat central nervous system 4.2. Distribution of orexin receptors in the rat central nervous system 4.2.1. Telencephalon 4.2.2. Diencephalon 4.2.3. Mesencephalon and rhombencephalon (midbrain and hindbrain) 4.2.4. Spinal cord Comparison of OX1R and OX2R distribution Comparison of OX1R mRNA and protein distribution Comparison between localization of orexin receptor and sites of c-Fos activation upon central administration of orexins in rat How many orexin receptors? Physiological and pathophysiological implications of orexin receptors 9.1. Feeding behavior 9.2. Regulation of water balance 9.3. Neuroendocrine regulation 9.4. Regulation of autonomic nervous system 9.5. Vigilance state control 9.6. Other functions Conclusion Abbreviations Acknowledgements References

NEUROTENSIN RECEPTORS IN THE CENTRAL NERVOUS SYSTEM P. SARRET AND A. BEAUDET 1. 2.

3. 4.

Introduction Discovery of NT 2.1. Neurotensin and related peptides 2.2. Structure of the neurotensin/neuromedin N gene 2.3. Translational and post-translational processing of the NT/NN precursor 2.4. Degradation of neurotensin and neuromedin N Distribution of NT in the CNS Central effects of NT

250 251 251 252 252 252 252 252 253 257 257 258 258 260 262 263 263 312 312 312 313 313 313 313 313 314 314 315 315 320 320

323 323 323 323 325 325 326 327 327 XV

5.

6. 7. 8. 9.

NT receptors in mammalian CNS 5.1. Identification of NT binding sites 5.2. NT receptor subtypes 5.3. NT agonists and antagonists 5.3.1. Agonists 5.3.2. Antagonists 5.4. Localization of NT receptor subtypes 5.4.1. Methods of study 5.4.2. NTS1 receptors 5.4.3. NTS2 receptors Summary and conclusions Abbreviations Acknowledgements References

Subject Index

xvi

328 328 332 334 334 335 336 336 336 367 383 384 387 387 401

CHAPTER I

Opioid receptors CATHERINE ABBADIE AND GAVRIL W. PASTERNAK

1. INTRODUCTION Opioids have long played a major role in pharmacology, representing one of the oldest classes of clinically important pharmaceuticals. Like many drugs, they act through receptors and the opioid receptors were among the first to be identified in binding assays. With the ability to label these receptors came the opportunity to identify precisely their localization within the central nervous system using autoradiographic approaches. These early studies defining their distributions used various opioid ligands and quickly established the presence of opioid binding sites in brain regions presumed to be important in mediating opioid actions. However, as our understanding of opioid receptors has expanded, it has become apparent that opioids act through a family of receptors, as described below. Equally important, many of the ligands initially thought to be 'selective' are now known not to be, complicating the interpretation of these earlier studies.

2. OPIOIDS

Morphine and its related alkaloids found in opium have been used for the control of pain and constipation for millennia (Pasternak, 1993; Reisine and Pasternak, 1996). The simplest preparations were tinctures of opium, which are still used today. Eventually, it was possible to isolate and purify morphine and codeine (Fig. 1), followed soon afterwards by the synthesis of analogs. Many of these were generated from thebaine, a natural product also found in opium, but there are now a large number of totally synthetic drugs with many diverse structures (Fig. 1). The vast number of opiate derivatives has led to insights into the pharmacology of these agents. Opioids are still used primarily for the control of pain (Pasternak, 1993; Reisine and Pasternak, 1996). They have the unique ability to eliminate the 'suffering' or 'hurt' of pain without interfering with primary sensations, such as light touch, temperature, sharp/dull sensations and position sense, distinguishing them from agents such as the local anesthetics. Along with their analgesic activity, opioids have a number of other potent actions. Some, such as the inhibition of gastrointestinal motility, can be used constructively to treat disorders such as diarrhea, but more often impede the utility of the drugs to treat pain. Respiratory depression is another potential difficulty with this class of drug. It is rarely an issue when used in an outpatient setting unless the patient has an underlying pulmonary disorder, but it can be problematic in a number of other situations. Opioids also have a number of less dramatic

Handbook of Chemical Neuroanatomy, Vol. 20: Peptide Receptors, Part H R. Quirion, A. Bj6rklund and T. H6kfelt, editors 92003 Elsevier Science B.V. All rights reserved.

Ch. I

C. Abbadie and G.W. Pasternak

CH3

CH3

/__~N

I

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o~ N - C ~

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H3

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"~-

OH -"'> caudate nucleus, putamen, striatum, substantia nigra, globus pallidus) and low in the thalamus, spinal cord, cerebellum and medulla oblongata. (C-F) mRNA in situ experiments confirmed human SLC-1 gene expression in the cortex (C), the hippocampus (D) and the cerebellum (F). Immunoreactive cells were also detected in the dentate gyms (E). Scale bars: C, F, 0.6 cm; D, 0.5 cm; E, 35 Ixm. Abbreviations: (C) Caud., caudate; Put, putamen; MOrG, medial orbital gyms; POrG, posterior orbital gyms; GR, gyms rectus; (D) ARG, Andreas Retzius gyms; sms, superficial medullary stratum; ICG, isthmus of the cingulate gyms; PHG, parahippocampal gyms; DG, dentate gyms. 82

The melanin-concentrating hormone

Ch. H

83

Ch. H

G.J. Hervieu et al.

SLC-1 immunoreactivity was present within the medial hypothalamus, a region where there is prepro-MCH mRNA and peptide novel expression in lactating rats (Knollema et al., 1992). Transgenic mice lacking the MCH gene have a lean and hypophagic phenotype and this is described as the first example that 'deletion of a gene encoding a single orexigenic peptide can result in leanness' (Shimada et al., 1998). Also targeted disruption of the melaninconcentrating hormone receptor-1 results in resistance to diet-induced obesity (Chen et al., 2002), hyperphagia (Chen et al., 2002; Marsh et al., 2002), leanness, hyperactivity, and hyperphagic and altered metabolism (Marsh et al., 2002). Physiological structure-activity studies with a variety of MCH peptide analogues indicated a strong correlation between their effects upon food intake and their potency obtained at the rat SLC-1 receptor. This would indicate the relevance of the SLC-1 receptor in feeding behaviour (Haynes et al., 2001; Suply et al., 2001). Finally, the anorectic property of SNAP-7941, a specific MCH-R1 antagonist (Borowsky et al., 2002), are all proof that MCH, at least through its signalling to MCH-R1, is essential to energy balance homeostasis. A parallel line of evidence reinforces the important role of MCH in the feeding response. MCH is a regulator of glucocorticoid secretion (see Section 5.1). It is well established that the nutritional status of mammals and activity of the HPA axis are inter-related. In pathological situations, the overactivity of the HPA axis (elevated circulating ACTH and glucocorticoid blood levels) is a hallmark of comorbidity with obesity (see Peeke and Chrousos, 1995). Glucocorticoid excess induces abdominal obesity, insulin resistance, diabetes and hypertension. All of this experimental evidence may suggest a pathophysiological role for centrally acting MCH being involved in the development of obesity. The SLC-1 immunostaining observed in the hypothalamic paraventricular nucleus could be associated with the neurodocrine effects of MCH on the stress response. MCH has indeed been found either to stimulate (Jezova et al., 1992; Ashmeade et al., 2000) or inhibit (Ludwig et al., 1998; Bluet-Pajot et al., 1995) the HPA axis through an action on pituitary ACTH and/or hypothalamic CRH neurones. Both i.c.v, and i.v. injection of MCH evoked changes in HPA activity and suggest an action at both hypothalamic and peripheral levels, i.c.v. administration of MCH in conscious rats potently activated the CRH-like immunoreactive neuronal population of the parvicellular paraventricular hypothalamic nucleus (Parkes et al., 1992) and both rat and human pituitary glands expressed quite strongly the SLC-1 receptor. Intriguingly, the pairing of MCH and ~-MSH again appears as an evolutionary-acquired functional antagonism feature: MCH antagonises the effects of the melanocortin on grooming and locomotor activities in the rat (Sanchez et al., 1997; see Baker, 1994; Tritos and MaratosFlier, 1999). Excessive grooming behaviour is induced by melanocortins and is observed during mild stress situations and following exposure to novel stimuli, translating very often as a whole into anxious behaviours. MCH is reported to be anxiogenic when injected into the hypothalamic preoptic area (Gonzalez et al., 1996) or anxiolytic following i.c.v, administration (Monzon et al., 1999, 2001). There might be an overall outcome of the implication of MCH through SLC-1 in the stress neuroendocrine pathway. The stress axis is a key component of body homeostasis, itself dependent on fuel/nutrients available to appropriately maintain vital vegetative functions as others. Dysregulation of the stress axis may lead humans and other animal models to become statistically highly sensitive to affective disorders, of which abnormal feeding behaviour (anorexia and bulimia) is a frequent comorbidity component. Human clinical data have long been accumulated about the effect of glucocorticoid treatment on inducing obesity and depression in patients as well as immune disorders, amongst many other dysregulations. Fuel-deficient animals are immunodepressed, as depressed animals including major depressive humans are. That MCH 84


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by itself already has wide physiological actions in the fish as a stress, immune and pigmentary modulator, may mean that dysregulation in one of the system could potentially translate in clinically relevant human disorders, and worsen because of cascading to other interelated systems. Depressive states are often associated with anxiety behaviours. As a cardinal link, CRH appears to be a key messenger in mood disorders and anxiety. CRH mediates very wide and profound stress-induced changes in the autonomic nervous system, neuroendocrine function and behaviour (see Koob, 1999). There is substantial evidence for the hyperactivity of the HPA axis in the aetiology of affective illnesses as shown by up to two-thirds of drug-free depressed patients (depression, post-traumatic stress disorder, anxiety and anorexia nervosa) having hypercortisolaemia, enlarged adrenal and pituitary glands, elevated cerebrospinal fluid levels of CRH, blunted neuroendocrine response to synthetic GC (dexamethasone) challenge, cognitive impairments which may be consistent with a toxic activity of the chronically high levels of brain cortisol and the down-regulation of its receptors in the hippocampal formation (see Koob, 1999). This endocrinopathy is largely related to the hypersecretion of CRH as also suggested by the down-regulation of receptor level. Also, key neuromodulators implicated in affective disorders are regulated by MCH: MCH affects amine release thereby reducing serotonergic activity and inhibiting dopamine release (Gonzalez et al., 1997b). Thus dysregulations in the MCH system could potentially impact on affective behaviours. The very recent report by Synaptic Inc. showing the antidepressant and anxiolytic actions of SNAP-7941, an MCH-R1 antagonist (Borowsky et al., 2002), gives a strong credential to that hypothesis. The presence of SLC-1 protein in other major neuroendocrine regions such as the hypothalamic supraoptic, arcuate and the medial preoptic nuclei is consistent with MCH regulating oxytocin (Parkes and Vale, 1992a) and luteinising hormone release (Gonzalez et al., 1997a). This may possibly translate into sexual behaviour regulation (see Sections 4.2 and 5.2). Both the nucleus accumbens and ventral tegmental area were SLC-l-immunoreactive. The former nucleus is a major recipient of the mesolimbic dopaminergic projection from the ventral tegmental area and plays a key role in reward mechanisms. This brain region may mediate the positive reinforcing effects of food and thus provide an additional control by MCH on feeding behaviour. Borowsky et al. (2002) have shown that the SNAP-7941 compound does not inhibit food intake because of a taste-aversion effect. It should be borne that leptin is part of the reward system by regulating the incentive value of food (Fulton, 2000; see also Filglewicz and Wood, 2000). Melanotropins are known to be implicated in drug-seeking behaviours (see Eberle, 1988; Adan and Gipsen, 1997) and the primary location of action seems to be in the peri-aqueductal grey matter, which receives an important but separate hypothalamic innervation of both MCH and MSH. Bittencourt noted that the presence of MCH fibres with varicosities indicate that the PAG is a site of MCH fibre ending and presumably peptide release and not just a location of fibres of passage. Also it is well-known that the CRH pathway is potently implicated in the physiology of addiction and withdrawal behaviour (e.g. CRH increases the predisposition to self-administer drugs as observed in a stressful context). It is conceivable that MCH may be a component within the numerous neuromediators regulating reward mechanisms.

6. CONCLUSION The phylogenetic distribution of MCH has provided an interesting problem for biologists who wish to reconcile the melanophore modulation of this peptide in lower vertebrates 85

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with heretofore unknown functions in mammalian brain. The pairing of MCH to its SLC-1 receptor intervened some 15 years after the isolation of the MCH peptide. While data had been gathered on the biology of MCH for that period (with a landmark study demonstrating that central MCH induces feeding intake), no information was available on the receptor. As alluded in the introduction, peptides are difficult molecular entities to work with. However, it is strongly suggested and suggested again 20 years after that peptides may only show significant up to dramatic biological relevance in a pathophysiological context (see Hockfelt, 1991; Hockfelt et al., 2000). This should provide impetus to unravel the functions of many other peptides. In particular, one eagerly awaits to know about the functions of the NGE, NEI, MGOP and potential human variant MCH. The main effect of MCH in teleost concerns pigmentary control. That is also a functional boundary between fish and mammals. MCH is not involved in pigmentary control in mammals. But is that as clear-cut? A human melanoma cell line SL-MEL-37 harboured MCH receptors (MCH-R1) (Saito et al., 2001b). Pigmentory cell of the eye express the SLC-1 receptor (Hintermann et al., 2001b). In fact, there is recent evidence for a role in pigmentogenesis as a very recent study has reported that the first discovered paralogue of both MCH receptors so far characterised, MCH-R1, is an auto-antigen associated with vitiligo, a common depigmenting disorder resulting from the loss of melanocytes in the skin. The study also reported that anti-MCH-R1 IgG were naturally inhibiting MCH binding to its receptor MCH-R1 (Kemp et al., 2002). This may be coincidental however as may peptidergic systems are present in melanocytes for no direct functions per se on pigmentory control. Lastly, does MCH act as a feeding factor in fish? A recent transgenic fish medaka strain overexpressing the MCH gene was established and its phenotypic features were examined (Kinoshita et al., 2001). Development, growth, feeding behaviour, and reproduction of transgenics did not differ significantly among transgenic and non-transgenic siblings. The result whereby enhanced MCH expression induced a change in body colour, but no remarkable abnormalities. The review has presented the characterisation of SLC-1 and MCH2 as being two MCH receptors and should open wide avenues for probing additional functions of the peptide, both in the brain and in the periphery.

7. ABBREVIATIONS

Anatomical (adapted from Paxinos and Watson, 1998 and Swanson, 1998) AAA

ac(o, t)

ACA(v,d) ACB ACT AD AH AHN(a, p) Al(d) (v) AM AMB Amygd. 86

anterior amygdaloid nucleus anterior commissure (olfactory, temporal) limb anterior cingulate area nucleus accumbens anterior corticothalamic tract anterodorsal nucleus thalamus anterior hypothalamus anterior hypothalamic nucleus (anterior, posterior part) agranular insular area (dorsal)(ventral) part anteromedial nucleus thalamus nucleus ambiguus amygdala


AN AOL AON APN AQ ARG AUDp AUDv AV BLA BMA BST C(M) (L) CA(l) (2) (3) CA(so) (sp) Caud. Put. CB(gr) cc(g) CEA1 CG CL CLA CLi CM COA cpd CS(m) cst

CTX DCO DG(s g, cr) dhc DLL DMH(p) DR DRG DTN ec

ECT ECU EP EPN(d) EW fa fi fr Fr FRP(am)

Ch. H

arcuate nucleus anterior olfactory nucleus, lateral part anterior olfactory nucleus anterior pretectal nucleus cerebral aqueduct Andreas Retzius gyrus primary auditory area ventral auditory area anteroventral nucleus thalamus basolateral nucleus amygdala basomedial basolateral nucleus amygdala bed nuclei of the stria terminalis (mediocentral) (laterocentral) nucleus thalamus Ammon's horn field (1) (2) (3) field CA stratum (oriens) (pyramidal) caudate putamen cerebellum (granular cell layer of the cerebellar cortex) corpus callosum (genu) central nucleus amygdala central grey central lateral nucleus claustrum caudal linear nucleus of raphe central medial nucleus thalamus cortical nucleus amygdala cerebral peduncle superior central nucleus raphe, medial part corticospinal tract neocortex dorsal cochlear nucleus dentate gyrus (granule cell, corona radiata) layer dorsal hippocampal commissure dorsal nucleus lateral lemniscus dorsomedial hypothalamus (posterior part) dorsal nucleus raphe dorsal root ganglion dorsal tegmental nuclei extermal commissure ectorhinal area external cuneate nucleus endopiriform nucleus entopeduncular nucleus (dorsal part) Edinger-Westphal nucleus corpus callosum, anterior forceps fimbria fasciculus retroflexus frontal cortex frontoparietal/frontal pole cortex (motor area) 87

Ch. II

FS GP(1) GRN GU Hab HDB HF hi HYP IA IA(D) (M) IC(c, d, e) ICG IG III ILL int Int(A) (P) IO IP, IPN islm KF LA Lat LAV LC lct LD LG(d, v, m, 1) LH LHA LL LM LPO LS(d) (v) lsc 1st LV MA Sam MARN mcp MD MDRNv ME(ex) MEA 88

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fundus of the striatum globus pallidus (lateral segment) gigantocellular reticular nucleus gustatory area habenula nucleus horizontal limb diagonal band hippocampal formation hippocampus hypothalamus intercalated nuclei amygdala interantero(dorsal) (medial) nucleus thalamus inferior colliculus (central, dorsal, external) nucleus isthmus of the cingulate gyrus indusium griseum principal oculomotor nucleus intermediate nucleus of the lateral lemniscus internal capsule interpositus cerebellar nucleus (Int) (anterior) (posterior) subdivisions inferior olivary complex interpeduncular nucleus central subnucleus major islands of Calleja Kolliker-Fuse subnucleus lateral nucleus amygdala lateral cerebellar nucleus lateral vestibular nucleus locus coeruleus laterocorticospinal tract laterodorsal thalamus lateral geniculate complex (dorsal, ventral, medial, lateral) part lateral habenula lateral hypothalamic area lateral leminisci lateral mammillary nucleus lateral preoptic area lateral septum (dorsal) (ventral) segments laterospinocortical tract lateral spinothalamic tract lateral vestibular nucleus magnocellular preoptic nucleus mammillary nuclei magnocellular reticular nucleus middle cerebellar peduncle mediodorsal nucleus thalamus medullary reticular nucleus, ventral part median eminence (external lamina) median nucleus amygdala


MEPO MG(d, v) MH MidThal ml MM NO(p) (s) MOB(opl) moV MPN MPO MRN MS MV(m) (v) NA NB NDB NLL NLOT NTS(i) (m) (rm) (vl) OCP OP OT (3) PA PAG(d, vl, dl, m) PAR PARN PB(1) (mm) PCG PCN PF PG PGRN(1) PH PHG PIR(2) PMd PO POLF POR PorG POST PP PPN PRE

Ch. H

median preoptic nucleus medial geniculate complex (dorsal, ventral element) medial habenula median part of the thalamus medial lemniscus medial mammillary nucleus (primary) (secondary) motor area main olfactory bulb (outer plexiform layer) motor root of the trigeminal nerve medial preoptic nucleus (MPN) medial preoptic area mesencephalic reticular nucleus septal nucleus (MS) medial vestibular nucleus (magnocellular)(parvicellular) parts nucleus accumbens nucleus brachium inferior colliculus nucleus of the diagonal band nucleus of lateral lemniscus nucleus of the lateral olfactory tract nucleus of the solitary tract (intermediate) (medial) (rostral zone of the rostral part) (ventrolateral) parts occipital lobe (neocortex) olivary pretectal nucleus olfactory tubercle (polymorph layer) posterior nucleus amygdala periaqueductal grey matter (dorsal, ventrolateral, dorsolateral, medial) parasubiculum parvicellular reticular nucleus parabrachial nucleus, (lateral) (mediomedial) part pontine central grey paracentral nucleus thalamus parafascicular nucleus thalamus pontine grey paragigantocellular reticular nucleus (lateral part) posterior hypothalamus nucleus parahippocampal gyrus piriform cortex (pyramidal layer) dorsal premammillary nucleus posterior complex thalamus primary olfactory cortex periolivary nuclei posterior orbital gyrus postsubiculum posterior pituitary (neurohypophysis) pedunculopontine nucleus presubiculum 89

Ch. H

PreCBL Pretect PRN(c) (r) PrS PSV PT PTLp PV(i) (p) PVH(dp, mpv, pml, pmd, pv)

PVT PY RE RH RN RPA RR RSP(d) (v) RT rust

SC (zo) (op) (sg) (ig) (dg) SEP

SEZ/RC SG SI sms

SN p(c) (r) so

SO SOC(1) Sp Cd sp, SP SPIV sptV SPV(o, i, 1, c) SPVO(rdm, vl) SS(p) (s) STN SUB(d) (v) SUMI SUV tb 90

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precerebellar nuclei pretectal areas pontine reticular nucleus (caudal) (rostral) part presubiculum principle sensory trigeminal nerve parathenial thalamus nucleus posterior-parietal region association area periventricular nucleus hypothalamus (intermediate) (posterior) part paraventricular nucleus hypothalamus (dorsal parvicellular) (medio-parvicellular) (posterior magnocellular- lateral zone) (medial parvicellular-dorsal zone) (periventricular zone) periventricular nucleus thalamus pyramidal tract nucleus reuniens rhomboid nucleus red nucleus nucleus raphe pallidus mesencephalic reticular nucleus, retrotuberal area retrosplenial cortex, (dorsal) (ventral) part reticular nucleus thalamus rubrospinal tract superior colliculus (zonal) (optic) (intermediate grey) (superficial grey) (deep grey) layer septum subependymal zone/rhinocele supragenual nucleus substantia innominata superficial medullary stratum substantia nigra pars (compacta) (reticulata) stratum oriens supraoptic nucleus superior olivary complex (lateral part) spinal cord pyramidal layer spinal vestibular nucleus spinal tract of the trigeminal nerve nucleus spinal tract trigeminal nerve (oral) (interpositus) (lateral) (caudal) part nucleus spinal tract trigeminal nerve, oral part (rostrodorsomedial, ventrolateral) (primary) (secondary) somatosensory area subthalamic nucleus subiculum (dorsal) (Bd) (ventral) parts supramammillary nucleus (lateral) superior vestibular nucleus trapezoid body


TEv TMv TR TRN TT(d)(v3) V (pc) (mo) v3 VAL VCO VDB Vest VII VIIn VIS(al) (am) (li) (11) (p) (pl) (pro)

VISC VL VLL VM VMH VP VP(L) (M) VTA VTN ZI(da)

Ch. H

ventral temporal association area tuberomammillary nucleus post piriform transition area tegmental reticular nucleus taenia tecta (dorsal)(ventral-layer 3) trigeminal nerve (parvicellular part of the motor nucleus) (motor root)/motor nucleus of the trigeminal nerve third ventricle ventral anterior-lateral complex thalamus ventral cochlear nucleus vertical limb of the diagonal band vestibular nuclei facial nerve facial nucleus visual area (anterolateral) (anteromedial) (intermediolateral) (laterolateral) primary) (posterolateral) (posteriomedial) visceral area ventrolateral nucleus thalamus ventral nucleus lateral lemniscus ventral medial nucleus thalamus ventromedial area hypothalamus ventroposterior thalamic complex ventral postero (lateral) (medial) nucleus thalamus ventral tegmental area ventral tegmental nucleus zona incerta (dopaminergic cell group of the)

Miscellaneous

BSA cDNA DAB EDTA GPCR HEK i.c.v. -ir i.v. KLH -li mRNA NGS PAGE PBS PMSF RPMA RT-PCR

bovine serum albumin complementary deoxyribonucleic acid 3,3'-diaminobenzidine ethylene diamine tetraacetate G-protein-coupled receptor human embryonic kidney intracerebroventicular -immunoreactive intravenous keyhole limpet haemocyanin -like messenger ribonucleic acid normal goat serum polyacrylamide gel electrophoresis phosphate-buffered saline phenylmethylsulfonylfluoride reverse pharmacology approach reverse transcription followed by polymerase chain reaction 91

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SDS

(T)TBS v/v w/v

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sodium dodecyl sulphate (Tween 20) Tris-buffered saline volume per volume weight per volume

8. A C K N O W L E D G E M E N T S

This study was supported by SmithKline Beecham Pharmaceuticals, Research and Development (G.J.H., J.K.C., J.E.C., S.W.) and the Centre National de la Recherche Scientifique (CNRS), the Institut de Recherche Servier, Nestle, and Danone Institute (J.-L.N., EE, L.M.-E). We wish to thank SmithKline Beecham Bioinformatics (Simon Topp), SmithKline Beecham Biopharmaceuticals (Paul Murdoch), SmithKline Beecham Neuroscience Research (David Harrison, Peter Maycox) for their contributions. 9. REFERENCES Abrahamson EE, Moore RY (2001): The posterior hypothalamic area: chemoarchitecture and afferent connections. Brain Res 889:1-22. Abrahamson EE, Leak RK, Moore RY (2001): The suprachiasmatic nucleus projects to posterior hypothalamic arousal systems. NeuroReport 12 (2):435-440. Adan RAH, Gipsen WH (1997): Brain melanocortin receptors: from cloning to function. Peptides 18 (8):12791287. An S, Cutler G, Zhao JJ, Huang SG, Tian H, Li W, Liang L, Rich M, Bakleh A, Juan Du J, Chen JL, Dai K (2001): Identification and characterization of a melanin-concentrating hormone receptor. Proc Natl Acad Sci USA 98:7576-7581. Anand BK, Brobeck JR (1951): Localisation of a 'feeding center' in the hypothalamus of the rat. Proc Soc Exp Biol Med 77:323-324. Ashmeade TE, Jones DNC, Munton RP, Shilliam C, Gartlon JE, Parker F, Hervieu G, Heidbreder CA (2000): An investigation into the effect of MCH on neuroendocrine markers in the rat. Eur J Neurosci 12 (Suppl 11):217.2, p. 476. Audinot V, Lahaye C, Suply T, Rovere-Johene C, Rodriguez M, Nicolas JP, Beauverger P, Cardinaud B, Galizzi JP, Fauchere JL, Nahon JL, Boutin JA (2001a): Structure-activity relationship studies of melanin concentrating hormone (MCH)-related peptide ligands at SLC-1, the human MCH receptor. J Biol Chem 276 (17):1355413562. Audinot V, Lahaye C, Suply T, Beauverger P, Rodriguez M, Galizzi JP, Fauchere JL, Boutin JA (2001b): [I-125]$36057: a new and highly potent radioligand for the melanin-concentrating hormone receptor. Br J Pharmacol 133 (3):371-378. Bachner D, Kreienkamp H, Weise C, Buck F, Richter D (1999): Identification of melanin concentrating hormone (MCH): as the natural ligand for the orphan somatostatin-like receptor 1 (SLC-1):. FEBS Lett 457 (3):522-524. Bahjaoui-Bouhaddi M, Fellmann D, Griffond B, Bugnon C (1994): Insulin treatment stimulates the rat melaninconcentrating hormone-producing neurons. Neuropeptides 24:251-258. Baker BI (1991): Melanin-concentrating hormone: a general vertebrate neuropeptide. Int Rev Cytol 126:1-47. Baker BI (1994): Melanin-concentrating hormone updated: functional considerations. Trends Endocrinol Metab 5 (3):120-126. Baker BI, Bird DJ (2002): Neuronal organization of the melanin-concentrating hormone system in primitive actinopterygians: evolutionary changes leading to teleosts. J Comp Neurol 442 (2):99-114. Baker BI, Bird DJ, Buckingham JC (1985): Salmonid melanin-concentrating hormone inhibits corticotropin release. J Endocrinol 106:R5-R8. Bayer L, Poncet F, Fellmann D, Griffond B (1999a): MCH expression in slice cultures of rat hypothalamus is not affected by 2-deoxyglucose. Neurosci Lett 267:77-80. Bayer L, Risold PY, Griffond B, Fellmann D (1999b): Rat diencephalic neurons producing melanin-concentrating hormone are influenced by ascending cholinergic projections. Neuroscience 91 (3): 1087-1101.

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Maratos-Flier E (1996): A role for melanin-concentrating hormone in the central regulation of feeding behaviour. Nature 380:243-244. Risold PY, Griffond B, Kilduff TS, Sutcliffe JG, Fellman D (1999): Preproorexin and PRL-like immunoreactivity are coexpressed by neurones of the rat lateral hypothalamus. Neurosci Lett 259:153-156. Rodriguez M, Beauverger P, Naime I, Rique H, Ouvry C, Souchaud S, Dromaint S, Nagel N, Suply T, Audinot V, Boutin JA, Galizzi JP (2002): Cloning and molecular characterization of the novel human melanin-concentrating hormone receptor MCH2. Mol Pharmacol 60 (4):632-639. Rossi M, Bloom SR (1997): MCH acutely stimulates feeding, but chronic administration has no effect on body weight. Endocrinology 138 (1):351-355. Rossi M, Beak SA, Choi S-J, Small CJ, Morgan DGA, Ghatei MA, Smith DM, Bloom SR (1999): Investigation of the feeding effects of MCH on food intake-action independent of galanin and the melanocortin receptors. Brain Res 846:164-170. Rov~re C, Viale A, Nahon JL, Kitabgi P (1996): Impaired processing of brain pro-neurotensin and pro-melaninconcentrating hormone in obese fat/fat mice. Endocrinology 137:2954-2958. Sahu A (1998): Evidence suggesting that galanin, MCH, neurotensin, POMC and NPY are targets of leptin signalliung in the hypothalamus. Endocrinology 139 (2):795-798. Sailer AW, Sano H, Zeng Z, McDonald TE Pan J, Pong SS, Feighner SD, Tan CP, Fukami T, Iwaasa H, Hreniuk DL, Morin NR, Sharon J, Sadowski SJ, Ito M, Ito M, Bansal A, Ky B, Figueroa DJ, Jiang Q, Austin CP, MacNeil DJ, Ishihara A, Ihara M, Kanatani A, Van der Ploeg LHT, Howard AD, Liu Q (2001): Identification and characterization of a second melanin-concentrating hormone receptor, MCH-2R. Proc Natl Acad Sci USA 98:7564-7569. Saito Y, Nothacker HP, Wang ZW, Lin SHS, Leslie F, Civelli O (1999): Molecular characterization of the melanin-concentrating-hormone receptor. Nature 400:265-269. Saito Y, Nothacker HP, Civelli O (2000): Melanin-concentrating-hormone receptor: an orphan receptor fits the key. Trends Endocr Metabol 11 (8):299-303. Saito Y, Cheng M, Leslie FM, Civelli O (2001a): Expression of the melanin-concentrating-hormone receptor mRNA in the rat brain. J Comp Neurol 435:26-40. Saito Y, Wang Z, Hagino-Yamagishi K, Civelli O, Kawashima S, Maruyama K (2001b): Endogenous MCH receptor SLC-1 in the human melanoma SK-MEL-37 cells. Biochem Biophys Res Commun 289:44-50. Sakuntabhai A, Ruiz-Perez V, Carter S, Jacobsen N, Burge S, Monk S, Smith M, Munro CS, O'Donovan M, Craddock N, Kucherlapati R, Rees JL, Owen M, Lathrop GM, Monaco AP, Strachan T, Hovnanian A (1999): Mutations in ATP2A2, encoding a Ca 2+ pump, cause Dafter disease. Nat Genet 21:271-277. Sakurai T, Amemiya A, Ishii M, Matsuzaki I, Chemelli RM, Anaka H, Williams SC, Richardson JA, Kozlowski GP, Wilson S, Arch JRS, Buckingham RE, Haynes AC, Carr SA, Annan RS, NcNulty DE, Liu W-S, Terret JA, Elshourbagy NA, Bergsma DJ, Yanagisawa M (1998): Orexins and orexin receptors: a family of hypothalamic neuropeptides and G-protein-coupled receptors that regulate feeding behavior. Cell 92:573-585. Sanchez M, Baker I, Celis M (1997): Melanin-concentrating hormone (MCH): antagonizes the effects of alphaMSH and neuropeptide E-I on grooming and locomotor activities in the rat. Peptides 18:393-396. Sanpei K, Takano H, Igarashi S, Sato T, Oyake M, Sasaki H, Wakisaka A, Tashiro K, Ishida Y, Ikeuchi T, Koide M, Saito M, Sato A, Tanaka T, Hanyu S, Takiyama Y, Nishizawa M, Shimizu N, Nomura Y, Segawa M, Iwabuchi K, Eguchi I, Tanaka H, Takahashi H, Tsuji S (1996): Identification of the spinocerebellar ataxia type 2 gene using a direct identification of repeat expansion and cloning technique DIRECT. Nat Genet 14:277-284. Sawchenko PE (1998): Toward a new neurobiology of energy balance, appetite and obesity. The anatomists weigh-in. J Comp Neurol 402:435-441. Schwartz MW, Gelling RW (2002): Rats lighten up with MCH antagonist. Nature Medicine 8(8): 779-781. Schwartz MW, Woods SC, Porte D, Seeley RJ, Baskin DG (2000): Central nervous system control of food intake. Nature 404:661-671. Sergeyev V, Broberger C, Hokfelt T (2001): Effect of LPS administration on the expression of POMC, NPY, galanin, CART and MCH mRNAs in the rat hypothalamus. Mol Brain Res 90:93-100. Shimada M, Tritos N, Bardford Lowell A, Flier J, Maratos-Flier E (1998): Mice lacking melanin-concentrating hormone are hypophagic and lean. Nature 396:670-674. Shimomura Y, Mori M, Sugo T, Ishibashi Y, Abe M, Kurokawa T, Onda H, Nishimura O, Sumino Y, Fujino M (1999): Isolation and identification of melanin-concentrating hormone as the endogenous ligand of the SLC-1 receptor. Biochem Biophys Res Commun 261:622-626. Sone M, Takahashi K, Murakami O, Totsune K, Arihara Z, Satoh F, Sasano H, Ito H, Mouri T (2000): Binding sites for melanin-concentrating hormone in the human brain. Peptides 21:245-250.

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Stadel JM, Wilson S, Bergsma DJ (1997): Orphan G protein-coupled receptors: a neglected opportunity for pioneer drug discovery. Trends Pharmacol Sci 18:430-437. Strand FL, Beckwith B, Chronwall B, Sandman CA (1994): Models of neuropeptide actions. Ann New York Acad Sci 739:000-000. Stricker-Krongrad A, Dimitrov T, Beck B (2001): Central and peripheral dysregulation of melanin-concentrating hormone in obese Zucker rats. Mol Brain Res 92 (1-2):43-48. Suply T, Della Zuana O, Audinot V, Rodriguez M, Beauverger P, Duhault J, Canet E, Galizzi JP, Nahon JL, Levens N, Boutin JA (2001): SLC-1 receptor mediates effect of melanin-concentrating hormone on feeding behavior in rat: a structure-activity study. J Pharmacol Exp Ther 299 (1):137-146. Swanson LW (1998): Brain Maps: Structure of the Rat Brain, 2nd edn., Elsevier, Amsterdam. Takahashi K, Suzuki H, Totsume K, Murakami O, Satoh F, Sone M, Sasano H, Mouri T, Shibahara S (1995): MCH in human and rat. Neuroendocrinology 61:493-498. Takahashi K, Totsume K, Murakami O, Sone M, Kitamuro T, Noshiro T, Hayashi Y, Sasano H, Shibahara S (2001): Expression of melanin-concentrating hormone receptor messenger ribonucleic acid in tumor tissues of pheochromocytoma, ganglioneuroblastoma, and neuroblastoma. J Clin Endocrinol Metab 86 (1):369-374. Takekawa S, Asami A, Ishihara Y, Terauchi J, Kato K, Shimomura Y, Mori M, Murakoshi H, Suzuki N, Nishimura O, Fujino M (2002): T-226296: a novel, orally active and selective melanin-concentrating hormone receptor antagonist. Eur J Pharmacol 438 (3):129-135. Tan CP, Sano H, Iwaasa H, Pan J, Sailer AW, Hreniuk DL, Feighner SD, Palyha OC, Pong SS, Figueroa DJ, Austin CP, Jiang MM, Yu H, Ito J, Ito M, Guan XM, MacNeil DJ, Kanatani A, Van der Ploeg LHT, Howard AD (2002): Melanin-concentrating hormone receptor subtypes 1 and 2: Species-specific gene expression. Genomics 79 (6):785-792. Toumaniantz G, Bittencourt JC, Nahon JL (1996): The rat MCH gene encodes an additional protein in a different reading frame. Endocrinology 137 (10):4518-4521. Toumaniantz G, Ferreira PC, Allaeys I, Bittencourt JC, Nahon JL (2000): Differential neuronal expression and projections of MCH and MGOP in the rat brain. Eur J Neurosci 12:4367-4380. Touzani K, Tramu G, Nahon JL, Velley L (1993): Hypothalamic MCH and alpha-neoendorphin-immunoreactive neurones project to the medial part of the rat parabranchial area. Neuroscience 53 (3):865-876. Tritos NA, Maratos-Flier E (1999): Two important systems in energy homeostasis: melanocortins and MCH. Neuropeptides 33:339-349. Tritos NA, Elmquist JK, Mastaitis JW, Flier JS, Maratos-Flier E (1998a): Characterization of expression of hypothalamic appetite-regulating peptides in obese hyperleptinemic brown adipose tissues-deficient (uncoupling protein-promoter-driven diphtheria toxin A) mice. Endocrinology 139:4634-4641. Tritos NA, Vicent D, Gillette J, Ludwig DS, Flier ES, Maratos-Flier E (1998b): Functional interactions between MCH, NPY and anorectic peptides in the rat hypothalamus. Diabetes 47:1687-1692. Tritos NA, Mastaitis JW, Kokkotou E, Maratos-Flier E (2001): Characterization of melanin concentrating hormone and preproorexin expression in the murine hypothalamus. Brain Res 895(1-2):160-166. Tsukamura H, Thompson RC, Tsukahara S, Ohkura S, Maekawa F, Moriyama R, Niwa Y, Foster DL, Maeda KI (2000): Intracerebroventricular administration of melanin-concentrating hormone suppresses pulsatile luteinizing hormone release in the female rat. J Neuroendocrinol 12 (6):529-534. Varas M, Perez M, Ramirez O, de Barioglio SR (2002): Melanin concentrating hormone increase hippocampal synaptic transmission in the rat. Peptides 23 (1):151-155. Vaughan JM, Fisher WH, Hoeger C, River J, Vale W (1989): Characterisation of melanin-concentrating hormone from rat hypothalamus. Endocrinology 125:1660-1665. Verlaeten O, Griffond B, Khuth ST, Giraudon P, Akaoka H, Belin MF, Fellmann D, Bernard A (2001): Down regulation of melanin concentrating hormone in virally-induced obesity. Mol Cell Endocrinol 181 (1-2):207-219. Viale A, Zhixing Y, Breton C, Pedeutour F, Coquerel A, Jordan D, Nahon JL (1997): The melanin-concentrating hormone gene in human: flanking region analysis, fine chromosome mapping, and tissue-specific expression. Mol Brain Res 46(1-2):243-255. Viale A, Ortola C, Richard F, Vernier P, Presse F, Schilling S, Dutrillaux B, Nahon JL (1998): Emergence of a brain-expressed variant melanin-concentrating hormone gene during higher primate evolution: a gene 'in search of a function'. Mol Biol Evol 15:196-214. Viale A, Kerdelhue B, Nahon JL (1999a): 17~-Estradiol regulation of melanin concentrating hormone and neuropeptide-E-I contents in cynomolgus monkeys: a preliminary study. Peptides 20:553-559. Viale A, Ortola C, Hervieu G, Furuta M, Barbero P, Steiner D, Seidah N, Nahon JL (1999b): Cellular localization and role of prohormone convertases in the processing of pro-melanin concentrating hormone in mammals. J Biol Chem 274:6536-6545. 100

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Viale A, Courseaux A, Presse F, Ortola C, Breton C, Jordan D, Nahon JL (2000): Structure and expression of the variant melanin-concentrating hormone genes: only PMCHL1 is transcribed in the developing human brain and encodes a putative protein. Mol Biol Evol 17:1626-1640. Wang SK, Behan J, O'Neill K, Weig B, Fried S, Laz T, Bayne M, Gustafson E, Hawes BE (2001): Identification and pharmacological characterization of a novel human melanin-concentrating hormone receptor, MCH-R2. J Biol Chem 276 (37):34664-34670. Witty DR, Hadley MS, Hervieu GJ, Jeffrey R Johnson CN, Jones M, Muir A, O'Hanlon PJ, O'Toole CR Riley GJ, Stemp G, Stevens AJ, Thewlis KM, Wilson S, Winborn KY, Wroblowski B (2002): Biphenyl carboxamide antagonists of the human melanin-concentrating hormone receptor 11CBy (SLC-1); discovery and SAR, Medicinal Chemistry Symposium, Barcelona, Spain, September 2002. Yamada M, Mikayama T, Duttaroy A, Yamanaka A, Moriguchi T, Makita R, Ogawa M, Chou CJ, Xia B, Crawley JN, Felder CC, Deng C-X, Wess J (2001): Mice lacking the M3 muscarinin receptor are hypophagic and lean. Nature 410:207-212. Zamir N, Skotfish G, Banson ML, Jacobowitz DM (1986a): MCH: unique peptide neuronal system in the rat brain and pituitary gland. Proc Natl Acad Sci USA 83:1528-1531. Zamir N, Skotfish G, Jacobowitz DM (1986b): Distribution of immunoreactive MCH in the rat CNS. Brain Res 373:240-245. Zhang R Liang JD, Sandusky GE, Burguera B, Considine RV, Hyde TM, Caro JF (1998): Hypothalamic MCH mRNA protein are increased in human obesity. Satellite Symposium: Ninth International Congress of Obesity, France, Proceedings of the Symposium, Int J Obesity, p. 51.

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Neuroanatomical studies of the opioid receptor-like-1 receptor and its endogenous neuropeptide orphanin FQ (nociceptin) CHARLES R. NEAL JR., HUDA AKIL AND STANLEY J. WATSON JR.

1. INTRODUCTION Following the cloning of the Ix, 3 and ~: receptors, a novel clone was isolated that encoded a putative membrane receptor with homology to the Ix, 3 and ~: receptors (Bunzow et al., 1994; Marchese et al., 1994; Wick et al., 1994). This orphan clone was soon characterized and named the opioid receptor-like (ORL1) receptor to emphasize its relationship to the known opioid receptors (Mollereau et al., 1994). Early studies of ORL1 demonstrated it to be a member of the G-protein family of seven transmembrane receptors, to have similar homology to opioid receptors in rat, mouse and human, but to be distinct in its structure and distribution (Chen et al., 1994; Fukuda et al., 1994; Lachowicz et al., 1994). The ORL1 receptor has a 47% amino acid homology when compared across the opioid receptors. Within the transmembrane domains, the level of identity increases to 61-64% (Bunzow et al., 1994), and the receptors share a high degree of identity in the three cytoplasmic loops. Other structural features conserved in the ORL1 and opioid receptors include multiple glycosylation sites in the Nterminal domain, aspartate residues in transmembrane regions 2 and 3, cyclic AMP-dependent phosphorylation sites in the third intracellular loop and several palmitoylation sites in the Cterminal extracellular domain. In addition, all four receptors are negatively linked to adenylate cyclase (Bunzow et al., 1994; Fukuda et al., 1994; Lachowicz et al., 1994). Northern analysis suggests the presence of three ORL1 receptor transcripts (Fukuda et al., 1994; Lachowicz et al., 1994; Wick et al., 1994), and splice variants of ORL1 have been reported (Wang et al., 1994; Mathis et al., 1997; Curro et al., 2001; Mogil and Pasternak, 2001). In situ hybridization studies of the ORL 1 receptor have demonstrated that it is widely distributed in the central nervous system (CNS) of the rat (Bunzow et al., 1994; Fukuda et al., 1994; Lachowicz et al., 1994; Mollereau et al., 1994; Neal et al., 1999b; Wick et al., 1994). Additionally, the presence of ORL1 outside of the CNS has been reported, with detectable levels in the intestine, vas deferens and spleen (Lachowicz et al., 1994; Wang et al., 1994; Halford et al., 1995). In the search for a neuropeptide that activates the ORL1 receptor, the heptadecapeptide referred to as nociceptin, or orphanin FQ (OFQ; FGGFTGARKSARKLANQ), was identified (Reinscheid et al., 1995). OFQ exhibits structural features suggestive of endogenous opioid peptides (Civelli et al., 1997). The presence of a Gly-Gly-Phe motif in amino acid positions

Handbook of Chemical Neuroanatomy, Vol. 20: Peptide Receptors, Part H R. Quirion, A. Bj6rklund and T. H6kfelt, editors 92003 Elsevier Science B.V. All fights reserved.

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2-4, an Asn-Gln sequence at the C-terminus, and several positively charged amino acids in the intervening sequence are very similar to dynorphin Al-17 (Meunier et al., 1995; Reinscheid et al., 1995). Similar to endogenous opioids, OFQ is derived from a larger precursor (preproorphanin or preproOFQ) which contains additional neuropeptides that may be biologically active (Houtani et al., 1996; Mollereau et al., 1996b; Nothacker et al., 1996; Pan et al., 1996; Vaughan et al., 2001). Immediately downstream from OFQ is a lysylarginine processing signal followed by a heptadecapeptide like OFQ, which also begins with a phenylalanine and ends with a glutamine. This peptide has been referred to as OFQ II. In contrast to the nociceptive effects observed with OFQ administration, OFQ II administration appears to provide analgesic activity in high doses in mice (Rossi et al., 1998; Mathis et al., 2001). Upstream from OFQ is an amino acid sequence that is flanked by double basic amino acids, possibly being liberated with post-translational processing. This molecule, referred to as nocistatin, has demonstrated analgesic activity and antagonism of OFQ nociceptive effects (Okuda-Ashitaka et al., 1998; Xu et al., 1999; Hiramatsu and Inoue, 1999; Yamamoto et al., 1999; Zhao et al., 1999; Nakano et al., 2000; Okuda-Ashitaka and Ito, 2000; Sun et al., 2001). Because preproorphanin shares close structural homology to the endogenous opioid peptide precursors prodynorphin and preproenkephalin, it has been suggested that a coordinated mechanism of evolution has separated the OFQ and opioid systems (Reinscheid et al., 1998; Danielson and Dores, 1999; Danielson et al., 2001). In spite of these similarities to the endogenous opioids, OFQ demonstrates specific binding characteristics with the ORL1 receptor (Meunier et al., 1995; Reinscheid et al., 1995, 1996; Saito et al., 1995, 1996, 1997; Shimohigashi et al., 1996; Dooley and Houghten, 1996; Ardati et al., 1997; Butour et al., 1997; Guerrini et al., 1997). The primary structure of rat and human preproorphanin has been elucidated (Mollereau et al., 1996b; Nothacker et al., 1996; Zaveri et al., 2001), as has tissue distribution of the preproorphanin mRNA in rat (Neal et al., 1999a) and mouse (Houtani et al., 1996; Pan et al., 1996). Antisera to OFQ have been produced and detection of OFQ-like immunoreactivity is wide spread in the rat (Riedl et al., 1996; Schulz et al., 1996; Neal et al., 1999a; Lai et al., 1997; Schuligoi et al., 1997). 2. GENERAL CHARACTERISTICS

2.1. KINETICS AND PHARMACOLOGY OFQ binds saturably and with high affinity to the ORL1 receptor and it inhibits cAMP formation in ORLl-transfected cells (Meunier et al., 1995; Reinscheid et al., 1995, 1996; Dooley and Houghten, 1996; Saito et al., 1996, 1997; Shimohigashi et al., 1996; Ardati et al., 1997; Butour et al., 1997; Civelli et al., 1997; Guerrini et al., 1997). Several OFQ fragments with high affinity binding to ORL1 have also been identified (Dooley et al., 1997). Although OFQ has an amino acid sequence very similar to dynorphin Al-17, it demonstrates no binding affinity for the endogenous opioid receptors (Meunier et al., 1995; Reinscheid et al., 1995). Functional studies of ORL1 have demonstrated that, similar to opioid receptors, its activation stimulates GTPyS binding and inhibits adenylate cyclase (Wu et al., 1997), but despite the degree of amino acid and structural conservation across these receptors, ORL1 does not bind any opioid peptide or alkaloid with high affinity (Bunzow et al., 1994; Chen et al., 1994; Fukuda et al., 1994; Lachowicz et al., 1994; Ma et al., 1997; Wick et al., 1994). Binding and mutation analyses have established that ORL1 has features that specifically 104

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exclude opioids and promote the high affinity binding of OFQ (Standifer et al., 1994; Meng et al., 1996; Mollereau et al., 1996a). Interestingly, with single amino acid changes in transmembranes 5, 6 and 7, ORL1 develops the capacity to bind opioid agonists (Meng et al., 1996; Mollereau et al., 1996a). 2.2. CELLULAR NEUROPHYSIOLOGICAL EFFECTS Orphanin activation of the ORL1 has been shown to inhibit cAMP accumulation in stably transfected cells and protein C activation (Meunier et al., 1995; Reinscheid et al., 1995; Saito et al., 1995, 1996, 1997; Civelli et al., 1997; Lou et al., 1997). Binding of OFQ to the ORL1 receptor has also been shown to inhibit both T-type and N-type Ca 2+ channel currents in rat sensory neurons and human neuroblastoma cells (Connor et al., 1996; Abdulla and Smith, 1997; Luo et al., 2001). This type of Ca 2+ current inhibition has been demonstrated in hippocampal, central gray and locus coeruleus neurons (Connor and Christie, 1998; Connor et al., 1999; Pu et al., 1999; Borgland et al., 2001). Inwardly rectifying potassium currents are also activated by orphanin. Examples of this effect have been reported in freshly dissociated neurons from cortex (Nicol et al., 1996), hippocampus (Knoflach et al., 1996; Madamba et al., 1999; Amano et al., 2000), arcuate nucleus (Wagner et al., 1998), supraoptic nucleus (Slugg et al., 1999), central gray (Vaughan et al., 1997; Connor and Christie, 1998), dorsal raphe (Vaughan and Christie, 19961) and locus coeruleus (Connor et al., 1996, 1999; Ikeda et al., 1997; Jennings, 2001). Modulation of glutamate- and kainic acid-induced currents in rat dorsal horn neurons has also been reported (Shu et al., 1998). 3. BIOLOGICAL EFFECTS OF OFQ BINDING AT THE ORL1 RECEPTOR

Binding of OFQ to the ORL1 receptor has been shown to mediate several physiologic and behavioral functions. At the cellular level, ORL1 activation can effect function of numerous neurotransmitter systems. Orphanin has been reported to suppress both excitatory (Faber et al., 1996; Liebel et al., 1997) and inhibitory (Zeilhofer et al., 2000) synaptic transmission in the rat spinal cord, suppress NMDA receptor-dependent long-term depression in the dentate gyms (Wei and Xie, 1999), inhibit oxytocin, vasopressin and GnRH secretion (Doi et al., 1998a,b; Dhandapani and Brann, 2002) and inhibit tachykinin transmission (Giuliani and Maggi, 1996; Inoue et al., 1999). OFQ activation of ORL1 has also been shown to modulate activity of suprachiasmatic nucleus neurons (Allen et al., 2000), lateral amygdala (Meis and Pape, 2001), enkephalin release (Gintzler et al., 1997), mesolimbic dopamine transmission (Murphy et al., 1996; Di Giannuario et al., 1999; Murphy and Maidment, 1999; Maidment et al., 2002; Norton et al., 2002; Zheng et al., 2002) and trigeminal neuronal response to excitatory amino acids (Wang et al., 1996). Orphanin is also shown to be antagonistic to endomorphin-1 induced analgesia (Wang et al., 1999). In the periphery, OFQ inhibits tonic nitric oxide release in the mouse colon (Menzies and Corbett, 2000). Administration of OFQ into the CNS induces variable modulatory effects on allodynia (Hara et al., 1997; Minami et al., 1997) and nociception (Mogil et al., 1996a,b, 1999; Grisel et al., 1996; Rossi et al., 1996, 1997; Stanfa et al., 1996; Xu et al., 1996; Dawson-Basoa and Gintzler, 1997; Heinricher et al., 1997; King et al., 1997; Kolesnikov and Pasternak, 1997; Liebel et al., 1997; Morgan et al., 1997; Nishi et al., 1997; Tian et al., 1997a,b; Yamamoto et al., 1997, 1999; Zhu et al., 1997; Vanderah et al., 1998; Pan et al., 2000; Mogil and Pasternak, 2001; Przewlocki and Przewlocka, 2001; Yu et al., 2002), enticing many to also refer to 105

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this molecule as nociceptin. Unlike opioid peptides, OFQ fails to produce conditioned place preference (Devine et al., 1996b; Kotlinska et al., 2002) or morphine cross-tolerance (Hao et al., 1997; Lutfy et al., 2001a), but it does inhibit acquisition of morphine place preference (Murphy et al., 1999) and morphine withdrawal (Kotlinska et al., 2000; Kest et al., 2001; Mamiya et al., 2001; Walker et al., 2002). The role of OFQ in drug-seeking behavior and sensitization to cocaine remains unclear (Narayanan and Maidment, 1999; Ciccocioppo et al., 2000, 2002; Lufty et al., 200 l b; Narayanan et al., 2002). OFQ has also has been implicated in many physiologic and behavioral processes. Physiologic processes include cardiac and peripheral vascular control (Champion and Kadowitz, 1997a,b; Champion et al., 1997, 1998; Gumusel et al., 1997; Bucher, 1998; Arndt et al., 1999; Chu et al., 1999; Kapusta and Kenigs, 1999; Maslov et al., 1999; Mao and Wang, 2000), diuresis and sodium balance (Kapusta et al., 1997; Kapusta and Kenigs, 1999), thermoregulation (Yakimova and Pierau, 1999; Chen et al., 2001), vestibular function (Sulaiman et al., 1999), colonic transit (Takahashi et al., 2000) and modulation the neural control of intestinal smooth muscle contractility and mucosal transport (Osinski et al., 1999). Behavioral processes include feeding (Olszewski et al., 2000; Pomonis et al., 1996; Ciccocioppo et al., 2001, 2002; Pietras and Rowland, 2002; Olszewski et al., 2002), locomotion (Devine et al., 1996b; Florin et al., 1996, 1997a; Rizzi et al., 2001a), learning and memory (Sandin et al., 1997; Hiramatsu and Inoue, 1999; Higgins et al., 2002), scratching, biting and licking (Sakurada et al., 2000), sexual behavior (Sinchak et al., 1997; Gupta et al., 2001; Dhandapani and Brann, 2002) and stress (Griebel et al., 1999; Devine et al., 2001; Gavioli et al., 2002; Redrobe et al., 2002). In the rat, orphanin appears to play a role as an anxiolytic (Griebel et al., 1999; Jenck et al., 1997, 2000). It has also been reported to prevent stress-induced ethanol-seeking behavior (Martin-Fardon et al., 2000). This has been further supported in the mouse where targeted disruption of the OFQ gene increases stress susceptibility and impairs stress adaptation (Koster et al., 1999). It should be noted that a possible role for OFQ in hypoxic-ischemic brain injury is emerging (Armstead, 2001, 2002; Jagolino and Armstead, 2001; Laudenbach et al., 2001). This role may be via its contribution to hypoxic-ischemic impairment of NMDA-induced cerebral vasodilation after hypoxic-ischemic brain injury (Armstead, 2000a,b,c), or by direct effect on pial arteries via potassium channel activation (Armstead, 1999, 2000d). 4. ANATOMICAL STUDIES OF THE ORPHANIN PEPTIDE-RECEPTOR SYSTEM

Several early studies reported the general distribution of OFQ and the ORL1 receptor in the C N S o f several species. Following the sequencing of the primary structure of the rat and human OFQ precursor (Mollereau et al., 1996b; Nothacker et al., 1996), the tissue distribution of preproorphanin mRNA was reported for the rat (Mollereau et al., 1996b; Nothacker et al., 1996) and mouse (Houtani et al., 1996; Pan et al., 1996). Using antisera to OFQ, detection of OFQ-like immunoreactivity has been reported in the spinal cord (Riedl et al., 1996; Lai et al., 1997; Schuligoi et al., 1997) and other structures within pain-modulatory regions in the rat (Schulz et al., 1996), and the hypothalamus of the rodent and monkey (Quigley et al., 1998). An extensive analysis of orphanin peptide and mRNA distribution in the rat brain and spinal cord has also been reported (Neal et al., 1999a). General descriptions of ORL 1 mRNA distribution in rat CNS and peripheral tissue has also been reported (Bunzow et al., 1994; Fukuda et al., 1994; Lachowicz et al., 1994; Mollereau et al., 1994; Wick et al., 1994). Binding studies of the orphanin receptor, including OFQ106


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stimulated GTPyS binding in the rat and guinea pig brain (Sim et al., 1996; Sim and Childers, 1997), [3H] orphanin receptor binding in the mouse and rat (Florin et al., 1997b; Kusaka et al., 2001) and 125I-labeled orphanin binding in the rat and human hypothalamus (Makman et al., 1997) have confirmed the specificity of OFQ for the ORL1 receptor, as well as the wide distribution of this system throughout the rat forebrain, brainstem and spinal cord. Complimenting these general descriptive studies, which provide a framework in evaluating the distribution of this system in the brain, detailed analyses of the OFQ and ORL1 receptor systems have been reported in both rat and mouse. In the mouse, the distribution of preproOFQ and ORL1 mRNA in the developing brain has been reported (Ikeda et al., 1998). Recently, in a study using in situ hybridization and X-gal histochemistry in ORL 1 receptor-deficient mice, the distribution of the OFQ precursor protein and ORL 1 receptor in brain and spinal cord was reported (Houtani et al., 2000). In the rat, a detailed distribution of the ORL1 receptor in the CNS has been reported, using immunocytochemistry (Anton et al., 1996), mRNA distribution, and receptor binding with 125I-[14Tyr]OFQ (Neal et al., 1999b). Additionally, a recent anatomical study comparing 125I-[14Tyr]OFQl_17 and 125I-[1~ binding in the rat brain has been reported (Letchworth et al., 2000). The authors of this paper report binding differences between the two ligands OFQ, with decreased binding levels noted at all anatomical levels when using 125I-[l~ The distinctive autoradiographic patterns of these two ligands are suggestive of different binding sites on the orphanin receptor (Mathis et al., 1999; Letchworth et al., 2000; Curro et al., 2001; Mogil and Pasternak, 2001). The pattern of binding reported by these authors for 125I-[14Tyr]OFQl_17 is similar to that reported earlier in a detailed review of this ligand in the rat CNS (Neal et al., 1999b), while the level of labeling detected using 125I-[l~ although decreased in many regions, is also similar to that of 125I[14Tyr]OFQl_17. This raises the distinctive possibility that the difference in binding observed with 125I-[l~ in many regions is due to decreased affinity for the same receptor binding site rather than a binding domain distinctive from that of ORL 1. Although immunocytochemical analyses of OFQ and ORL1 distribution in the rat brain have been reported, the specificity of the antiserum used to characterize ORL1 receptor distribution has recently come into question (Evans, 1999). The descriptions that follow are a compilation of a number of neuroanatomical approaches used to delineate the distribution of the orphanin system in the rat brain and spinal cord. OFQ distribution has been reported in detail using immunohistochemistry and in situ hybridization, allowing us to examine the distribution of both preproOFQ mRNA expression and its peptide product, OFQ1_17. The distribution of the ORL1 receptor will be discussed in terms of detailed distribution of ORL1 mRNA expression and 125I-[14Tyr]OFQ binding. A brief discussion of distribution of OFQ-stimulated GTPyS binding will also be undertaken, followed by brief descriptions of OFQ and ORL 1 distribution in the developing rat and human brain. 5. IN SITU HYBRIDIZATION AND IMMUNOHISTOCHEMISTRY STUDIES

5.1. METHODS 5.1.1. Animals

Adult male Sprague-Dawley rats (Charles River; 250-300 g) were used for all in situ hybridization and immunocytochemistry studies. Handling and use of all animals strictly conformed to NIH guidelines. 107

Ch. III


5.1.2. Tissue preparation For in situ hybridization, adult male Sprague-Dawley rats were decapitated and their brains, pituitary, and cervical and thoracic spinal cords removed. All tissue was quick-frozen at -30~ Tissue was sectioned in the coronal plane at 15 txm and thaw-mounted on polylysinetreated microscope slides, then stored at - 8 0 ~ until used. For immunohistochemistry, adult male Sprague-Dawley rats were deeply anesthetized with sodium pentobarbital and transcardially perfused with 0.9% NaC1 containing 2% sodium nitrite, followed with Zamboni's fixative (Zamboni and DeMartino, 1967). Several animals were treated with intraventricular colchicine prior to perfusion. After perfusion was completed, the brain, pituitary and portions of the thoracic and cervical spinal cord were removed, post-fixed for 1 h, soaked in a buffered 10% sucrose solution, then quick-frozen at -30~ All tissue was sectioned in the coronal plane at 30 txm and stored in at - 2 0 ~ until used.

5.1.3. Preproorphanin and ORL1 cRNA probes Hybridization of CNS tissue was performed using 35S-UTP and 35S-CTP-labeled riboprobes generated against the rat preproOFQ mRNA sequence and the 5' region of the rat ORL1 receptor. A PCR fragment corresponding to the 5' portion of the preproOFQ sequence was used to prepare the 35S-labeled cRNA probe. This cDNA fragment spans 580 base pairs (bp) and contains the entire open reading frame of the preproOFQ precursor molecule (Houtani et al., 1996; Mollereau et al., 1996b; Nothacker et al., 1996). The orphanin receptor cRNA riboprobe was generated from a 700-base cDNA that extended from the 5' UT and protein coding regions of the ORL1 receptor (Bunzow et al., 1994; Chen et al., 1994; Fukuda et al., 1994; Lachowicz et al., 1994; Mollereau et al., 1994; Wang et al., 1994; Wick et al., 1994).

5.1.4. OFQ antibody production The orphanin antiserum used to analyze OFQ peptide distribution in the rat brain was manufactured in our laboratory, in collaboration with HRP Inc. (Denver, PA) where the rabbit anti-OFQ antiserum was produced (Neal et al., 1999a). Briefly, the entire 17-amino acid OFQ peptide sequence was conjugated to thyroglobulin and suspended in complete Freund's adjuvant. New Zealand White Rabbits (n = 2) were inoculated with the thyroglobulinconjugated OFQ peptide. A total of eight bleeds were obtained, after-which the rabbits were exsanguinated. Serum from each bleed, from each rabbit, was tested immunohistochemically and antisera from two optimal bleeds were affinity purified. Affinity purified antisera from the same bleed was then used in all immunocytochemical studies.

5.1.5. Immunohistochemistry The immunohistochemistry technique employed in the analysis of OFQ distribution in the rat brain has been described in detail (Neal et al., 1999a). Floating rat brain and spinal cord sections were initially washed in potassium phosphate buffered saline (KPBS) to remove cryoprotectant, then incubated in 0.3% H202. Next, tissue was incubated with an Avidin D blocking agent, followed by a biotin blocking solution (Vector Laboratories, Burlingame, CA). Sections were next incubated in a bovine serum albumin (BSA) diluent, then transferred to a solution containing affinity purified OFQ antibody in BSA diluent for 36-48 h. After primary 108


Ch. III

antibody incubation, sections were washed in KPBS and incubated with biotinylated goat antirabbit IgG, followed by an avidin-biotin complex (ABC) coupled to horseradish peroxidase (Vector Elite, Burlingame, CA). Immunostaining was visualized with a diaminobenzidinenickel chloride reaction, and sections were mounted onto polylysine-subbed microscope slides and prepared for brightfield analysis.

5.1.6. In situ hybridization The in situ hybridization technique employed in the analysis of OFQ and ORL1 mRNA distribution has been described in detail (Neal et al., 1999a,b), and used previously for the detection of opioid receptor mRNA in the rat CNS (Mansour et al., 1993, 1994). Sections of frozen brain, pituitary and spinal cord (with dorsal root ganglia) were placed directly from -80~ storage into 4% paraformaldehyde for fixation. Sections were washed in a sodium chloride-sodium citrate (SSC) solution, treated with triethanolamine and acetic anhydride, rinsed in water, dehydrated and air-dried. Prepared tissue was then hybridized overnight with a 35S-UTP and 35S-CTP-labeled riboprobe generated to the rat OFQ peptide precursor or ORL1 receptor. On day 2, the 35S-cRNA solution was removed, tissue was rinsed in SSC, then treated with RNase A. Following RNase A treatment, sections were treated with decreasing salt washes, rinsed in water, dehydrated and air-dried. Upon completion of hybridization, slide-mounted sections were opposed to Kodak XAR-5 X-ray film for 5 days, then dipped in NTB2 film emulsion. Slides were developed following a 30-day exposure to NTB2, Nissl counterstained with Cresyl violet and prepared for darkfield analysis and photography.

5.1.7. Immunohistochemistry and in situ hybridization controls For immunohistochemical preabsorption controls, OFQ antibody was treated with a 25txM concentration of OFQI_17 overnight prior to its addition to floating tissue sections. Preabsorption controls and normal immunohistochemical studies were performed on adjacent tissues from both normal and colchicine-treated animals. The specificity of the preproOFQ and ORL 1 cRNA sequences used for in situ hybridization was determined with two separate control conditions. After fixation with 4% paraformaldehyde, sections from representative brain regions were incubated in RNase A for 60 min. They were then run through the same hybridization procedure described above. A separate set of sections were run through the entire hybridization procedure as described above with the exception that a 35S-labeled mRNA (sense strand) was used for the hybridization. All control tissue was treated identical to, and run with adjacent sections under normal conditions. 5.2. CONTROL RESULTS

5.2.1. Immunocytochemistry controls 5.2.1.1. Colchicine treatment

Colchicine injection into the lateral ventricle markedly altered OFQ immunohistochemical staining in the rat brain. In untreated animals, neuronal labeling was observed in several regions (e.g. septum, hypothalamus, reticular thalamus), but intensity of labeling was significantly diminished. In contrast, fiber and terminal labeling was more pronounced in untreated rats. This pattern of immunolabeling is expected in animals treated with an inhibitor of microtubule-dependent axonal transport such as colchicine. 109

Ch. III


Although used extensively in immunohistochemical mapping studies, reports have raised the possibility that colchicine treatment induces an increase in mRNA levels for neuropeptides in the rat brain (Cortes et al., 1990; Ceccatelli et al., 1991; Kiyama and Emson, 1991). If true, one could argue that some immunolabeling observed in colchicine-treated animals could represent artifact, rather than the product of true message expression. In our experiences we have not found colchicine treatment to produce such an effect. An excellent correlation was observed between OFQ mRNA distribution in untreated animals and that of the OFQ peptide in colchicine-treated and untreated animals. Areas that contain mRNA expression also demonstrate some degree of immunolabeling. In no brain regions are immunolabeled neurons detected where mRNA-expressing cells were not localized. The brains of animals not receiving colchicine treatment provide minimal cell labeling but provide an added source of information concerning the distribution of OFQ fiber and terminal immunoreactivity, complimenting immunostaining observed in colchicine-treated animals. Therefore, recent descriptions of OFQ peptide distribution (Neal et al., 1999a) are a synthesis of immunolabeling observed in both colchicine-treated and normal animals. 5.2.1.2. Preabsorption controls

Preabsorption of the primary OFQ antibody with orphanin peptide eliminates all immunolabeling (Fig. 1). However, there were two regions where immunolabeling did not block completely. A group of densely immunolabeled cell bodies clustered lateral to the paraventricular hypothalamus contain a very small population of neurons not completely blocked in preabsorption controls. Unblocked perikarya are visible but lightly labeled. In the cerebellum dense staining of perikarya, observed throughout the Purkinje cell layer, are present in both colchicine-treated and untreated animals. They are confined to the Purkinje layer and are completely unblocked in preabsorption control studies. Additionally, they demonstrate no OFQ mRNA expression. Therefore, immunolabeling in the cerebellar Purkinje cell layer is considered non-specific. In contrast, immunolabeling in the granular layer and deep cerebellar nuclei is specific, with immunolabeling in these areas blocked completely in preabsorption controls. 5.2.2. In situ hybridization controls Virtually no mRNA-expressing cells are detected in tissues hybridized with a 35S-labeled mRNA (sense strand) directed to the 5' portion of the preproOFQ cDNA sequence or the 5' UT portion of the ORL1 cDNA sequence (Fig. 2). Messenger RNA levels in these tissues are negligible in all levels of the brain and spinal cord, with exceptions being non-specific 35S-ORL1 labeling in the cerebellar lobules and area CA1 of Ammon's horn. Additionally, no mRNA expression is detected in tissues pretreated with RNase A prior to in situ hybridization using 35S-labeled cRNA (antisense) directed to the 5' UT portion of the ORL1 cDNA sequence (Fig. 2) or the 5' portion of the preproOFQ cDNA sequence. 5.3. DISTRIBUTION OF OFQ AND THE ORL1 RECEPTOR IN THE RAT FOREBRAIN A detailed presentation of the distribution of preproOFQ and ORL1 in the rat CNS is provided in Table 1. The table is organized regionally, providing a comparison of OFQ peptide and preproOFQ mRNA distribution to that of ORL 1 mRNA expression. Examples of OFQ mRNA distribution are provided in Figs. 3 and 4. Examples of ORL1 mRNA distribution compared to orphanin binding (see below) are provided in Figs. 5-9. 110


Ch. III

Fig. 1. Brightfield photomicrographs demonstrating immunohistochemistry controls in colchicine-treated animals: immunolabeling in area CA1 of Ammon's horn in the hippocampal formation after incubation with primary OFQ antiserum (A) is absent in the same region when incubated in primary antiserum after preabsorption with 25 ~M OFQ peptide (B); densely immunolabeled neurons within the substantia nigra, pars reticulata (C) are unlabeled in adjacent control tissue (D); cell and fiber immunolabeling within the dorsal horn of the spinal cord (E) is also completely blocked in adjacent control tissue (F). Scale bar: 200 ~m (C-F); 75 Ixm (A, B).

5.3.1. Cortex

5.3.1.1. PreproOFQ in situ hybridization PreproOFQ mRNA expression is moderate in the neocortex, found consistently throughout its rostral to caudal extent. Cells containing OFQ mRNA are observed in layers II, III, V and strongest in layer VI. Expression is more pronounced in frontal versus parietal cortex, and is weakest in temporal cortex. In the occipital cortex mRNA-expressing neurons are equally distributed in layers II, III and VI. In other cortical regions, strong expression is observed in agranular insular cortex, tenia tecta, and the cingulate and retrosplenial cortices. Moderate mRNA expression is seen in medial, lateral and ventral orbital, infralimbic, dorsal peduncular, granular insular, piriform and entorhinal cortices. 111

Ch. III


Fig. 2. Darkfield images of in situ hybridization controls. (A) Orphanin FQ receptor mRNA expression obtained after hybridization with a 35S-labeled cRNA (antisense strand) generated against the 5' UT portion of the ORL1 sequence. (B) Labeling is absent in an adjacent section hybridized with a 35S-labeled mRNA (sense strand) generated against the same region of the orphanin receptor. Note non-specific labeling in area CA1 of Ammon's horn. (C) Orphanin FQ receptor mRNA expression obtained after hybridization with a 35S-labeled riboprobe generated against the transmembrane 3-6 region of the ORL1 sequence. (D) Labeling is absent in an adjacent section treated with RNase A prior to in situ hybridization. Scale bar: 500 ~m. 112


Ch. III

Distribution of orphanin FQ immunoreactivity, preproorphanin mRNA expression, ORL1 mRNA expression, and 125I-[Tyr14]OFQ binding in the rat central nervous system T A B L E 1.

CNS region

OFQ peptide

OFQ mRNA

Cells

ORL1 mRNA

OFQ binding

F/T

Neocortex Frontal Layer I

.

L a y e r II

+

. +

.

+++

. +++

+ +

L a y e r III

++

+

+++

++

++

Layer IV

-

-

-

++++

++++

Layer V

+

+

++

+

++

Layer VI

++

+

+++

+++

+++

Parietal Layer I

.

L a y e r II

+

. -

.

+++

. ++

+ +

L a y e r III

+

-

++

++

++

Layer IV

-

-

-

+++

+++

Layer V

+

+

+

++

++

Layer VI

++

+

+++

+++

++

Temporal Layer I

.

L a y e r II

-

. +

. +

.

. +++

++

L a y e r III

-

+

+

++

++

Layer IV

-

-

-

++++

+

Layer V

-

+

+

++

+++

Layer VI

+

+

++

+++

++

Occipital Layer I

.

L a y e r II

-

. -

.

++

. +++

++ +

L a y e r III

-

-

++

++

++

Layer IV

-

-

-

+++++

++

Layer V

+

+

+

++

+++++

Layer VI

++

+

++

+++

++++

+++

++++

+++ +

++++ ++

+++++ +++

+++

O~erco~icalregions AI

+

CC

.

Cg DP

++ -

+ -

-

-

Ent

.

+++ .

.

.

++++

+++

Epl

-

-

Ipl

-

-

++

+++

+++

+++

GI

+

-

G1

-

++++

GrO

-

-

IL

+

-

++

++

LO

+

-

+++

++

+++

++

++

Mi MO

-

-

++

+++

+++

Pir

+

++

++

++++

++++

RSA

++

+

+++

++++

+++++

RSG

++

+

++

++++

+++++

TT

++

-

++

+++

++

VO

++

-

+++

+++

+++

113

Ch. III


T A B L E 1. (continued) CNS region

O F Q peptide Cells

OFQ mRNA

ORL1 m R N A

O F Q binding

+ + +++ ++ ++ ++ ++ ++

++ +++ +++ ++++ +++ ++++ ++ +++++

+++

+++

+

+++

+++ ++ +++ ++

++ ++ ++ ++

++++ +++ + ++

++ ++ +++

+++++ ++++ +++

+ +++ + +++ ++

F/T

Ventral forebrain ac

.

AcbC AcbSh AOD AOE AOL AOM AOP AOV FStr HDB ICj IPAC lo SI Tu VDB VP

+ + -

.

++++ ++ -

.

++ + -

.

++ + ++++ .

+ +++ ++ .

.

+ ++ + ++ ++++ .

. +++ + + +

.

.

.

df Ld LSD LSI LSV

. +++ ++ +

.

++ + ++ +++

m

Septum .

.

m

MS

+

-

+

PLd SFi

-

-

-

++ +++ ++ +++ +++ +

SHi

++

++

++

++++

SHy

+++

+++

++++

++

+++

++

++

+++

++ + ++ ++ +++ ++ ++ +++ +

++ + ++ + ++ + + +

+++ + +++ ++ +++ +++ ++ +++ +

++ + ++++ ++ +++ ++++ ++ +++ +++

++

-

++

++++

++

+ +++

++++ +++

+ ++++

++ +++ + +++ +

++++ +++ ++ +++ ++++

++ ++++ + ++++ +++

++++ +++ +++ + +++

++ +++ +++ +++ +++

++++ ++

+++

Basal ganglia B C1 CPu Den EP GP SNC SNL SNR STh VEn

++ ++ +

++++ ++++ +++ +++ ++ +++ ++

Basal telencephalon AMPO AVPO BSTi BST1 BSTld BSTlj BSTlp BSTlv

114

m

Neuroanatomical studies of the ORL1 receptor and OFQ T A B L E 1.

Ch. III

(continued) OFQ mRNA

ORL1 m R N A

O F Q binding

++++

+++++

++

+

++

+++++

++++

+++

++++

++

+++++

++++

-

+++

++++

++++

++

+

++

+

Layer I

++

+++++

L a y e r II

++++

++

L a y e r III

++

++

CNS region

O F Q peptide Cells

F/T

BSTma

+++

BSTmpl

++++

BSTmpm BSTmv BSTv f

.

LOT

.

++

. +

.

.

++

LPO

++

+++

+++

++/+++

++

MCPO

+++

+

+++

++

++

++++

++++

MnPO MPA

++

++++

++++

+++

++

MPO

+++

++++

++++

++++

-

++

-

MPOC Pe

+

+++

+

+++

-

st

+

++

-

-

-

AHA

++

+

+++

++

++

Arc

+++

+++

+++

++++

++

DA

+

++

++

+

-

DM

++

+++

+++

+++

+

LA

++

+

+

++

+

LH

+++

+

++

+++

++

LM

++++

++

++

++

-

ME

-

++++

-

-

ML

.

Hypothalamus

.

.

.

+++

MM

-

-

-

+++

+++

MMn

++

+++

+

-

-

Mp

.

MTu

++

-

++

++

+++

Pa

++

++

++

+++

++

PaAP PaDC PaLM

++

++

++

+++ ++ ++++

+ ++ ++

.

.

.

.

+

+

+

PaPo PaV

+

+

+

++ ++++

++ +++

Pe PeF

+ +

+++ -

+ +

+++ +

+ +++

PH Pin

++ .

+++

++

.

Pit

.

.

PMD

++

+++

+++

+

++

PMV

++

+++

++

++++

+

RCh

+++

+

+

+

++

SCh

-

-

-

+

++++

SO

-

++

-

++++

+

+++

-

+++

-

++

+++

+++

+++

+

+++

SOR SuM

-

-

++

s u m x

.

TC

++

+++ . .

.

.

++

.

.

.

.

.

++

.

115

Ch. III T A B L E 1.


(continued)

CNS region

O F Q peptide Cells

Te

++

TM

++

TMC

+

VMH

+

OFQ mRNA

ORL1 m R N A

OFQ binding

F/T

+ +++

++

+++

++

+++

++

+

+

-/+

+++++

++++

VMHC VMHDM

+++++

++++

+++++

++++

VMHVL

++++

++++

+ +

+

+

++

+ ++ +++

Amygdala AAA

+

ACo

+

AHi APir ++ +

BAOT BL BLA

+ ++

BLP +

BM

BMA

+++ ++ ++

++++ +++

++

+++

++

+

+++ +++ ++

BMP BSTIA

+

CeL

+

CeM CxA

+++

++ +++ ++++

I

+

++

La MeAD

++

++ ++

+ +

+

++++ ++

++

MeAV

+++

++

+ + ++

+

+

++++ ++

MePD

++++

++++

MePV

+

+++

+++++ +++

++

+

+ ++ +++ ++

++++

+++

++

++ ++

+++

opt

PLCo PMCo

Hippocampalformation CAlso CAlsp CAlsr CA2so CA2sp CA2sr

+ +++ ++ + ++ -

++ + ++ -

+ +++ + ++ +

-/+ ++ ++ +

+ ++ +

CA3sl

+++

++

+++

+

++

CA3so

-

+

-/+

+++

++++

CA3sp

++

+

++

++++

-

CA3sr DGgr

++

+ +

+ +++

+ ++++

-

DGhi

-

-

-

+

-

DGmo DGpo

+ +

++ +

+ +

++

+++ -

IG PaS PrS

+++ +

+ -

+++ ++

+ +

+ +++

+

+ +++

+ +++

+++ ++ ++

S

116

++++

Ch. III

Neuroanatomical studies of the ORL1 receptor and OFQ TABLE

1.

CNS region

(continued) OFQ peptide

OFQ mRNA

ORL1

mRNA

OFQ binding

Cells

F/T

SFO

+

++

++

+++

++

SHi

++

++

++

++++

++

AD

-

++

-

+

+++

AM

-

-

-

+

Ar

.

AV

-

Thalamus

.

.

.

m

+

++++

AVDM

+

++

AVVL

+ +

+++

+

++

+++

BSTS -

-

CM

.

F

-

-

-

+

++

-

+++

-

+

++

.

.

+

++

CL

++

.

G IAD

m

++

IAM IMD LD

+ -

-

+

LDDM

++

+

+++

+

+++

+

+++

LGN

-

-

+

++

+++

LHb

-

++

++

++

++

LP

-

-

-

+

++

MD

-

-

+

-

MDDC

.

.

.

MDDL

.

.

.

.

MDDM

.

.

.

.

LDVL

.

MGN

-

-

-

+

+

MHb

++

+++

+

+++

++++

Mt

.

pc

-

.

. + +

. -

PF

-/+

+++

+++

+++

PLi

+

-

++

Po

+

-

++

-

++

PR

-

-

+

+++

++

PrC

++

+++

++

+++

PT

-

+++

+++

++

+++

PVA

+

+++

++

++++

++++

PVP

-

+

++

+++

+++

Re

-

+

+

++

+++

Rh

-

+

-

++

++

RI

-

-

+

+++

++

Rt

++++

++

+++++

++

+++

SG

+++

-

++

+

+

SPFPC

++

+++

+++

++

SubG

-

-

++ +++

+

VL

-

-

-

+

++

VM

+

+++

-

-

VPL

++

+

-

-

VPM

++

-

-

-

ZI

++

++

+++

++

SubI

+

+++

117

Ch. III


TABLE 1. (continued) CNS region

OFQ peptide Cells

OFQ mRNA

+++ ++ +++ +

OFQ binding

F/T

Mesencephalon 3 4 APT APTD APTV ATg bic BIC bsc CG CGD cic CIC CLi (B8) CnF cp

ORL1 mRNA

++ ++ + +

++ ++ +++

++

+++ ++++ ++ ++ ++ ++

++

++

+

m

D

+ +

++++ +

+++ +

++++ ++

+++ ++++

+++

+ ++

++

+ ++++

§

+

+ +++++

+ ++

+ +++++

+++ ++

+ ++ +++ ++

+ ++ + +

++ ++ ++++ +++

CSC

ctg DCIC Dk dlf DLL DpMe

DR (B7) DTg ECIC

EW

++ ++ +++++

+§

++ ++++

+

++ + ++ ++ +++

++

fr

lfp IF ILL IMLF InCo IPC IPD IPL IPR LC LDTg L1 MA3 MCPC Me5 MiTg ml MnR (B8) mp MPT MT OPT OT

Pa4

118

++

+

+++

+ ++

+ +++ + + ++ ++

++ ++++ +++ +++ +++ +++

+++ ++ +++

+

+++

++ ++

++

++ +

+

+

++ + +++++ ++ +++ +++ ++++ +

++

++++

+ ++

++ +++ + +

++

+§

Neuroanatomical studies of the ORL1 receptor and OFQ TABLE CNS

1.

Ch. III

(continued)

region

OFQ

peptide

OFQ

Cells

F/T +

PBG

+++

PBG

-

pc

.

.

PCom

+++

-

++++

+++

ORL1

-

mRNA

OFQ

+++

+

+++

++

++

+++

-

.

PF

mRNA

.

PL

+

-

-

PLi

+

-

++

.

PMR

++

-

+

+++

+++

Pn

-

+

-

++++

++++

PN

+++

+

++++

++++

+

PnO

+

++

++

+

++

PP

+++

++

++++

++

+++

PPT

++

-

++

+

++

PPTg

++

+

+++

++

+

PR

-

-

-

+++

++

Rbd

-

-

-

+++

-

RLi

++

+

++

+++

+++

RMC

+

+

++

+++++

+

RPC

-

+

+

++++

+

RR

++

-

+

++

-

RRF

++

+

++

++

-

rs

.

RtTg

-

-

-

++

+

SC(DpG)

-

-

+

+

++

.

.

.

.

SC(DpWh)

-

-

-

+

-

SC(InG)

+++

+

+++

++

+ -

SC(InWh)

-

-

-

++

SC(Op)

++

+

++

++

-

SC(SuG)

+

+

+

-

++++

-

++++

+

SC(Zo)

-

Scp

.

+

SG

++

-

+++

+

SPTg

-

-

-

++

-

Su3

++

+

++

+++

++++ -

.

.

.

binding

.

VLL

-

-

-

+

VLTg

-

-

-

+++

-

VTA

+++

++

+++

+++

+/++

VTg

+

+

++

+

-

Lobules

+

-

+

-

-

IntA

-

-

-

+++

-

IntDL

-

-

-

++

-

IntDM

-

-

-

++

-

IntP

-

-

-

++

-

Lat

-

-

-

++++

++

LatPC

-

-

-

+++

-

Med

+++

++

++

+++

+

MedDL

++

++

++

++

+

++++

++

+++++

+++

Cerebellum

Metencephalon 6

-

6n

.

7

-

.

.

-

. -

.

119

Ch. III


TABLE 1. (continued) CNS region

OFQ peptide Cells

7

OFQ mRNA

120

OFQ binding

u

n

m

m

m

m

++

++

+++ ++

++

++ ++ ++

++ ++ + +++ ++ ++++

+++

+

++ +++ ++++

++

n

8n A5 A7 Bar CGPn CPO DC DMTg DPO DTg g7 Gi GiA GiV icp IRt KF LC LPB LPGi LSO LVe LVPO Me5 Mlf Mo5 MPB MSO MVeV MVe MVPO Pa5 Pa6 PCRt PDTg PnC PnR PnV Pr5 RPO s5 scp SGe Sp50 SPO SpVe SubC (or) SuVe Tz tz VCA VCP

ORL 1 mRNA

F/T

++ ++ + + ++

+ + +

+ ++ +++

++ ++ ++ + ++ ++

++ ++ ++ +

++ ++ +++

++ + ++ + +

+ ++ +

++ + ++ ++ ++

+ ++ ++ ++

+++ +++ ++++ ++ +++ ++

++ ++ +++ ++

+ + +

+ +++ ++ +++ +

++++ + ++ +

+ ++ ++

+

++ + +++ ++

+

++ +++

m

++ + +++++ ++++ -/+ ++++ +++ +++ ++++

+++

++ ++++ +++

++++ +

++ +

+++ +++ +++ +++ +++ + + ++++ +++ +++ +++++ ++

++ +++ +++ ++ + ++ ++++ +++

++++ +++ +++ ++ ++ +++

+++ + ++ ++ ++ +++

+ +++

+++ ++++

m

Neuroanatomical studies of the ORL1 receptor and OFQ TABLE

1.

Ch. III

(continued)

CNS region

OFQ peptide

OFQ mRNA

Cells

ORL1 mRNA

OFQ binding

++++

+++

+++++

+

F/T

Myelenc~halon 9n

.

10

-

.

.

10n

.

12

-

12n

.

A1

++++

++

A2

+++

++

A7

-

-

-

++

++++

Amb

+++

++

++++

+++++

+++++

-

+++

-

.

.

.

.

.

.

-

.

. -

-

.

.

AP C1

-

-

-

++++

++

C2

-

-

-

+++

++

C3

.

CI

++

-

+++

-

-

Cu

-

-

-

+++

+++

DMSp5

+++

+++

+++++

+++

+++

DPGi

+

-

+

+

+

ECu

-

-

+

+

+++

Gi

+

-

++

++

++

GiV

+++

-

+++

++++

+++

Gr

-

-

-

+

+

.

.

.

.

In IOA

+

-

-

-

++

+

+++++

IOB

-

-

+

+

+++

IOD

+++

++

++

+++

+++

IODM

-

-

-

++

+++

IOM

-

-

-

++

+++++

IOPr

+++

+

+++

+++

++++

IRt

++

-

+++

++

-

LRt

+

-

++

++++

++

+

LVe

++

+

+++

+++

-

MdD

++

-

++

++

++++

MdV

++

++

++

mlf

.

-

++

.

.

.

.

MnA

-

++++

M~V

-

-

-

+++

++

M~

++

+

++

+++

+++

Pa5

+

-

++

+++

+++

PrH py

++++ .

+++

++++

++++

++++

RAmb

+++

-

+++++

++++

+++

RMg

+++

++

++++

+++++

+++

ROb (B2)

+

+

++

+++

++

R P a (B 1)

++

+

++

+++

++++

RVL

++++

++

SGe

++

-

++++

sol

.

Sol

++

++

++++

+++

++++

SolC

+++

++

++

++++

++++

SolL

+++

+++

+++

+++

++

SolM

++

+++

+++

+++

++

sp5

.

.

.

+++

.

+++

.

.

.

.

.

.

.

.

++++

121

Ch. III T A B L E 1.

C.R. Neal Jr. et al. (continued)

CNS region

OFQ peptide

OFQ m R N A

ORL1 m R N A

OFQ binding

§247247247247 § §

§247247 §247247247 §247247

§247247247 §247247 §

Cells

F/T

§247247 §247

§247247247 §247247 §

I

-

§ 2 4 7

-

-

-

II

+§ ++ + § ++ §

+++§ +++ +++ + § + § +++

§247247 §247247 + § ++ + +§ §247247

+ ++ § +++ §247 ++ +++§ §247247 §247

++++ +++ ++ + +§247 ++ § §247 §247247

Sp5C Sp5I SpVe

Spinal cord Cervical

III IV V VI VII VIII IX X Thoracic I

-

+

-

-

-

II

§247 -

++§ +§ § §247 ++ §247 +++ +

§247 +++ + + ++ ++

+ + +++ § +++ §247247 §247247247 +++

§ § ++++ § +§ + ++++

++§247

-

+++§ §247247 +

§ §247247 -

§

-

III IV V VII VIII IX X CeCv dcs DRG Gr IML

§ ++ ++ .

.

.

.

.

.

.

+ . -

I M M

§

LatC lfu LSp vfu

+++ . ++ .

.

. -

+++ .

. +++

.

.

§247247 + . .

. .

Degree of immunoreactivity, m R N A expression and OFQ binding were arbitrarily graded, based on density and intensity of immunostaining or binding, and intensity of microscopic m R N A expression on emulsion-dipped sections. Gradations used for immunostaining were: intense ( + + + + ) ; moderate ( + + + ) ; light to moderate ( + + ) ; light or sparse (+); undetectable ( - ) . Gradations used for m R N A expression were: highest signal intensity (++§ high ( + § moderate ( + + + ) ; low to moderate ( + + ) ; low or sparse ( § undetectable ( - ) . Gradations used for OFQ binding were: densest signal intensity ( § 2 4 7 2 4 7 2 4 7dense 2 4 7 ( § 2 4 7 2 4 7 2moderate 47 (§247247 low to moderate ( § 2 4 7 low or sparse ( § undetectable ( - ) . For abbreviations, see Section 10.

5.3.1.2. OFQ immunohistochemistry

Moderately immunostained cells are scattered throughout the neocortex and allocortex, and fiber labeling is sparse. Frontal and parietal cortex contain light to moderately immunolabeled neurons in layers II, III and VI. In the temporal cortex, OFQ immunolabeling is weak. The occipital cortex contains scattered, moderately labeled neurons in layers V and VI. 122


Ch. III

Fig. 3. Distribution of preproOFQ mRNA in the rat brain at representative coronal sections through the forebrain (A-J). Scale bar: 2000 Ixm.

123

Ch. III


Fig. 4. Distribution of preproOFQ mRNA in the rat brain at representative coronal sections through the brainstem

and spinal cord (A-H). Scale bar: 500 ~m.

Numerous darkly immunolabeled cells are observed in the cingulate and retrosplenial cortices. Moderately labeled cells are observed in the dorsal peduncular and infralimbic cortices, the tenia tecta and indusium griseum. Moderately stained cells, fibers and puncta are observed in layer III of piriform cortex. Scattered, lightly labeled cells are noted in the ventral orbital and insular cortices. The entorhinal and perirhinal cortices exhibit no immunolabeling. 124


Ch. III

Fig. 5. Darkfield autoradiograms comparing 125I-[14Tyr]OFQ binding (A,C) and orphanin receptor mRNA expression (B,D) at representative rostral forebrain levels. Scale bar: 1000 Ixm. 125

Ch. III


Fig. 6. Darkfield autoradiograms comparing 125I-[14Tyr]OFQbinding (A,C) and orphanin receptor mRNA expression (B,D) at representative levels of the mid- and mid-caudal forebrain. Scale bar: 1000 ~tm. 5.3.1.3. ORL1 in situ hybridization

Orphanin receptor mRNA expression is strong throughout layers II, IV and VI of the neocortex, densest in the frontal and occipital regions. High expression is observed in the 126


Ch. III

Fig. 7. Darkfield autoradiograms comparing 125I-[14Tyr]OFQbinding (A,C) and orphanin receptor mRNA expression (B,D) at representative levels of the midbrain. Scale bar: 1000 txm. granular and agranular parts of the insular cortex, layer III of the piriform cortex, the cingulate cortex, the granular and agranular parts of the retrosplenial cortex and the entorhinal cortex. Moderate mRNA expression is seen in the medial, lateral and ventral orbital cortices. Low expression is found in the mitral cell layer of the olfactory bulb, external anterior olfactory nucleus, ventral tenia tecta, infralimbic cortex and the dorsal peduncular cortex. 127

Ch. III

128



Ch. III

Fig. 9. Darkfield autoradiograms comparing 125I-[14Tyr]OFQbinding (A,C) and orphanin receptor mRNA expression (B,D) in the caudal medulla and spinal cord with dorsal root ganglion. Scale bar: 1000 Ixm. 5.3.2. Ventral forebrain

5.3.2.1. PreproOFQ in situ hybridization Preproorphanin mRNA expression in the ventral forebrain is strong in the ventral division of the anterior olfactory nucleus and heaviest in the horizontal limb of the diagonal band of Broca. Messenger RNA expression is moderate in the lateral ventral pallidum, olfactory tubercle and vertical limb of the diagonal band of Broca, and weak in the ventral pallidum and the medial, lateral and posterior parts of the anterior olfactory nucleus. In nucleus accumbens, scattered OFQ-containing neurons are observed in the shell, with mRNA expression very light throughout the core. The rostral pole of nucleus accumbens, islands of Calleja and fundus striati are devoid of mRNA expression.

5.3.2.2. OFQ immunohistochemistry Neuronal immunolabeling in the anterior olfactory nucleus is light to moderate, with diffuse fibers and puncta observed in its caudal aspect. Immunolabeling in the rostral pole of nucleus accumbens is limited to diffuse, moderately labeled fibers. The accumbens core contains

.85%) of GALP neurons within the arcuate nucleus coqocalized:with the leptin receptor, but not with c~-melanotropin-stimulating hormone, somatostatin, neuropeptide Y, agouti,related protein, or galanin ~(Takatsu et al., 2001). In~ addition, dense staining GALPcontaining fibers are present in the p~avemricular hypothalamic nucleus, lateral septum as well as in the bed nucleus of the stria terminalis and medial preoptic area where they are in close contact to gonadotropin=releasing hormone-immunoreactive fibers (Takatsu et al,, 2001). Interestingly, the overall distribution of GALP-i~unoreactive profiles coincides with the expresfion of GALR3 mRNA that we reported (Mennicken et al., 2002; herein). Although thephysiological roles of GALP remainto be elucidated, these neuroanatomical data suggest that GALP, like galanin, may play a role in regulation of feeding behavior and reproduction. Furthermore, the &scovery of a novel galanin-like peptide reinforces the notion that other 202

Localization of galanin receptor subtypes in the rat CNS

Ch. IV

yet un-identified endogenous ligands may also exist and may be capable of preferentially activating the different galanin receptor subtypes. 3. GALANIN RECEPTOR SUBTYPES

3.1. CHARACTERIZATION OF GALRs The actions of galanin are mediated by specific cell surface receptors that belong to the seven transmembrane, G-protein-coupled receptor (GPCR) family. High affinity binding sites for galanin were first identified and characterized in membranes from a hamster pancreatic ~-cell tumor using radiolabeled 125I-galanin (Amiranoff et al., 1987). Biochemical characterization revealed that this pancreatic galanin receptor was a 54-kDa glycoprotein and that it was associated with a pertussis toxin-sensitive G-protein (Amiranoff et al., 1989). Specific galanin-binding sites were subsequently characterized pharmacologically from rat and pig brain (Servin et al., 1987; Chen et al., 1993) and from the human Bowes melanoma cell line (Heuillet et al., 1994), which was subsequently used to clone the first galanin receptor (Habert-Ortoli et al., 1994). Radiolabeled galanin has also been used extensively in classical receptor autoradiography studies to map the anatomical localization of 125I-galanin-binding sites in several species including rat (Skofitsch et al., 1986; Melander et al., 1988, 1992), man (Kohler et al., 1989b; Mantyh et al., 1989a; Kohler and Chan-Palay, 1990; Ikeda et al., 1991, 1995; Deecher et al., 1998; Mufson et al., 2000), monkey (Kohler et al., 1989a,b; Rosier et al., 1991), cat (Arvidsson et al., 1991; Rosier et al., 1991), rabbit (King et al., 1989; Mantyh et al., 1989b, 1992), guinea pig (King et al., 1989; Dutriez et al., 1996, 1997), Atlantic salmon (Holmqvist and Carlberg, 1992), quail (Azumaya and Tsutsui, 1996) and blowfly (Johard et al., 1992; Lundquist et al., 1993). A detailed description of the distribution of galanin-binding sites in the CNS of the rat is provided below (Section 4.1). The possibility of a multi-receptor system for galanin was recognized early on by several groups based on pharmacological and physiological findings using galanin fragments, galanin agonists and galanin antagonists (for a review see Bartfai et al., 1992 and Bedecs et al., 1995). Studies on the pituitary gland provided further support for the existence of different galanin receptor subtypes. Galanin is abundantly expressed in the pituitary and several of galanin's neuroendocrine effects are exerted at the level of the pituitary. ~However, surprisingly,~ initial studies failed to demonstrate the presence of specific 125I-galanin-binding sites in this structure (Gaymann and Falke, 1990; Hulting et al., 1991). In an attempt to specifically address this issue~ Wynick et al. (1993) used a novel N-terminally labeled 125I-galanin and successfully demonstrated the presence of a high affinity galanin receptor in the rat pituitary gland, which they designated as GAL-R2. Based on their findings, they proposed that incontrast tothe previously characterized gut/brai'n receptor (designated as GALR1), regions 3~10 and amino acid 25 were critical for binding activity of this novel pituitary~galanin receptor (Wynick et al., 1993). Cloning of t~ee distinct galanin receptors h ~ subsequently revealed that it is in fact GALR2 transcripts, but not GALR1 or GALR3, which are present in the pituitary~ gland (Fathi et al,, 1997; Depczynski et al., 1998; O'Donnell et al., unpubfished data), thus substantiating Wynick's earlier conclusionsthat a pharmacologically distinct galanin receptor subtype exists in ~the pituitary. 9 ~ ~ ~ ~ : , :

, ,

,

203

Ch. I V

D. O ' D o n n e l l et al. I

I

i

..........[

,,

SSR3.PRO SBRS.PRO BSR2.PRO SBR1 .PRO BSR4.PRO OPRK.PRO OPRM.PRO OPRD.PRO OPRX.PRO MCHR1 .PRO GALR2.PRO GALR3.PRO GALRI.PRO OPR54.PRO UTIIR.PRO

Fig. 1. Phylogenetic tree of the galanin receptor subfamily and GPR54, somatostatin, opioid, melanin concentrating

hormone, urotensin subfamilies in the rat. The alignment and phylogenetic tree were prepared using MegAlign v4.03 (DNASTARInc). 3.2. CLONING OF GALR SUBTYPES To date, three distinct galanin receptor subtypes have been cloned, each encoded by separate genes and located on different chromosomes. Human GALR1, GALR2 and GALR3 are located on chromosomes 18q23, 17q25.3 and 22q13.1, respectively and the structural organization of these genes has been elucidated (for review see Iismaa et al., 1998). Sequence analysis indicates that the three galanin receptor subtypes share only modest homology amongst each other, ranging from 40 to 50% identity. Dendrogram analysis indicates that galanin receptors are phylogenically closely related to two novel and distinct melaninconcentrating hormone (MCH) receptor subtypes, MCHR1 (Lakaye et al., 1998; Chambers et al., 1999; Lembo et al., 1999; Saito et al., 1999) and MCHR2 (An et al., 2001; Hill et al., 2001; Sailer et al., 2001), and to the novel urotensin type II receptor (Liu et al., 1999), followed by the opioid and somatostatin receptor subfamilies (see Fig. 1). 3.2.1. GALR1 The first galanin receptor subtype, GALR1, was cloned from a human Bowes melanoma cell line cDNA library by expression cloning in the mid 1990s (Habert-Ortoli et al., 1994). The nucleotide sequence of the cloned receptor revealed a 349 amino acid protein with seven putative hydrophobic transmembrane domains, a characteristic feature of the G-proteincoupled receptor family. Pharmacological characterization of membranes prepared from COS cells transfected with hGALR1 clone revealed a single class of high affinity 125I-galaninbinding sites (Kd = 0.8 nM) that could be displaced by human, pig and rat galanin with similar inhibition constants in the nanomolar range, Ki -- 0.2-0.8 nM (Habert-Ortoli et al., 1994). Cloning of the rat homologue from brain (Burgevin et al., 1995) and Rinl4B insulinoma cells (Parker et al., 1995) ensued shortly thereafter. The rat GALR1 is slightly shorter, 346 amino acids in length, sharing 91% identity with its human equivalent and a similar pharmacological profile (Burgevin et al., 1995; Parker et al., 1995). It binds N-terminal galanin fragments (galanin(1-15) and galanin(1-16)) and putative galanin receptor antagonists C7, M35, M40 and galantide with high affinity (Parker et al., 1995). GALR1 couples via Gia to inhibit adenylyl cyclase activity and lower intracellular levels of cAMP as well as via the Gif~/ 204


Ch. IV

complex activating the mitogen-activated protein kinase (MAPK) pathway (see Iismaa et al., 1998; Wang et al., 2000). Northern blot, RT-PCR, and RNase protection assay analyses have shown that GALR1 transcripts are predominantly expressed in brain and spinal cord with little or no expression in peripheral tissues (Habert-Ortoli et al., 1994; Parker et al., 1995; Wang et al., 1997c; Waters and Krause, 2000). However, Sullivan et al. (1997) reported a broader tissue distribution in their Northem blot analyses. In contrast to the contradictory data with respect to expression of GALR1 in peripheral tissues, the precise distribution of GALR1 mRNA in the rat CNS and sensory ganglia has been extensively studied using in situ hybridization and is corroborated by several groups (Burgevin et al., 1995; Parker et al., 1995; Gustafson et al., 1996; Xu et al., 1996a,b; Ahmad et al., 1998; O'Donnell et al., 1999; Burazin et al., 2000) and is described in more detail in Section 4.2. 3.2.2. GALR2

Six different groups reported in parallel the cloning of the second galanin receptor subtype, GALR2 (Ahmad et al., 1996, 1998; Fathi et al., 1997; Howard et al., 1997; Smith et al., 1997; Wang et al., 1997a; Bloomquist et al., 1998; Borowsky et al., 1998). Rat (Ahmad et al., 1996, 1998; Fathi et al., 1997; Howard et al., 1997; Smith et al., 1997; Wang et al., 1997a) and human (Bloomquist et al., 1998; Borowsky et al., 1998; Fathi et al., 1998; Kolakowski et al., 1998) GALR2 receptors contain 372 and 387 amino acids, respectively and share 92% identity. By contrast, however, the homology of GALR2 with the GALR1 is surprisingly low, sharing only 33% homology. Pharmacologically, GALR2 binds galanin with high affinity (Kd = 0.1-0.6 nM) as well as several galanin-related peptides with a rank order of potency as follows: galanin(1-29)~ galanin(2-29) > galanin(1-16) > [D-Trpe]galanin(1-29) >>> galanin(3-29) (Iismaa and Shine, 1999). GALR2 coupling is primarily via Gq however it also couples to Gi~ to inhibit the activity of adenylyl cyclase and to MAPK pathway through as yet unknown mechanism, perhaps through Go (see Iismaa et al., 1998; Wang et al., 2000). Northem blot analyses and RNase protection assays revealed that GALR2 mRNA tissue expression profile differed considerably from that of GALR1. In contrast to the rather restricted tissue distribution of GALR1 mRNA, which is found predominantly in the CNS and to a lesser extent in heart and skeletal muscle (see Section 3.2.1), GALR2 transcripts are widely distributed, detected in several peripheral tissues including lung, heart, kidney, liver, skeletal muscle, spleen, testis, uterus, stomach, large intestine, pituitary as well as in brain, spinal cord and DRG (Ahmad et al., 1996, 1998; Fathi et al., 1997, 1998; Howard et al., 1997; Smith et al., 1997; Wang et al., 1997a; Bloomquist et al., 1998). Likewise, in situ hybridization analyses have demonstrated that although there is some overlap, the anatomical localization of GALR2 mRNA in the rat CNS is distinct from that of GALR1 (Ahmad et al., 1998; O'Donnell et al., 1999) and is discussed in Section 4.3. 3.2.3. GALR3

As reports were emerging on the cloning of GALR2, a third galanin receptor subtype, GALR3, was isolated from rat hypothalamic cDNA libraries by both homology and expression cloning techniques (Wang et al., 1997b; Smith et al., 1998). The rat GALR3 has 370 amino acids and shares higher sequence identity with the rat GALR2 (52-54%) than with the rat GALR1 (35-36%). The human GALR3 was also cloned, containing 368 amino acids and sharing 90% identity with the rat GALR3 (Kolakowski et al., 1998; Smith et al., 1998). Analogous to the rat sequence, human GALR3 shares 53% and 30% identity with the human GALR2 205

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D. O'Donnell et al.

and GALR1 receptors, respectively. Galanin binds to GALR3 with high affinity and the rank order of potency for galanin and galanin-related peptides is: galanin(1-29) > galanin(2-29) > galanin(1-16) >>> galanin(3-29) (Iismaa and Shine, 1999). Interestingly, however, the affinity of galanin for the cloned rat and human GALR3 receptors is reportedly lower than that observed for GALR2 and GALR1 (Smith et al., 1998), suggesting the possibility that a novel substance related to galanin, such as GALP (see Section 2.7.2), may in fact be the preferred ligand for GALR3. GALR3 couples to Gi/Go and it can activate inward K + currents in Xenopus oocytes when co-expressed with potassium channel subunits GIRK1 and GIRK4 (Smith et al., 1998). Reports to date on the tissue distribution of GALR3 are highly contradictory. Northern blot analysis showed expression of GALR3 in heart, spleen, and testis with much lower levels detected in brain and spinal cord (Wang et al., 1997b) whereas RNase protection assays revealed that GALR3 transcripts were widely distributed but expressed at low abundance, with highest levels observed in hypothalamus and pituitary and lower levels seen in spinal cord, pancreas, liver, kidney, stomach and adrenal gland (Smith et al., 1998). The situation in the rat CNS is equally contradictory, with two different studies using in situ hybridization demonstrating both widespread (Kolakowski et al., 1998) and highly restricted (Mennicken et al., 2002) patterns of GALR3 expression (see Section 4.4). In summary, extensive pharmacological characterization of the three cloned galanin receptors has been performed by several groups largely with galanin fragments and chimeric peptides and there is a paucity in the literature of the non-peptide small molecules, both agonists and antagonists, active at these receptors. In general, all three galanin receptor subtypes demonstrate similar structure activity relationships to these peptides; however, some subtle differences have been observed. For example, the affinity of galanin at GALR3 is close to the 10-nM range whereas it displays subnanomolar affinity at both GALR1 and GALR2. C-Terminal truncation of galanin (galanin(1-16)) does not change substantially the affinity or the efficacy of this peptide at either GALR1 or GALR2; however, there is a significant decrease in its affinity for GALR3. Similarly, [D-Trp2]galanin has relatively lower affinity for GALR3 than for either GALR1 or GALR2. Many of the chimeric peptides have also been tested at cloned galanin receptors and display similar affinity at GALR1 and GALR2 but display slightly lower affinity at GALR3, which in general displays lower affinity for many peptides. In addition, these chimeric peptides demonstrate agonistic activity at cloned galanin receptors but generally work as antagonists in in vivo situations (see Section 2.5). The cloned galanin receptors differ significantly both in their intracellular signaling pathways and in their overall tissue distribution. 3.3. THE ELUSIVE GALANIN FRAGMENT RECEPTOR Several groups have proposed the existence of a putative 'galanin fragment receptor' based on a variety of pharmacological, functional and anatomical lines of evidence. For example, galanin(1-16) and galanin(1-15) have been shown to modulate the expression of 5-hydroxytryptamine 1A (5-HT1A) receptors in membrane preparations from dorsal hippocampus and ventral limbic cortex of the rat whereas rat galanin(1-29) was less potent or had no effect, suggesting the existence of a galanin receptor subtype in these brain regions mainly recognizing N-terminal galanin fragments (Fisone et al., 1989; Hedlund et al., 1994; Diaz-Cabiale et al., 2000). Likewise, galanin fragment (1-15), but not galanin(1-29), decreases the baroreceptor reflex sensitivity, suggesting the existence of a specific receptor subtype which exclusively recognizes N-terminal fragments of galanin, and mediates the cardiovascular response of galanin (Diaz et al., 1996; Narvaez et al., 2000). 206


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Neuroanatomical studies have provided likely the most convincing data for the existence of this receptor. Hedlund et al. (1992) demonstrated the widespread presence of novel high affinity 125I-galanin(1-15)-binding sites in rat brain. Surprisingly, 125I-galanin(1-15)-binding sites were detected in several brain regions known to be devoid of or having very few 125I-galanin(1-29)-binding sites, namely the dorsal hippocampal formation, the neocortex and the neostriatum. Moreover, when used as a competitor galanin(1-15) displaced the majority ('-,80%) of these novel 125I-galanin(1-15) sites whereas galanin(1-29) only competed for 30% of these sites (Hedlund et al., 1992), suggesting the existence of a novel galanin receptor that preferentially binds the galanin fragment galanin (1-15). Unexpectedly, though we have tried on several occasions to map the distribution of putative galanin(1-15)-binding sites according to the methods described in Hedlund et al. (1992), we have been unable to reproduce their findings. In our hands, the distribution of 125I-galanin(1-15)-binding sites in the rat CNS was identical to that obtained using porcine 125I-galanin(1-29) or human 125I-galanin(1-30). At this time, the reason for this discrepancy is not known. Nonetheless, the molecular identity of this elusive galanin fragment receptor remains to be elucidated since the cloned galanin receptors exhibit greater or similar binding affinities for full-length galanin as compared to galanin(1-15) and galanin(1-16) fragments and therefore could not account for these reported galanin fragment-mediated effects (Fathi et al., 1997; Howard et al., 1997; Smith et al., 1997; Wang et al., 1997a; Bloomquist et al., 1998; and see reviews by Branchek et al., 2000 and by Floren et al., 2000). 3.4. GALANIN-LIKE RECEPTORS The cloning of three distinct galanin receptors sparked considerable speculation as to the potential existence of additional subtypes. Lee et al. (1999) recently identified a novel cDNA from rat brain, named GPR54, which shared significant identity in the transmembrane regions with the rat GALR1 (45%), GALR3 (45%) and GALR2 (44%). As illustrated in the phylogenetic tree in Fig. 1, GPR54 is most closely related to the galanin receptor family. In situ hybridization analyses revealed that GPR54 mRNA CNS expression pattern resembled that of GALRs (Lee et al., 1999; O'Donnell et al., unpublished data). In particular, high levels of GPR54 expression were detected within the hypothalamus, amygdala and pons, coinciding with regions known for high 125I-galanin-binding densities. However, when GPR54 was transfected into a heterologous system, it did not appear to bind human 125I-galanin (Lee et al., 1999) or to be activated by galanin or galanin-related peptides (P. Lembo, personal communication). While this manuscript was in preparation, a somewhat unexpected mate, the gene product of a human metastasis suppressor gene KiSS-l, a 54 amino acid peptide also named 'mestatin', was identified as the ligand for GPR54 (Ohtaki et al., 2001). The role of mestatin in the CNS and the significance of this discovery are unclear at this time. Anecdotally, a cDNA encoding for a novel GPCR from Drosophila melanogaster was recently identified by two different groups (Birgtil et al., 1999; Lenz et al., 2000a), and was the first invertebrate receptor that shared close sequence identity (29-30%) with the three rat galanin receptors. Although structurally related to the mammalian galanin receptors, its cognate ligand was identified shortly thereafter and was found to be a novel octapeptide, member of the allatostatin peptide family (Birgtil et al., 1999). A second putative Drosophila allatostatin-like receptor subtype was also identified sharing 47% identity to the first Drosophila receptor and 30% identity to the rat GALR1 (Lenz et al., 2000b), suggesting a multi-receptor system for this novel peptide. Although allatostatin has been shown to control diverse functions, such as juvenile hormone metamorphosis and visceral muscle contraction 207

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in insects (see references in BirgiJl et al., 1999 and Lenz et al., 2000b), the role of this novel octapeptide is unknown. Neither this novel octapeptide nor allatostatin share any sequence homology with galanin nor does there appear to be mammalian counterparts for these peptides. The identification of novel GALRs remains a critical factor to fully understand galanin's numerous physiological effects and has significant therapeutic implications for designing receptor subtype selective agonists and antagonists. Hopefully, debate on the existence of novel human galanin receptors will soon be resolved with the imminent completion of the Human Genome Sequencing project.

4. L O C A L I Z A T I O N OF GALANIN R E C E P T O R S IN THE RAT CNS The following section on the localization of GALRs in the rat CNS is a compilation of our results and those reported in the literature. For this review, series of adjacent coronal brain sections spanning the entire rat brain were specifically generated in order to determine and compare in parallel the distribution of 125I-galanin-binding sites with the mRNA distribution patterns of the rat GALR1, GALR2 and GALR3 receptors. These data are illustrated in Fig. 2 and the relative signal intensities, derived from both film autoradiograms and emulsionprocessed sections, are summarized in Table 1. It is important to mention that although the in situ hybridization data presented herein are similar to those we published previously (O'Donnell et al., 1999), they were generated from entirely different sets of animals, thus corroborating our earlier findings. Galanin receptor autoradiography was carried out according to previously published protocols (Skofitsch et al., 1986; Melander et al., 1988; Kar and Quirion, 1994, 1995). Briefly, coronal rat brain sections were incubated for 60 min at room temperature with 50 pM human 125I-galanin. Non-specific binding was determined in the presence of 1 IxM galanin. Sections were washed in ice-cold Tris-HC1 buffer, air dried and exposed to Kodak BioMax MS film for 3 days. The GALR1, GALR2, and GALR3 constructs used for riboprobe synthesis as well as in situ hybridization protocol have been described elsewhere (O'Donnell et al., 1999; Mennicken et al., 2002). In situ hybridization processed sections were exposed to Kodak Biomax MR film for 12, 14 or 20 days, respectively, for GALR1, GALR2 or GALR3. Sections were then dipped in Kodak NTB2 emulsion diluted 1:1 with distilled water and exposed, respectively, for 4, 6 or 10 weeks for GALR1, GALR2 or GALR3 prior to development and counterstaining. Neuroanatomical structures were identified according to the rat brain atlas of Paxinos and Watson (1998). 4.1. DISTRIBUTION OF 125I-GALANIN-BINDING SITES IN THE RAT CNS The anatomical distribution of 125I-galanin-binding sites in the rat CNS is well characterized (Skofitsch et al., 1986; Melander et al., 1988, 1992) and for the most part overlaps

Fig. 2. Film autoradiographs showing the distribution of galanin binding sites and GALR1, GALR2 and GALR3 mRNA in rat brain. Series of adjacent coronal brain sections (A-R) were processed in parallel for receptor

autoradiography using 125I-galanin (first column) or in situ hybridization with 35S-labeled riboprobes directed to GALR1 (second column), GALR2 (third column) or GALR3 (fourth column) as described in Section 4. Neuroanatomical structures were identified according to the rat brain atlas of Paxinos and Watson (1998). See Section 9 for abbreviations. 208


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Localization of galanin receptor subtypes in the rat CNS T A B L E 1.

rat CNS

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Distribution of 125I-galanin binding sites and of GALR1, GALR2 and GALR3 receptor mRNA in adult

Region

125I-Galanin

GALR 1

GALR2

+

GALR3

Olfactory system Olfactory bulb G l o m e m l a r layer

++++

+++

External plexiform layer

+++

-

-

Internal plexiform layer

++

++

+

Granular layer

++++

++

+

++

++

+

Islands of Calleja

+++

(+)

(+)

M o l e c u l a r layer

+

-

-

++++

++++

-

(+)

Anterior olfactory nucleus Olfactory tubercle

Nucleus of the accessory olfactory tract

Cerebral cortex Neocortex

(+)

-

Entorhinal cortex

+++

++

-

Deep p e d u n c u l a r cortex

+++

+++

-

Insular cortex

++

++

-

Piriform cortex

+++

++

+

Retrosplenial cortex

+

-

++

Limbic and basal forebrain Lateral septum

+++

++

-

Medial septum

+

+

-

Bed nucleus of the stria terminalis

+++

++

+

Stria terminalis

++++

-

-

Nucleus vertical limb of the diagonal band

+++

++

+

Nucleus horizontal limb of the diagonal band

+++

++

+

A m y g d a l o i d nuclei Basal nucleus (lateral and median)

+++

++

-

Medial nucleus

+++

++

+

Central nucleus

+++

+++

+

Cortical nucleus

+++

++

-

Shell

+++

+

-

Core

++

-

-

+ +

+

-

Dorsal C A cell fields

-

-

-

Dorsal dentate g y m s Dorsal subiculum Ventral CA1

++ +++

+++

+++ +

Ventral CA2, CA3 Ventral dentate g y m s

+ +

+ -

+ ++

Ventral subicu lum

+++

-

-

Centrolateral nucleus

++++

+++

-

C e n t r o m e d i a n nucleus

++++

+++

-

Intermediodorsal nucleus

++

++

-

Laterodorsal nucleus

++

+

+

Paracentral nucleus

++

+++

+

Paraventricular nucleus

++

++++

-

Reuniens nucleus

++

++

+

(+)

A c c u m b e n s nucleus

Striatum Globus pallidus

Hippocampus

Thalamus

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TABLE 1 (continued) Region

125i_Galanin

GALR1

GALR2

GALR3

Habenula

+++

Zona incerta

+++

+++

+

(+)

++

+

-

Arcuate nucleus Paraventricular hypothalamic nucleus

+

+

++

(+)

+

+++

++

P e r i v e n t r i c u l a r h y p o t h a l a m i c nuclei

(+)

+

+

+

++

Hypothalamus

Supraoptic nucleus

+++

+++

-

-

Ventromedial hypothalamic nucleus

+++

++

++

++

P r e o p t i c area

+

++

+

++

A n t e r i o r h y p o t h a l a m i c area

++

++

+

(+)

L a t e r a l h y p o t h a l a m i c area

+++

+++

++

+

D o r s a l h y p o t h a l a m i c area

++

++

++

++

P r e m a m m i l l a r y nucleus, ventral part

++

++

++

+

P r e m a m m i l l a r y n u c l e u s , dorsal part

++

-

+++

+

Medial mammillary nucleus

+

-

+++

-

Supramammillary nucleus

++

++

+

(+)

S u b s t a n t i a n i g r a pars c o m p a c t a

+++

-

++

-

S u b s t a n t i a n i g r a pars reticularis

-

-

+

-

Ventral t e g m e n t a l area

+++

-

++

-

R a p h e linearis

++

-

+

-

Midbrain

Central gray

+++

++

+

+

Dorsal raphe

+++

+

+

-

Mesencephalic V nucleus

+

+

+

-

Superficial layers

++

+

(+)

-

Optic l a y e r

+++

++

(+)

-

-

-

Superior colliculus

C o r t e x of the inferior colliculus

++

M e d i a l and lateral g e n i c u l a t e

.

+ .

.

.

Pons and medulla Parabrachial nucleus

+++

+++

++

+

Locus coeruleus

+

++

+

(+)

Sensory trigeminal nucleus

++

-

++

-

Motor trigeminal nucleus

++

++

+

-

Pontine reticular nucleus M e d i a l m e d u l l a r y reticular f o r m a t i o n Raphe magnus nucleus

++ + ++

++ ++

(+) (+) +

(+) + -

Raphe obscursus nucleus

++

++

(+)

-

R a p h e pallidus n u c l e u s

++

-

(+)

-

Spinal t r i g e m i n a l n u c l e u s

++

-

++

-

D o r s a l m o t o r n u c l e u s of v a g u s

+

+

++

Hypoglossal nucleus

+

-

++

-

Vestibular c o m p l e x

+

(+)

+

-

Facial n u c l e u s

-

-

+

-

Ambiguus nucleus

+

-

++

-

Lateral reticular nucleus

++

+

+

-

External cuneate nucleus

-

-

++

-

N u c l e u s of the solitary tract

+++

++

-

-

Cerebellum Molecular layer

.

P y r a m i d a l cell l a y e r

-

-

+++

-

G r a n u l a r cell l a y e r

-

-

(+)

-

214

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125i_Galanin

GALR1

GALR2

GALR3

++++ +++ ++

+++ + ++

+++ ++ ++-t++

(+) + +

+++ +++ ++ + . . .

+ ++ . . .

+at++ -

++ -

Spinal cord Laminae I and II Laminae III-VII Laminae IX (motoneurons) Laminae X

Circumventricular organs Subfomical organ Median eminence Subcommissural organ Area postrema Vascular organ of the laminae terminalis Pineal gland Choroid p l e x u s

. . .

. . .

Dorsal root ganglia Small cells Medium cells Large cells

* * *

++ ++ ++++

++++ at-++ +

The presence of 125I-galanin binding sites was determined by autoradiography and the presence of galanin receptor mRNA-expressing cells was determined by in situ hybridization using both autoradiographs and emulsion-coated sections as described in the text. The relative level of expression is denoted by '+' signs and reflects the intensity of the labeling for binding study and both the number of cells expressing GALR mRNA and the intensity by cell for in situ hybridization. ' - ' signs denote absence of signal. '(+)' signs denote a very low labeling or very few cells. *, Sections for autoradiographic studies were not processed for emulsion autoradiography, therefore information on cellular distribution was not available.

with the distribution of galanin-immunoreactive cell bodies and terminals described above (Section 2.2). Results from our receptor autoradiography studies are illustrated in Fig. 2 and Table 1 and revealed the presence of high levels of 125I-galanin distributed throughout the rostrocaudal extent of the rat brain. Highest densities of galanin-binding were observed throughout the olfactory system, limbic and basal forebrain, ventral hippocampus, pons and medulla. It is noteworthy that very little or no binding was detected in the dorsal hippocampus and cerebellum of the rat. The overall pattern of distribution observed herein using h u m a n 125I-galanin is identical to that previously reported using porcine 125I-galanin (Skofitsch et al., 1986; M e l a n d e r et al., 1988, 1992) and is described in more detail below.

4.1.1. Telencephalon A very dense labeling was observed in the insular, piriform, retrosplenial and entorhinal cortices (Fig. 2 C - N ) , whereas only a sparse labeling was observed throughout the neocortex (Fig. 2 B - N ) . Interestingly, though the overall pattern of distribution of galanin-binding sites in the rat brain closely resembles that observed in h u m a n and m o n k e y brains, it differs in that, unlike the rat, m o n k e y and h u m a n neocortex exhibit high densities of 125I-galanin-binding sites in all areas of the neocortex (Kohler et al., 1989a,b). The olfactory system, basal forebrain and limbic system were all highly enriched in galanin-binding sites. Very high levels were observed in the glomerular and granular layers of the olfactory bulb, as well as in the islands of Calleja, the nucleus of the accessory olfactory 215

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tract (Fig. 2A-H), whereas more moderate levels were seen in the anterior olfactory nucleus (Fig. 2B). In the basal forebrain, the highest densities were found in the stria terminalis (Fig. 2H,I), the lateral septum, the bed nucleus of the stria terminalis, the vertical and horizontal limbs of the diagonal band, in specific amygdaloid nuclei (such as laterobasal, mediobasal, central and cortical nuclei) and the shell of the nucleus accumbens (Fig. 2C-J). In contrast, only low levels of 125I-galanin-binding were observed in the striatum and the medial septum (Fig. 2C-H). Within the rat hippocampal formation, labeling was restricted to the ventral part. Specifically, very high binding densities were present in the ventral CA1 and ventral subiculum (Fig. 2K-M), whereas only the dorsal subiculum was labeled, exhibiting moderate levels of binding sites (Fig. 2K-L). The ventral parts of the CA2, CA3 and dentate gyrus also displayed a low level of binding sites whereas all the dorsal CA1, CA2, CA3 subfields and dorsal dentate gyrus were devoid of labeling (Fig. 2H-M). In contrast, 125I-galanin-binding sites were observed throughout the hippocampus and dentate gyrus of the monkey brain (Melander et al., 1992).

4.1.2. Diencephalon An abundance of galanin-binding densities were observed throughout the hypothalamus and in some thalamic nuclei (Fig. 2H-K). In the hypothalamus, the highest levels of 125I-galanin labeling were observed in the ventromedial and lateral nuclei, whereas moderate levels were found in the anterior and dorsal areas and low levels in the arcuate, paraventricular and periventricular nuclei. The preoptic areas and the premammillary and supramammillary nuclei also displayed sustained amount of labeling. In the thalamus, galanin-binding sites were restricted to the medial nuclei, with very high levels in the centrolateral and centromedian nuclei, high levels in the intermediodorsal, laterodorsal, paracentral, paraventricular and reuniens nuclei. The habenula and the zona incerta also displayed high amounts of binding.

4.1.3. Mesencephalon Galanin-binding sites were seen in several mesencephalic nuclei, with high amounts in the pars compacta of the substantia nigra and the ventral tegmental area, the dorsal raphe nucleus and the periaqueductal gray (Fig. 2L,M). More moderate levels of binding sites were observed in the superior colliculi, the cortex of the inferior colliculi and the raphe linearis (Fig. 2L-N).

4.1.4. Rhombencephalon High densities of binding sites were observed in the locus coeruleus, parabrachial nuclei and nucleus of the solitary tract (Fig. 20-R). Moderate levels were also observed in various nuclei as described in the Table 1 and as previously reported (Melander et al., 1988, 1992; Skofitsch et al., 1986). The overall restricted distribution of galanin-binding sites within the rhombencephalon is consistent with the limited expression of galanin observed within this region.

4.1.5. Spinal cord Several groups have reported the localization of high density binding sites in rat spinal cord (Kar and Quirion, 1994; Zhang et al., 1995a) and the pattern is identical to our receptor 216


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autoradiography findings (Table 1). Galanin-binding sites are essentially concentrated in laminae I, II and X with more modest binding densities detected throughout the medial lateral portion of the dorsal horn. Very little or no labeling was observed in the ventral horn. 4.2. DISTRIBUTION OF GALR1 mRNA IN THE RAT CNS The precise anatomical distribution of GALR1 mRNA in the adult rat CNS and sensory ganglia has been extensively studied by in situ hybridization and is corroborated by several groups (Burgevin et al., 1995; Parker et al., 1995; Gustafson et al., 1996; Ahmad et al., 1998; O'Donnell et al., 1999; Burazin et al., 2000). As seen in Fig. 2, GALR1 mRNA is widely expressed throughout the basal extent of the rat brain. Likely the most striking feature of GALR1 mRNA expression in the rat CNS is that it coincides with the distribution of 125I-galanin-binding sites (see Fig. 2 and Table 1).

4.2.1. Telencephalon The overall prevalence of GALR1 mRNA within the olfactory system is striking with highest levels observed in the olfactory bulb as well as in the cortical and subcortical associated structures. Moderate to high levels of GALR1 were observed in the glomerular and mitral cell layers of the olfactory bulb, whereas lower levels were present in the internal plexiform layer and the anterior olfactory nucleus (Fig. 2A,B). Several structures functionally related to the olfactory bulb also express GALR1 mRNA. These include the bed nucleus of the accessory olfactory tract (Fig. 2H), which displayed very high levels of GALR1 mRNA, as compared to the primary olfactory cortex including the piriform, entorhinal, insular and deep peduncular cortices, the supraoptic nucleus and the cortical amygdaloid nucleus, which exhibited somewhat more moderate levels (Fig. 2D-M; Table 1). The limbic structures associated with the olfactory system such as the diagonal band of Broca (vertical and horizontal limbs; Figs. 2E and 3C) and the bed nucleus of the stria terminalis (Fig. 2F, G) also expressed GALR1. Within the limbic system, the ventral part of the hippocampal CA1 field displayed the highest level, with the majority of cells expressing very high levels of GALR1 mRNA (Fig. 2K-M). In sharp contrast, the dorsal hippocampus and dentate gyms were devoid of GALR1 hybridization signal. Moderate to low levels of GALR1 mRNA were present predominantly in the lateral septum with lower levels detected in the medial septum (Fig. 2DF and 3A), the bed nucleus of the stria terminalis/substantia innominata (Fig. 2F,G), the septohippocampal nucleus and the shell of the nucleus accumbens (Fig. 2C-E). GALR1 mRNA was also prevalent in several nuclei of the amygdala. The central and medial nuclei (Fig. 4D) displayed higher levels of expression than the basolateral, lateral and cortical nuclei (Fig. 2H-J). A finding which has not previously been reported was the specific labeling observed in the lateral globus pallidus on film autoradiograms (Fig. 2G), and was confirmed by emulsion autoradiography, which revealed the presence of several large moderately labeled cells (Fig. 3B). With the possible exception of randomly labeled cells, neo- and limbic cortices were devoid of GALR1 mRNA expression.

4.2.2. Diencephalon Expression of GALR1 mRNA within the thalamus was mostly restricted to the midline and intralaminar nuclei, which are known to project to the striatum, cortex and the amygdala, 217

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Fig. 3. High magnification brightfield photomicrographs illustrating the distribution of GALR1 mRNA-expressing cells in the lateral septum (A) and the lateral globus pallidus (B); and the distribution of GALRI-, GALR2and GALR3-expressing cells in the nucleus of the horizontal limb of diagonal band (HDB, C-E). Adjacent coronal rat brain sections were hybridized with 35S-labeled riboprobes directed to GALR1, GALR2 or GALR3 and counterstained with hematoxylin and eosin. Neuroanatomical structures were identified according to the rat brain atlas of Paxinos and Watson (1998) as represented in schematic drawings (a,b). GALRI-, but not GALR2- or GALR3-, mRNA expressing cells were detected in the lateral septum and the lateral globus pallidus. In contrast, all three GALR subtypes are expressed to some extent in the HBD. See Section 9 for abbreviations. Scale bar: 50 txm.

suggesting a role for GALR1 in various motor, somatic and sensory functions. The highest amounts of GALR1 mRNA were found in the paraventricular thalamic nucleus (anterior and posterior parts) and the lateral habenula (Fig. 2H,I) with labeling coveting all cell bodies. Low to moderate levels of GALR1 mRNA were detected in the centrolateral, centromedian, intermediodorsal, laterodorsal, paracentral and reuniens nuclei (Fig. 2H-J) as well as in the zona incerta (Fig. 2I-K). In contrast to the thalamus, GALR1 transcripts were widely distributed throughout the preoptic area and the hypothalamus (Fig. 2F-I), suggesting a prominent role for this receptor in endocrine, autonomic and somatomotor systems. Several heavily labeled GALR1expressing cells were observed throughout the medial preoptic nucleus and medial preoptic 218


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area (Fig. 4C). High levels of GALR1 expression were also observed in hypothalamic nuclei such as the paraventricular (Fig. 5B), dorsomedial, lateral and supraoptic nuclei (Fig. 2H-J). Landry et al. (1998) demonstrated that some of the GALR1 neurons within the paraventricular and supraoptic nuclei contain vasopressin. Moreover, GALR1 mRNA in these hypothalamic nuclei is increased following salt loading, but decreased during lactation and following hypophysectomy (Landry et al., 1998), suggesting that GALR1 expression in the hypothalamus is tightly regulated. Low to moderate expression of GALR1 mRNA was seen in other hypothalamic nuclei including the ventromedial hypothalamic nucleus (Figs. 2H-J and 5F, Table 1). Overall, the pattern of GALR1 expression in the hypothalamus described herein is similar to that reported by several other groups (Parker et al., 1995; Mitchell et al., 1997; Gundlach and Burazin, 1998; Landry et al., 1998). We as well as others (Mitchell et al., 1997) observed that the expression of GALR1 in the mammillary bodies was restricted to the ventral part of the premammillary and supramammillary nuclei (Fig. 2K-L); however, we did not observe any labeling in the medial mammillary nucleus (Fig. 2L) as was reported by Gustafson et al. (1996).

4.2.3. Mesencephalon GALR1 mRNA labeling in the midbrain was discrete. Clearly discernible clusters of cells expressing high levels of GALR1 were located in dorsolateral and ventrolateral aspects of the periaqueductal gray (Fig. 2M,N). Moderate labeling was observed in the superior colliculi with a higher number of labeled cells in the optic layer than the superficial layer (Fig. 2L,M). The mesencephalic V nucleus, the medial pretectal nucleus, and the lateral part of the substantia nigra pars compacta, the peripeduncular nucleus and the posterior intralaminar nucleus were also moderately labeled (Fig. 2K-M). In comparison, fewer GALRl-expressing cells were detected in the dorsal raphe, and the inferior colliculi (Fig. 2N).

4.2.4. Rhombencephalon Highest levels of GALR1 mRNA were found in the external part of the lateral parabrachial nucleus, whereas the other subdivisions of the parabrachial nucleus presented low to moderate labeling (Figs. 2 0 - P and 6C). In the locus coeruleus, a moderate level of GALR1 expression was observed with the majority of cells being weakly labeled (Fig. 7A) as compared to the high density observed over cells in the parabrachial nucleus (Figs. 2P and 6C). Moderate to high amounts of GALR1 mRNA were also observed in the reticular nuclei (pontine and lateral). Similarly, several moderately to highly labeled cells were detected in the dorsal tegmental nucleus, the motor trigeminal nucleus, the nucleus of the solitary tract, the dorsal motor nucleus of the vagus, the raphe magnus and obscursus nuclei, the reticular formation (Fig. 6D) as well as the inferior olive (Fig. 2N-R). A few weakly labeled GALR1 mRNAexpressing cells could also be seen in the vestibular complex. No GALR1 mRNA expression was detected within the cerebellum (Fig. 20-R).

4.2.5. Spinal cord GALR1 mRNA expression in rat spinal cord was dense and restricted. The majority of labeled cells were localized in laminae I and II; with a few moderately labeled cells observed in the medial dorsal aspect of the dorsal horn and surrounding the central canal in lamina X (Gustafson et al., 1996; O'Donnell et al., 1999). As a rule, the ventral horn was devoid of 219

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GALR1 mRNA-expressing cells. Thus, the overall distribution of GALR1 mRNA-expressing cells in the rat spinal cord parallels that of galanin-binding sites. 4.3. D I S T R I B U T I O N OF G A L R 2 m R N A IN T H E RAT CNS Although widely distributed throughout the extent of the adult rat brain, the overall levels of G A L R 2 m R N A were noticeably lower relative to those of GALR1 (see Fig. 2 and Table 1). The highest levels of G A L R 2 m R N A were present in the hippocampal formation, hypothalamus and cerebellar cortex with low levels detected in several other brain regions. This overall pattern of G A L R 2 expression is in agreement with previous reports describing the anatomical distribution of G A L R 2 m R N A in the adult rat brain (Ahmad et al., 1998; Fathi et al., 1998; Kolakowski et al., 1998; Xu et al., 1998; O'Donnell et al., 1999) with only some minor exceptions cited below. Moreover, the overall patterns of G A L R 2 and GALR1 m R N A expression in the developing rat brain are similar to those in the adult (Burazin et al., 2000). Interestingly, levels of G A L R 2 transcripts are higher during the first postnatal week than in the adult, whereas the relative abundance of GALR1 m R N A did not change during postnatal development, suggesting that G A L R 2 may be preferentially involved in maturation of synaptic connections in the developing brain (Burazin et al., 2000).

4.3.1. Telencephalon In the olfactory system, GALR2-expressing cells were most numerous in the olfactory bulb (Fig. 2A,B) where they pervaded the granule cell layer. Cells expressing G A L R 2 were observed in the anterior olfactory nucleus as well as within the dense cellular part of the olfactory tubercle and of the piriform cortex (Fig. 2B,C). Within the basal forebrain, GALR2expressing neurons were evident along the medial border of the vertical and horizontal limbs of the diagonal band of Broca, exhibiting a pattern of distribution distinct from that observed for G A L R l - e x p r e s s i n g cells in this region (Figs. 2E and 3C,D). In contrast to the widespread distribution of G A L R l - e x p r e s s i n g cells in the medial septum, G A L R 2 expression was confined to a few scattered neurons in this area. Several moderately labeled G A L R 2 cells were also detected within the magnocellular preoptic area of the basal forebrain. Within the limbic cortex, specific cellular G A L R 2 labeling was observed in the outer part of layer II and in a few scattered cells within layer III of the retrosplenial area (Fig. 2 H - L ) .

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Fig. 4. Brightfield (A,B) and darkfield photomicrographs (C-H) showing distinct cellular localization of GALR1 (C,D), GALR2 (E,F) and GALR3 (G,H) mRNA in the medial preoptic region (left panel) and the medial amygdaloid body (right panel). Adjacent coronal rat brain sections were hybridized with 35S-labeled riboprobes directed to GALR1, GALR2 or GALR3. Neuroanatomical structures were identified using hematoxylin and eosin counterstained sections (A,B), according to the rat brain atlas of Paxinos and Watson (1998). In the ventromedial preoptic region, GALR1 and GALR2 mRNA transcripts show widespread but distinctive patterns of expression; GALR1 is expressed at very high levels in many cells throughout the MPO and MPA (C) whereas GALR2 is expressed in the majority of cells but at very low levels (E). In contrast to this widespread distribution, GALR3 mRNA expressing cells were sparse and moderately labeled, located mainly on the border of the medial preoptic region in the suprachiasmatic nucleus (Sch) and the ventrolateral preoptic nucleus (VLPO) (G). Within the medial amygdala, GALR1 mRNA is highly expressed in a few cells scattered throughout the medial amygdaloid nuclei (D) whereas GALR2 mRNA is expressed rather diffusively at low levels in the majority of cells (F). Only a few weakly labeled GALR3 cells were detected, located selectively on the lateral border of the medial amygdala (H). See Section 9 for abbreviations. Scale bar: 200 Ixm. 221

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Fig. 5. Brightfield (A,E) and darkfield photomicrographs (B-D and F-H) showing the distinct cellular localization of GALR1 (B,F), GALR2 (C,G) and GALR3 (D,H) mRNA in the paraventricular hypothalamic nucleus (PVN, A-D) and ventromedial hypothalamic nucleus (VMH, E-H). Adjacent coronal rat brain sections were hybridized with 35S-labeled riboprobes directed to GALR1, GALR2 or GALR3. Neuroanatomical structures were identified using hematoxylin and eosin counterstained sections (A,E), according to the rat brain atlas of Paxinos and Watson (1998) as indicated by the schematic drawings (a,b). In the PVN, GALR1 mRNA is preferentially observed in the medial parvicellular (PaMP) and lateral magnocellular (PALM) parts of the PVN (B) whereas GALR2 mRNA expression is largely restricted to the ventral aspect (PaV) of the PVN (C). Similarly, a few specifically labeled GALR3 cells are observed only in the ventral part of the PVN (PaV) (D). Several highly labeled GALR1 cells are discretely and preferentially distributed within the ventrolateral and central portions of the VMH, whereas expression of GALR2 and GALR3 is weaker but found more uniformly distributed throughout the VMH (G,H). See Section 9 for abbreviations. Scale bar: 200 Ixm.

A weak but distinct signal was also observed within the neocortex where cells expressing low levels of GALR2 mRNA were mainly localized within layers II-III and VI (O'Donnell et al., 1999). Highest levels within the telencephalon were observed in the hippocampal formation where GALR2 labeling was intense and selectively distributed over the granule cell layer of the dentate gyms (Fig. 2G-H). At the light microscopic level, the vast majority (approximately 70-80%) of dentate granule cells were labeled with little or no GALR2 hybridization signal observed in the CA cell fields (Xu et al., 1998; O'Donnell et al., 1999). 222


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4.3.2. Diencephalon Overall, expression of GALR2 mRNA in the thalamus was low (Fig. 2H-K). Only a few moderately labeled cells were detected scattered within the laterodorsal, paracentral and reuniens nuclei whereas a slightly greater number of labeled cells were detected in the habenula and zona incerta. GALR2 distribution within the hypothalamus was restricted as compared to that of GALR1. Within the hypothalamus, the highest levels of GALR2 were observed in the mammillary body with all mammillary nuclei being labeled with equal intensity (Fig. 2L). As revealed by light microscopy, virtually every neuron within the supramammillary, medial and lateral mammillary nuclei was intensely labeled. Moderate levels of GALR2 were detected in the preoptic area (Fig. 4E), arcuate nucleus, and posterior hypothalamic area (Fig. 2FK). In the arcuate nucleus, moderately labeled neurons were concentrated ventrolaterally throughout the rostrocaudal extent of the nucleus (Fig. 2I-K). By contrast, within the posterior hypothalamic area GALR2 hybridizing cells were more dispersed. Other hypothalamic regions also expressed GALR2, but at lower levels. These included the periventricular nucleus, anterior hypothalamic area, a selective subpopulation of neurons in the paraventricular nucleus, and scattered neurons within the more caudal aspects of the ventromedial nucleus as well as the lateral hypothalamic area (Fig. 5C,G; Table 1; O'Donnell et al., 1999). The entire preoptic region displayed widespread, moderately labeled cells (Figs. 2F, G and 4E). Mitchell et al. (1999) found a similar distribution pattern of GALR2 mRNA-expressing cells within the hypothalamus. Based on the moderate distribution of GALR2 in the hypothalamus, GALR2 is likely to play role in neuroendocrine regulation and feeding behavior.

4.3.3. Mesencephalon Within the midbrain, a weak but distinct signal was observed within the periaqueductal gray. GALR2 labeling was low and uniformly distributed around the central canal with no apparent difference in ventral versus dorsal labeling intensities. GALR2 labeling was most prominent over the substantia nigra where it overlaid the vast majority of medium to large, presumably dopaminergic neurons in the pars compacta and lateralis (Fig. 2L,M). Although apparently devoid of labeling at the level of autoradiograms, upon microscopic examination, the pars reticulata and ventral tegmental area displayed a few dispersed labeled cells (O'Donnell et al., 1999). GALR2 labeling was also observed over a select population of cells in the raphe linearis.

4.3.4. Rhombencephalon Several nuclei within the pons and medulla, including the lateral parabrachial (Fig. 6E), motor trigeminal, hypoglossal, vestibular, ambiguus, facial and reticular (Fig. 6F) nuclei contained a few labeled GALR2 neurons (Table 1, Fig. 2N-R). A large proportion of cells within the locus coeruleus (Fig. 7B) as well as in the main sensory nucleus of the trigeminal nerve were also found to express moderate levels of GALR2 mRNA. Within the cerebellar cortex, intense GALR2 labeling was observed in the lower tier of the molecular layer in all lobules (Fig. 20-R). In emulsion-coated sections, grains were specifically observed over neurons in the internal third of the molecular layer, in the position of basket cells (O'Donnell et al., 1999). A few small labeled neurons were also observed scattered throughout the more dorsal aspects of the molecular layer, but not within Purkinje 223

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or granule cell layers. The cerebellar localization described herein differs from that described by Fathi et al. (1997) who reported the presence of GALR2 mRNA on Purkinje cells using a non-radioactive digoxigenin in situ hybridization method.

4.3.5. Spinal cord GALR2 was expressed at low levels in spinal cord of the normal rat. However, unlike the dense and restricted pattern of GALR1 expression, GALR2 was sparse and diffuse. Examination of emulsion-processed sections indicated that this signal originated from specifically labeled neurons scattered throughout both dorsal and ventral horns (Table 1). Within the dorsal horn, labeled neurons were small and present within all lamina (I-VII) while in the ventral horn, some but not all large c~-motoneurons were moderately labeled (O'Donnell et al., 1999). 4.4. DISTRIBUTION OF GALR3 mRNA IN THE RAT CNS In contrast to GALR1 and GALR2, the precise neuroanatomical distribution of GALR3 mRNA is somewhat more controversial. To date, only two in situ hybridization studies have been reported and their findings differ entirely. Our results (Mennicken et al., 2002; herein, see Fig. 2 and Table 1) revealed GALR3 mRNA expression in the rat brain to be low and highly restricted, detected rather exclusively in the preoptic/hypothalamic area (see detailed description below). These findings contradict an earlier report by Kolakowski et al. (1998), describing a widespread distribution of GALR3 mRNA in rat CNS. According to the findings of Kolakowski et al. (1998), GALR3 mRNA expression in the rat brain is very abundant, with highest levels observed in many regions including the primary olfactory cortex, olfactory tubercle, islands of Calleja, hippocampal CA cell fields and dentate gyms. More moderate GALR3 expression was detected throughout the cerebral cortex, as well as in the tenia tecta, caudate putamen, nucleus accumbens, lateral septum, medial habenular nucleus, and several hypothalamic nuclei (Kolakowski et al., 1998). The reason for the discrepancy between these two in situ hybridization studies is unclear, but in all likelihood, given the extent of dissimilarity, may reflect experimental factors and is discussed at length elsewhere (Mennicken et al., 2002).

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Fig. 6. Brightfield (A,B) and darkfield photomicrographs (C-H) showing the cellular distribution of GALR1 (C,D), GALR2 (E,F) and GALR3 (G,H) mRNAs in the parabrachial nucleus (left panels) and the medial reticular formation of the medulla (right panels). Adjacent coronal rat brain sections were hybridized with 35S-labeled riboprobes directed to GALR1, GALR2 or GALR3. Neuroanatomical structures were identified using hematoxylin and eosin counterstained sections (A,B), according to the rat brain atlas of Paxinos and Watson (1998). GALR1 mRNA-expressing cells are distributed throughout the parabrachial nucleus with the highest levels found in the external lateral parabrachial nucleus (LPBE) (C). A similar pattern of expression but less intense was found for GALR2 (E). In contrast, GALR3 mRNA expressing cells within LPBE are sparse and faintly labeled (G). The medial medullary reticular formation was largely devoid of GALR1 mRNA expression with the exception of one or two moderately labeled cells (D) and displayed only very low levels of GALR2 (F). A few moderately labeled GALR3 neurons are specifically and discretely localized between the medial longitudinal fasciculus and the lateral reticular formation, at the level of the dorsal paragigantocellular nucleus (H; see also Fig. 2Q). See Section 9 for abbreviations. Scale bar: 200 ~m.

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4.4.1. Telencephalon As seen in Fig. 2, GALR3 expression was virtually absent within the telencephalon with the exception of the diagonal band of Broca (Fig. 2E-F). Several GALR3 mRNA-expressing cells were evident along the medial border of the vertical and horizontal limbs of the diagonal band of Broca (Fig. 3E). Although not apparent on film, a few specifically labeled cells were localized in the bed nucleus of the stria terminalis and in the posterodorsal part of the medial amygdaloid nucleus (Fig. 4H; Table 1). According to our analyses, the hippocampal formation was devoid of GALR3 labeling.

4.4.2. Diencephalon With the exception of a few moderately labeled cells detected in the border of the lateral habenula, the thalamus was essentially devoid of GALR3 expression. Interestingly, GALR3 expression within the CNS appears to be largely restricted to the preoptic-hypothalamic complex (Fig. 2H-K). High levels of GALR3 were observed in the medial and ventral parts of the preoptic area (Fig. 2F, G). As revealed by microscopic examination, several intensely labeled GALR3-expressing cells were observed mainly on the border of the medial preoptic region, in the suprachiasmatic nucleus and in the ventrolateral preoptic nucleus (Fig. 4G; Mennicken et al., 2002). A few moderately labeled cells were also observed scattered throughout the lateral preoptic region. Within the hypothalamus, highest levels were seen in the dorsomedial and ventromedial nuclei, whereas the anterior, lateral and posterior hypothalamic areas displayed moderate to low levels of expression (Figs. 2 H - K and 5D,H). A few weakly labeled GALR3 mRNA-expressing cells were scattered within the premammillary bodies (Fig. 2K).

4.4.3. Mesencephalon As revealed on film autoradiograms, a faint GALR3 hybridization signal was observed overlying the periaqueductal gray (Fig. 2L-N). Microscopic examination revealed some additional GALR3 mRNA-expressing cells within the dorsal tegmentum and the dorsolateral part of the substantia nigra, pars lateralis.

4.4.4. Rhombencephalon Expression of GALR3 within the rhombencephalon was highly restricted being detected exclusively in the lateral parabrachial nucleus (Fig. 20) as well as a subregion of the medial medullary reticular formation (Fig. 2Q). At the light microscope level, the labeling in the medial medullary reticular formation appears to be mostly localized over the medium-sized

GalR1, Figs. 2G and 8). Even more striking is the observation that among the few structures within the CNS that express GALR3 mRNA, the SFO contains amongst the highest levels of GALR3 mRNA observed (see Figs. 2G and 8C, and Table 1). The overall preponderance of GALR3 mRNA in the SFO suggests that GALR3 may play a prominent role in body fluid homeostasis and cardiovascular regulation. The median eminence also displayed high levels of galanin-binding sites along with moderate levels of GALR1 and GALR2 mRNA, but was devoid of GALR3 mRNA expression (Fig. 2I). Moderate to low 125I-galanin-binding site densities were observed over the subcommissural organ and area postrema; however, expression of the different GALR subtypes mRNAs was not detected in these structures. No significant amounts of galanin-binding or GALR subtype hybridization signal were detected in the three remaining circumventricular organs, namely the vascular organ of the lamina terminalis (VOLT), the pineal gland and the choroid plexus.

7. L O C A L I Z A T I O N OF GALANIN R E C E P T O R S IN DORSAL ROOT GANGLIA OF T H E RAT Galanin is present in low levels in a discrete population of primary sensory neurons in the normal adult rat (Ch'ng et al., 1985; Skofitsch and Jacobowitz, 1985). Although the exact role of galanin in DRG neurons under basal conditions is unclear, following peripheral nerve injury 229

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Fig. 8. Brightfield photomicrographs showing the cellular expression of GALR1 (A), GALR2 (B) and GALR3 (C)

mRNAs in the subfornical organ (SFO). Neuroanatomical structures were identified using hematoxylin and eosin counterstained sections (A,B), according to the rat brain atlas of Paxinos and Watson (1998) as illustrated on the schematic drawing. Note that GALR2 (B) and GALR3 (C) are expressed at higher levels than GALR1 (A). See Section 9 for abbreviations. Scale bar: 50 Ixm.

its synthesis is substantially up-regulated within several small and some large primary sensory neurons (Hokfelt et al., 1987) suggesting that galanin may be involved in the modulation of primary sensory transmission under specific pathophysiological conditions (Villar et al., 1989, 1991; Wiesenfeld-Hallin et al., 1989a,b, 1992a; Xu et al., 1990). 7.1. BINDING SITES The presence of 125I-galanin-binding sites in DRG is controversial. Galanin-binding sites have been observed by receptor autoradiography in monkey lumbar DRGs but were not detected in lumbar DRGs of the rat (Zhang et al., 1995a,b). In contrast to the latter finding, our receptor autoradiographic studies have consistently revealed moderate levels of specific 125I-galanin labeling over rat DRGs (see Table 1), which was completely displaced by cold galanin. The reason for this discrepancy is unclear. The fact remains, however, that several groups have demonstrated high levels of GALR1 and GALR2 mRNAs in DRGs of naive rats (see below), and therefore it is reasonable to assume that galanin-binding sites would be present on rat primary sensory neurons.

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7.2. EXPRESSION OF DIFFERENT RECEPTOR SUBTYPES The presence of high levels of GALR1 and GALR2 mRNA in dorsal root ganglia of normal rats has been demonstrated by several groups using a variety of different techniques including Northern blot, RNase protection assays and in situ hybridization (Xu et al., 1996; Sten Shi et al., 1997; Zhang et al., 1998; O'Donnell et al., 1999; Waters and Krause, 2000). However, the expression of GALR3 mRNA in rat DRG is more contentious. RNase protection assay and RT-PCR analysis have yielded contradictory data with respect to GALR3's expression in rat DRGs. In a recent paper, Waters and Krause (2000) failed to detect the presence of GALR3 message using highly sensitive RT-PCR analysis, yet unexplicably they observed moderate levels of GALR3 mRNA using RNase protection assay, which inherently is less sensitive than RT-PCR. Moreover, this latter finding contradicts an earlier study, which showed an absence of GALR3 transcripts in RNA extracted from rat DRG using the same RNase protection assay (Smith et al., 1998). To the best of our knowledge, in situ hybridization studies examining the cellular localization of GALR3 in primary sensory neurons have not been reported. In the present study, expression of all three subtypes was examined by in situ hybridization in parallel on a series of consecutive rat DRG sections and the results are summarized in Table 1. Consistent with earlier studies (O'Donnell et al., 1999), GALR2 mRNA was preferentially expressed in small to medium-sized cells whereas GALR1 expression was observed predominantly in larger diameter neurons. Satellite cells were devoid of any specific GALR1 or GALR2 hybridization signal (Table 1). Since small and medium sensory neurons (unmyelinated C-fibers or thin myelinated A~-fibers) mediate nociceptive information and large neurons (A~-fibers) mediate mainly proprioception, GALR2 is likely to play a more important role than GALR1 in processing nociceptive information at the level of primary afferents. However, it should be noted that GALR2 is also expressed, albeit at much lower levels, in a few large neurons and that some small and medium neurons express GALR1 mRNA (Xu et al., 1996; Sten Shi et al., 1997; Table 1) indicating a possible overlap in function between these receptors and the possibility that both receptors may be co-localized in certain DRG neurons (Sten Shi et al., 1997). In contrast to the abundant expression of GALR1 and GALR2 transcripts, rat DRGs were devoid of GALR3 mRNA expression (Table 1). Although a faint hybridization signal was apparent over DRGs on GALR3 film autoradiograms, microscopic examination of emulsionprocessed sections failed to reveal any specific accumulation of grains over individual cells (Table 1), suggesting that GALR3 transcripts are not expressed or are present in extremely low abundance in DRGs of the normal rat. These in situ hybridization data concur with those obtained using RNase protection assays (Smith et al., 1998) and RT-PCR analysis (Waters and Krause, 2000).

8. CONCLUDING REMARKS

The widespread distribution of galanin receptor binding sites and mRNA transcripts in the brain is consistent with the numerous central actions of galanin. Although there are some areas of overlap, each cloned galanin receptor differs considerably in its relative abundance and exhibits its own unique spatial pattern of CNS expression. For instance, GALR1 mRNA is expressed at high levels but is discretely distributed in specific regions of the CNS whereas GALR2 expression overall is low and diffuse but more widespread. In contrast, GALR3 expression is very low and restricted, expressed almost exclusively in the preoptic area, the 231

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hypothalamus and specific circumventricular organs. Thus, the graded abundance of GALRs in the CNS is GALR1 > R2 >> R3, suggesting that of the three, GALR1 may play a more prominent role in mediating most of galanin's central effects. This idea is strengthened by the observation that distribution of galanin-binding sites in the CNS coincides most closely with that of GALR1 mRNA expression. Whether the relative abundance of each receptor has functional implications remains to be determined. It is nonetheless tempting to want to ascribe specific galanin-mediated biological effects to each of the receptor subtypes based on their neuroanatomical localization. The hypothalamus represents a major target for galanin's central actions. Not surprisingly, high levels of galanin-binding sites are present throughout this structure. Although their overall hypothalamic patterns of expression differ, all three receptor subtypes are expressed to varying extents in the paraventricular, lateral and ventromedial hypothalamus suggesting that all three receptors may mediate galanin's stimulatory effects on food consumption and fat intake. Galanin's mnemonic effects are thought to be mediated in part by galanin inhibitory effects on the medial septum diagonal band cholinergic neurons that project to the hippocampus. Despite the abundance of GALR1 mRNA-expressing neurons in the medial septum-diagonal band complex, co-localization studies found no evidence that cholinergic neurons express GALR1. Our data indicate that both GALR2- and GALR3-expressing cells are sparsely distributed within the diagonal band suggesting that one or both of these subtypes may be involved in mediating galanin's inhibitory effects on learning and memory. There is strong evidence suggesting that galanin may also exert modulatory effects on cognition at the level of the hippocampus. A recent study has convincingly demonstrated that infusion of galanin in the dorsal and ventral dentate gyms, areas that express only GALR2, hampered spatial acquisition in Morris swim maze test (Schott et al., 2000). In contrast, infusion of galanin into the ventral CA1 region, which contains only GALR1 mRNA-expressing cells, did not produce any deficits in spatial learning as compared to control animals. Thus, these findings strongly suggest that GALR2 is important in mediating galanin effects on spatial learning. Lastly, several behavioral studies support the notion that more than one galanin receptor subtype is involved in modulating pain transmission. The expression of GALR1, GALR2 and GALR3, to different degrees, in DRGs as well as in key spinal and supra-spinal pain structures indicates that this is in fact the case. The relative contribution of each galanin receptor in mediating pain transmission under normal and pathophysiological conditions, however, remains to be determined. It is noteworthy that, although all three GALRs are expressed to some extent in the dorsal horn of the spinal cord, only GALR2 is expressed in a subpopulation of et-motoneurons in the ventral horn, suggesting that spinally mediated motor effects are specific to GALR2. Clearly, additional studies, using subtype-selective radioligands and/or galanin receptor subtype-specific antibodies, are warranted in order to determine the distribution and relative protein abundance of each of the galanin receptor and whether it is similar to that observed for the message. However, the availability of receptor subtype-selective ligands has been quite limited to date. Likewise, there has been a lack of specific galanin receptor antibodies presumably reflecting the inherent difficulties associated with raising antibodies to seven transmembrane protein receptors and in particular to the galanin receptor subfamily. Sullivan et al. (1997) generated antisera to the human GALR1; however, its use was restricted to Western blotting analysis of CHO cells transiently transfected with hGALR1 or the stable CHO cell line. The authors did not specify whether the antisera could recognize endogenous GALR1 receptors in human tissues or if it cross-reacted with the rat sequence. To the best 232


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of our knowledge, there is one immunohistochemical study using a galanin receptor specific antibody and it was reported only recently (Burazin et al., 2001). Thus, there is still a pressing need for receptor subtype specific tools in order to further elucidate the role galanin as well as the exact role of each receptor in mediating the many known biological effects of galanin in the CNS. On a final note, although our knowledge of the anatomy and physiology of central galaninergic systems in the rat CNS is rapidly growing, there is a paucity of information with respect to the distribution of GALRs in human CNS tissues. In light of reported species differences between rat and primate and the therapeutic potential of drugs acting on galanin receptors, it is essential to perform similar neuroanatomical mapping studies in man. 9. ABBREVIATIONS

12 aci AcbSh AH AI Amb AO Apir Arc BAOT BL BLP BMA BST CA1-3 Ce Cer CL CM Co CPu Cx DA DCIC DG DM DP DR DRG DRI DTg

E/or

hypoglossal nucleus anterior commissure, intrabulbar shell of the accumbens anterior hypothalamic area agranular insular cortex ambiguus nucleus anterior olfactory nucleus (AOD, AOL, AOM and AOV: respectively dorsal, lateral, medial and ventral parts) amygdalopiriform transition area arcuate hypothalamic nucleus bed nucleus of the accessory olfactory tract basolateral amygdaloid nucleus basolateral amygdaloid nucleus, posterior part basomedial amygdaloid nucleus, anterior part bed nucleus of the stria terminalis fields of Ammon's horn central amygdaloid nucleus cerebellum centrolateral thalamic nucleus central medial thalamic nucleus cortical amygdaloid nucleus caudate putamen (striatum) cortex (CxS, CxI and CxD: respectively superficial, intermediate and deep layers of the cortex) dorsal hypothalamic area dorsal cortex of the inferior colliculus dentate gyrus dorsomedial hypothalamic nucleus dorsal peduncular cortex dorsal raphe nucleus dorsal root ganglia dorsal raphe nucleus, interfascicular part dorsal tegmental nucleus ependyma/olfactory ventricle 233

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ECu EF Ent EP1 EW f fi GALR G1 GMAP GP Gr GrCb GrDG Hb HDB

icj IMD IO LC LD LH LO LPB LPO LRt LS MCH MD Me Mi MM MnPO MnR Mo5 MolCb MPA MPB MPT MRe MS MVe mt O Op PAG PDTg Pir 234

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external cuneate nucleus epifascicular nucleus entorhinal cortex external plexiform layer of the olfactory bulb Edinger-Westphal nucleus fornix fimbria galanin receptor glomerular layer of the olfactory bulb galanin message-associated peptide globus pallidum granular cell layer of olfactory bulb granular cell layer of the cerebellum granular cell layer of the dentate gyrus habenula nucleus of the horizontal limb of the diagonal band islands of Calleja intermediodorsal thalamic nucleus inferior olive locus coeruleus laterodorsal thalamic nucleus lateral hypothalamic area lateral orbital cortex lateral parabrachial nucleus lateral preoptic area lateral reticular formation lateral septum melanin-concentrating hormone mediodorsal thalamic nucleus medial amygdaloid nucleus mitral cell layer of the olfactory bulb medial mammillary nucleus, medial part medial preoptic nucleus median raphe nucleus motor trigeminal nucleus molecular layer of the cerebellum medial preoptic area medial parabrachial nucleus medial pretectal nucleus mammillary recess of the third ventricle medial septum medial vestibular nucleus mammillothalamic tract nucleus O optic nerve layer of the superior colliculus periaqueductal gray posterodorsal tegmental nucleus piriform cortex


PH PMD PMV PnC PnO Pr Pr5 PVA PVN PVP PY Py PyCb Re Rh RMg ROb RPa rs

Rt RtTg RSG RtTg S SFi SFO SHi SNc SO Sol Sp5 st STh SuG SuMM TC VDB VLH VMH VMPO VTA ZI Zo

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posterior hypothalamic area premammillary nucleus, dorsal part premammillary nucleus, ventral part pontine reticular nucleus, caudal part pontine reticular nucleus, oral part prepositus nucleus principal sensory trigeminal nucleus paraventricular thalamic nucleus, anterior part paraventricular hypothalamic nucleus paraventricular thalamic nucleus, posterior part pyramidal tract pyramidal cell layer of the hippocampus pyramidal cell layer of the cerebellum reuniens thalamic nucleus rhomboid nucleus raphe magnus nucleus raphe obscursus nucleus raphe pallidus nucleus rubrospinal tract reticular formation reticulotegmental nucleus of the pons retrosplenial granular cortex reticulotegmental nucleus of the pons subiculum septofimbrial nucleus subfornical organ septohippocampal nucleus substantia nigra, compact part supraoptic nucleus solitary nucleus spinal trigeminal nucleus stria terminalis subthalamic nucleus superficial gray layer of superior colliculus supramammillary nucleus, medial part tuber cinereum area nucleus of the vertical limb of the diagonal band ventrolateral hypothalamic nucleus ventromedial hypothalamic nucleus ventromedial preoptic nucleus ventral tegmental area zona incerta zonal layer of the superior colliculus

10. ACKNOWLEDGEMENTS

The authors thank Dr. Alain Beaudet for insightful comments and suggestions in reviewing the manuscript. 235

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neuroendocrine functions in the male rat. Possible involvement of hypothalamic catecholamine neuronal systems. Acta Physiol Scand 131:25-32. Melander T, Kohler C, Nilsson S, Hokfelt T, Brodin E, Theodorsson E, Bartfai T (1988): Autoradiographic quantitation and anatomical mapping of 125I-galanin binding sites in the rat central nervous system. J Chem Neuroanat 1:213-233. Melander T, Khler C, Nilsson S, Fisone G, Bartfai T, H6kfelt T (1992): 125I-Galanin binding sites in the rat central nervous system. In: Bj6rklund A, H6kfelt T and Kuhar MJ (Eds.), Handbook of Chemical Neuroanatomy, Vol 11. Amsterdam: Elsevier, pp. 187-221. Mennicken F, Hoffert C, Pelletier M, Ahmad S, O'Donnell D (2002): Restricted distribution of Galanin receptor 3 (GALR3) mRNA in the adult rat central nervous system. J Chem Neuroanat, in press. Merchenthaler I, Lopez FJ, Negro-Vilar A (1990): Colocalization of galanin and luteinizing hormone-releasing hormone in a subset of preoptic hypothalamic neurons: anatomical and functional correlates. Proc Natl Acad Sci USA 87:6326-6330. Merchenthaler I, Lopez FJ, Negro-Vilar A (1993): Anatomy and physiology of central galanin-containing pathways. Prog Neurobiol 40:711-769. Michener SR, Aimone LD, Yaksh TL, Go VL (1990): Distribution of galanin-like immunoreactivity in the pig, rat and human central nervous system. Peptides 11:1217-1223. Miller MA, Kolb PE, Planas B, Raskind MA (1998): Few cholinergic neurons in the rat basal forebrain coexpress galanin messenger RNA. J Comp Neurol 391:248-258. Mitchell V, Habert-Ortoli E, Epelbaum J, Aubert JP, Beauvillain JC (1997): Semiquantitative distribution of galanin-receptor (GAL-R1) mRNA-containing cells in the male rat hypothalamus. Neuroendocrinology 66:160172. Mitchell V, Bouret S, Howard AD, Beauvillain JC (1999): Expression of the galanin receptor subtype Gal-R2 mRNA in the rat hypothalamus. J Chem Neuroanat 16:265-277. Mufson EJ, Kahl U, Bowser R, Mash DC, Kordower JH, Deecher DC (1998): Galanin expression within the basal forebrain in Alzheimer's disease. Comments on therapeutic potential. Ann NY Acad Sci 863:291-304. Mufson EJ, Deecher DC, Basile M, Izenwasse S, Mash DC (2000): Galanin receptor plasticity within the nucleus basalis in early and late Alzheimer's disease: an in vitro autoradiographic analysis. Neuropharmacology 39:14041412. Narvaez JA, Diaz Z, Aguirre JA, Gonzalez-Baron S, Yanaihara N, Fuxe K, Hedlund PB (1994): Intracisternally injected galanin-(1-15) modulates the cardiovascular responses of galanin-(1-29) and the 5-HT1A receptor agonist 8-OH-DPAT. Eur J Pharmacol 257:257-265. Narvaez JA, Diaz-Cabiale Z, Hedlund PB, Aguirre JA, Covenas R, Gonzalez-Baron S, Fuxe K (2000): The galanin receptor antagonist M40 blocks the central cardiovascular actions of the galanin N-terminal fragment (1-15). Eur J Pharmaco1399:197-203. Nouel D, Sarret P, Vincent JP, Mazella J, Beaudet A (1999): Pharmacological, molecular and functional characterization of glial neurotensin receptors. Neuroscience 94:1189-1197. O'Donnell D, Ahmad S, Wahlestedt C, Walker P (1999): Expression of the novel galanin receptor subtype GALR2 in the adult rat CNS: distinct distribution from GALR1. J Comp Neurol 409:469-481. Ogren SO, Hokfelt T, Kask K, Langel U, Bartfai T (1992): Evidence for a role of the neuropeptide galanin in spatial learning. Neuroscience 51:1-5. Ohtaki T, Kumano S, Ishibashi Y, Ogi K, Matsui H, Harada M, Kitada C, Kurokawa T, Onda H, Fujino M (1999): Isolation and cDNA cloning of a novel galanin-like peptide (GALP) from porcine hypothalamus. J Biol Chem 274:37041-37045. Ohtaki T, Shintani Y, Honda S, Matsumoto H, Hori A, Kanehashi K, Terao Y, Kumano S, Takatsu Y, Masuda Y, Ishibashi Y, Watanabe T, Asada M, Yamada T, Suenaga M, Kitada C, Usuki S, Kurokawa T, Onda H, Nishimura O, Fujino M (2001): Metastasis suppressor gene KISS-1 encodes peptide ligand of a G-protein-coupled receptor. Nature 411:613-617. Oldfield BJ and Mckinley MJ (1995): Circumventricular organs. In: Paxinos G (Ed), The Rat Nervous System, 2nd ed. San Diego: Academic Press, pp. 391-404. Ottlecz A, Snyder GD, McCann SM (1988): Regulatory role of galanin in control of hypothalamic-anterior pituitary function. Proc Natl Acad Sci USA 85:9861-9865. Parker EM, Izzarelli DG, Nowak HP, Mahle CD, Iben LG, Wang J, Goldstein ME (1995): Cloning and characterization of the rat GALR1 galanin receptor from Rinl4B insulinoma cells. Mol Brain Res 34:179-189. Paxinos G, Watson C (1998): The Rat Brain in Streotaxic Coordinates, 4th ed. San Diego: Academic Press. P6rez SE, Wynick D, Steiner RA, Mufson EJ (2001): Distribution of galaninergic immunoreactivity in the brain of the mouse. J Comp Neurol 434:158-185.

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Pooga M, Soomets U, Hallbrink M, Valkna A, Saar K, Rezaei K, Kahl U, Hao JX, Xu XJ, Wiesenfeld-Hallin Z, Hokfelt T, Bartfai T, Langel U (1998): Cell penetrating PNA constructs regulate galanin receptor levels and modify pain transmission in vivo. Nat Biotechnol 16:857-861. Post C, Alari L, Hokfelt T (1988): Intrathecal galanin increases the latency in the tail-flick and hot-plate test in mouse. Acta Physiol Scand 132:583-584. Priller J, Haas CA, Reddington M, Kreutzberg GW (1998): Cultured astrocytes express functional receptors for galanin. Glia 24:323-328. Rada P, Mark GP, Hoebel BG (1998): Galanin in the hypothalamus raises dopamine and lowers acetylcholine release in the nucleus accumbens: a possible mechanism for hypothalamic initiation of feeding behavior. Brain Res 798:1-6. Rokaeus A, Brownstein MJ (1986): Construction of a porcine adrenal medullary cDNA library and nucleotide sequence analysis of two clones encoding a galanin precursor. Proc Natl Acad Sci USA 83:6287-6291. Rokaeus A, Melander T, Hokfelt T, Lundberg JM, Tatemoto K, Carlquist M, Mutt V (1984): A galanin-like peptide in the central nervous system and intestine of the rat. Neurosci Lett 47:161-166. Rosier AM, Vandesande F, Orban GA (1991): Laminar and regional distribution of galanin binding sites in cat and monkey visual cortex determined by in vitro receptor autoradiography. J Comp Neurol 305:264-272. Sailer AW, Sano H, Zeng ZZ, McDonald TP, Pan J, Pong SS, Feighner SD, Tan CE Fukami T, Iwaasa H, Hreniuk DL, Morin NR, Sadowski SJ, Ito M, Bansal A, Ky B, Figueroa DJ, Jiang QE Austin CE MacNeil DJ, Ishihara A, Ihara M, Kanatani A, Van der Ploeg LHT, Howard AD (2001): Identification and characterization of a second melanin-concentrating hormone receptor, MCH-2R. Proc Natl Acad Sci USA 98:7564-7569. Saito Y, Nothacker HE Wang Z, Lin SH, Leslie F, Civelli O (1999): Molecular characterization of the melaninconcentrating-hormone receptor. Nature 400:265-269. Saper CB (1995): Central autonomic system. In: Paxinos G (Ed), The Rat Nervous System, 2nd ed., San Diego: Academic Press, pp. 107-135. Schick RR, Samsami S, Zimmermann JE Eberl T, Endres C, Schusdziarra V, Classen M (1993): Effect of galanin on food intake in rats: involvement of lateral and ventromedial hypothalamic sites. Am J Physiol 264:61. Schott PA, Hokfelt T, Ogren SO (2000): Galanin and spatial learning in the rat. Evidence for a differential role for galanin in subregions of the hippocampal formation. Neuropharmacology 39:1386-1403. Servin AL, Amiranoff B, Rouyer-Fessard C, Tatemoto K, Laburthe M (1987): Identification and molecular characterization of galanin receptor sites in rat brain. Biochem Biophys Res Commun 144:298-306. Sillard R, Rokaeus A, Xu Y, Carlquist M, Bergman T, Jornvall H, Mutt V (1992): Variant forms of galanin isolated from porcine brain. Peptides 13:1055-1060. Skofitsch G, Jacobowitz DM (1985): Immunohistochemical mapping of galanin-like neurons in the rat central nervous system. Peptides 6:509-546. Skofitsch G, Jacobowitz DM (1986): Quantitative distribution of galanin-like immunoreactivity in the rat central nervous system. Peptides 7:609-613. Skofitsch G, Sills MA, Jacobowitz DM (1986): Autoradiographic distribution of 125I-galanin binding sites in the rat central nervous system. Peptides 7:1029-1042. Smith KE, Forray C, Walker MW, Jones KA, Tamm JA, Bard J, Branchek TA, Linemeyer DL, Gerald C (1997): Expression cloning of a rat hypothalamic galanin receptor coupled to phosphoinositide turnover. J Biol Chem 272:24612-24616. Smith KE, Walker MW, Artymyshyn R, Bard J, Borowsky B, Tamm JA, Yao WJ, Vaysse PJ, Branchek TA, Gerald C, Jones KA (1998): Cloned human and rat galanin GALR3 receptors. Pharmacology and activation of G-protein inwardly rectifying K + channels. J Biol Chem 273:23321-23326. Steiner RA, Hohmann JG, Holmes A, Wrenn CC, Cadd G, Jureus A, Clifton DK, Luo M, Gutshall M, Ma SY, Mufson EJ, Crawley JN (2001): Galanin transgenic mice display cognitive and neurochemical deficits characteristic of Alzheimer's disease. Proc Natl Acad Sci USA 98:4184-4189. Sten Shi TJ, Zhang X, Holmberg K, Xu ZQ, Hokfelt T (1997): Expression and regulation of galanin-R2 receptors in rat primary sensory neurons: effect of axotomy and inflammation. Neurosci Lett 237:57-60. Sullivan KA, Shiao LL, Cascieri MA (1997): Pharmacological characterization and tissue distribution of the human and rat GALR1 receptors. Biochem Biophys Res Commun 233:823-828. Sundstrom E, Archer T, Melander T, Hokfelt T (1988): Galanin impairs acquisition but not retrieval of spatial memory in rats studied in the Morris swim maze. Neurosci Lett 88:331-335. Takatsu Y, Matsumoto H, Ohtaki T, Kumano S, Kitada C, Onda H, Nishimura O, Fujino M (2001): Distribution of galanin-like peptide in the rat brain. Endocrinology 142:1626-1634. Tatemoto K, Rokaeus A, Jornvall H, McDonald TJ, Mutt V (1983): Galanin: a novel biologically active peptide from porcine intestine. FEBS Lett 164:124-128.

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Tempel DL, Leibowitz SF (1990): Diurnal variations in the feeding responses to norepinephrine, neuropeptide Y and galanin in the PVN. Brain Res Bull 25:821-825. Tempel DL, Leibowitz SF (1993): Glucocorticoid receptors in PVN: interactions with NE, NPY, and Gal in relation to feeding. Am J Physiol 265:800. Tempel DL, Leibowitz KJ, Leibowitz SF (1988): Effects of PVN galanin on macronutrient selection. Peptides 9:309-314. Verge VM, Xu XJ, Langel U, Hokfelt T, Wiesenfeld-Hallin Z, Bartfai T (1993): Evidence for endogenous inhibition of autotomy by galanin in the rat after sciatic nerve section: demonstrated by chronic intrathecal infusion of a high affinity galanin receptor antagonist. Neurosci Lett 149:193-197. Villar MJ, Cortes R, Theodorsson E, Wiesenfeld-Hallin Z, Schalling M, Fahrenkrug J, Emson PC, Hokfelt T (1989): Neuropeptide expression in rat dorsal root ganglion cells and spinal cord after peripheral nerve injury with special reference to galanin. Neuroscience 33:587-604. Villar MJ, Wiesenfeld-Hallin Z, Xu XJ, Theodorsson E, Emson PC, Hokfelt T (1991): Further studies on galanin-, substance P-, and CGRP-like immunoreactivities in primary sensory neurons and spinal cord: effects of dorsal rhizotomies and sciatic nerve lesions. Exp Neurol 112:29-39. Wang S, Hashemi T, He C, Strader C, Bayne M (1997a): Molecular cloning and pharmacological characterization of a new galanin receptor subtype. Mol Pharmacol 52:337-343. Wang S, He C, Hashemi T, Bayne M (1997b): Cloning and expressional characterization of a novel galanin receptor. Identification of different pharmacophores within galanin for the three galanin receptor subtypes. J Biol Chem 272:31949-31952. Wang S, He C, Maguire MT, Clemmons AL, Burrier RE, Guzzi ME Strader CD, Parker EM, Bayne ML (1997c): Genomic organization and functional characterization of the mouse GalR1 galanin receptor. FEBS Lett 411:225230. Wang S, Hwa J, Varty G (2000): Galanin receptors and their therapeutic potential. Emerging Drugs 5:415-440. Waters SM, Krause JE (2000): Distribution of galanin-1, -2 and -3 receptor messenger RNAs in central and peripheral rat tissues. Neuroscience 95:265-271. Wiesenfeld-Hallin Z, Villar MJ, Hokfelt T (1989a): The effects of intrathecal galanin and C-fiber stimulation on the flexor reflex in the rat. Brain Res 486:205-213. Wiesenfeld-Hallin Z, Xu XJ, Villar MJ, Hokfelt T (1989b): The effect of intrathecal galanin on the flexor reflex in rat: increased depression after sciatic nerve section. Neurosci Lett 105:149-154. Wiesenfeld-Hallin Z, Xu XJ, Villar MJ, Hokfelt T (1990): Intrathecal galanin potentiates the spinal analgesic effect of morphine: electrophysiological and behavioural studies. Neurosci Lett 109:217-221. Wiesenfeld-Hallin Z, Bartfai T, Hokfelt T (1992a): Galanin in sensory neurons in the spinal cord. Front Neuroendocrinol 13:319-343. Wiesenfeld-Hallin Z, Xu XJ, Langel U, Bedecs K, Hokfelt T, Bartfai T (1992b): Galanin-mediated control of pain: enhanced role after nerve injury. Proc Natl Acad Sci USA 89:3334-3337. Wiesenfeld-Hallin Z, Xu XJ, Hao JX, Hokfelt T (1993): The behavioural effects of intrathecal galanin on tests of thermal and mechanical nociception in the rat. Acta Physiol Scand 147:457-458. Wynick D, Smith DM, Ghatei M, Akinsanya K, Bhogal R, Purkiss P, Byfield P, Yanaihara N, Bloom SR (1993): Characterization of a high-affinity galanin receptor in the rat anterior pituitary: absence of biological effect and reduced membrane binding of the antagonist M15 differentiate it from the brain/gut receptor. Proc Natl Acad Sci USA 90:4231-4235. Wynick D, Small CJ, Bacon A, Holmes FE, Norman M, Ormandy CJ, Kilic E, Kerr NC, Ghatei M, Talamantes F, Bloom SR, Pachnis V (1998a): Galanin regulates prolactin release and lactotroph proliferation. Proc Natl Acad Sci USA 95:12671-12676. Wynick D, Small CJ, Bloom SR, Pachnis V (1998b): Targeted disruption of the murine galanin gene. Ann NY Acad Sci 863:22-47. Xu XJ, Wiesenfeld-Hallin Z, Fisone G, Bartfai T, Hokfelt T (1990): The N-terminal 1-16, but not C-terminal 17-29, galanin fragment affects the flexor reflex in rats. Eur J Pharmacol 182:137-141. Xu XJ, Andell S, Zhang X, Wiesenfeld-Hallin Z, Langel U, Bedecs K, Hokfelt T, Bartfai T (1995): Peripheral axotomy increases the expression of galanin message-associated peptide (GMAP) in dorsal root ganglion cells and alters the effects of intrathecal GMAP on the flexor reflex in the rat. Neuropeptides 28:299-307. Xu XJ, Andell S, Bartfai T, Wiesenfeld-Hallin Z (1996): Fragments of galanin message-associated peptide (GMAP) modulate the spinal flexor reflex in rat. Eur J Pharmacol 318:301-306. Xu Y, Rokaeus A, Johansson O (1994): Distribution and chromatographic analysis of galanin message-associated peptide (GMAP)-like immunoreactivity in the rat. Regul Pept 51:1-16. 243

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Xu ZQ, Shi TJ, Hokfelt T (1996a): Expression of galanin and a galanin receptor in several sensory systems and bone anlage of rat embryos. Proc Natl Acad Sci USA 93:14901-14905. Xu ZQ, Shi TJ, Landry M, Hokfelt T (1996b): Evidence for galanin receptors in primary sensory neurones and effect of axotomy and inflammation. NeuroReport 8:237-242. Xu ZQ, Shi TJ, Hokfelt T (1998): Galanin/GMAP- and NPY-like immunoreactivities in locus coeruleus and noradrenergic nerve terminals in the hippocampal formation and cortex with notes on the galanin-R1 and -R2 receptors. J Comp Neurol 392:227-251. Zhang X, Ji RR, Nilsson S, Villar M, Ubink R, Ju G, Wiesenfeld-Hallin Z, Hokfelt T (1995a): Neuropeptide, Y and galanin binding sites in rat and monkey lumbar dorsal root ganglia and spinal cord and effect of peripheral axotomy. Eur J Neurosci 7:367-380. Zhang X, Aman K, Hokfelt T (1995b): Secretory pathways of neuropeptides in rat lumbar dorsal root ganglion neurons and effects of peripheral axotomy. J Comp Neurol 352:481-500. Zhang X, Xu ZO, Shi TJ, Landry M, Holmberg K, Ju G, Tong YG, Bao L, Cheng XP, Wiesenfeld-Hallin Z, Lozano A, Dostrovsky J, Hokfelt T (1998): Regulation of expression of galanin and galanin receptors in dorsal root ganglia and spinal cord after axotomy and inflammation. Ann NY Acad Sci 863:402-413.

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Orexin receptors TAKESHI SAKURAI, GUILLAUME HERVIEU AND MASASHI YANAGISAWA

1. INTRODUCTION 1.1. DISCOVERY AND IDENTIFICATION OF OREXINS/HYPOCRETINS In 1996, we were systematically searching endogenous peptide ligands for multiple orphan G-protein coupled receptors (GPCRs), using a cell-based reporter system, because we thought many of these orphan GPCRs are likely to be receptors for unidentified signaling molecules, including new biologically important peptide hormones and neuropeptides. These screening experiments led us to the identification of two neuropeptides that bind to two closely related orphan GPCRs. We named these peptides orexin-A and -B, after the Greek word orexis, which means appetite, because we found that these peptides increase food intake when administered centrally (Sakurai et al., 1998). On the other hand, de Lecea et al. (1998) identified a hypothalamus-specific mRNA by PCR-subtraction method and found that the identified transcript encoded a precursor for two novel neuropeptides. They named these peptides hypocretin-1 and-2. Some confusion has arisen in the literature because orexins and hypocretins are alternative names for the same peptides. The structure of the hypocretin-1 peptide was not precisely determined in the original description, but the hypocretin gene is identical to the orexin gene. Basically, we use here the name 'orexins' for convenience, but please note that orexin-1 is identical with a peptide termed hypocretin-1 and orexin-B is identical with hypocretin-2. 1.2. STRUCTURES OF OREXIN-A AND -B Structures of orexins were chemically determined by biochemical purification and sequence analysis by Edman sequencing and mass spectrometry. Orexin-A is a 33-amino-acid peptide of 3562 Da, with an N-terminal pyroglutamyl residue and C-terminal amidation (Fig. 1). Molecular mass of the purified peptide as well as its sequencing analyses indicated that the four Cys residues of orexin-A formed two sets of intrachain disulfide bonds. The topology of the disulfide bonds was chemically determined to be [Cys6-Cysl2, Cys7-Cysl4] (Sakurai et al., 1998). This structure is completely conserved among several mammalian species (human, rat, mouse, cow, pig and dog). On the other hand, rat orexin-B is a 28-amino acid, C-terminally amidated linear peptide of 2937 Da, which was 46% (13/28) identical in sequence to orexin-A (Fig. 1). The C-terminal half of orexin-B is especially similar to that of orexin-A, while the N-terminal half is more variable. The mouse orexin-B was predicted to be identical to rat orexin-B. The human orexin-B has two amino acid substitutions from the rodent sequence within the 28-residue stretch. Pig and dog orexin-B have one amino acid substitution from Handbook of Chemical Neuroanatomy, Vol. 20: Peptide Receptors, Part H R. Quirion, A. Bj6rklund and T. H6kfelt, editors 92003 Elsevier Science B.V. All rights reserved.

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human*/bovine/rat/mouse*/dog* orexin-A rat/mouse* orexin-B pig*Idog* orexin-B human*orexin-B

Ii Iq <EPLPOOOROKTOS 0NHO,L-NH2 ]RPGPPGLQGRLQRLLQAL~GNHAAGILTMtNH2 IRPGPPGLQGRLQRLLQASGNHAAGILTM~NH2 L~GPPGLQGRLQRLLQASGNHAAGILTM~NH2

Fig. 1. Structures of mature orexin-A and -B peptides. The topology of the two intrachain disulfide bonds in orexin-A is indicated above the sequence. Amino acid identities are indicated by boxes. Asterisks indicate that human, pig, dog and mouse sequences are deduced from the respective cDNA sequences and not from purified peptides. the human or rodent sequence (Fig. 1). Orexins have no relevant structural similarities to any other known biologically active peptides. The prepro-orexin c D N A sequences revealed that both orexins are produced from the same 130-residue (rodent) or 131-residue (human) polypeptide, prepro-orexin (Fig. 2). Overall, the human and m o u s e prepro-orexin sequences are 83% and 95% identical to the rat counterpart, respectively (Sakurai et al., 1998). The majority of amino acid substitutions were found in the

human

MNLPSTKVSW

AAVTLLLLLL

LLPPALLSSG

AAA Q P L P D C C

RQKTCSCRLY

ELLHGAGNHA

60

pig

MNPPFAKVSW

ATVTLLLLLL

LLPPAVLSPG

AAA Q P L P D C C

RQKTCSCRLY

ELLHGAGNHA

60

dog

MNPPSTKVPW

AAVTLLLLLL

L-PPALLSPG

AAA ~ P L P D C C

RQKTCSCRLY

ELLHGAGNHA

59

rat

FINLPSTKVPW A A V T L L L L L L

L-PPALLSLG

VDA ~ P L P D C C

RQKTCSCRLY

ELLHGAGNHA

59

mouse

MNFPSTKVPW

AAVTLLLLLL

L-PPALLSLG

VDA ~ P L P D C C

RQKTCSCRLY

ELLHGAGNHA

59

.

*

* * * * * * * * * *

* * * * * * * * * *

**

.

********

***

**

.

********

s i g n a l peptide human

AGILTL3KR

SGPPGLQGRL

QRLLQASGNH

A A G I L T M ;RR A G A E P A P R P C

LGRRCSAPAA

120

pig

AGILTLZKR

PGPPGLQGRL

QRLLQASGNH

A A G I L T M ;RR A G A E P A P R L C

PGRRCLAAAA

120

dog

AGILT/3KR

PGPPGLQGRL

QRLLQASGNH

A A G I L T M ;RR A G A E P A P R P C

PGRRCPVVAV

119

rat

A G I L T L 3KR R P G P P G L Q G R L

QRLLQANGNH

A A G I L T M ;RR A G A E L E P Y P C

PGRRCPTATA

119

mouse

A G I L T L 3KR R P G P P G L Q G R L

QRLLQANGNH

A A G I L T M ;RR A G A E L E P H P C

SGRGCPTVTT

119

* * * * * * * * * *

******

* * * * * * * * * *

* * * * * * * * *

human

ASVAPGGQSG

I

131

pig

SSVAPGGRSG

I

131

dog

PSAAPGGRSG V

130

rat

TALAPRGGSR V

130

mouse

TALAPRGGSG

130

V

***

****

*

*

****

Fig. 2. Deduced amino acid sequences of human, rat, and mouse prepro-orexin precursor polypeptides. Orexin-A and -B sequences are boxed. Predicted secretory signal sequences are underlined. Interspecifically identical residues are indicated by vertical bars.

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C-terminal part of the precursor, which appears unlikely to encode another bioactive peptide (Fig. 2). We reported orexins initially as orexigenic peptides (Sakurai et al., 1998). Subsequently they have been reported to have a variety of pharmacological actions (Willie et al., 2001). Especially, recent observations implicate orexins/hypocretins in sleep disorder narcolepsy and potentially, in the regulation of the normal sleep process (Kilduff and Peyron, 2000). We review here the distribution and biological effects of orexins and their receptors in the brain, focusing on the characterization and distribution of orexin receptors in the adult rat brain. We also discuss the possible physiological and pathophysiological implications of orexin receptors.

2. BIOLOGY OF OREXINS 2.1. PREPRO-OREXIN GENE, STRUCTURE AND REGULATION OF EXPRESSION Radiation hybrid mapping showed that the human prepro-orexin gene is most closely linked to the MIT STS markers WI-6595 and UTR9641 (Sakurai et al., 1998). The inferred cytogenetic location between these markers is 17q21. The human prepro-orexin gene consists of two exons and one intron distributed over 1432 bp (Sakurai et al., 1999). The 143-bp exon 1 includes the 5'-untranslated region and the coding region that encodes the first seven residues of the secretory signal sequence. Intron 1 is 818-bp long. Exon 2 contains the remaining portion of the open reading frame and the T-untranslated region. The human prepro-orexin gene fragment, which contains the 3149-bp 5'-flanking region and 122-bp 5'-non-coding region of exon 1, has the ability to express exogenous genes (lacZ or green fluorescent protein) in orexin neurons in transgenic mice, suggesting that this genomic fragment contains all the necessary elements for appropriate expression of the gene (Sakurai et al., 1999). This promoter might be useful to examine the consequences of expression of exogenous molecules in orexin neurons of transgenic mice, thereby manipulating the cellular environment in vivo (Sakurai et al., 1999; Hara et al., 2001). The regulation of expression of the prepro-orexin gene still remains unclear. Prepro-orexin mRNA was shown to be upregulated under fasting conditions, indicating that these neurons somehow sense the animal's nutritional state (Sakurai et al., 1998). Several reports have shown that orexin neurons express leptin receptor- and STAT-3-1ike immunoreactivity, suggesting that orexin neurons are regulated by leptin (Hakansson et al., 1998; Horvath et al., 1999). Indeed, we found that continuous infusion of leptin into the third ventricle of mice for 2 weeks resulted in marked down-regulation of prepro-orexin mRNA level (Yamanaka et al., submitted). Therefore, reduced leptin signaling may be a possible factor that up-regulates expression of prepro-orexin mRNA during starvation. Prepro-orexin levels were also increased in hypoglycemic conditions, suggesting that expression of the prepro-orexin gene is regulated by plasma glucose levels (Griffond et al., 1999). 2.2. FEATURES OF OREXIN SYSTEM IN MAMMALS

2.2.1. Striking hypothalamic localization of orexin-containing neurons Prepro-orexin is specifically expressed by neurons located in the lateral hypothalamic area (LHA) (de Lecea et al., 1998; Sakurai et al., 1998). The LHA, traditionally viewed as 247

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T. Sakurai et al.

Fig. 3. Schematic drawing of sagittal section through the rat brain to summarize the organization of the orexin neuronal system. Dots indicate the relative location of orexin-immunoreactive neurons, and arrows show some of the more prominent terminal fields.

a phylogenetic continuation of the reticular formation, governs many functions such as feeding, blood pressure, neuroendocrine axis, thermoregulation, sleep-waking cycle, emotion, sensorimotor integration and reward processes. This reflects extensive projections from the LHA to a variety of regions throughout the central nervous system. Many anatomical studies, using retro- or anterograde tracers, demonstrated that the LHA has many efferent projections, including monosynaptic projections to several regions of the cerebral cortex, limbic system, and brainstem (Saper et al., 1979; Hosoya and Matsushita, 1981; Touzani et al., 1990; Villalobos et al., 1987a,b). Immunohistochemical studies suggested that orexin-containing neurons (orexin neurons) are included in these projections (Fig. 3) (Peyron et al., 1998; Date et al., 1999; Nambu et al., 1999). Of these diverse roles of the LHA, regulation of feeding behavior is a major one: the so-called lateral hypothalamic feeding syndrome, due to lesions of the LHA, causes hypophagia and loss of weight. Within the hypothalamus, lesion experiments have suggested two areas that have traditionally been associated with the regulation of feeding and energy balance, the ventromedial hypothalamus (VMH) and the LHA (Anand and Brobeck, 1951; Winn et al., 1984). Understanding of the roles of the mediobasal hypothalamus and several neuropeptides in this region in regulating food intake and body weight is increasing recently. On the other hand, the LHA has not been well understood on a molecular/neurotransmitter basis until very recently. Orexins and melaninconcentrating hormone (MCH) were recently shown to be expressed in the LHA and the adjacent regions (Bittencourt et al., 1992; de Lecea et al., 1998; Sakurai et al., 1998). MCH neurons and orexin neurons have been shown to be distinct neuronal populations in the LHA, although their distribution and projections are very similar to each other (Broberger et al., 1998; Elias et al., 1998). Both orexins and MCH have been shown to increase food intake when administered centrally, further supporting the role of the LHA in the regulation of feeding behavior (Sakurai et al., 1998; Qu et al., 1996). The LHA contains a population of neurons that is sensitive to glucose level and is activated by hypoglycemia. These neurons are termed glucose-sensitive (GS) neurons. The identity of 248

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these cells has remained elusive, because the LHA is not as topologically organized as other major nuclei of the hypothalamus. Some orexin neurons are thought to be GS neurons rather than the formerly suspected MCH neurons (Moriguchi et al., 1999). The fact that insulininduced hypoglycemia increases the level of prepro-orexin precursor protein further supports this idea (Griffond et al., 1999). Indeed our recent electrophysiological examination showed that isolated orexin neurons were inhibited by a high extracellular glucose concentration (Yamanaka et al., submitted). Some orexin neurons are located in the posterior hypothalamus, a region that has been implicated in arousal state control (Nauta, 1946), consistent with recent observations that suggest orexins have roles in arousal and vigilance state control as well as in sleep/wakefulness state (Chemelli et al., 1999; Lin et al., 1999; Kilduff and Peyron, 2000; Peyron et al., 2000; Thannkikal et al., 2000). 2.2.2. Features of orexin innervation within mammalian brain

Although cell bodies of orexin neurons are exclusively localized to the LHA, these neurons innervate regions throughout the entire brain and spinal cord (Fig. 3) (Peyron et al., 1998; Date et al., 1999; Nambu et al., 1999; Van den Pol, 1999). Orexin-immunoreactive nerve terminals are observed throughout the hypothalamus, including the arcuate nucleus and paraventricular hypothalamic nucleus, regions implicated in the regulation of feeding behavior. Strong staining of orexin-immunoreactive varicose terminals is also observed outside the hypothalamus, including the cerebral cortex, medial groups of the thalamus, limbic system (hippocampus, amygdala, and indusium griseum), and brainstem (raphe nuclei, locus coeruleus and septal regions) (Fig. 3). Thus, the orexin system may provide a link between the hypothalamus and the cerebral cortex, limbic system and key monoaminergic systems. The projections of orexin-producing neurons suggest that orexins play an important role in cognitive, emotional, and motivational aspects of brain function. 2.2.3. Neuroanatomical colocalization with other factors

The majority of orexin neurons possess leptin receptors and are immunoreactive for STAT3, a transcription factor activated by leptin (Hakansson et al., 1998; Horvath et al., 1999). Isolated orexin neurons are galanin-immunoreactive (Hakansson et al., 1998). An antiserum raised against ovine prolactin was shown to stain orexin neurons in the rat hypothalamus (Risold et al., 1999). As ovine prolactin-like immunoreactivity co-localized with dynorphin B (Griffond et al., 1993) and bradykinin (Griffond et al., 1994), these latter substances might also colocalize with orexin. Recently, Chou et al. (2001) clearly showed that nearly all neurons expressing prepro-orexin mRNA also expressed prodynorphin mRNA. We recently found that angiotensin II is also co-localized in orexin neurons (Nambu et al., unpublished observation). 2.2.4. Neuronal and humoral input to orexin neurons

Orexin neurons in the LHA have been shown to receive terminal appositions from neuropeptide Y (NPY)-, Agouti-related peptide (AgRP)-, and ct-melanocyte-stimulating hormone (0t-MSH)-immunoreactive fibers (Broberger et al., 1998; Elias et al., 1998). The innervation of orexin neurons by these peptidergic fibers corresponding to leptin-responsive cell types that reside in the arcuate nucleus may have a role in linking peripheral metabolic cues to autonomic regulatory sites and the cerebral cortical mantle. Moreover, about 50% of orexin 249

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T. Sakurai et al.

neurons express the leptin receptor, suggesting that orexin neurons themselves might be regulated by plasma leptin levels (Hakansson et al., 1998; Horvath et al., 1999). Almost 30% of orexin neurons are activated by insulin-induced hypoglycemia, suggesting that orexin neurons are also regulated by plasma glucose levels (Moriguchi et al., 1999). In accordance with the previous report that prepro-orexin mRNA is up-regulated by fasting (Sakurai et al., 1998), these observations suggest that orexin neurons are sensing the animal's nutritional state by monitoring plasma leptin and glucose levels. Indeed, our recent electrophysiological experiments using isolated orexin showed that leptin-induced hyperpolarization and cessation of action potentials (Yamanaka et al., submitted). 2.3. CENTRAL EFFECTS OF OREXINS IN MAMMALS

2.3.1. Feeding behavior The striking localization of orexin-containing neurons in the LHA and some of its adjacent areas suggests that orexins may be involved in the regulation of food intake. Therefore, the initial studies investigating the physiological role of the orexins focused mainly on feeding behavior. When administered intracerebroventricularly (i.c.v.) in the early light phase, orexinA stimulated food consumption in a dose-dependent manner within 1 h (Sakurai et al., 1998). The magnitude of stimulation with 3 and 30 nmol orexin-A at the 2 h time point was 6- and 10-fold, respectively. The effect persisted at 6 h; the amount of food consumed during the interval from 2 to 4 h post-injection was increased approximately 3-fold with either dose as compared to vehicle control. Human orexin-B also significantly augmented food intake; at the 2-h time point, 5- and 12-fold stimulation of food consumption was observed with 3 and 30 nmol orexin-B, respectively, as compared with vehicle control. The effect of orexin-B did not last as long as that of orexin-A; there was little stimulation of food intake after 2 h even with the higher dose (Sakurai et al., 1998). Chronic administration of orexin-A (0.5 nmol/h) for 7 days resulted in a significant increase in food intake in the daytime, which increased to 180% of the control value (Yamanaka et al., 1999). However, it resulted in a slight decrease of nighttime food intake. Thus, the total food intake per day was the almost same as that of vehicle-administered rats. The gain of body weight and blood glucose, total cholesterol and free fatty acid levels remained normal. Thus, chronic orexin-A treatment did not cause obesity in rats. These observations suggest that continuous administration of orexin-A could not overcome the regulation of energy homeostasis and body weight. Orexins might be implicated in short-term, immediate regulation of feeding behavior rather than long-term regulation of energy balance. The orexin-A-induced increase in food intake was partly inhibited by prior administration of BIBO3340, a NPY-Y1 receptor antagonist, in a dose-dependent manner (Yamanaka et al., 2000). However, BIBO3304 did not completely abolish the effect of orexin-A. These observations suggest that orexin-A elicits feeding behavior partially via the NPY pathway. The NPY system could be the one of downstream pathways by which orexin-A induces feeding behavior. Another pathway may also be involved in orexin-A-induced feeding behavior, because BIBO3304 did not completely abolish orexin-A-induced feeding behavior (Yamanaka et al., 2000).

2.3.2. Behavioral studies In behavioral studies, orexin-A-induced stereotypy and hyperlocomotion when administered centrally in rats (Ida et al., 1999; Nakamura et al., 2000). Orexin-A-induced grooming behav250

Orexin receptors

Ch. V

ior and hyperlocomotion were inhibited by dopamine 1 receptor or dopamine 2 receptor antagonists (Nakamura et al., 2000). On the other hand, in double-label immunohistochemical study of rat brain, tyrosine hydroxylase (TH)-immunoreactive cells in the ventral tegmental area (VTA) received innervation from orexin-immunoreactive fibers. Moreover, orexin-A induced an increase in [Ca2+]i in isolated A10 dopamine neurons in a dose-dependent manner (Nakamura et al., 2000). These results suggest that the orexin system interacts with the dopaminergic system in the VTA, which has been thought to be implicated in the reward system. Since orexin neurons also densly innervate the serotonergic, noradrenergic systems and histaminergic system (Chemelli et al., 1999; Yamanaka et al., 2001), these pathways might also have some roles in orexin-induced behavioral responses. These pathways might be involved in emotional aspects of orexin-induced biological effects.

2.3.3. Water intake Orexins increased water intake when administered intracerebroventricularly to rats (Kunii et al., 1999). The effect of orexin-A was more potent as compared with orexin-B, suggesting the possible involvement of OX1R. The efficacy of orexin-A was comparable with that of angiotensin II, and the effect lasted more than 3 h. Prepro-orexin mRNA level was upregulated when rats were deprived of water. Some orexin neurons were found in the zona incerta, a region implicated in water intake, and orexin-immunoreactive fibers were observed in the subfornical organ and area postrema, regions implicated in drinking behavior. These observations suggest a role for orexins as mediators that regulate drinking behavior (Kunii et al., 1999).

2.3.4. Regulation of vigilance state and sleep process Dysfunction of the orexin system result in the sleep disorder nacolepsy (Chemelli et al., 1999; Lin et al., 1999; Nishino et al., 2000; Peyron et al., 2000; Thannkikal et al., 2000), which is a disabling sleep disorder characterized by excessive daytime sleepiness, cataplexy (a sudden weakening of muscles tone usually triggered by emotions) and an alteration in the expression of and entry into rapid eye movement (REM) sleep (Mignot et al., 1998). Positional cloning has identified orexin receptor-2 gene mutations as the cause of narcolepsy in a canine model (Lin et al., 1999). Moreover, mice with targeted deletion of the prepro-orexin gene demonstrated a phenotype strikingly similar to human narcolepsy (Chemelli et al., 1999). In contrast to monogenic canine and murine narcolepsy models, however, human narcolepsy is rarely familial and may result from undefined environmental factors acting on a susceptible genetic background (Mignot et al., 1998). Recently, it was shown that orexin-A was undetectable in cerebrospinal fluid of seven out of nine patients with narcolepsy, indicating that abnormal orexin neurotransmission also exists in human narcolepsy (Nishino et al., 2000). Since these patients do not carry mutations in the prepro-orexin gene, decreased orexin levels in these patients are thus not likely to be due to highly penetrant orexin gene mutations. Rather, degeneration of orexin neurons might produce narcolepsy in these patients. Indeed, Peyron et al. recently showed global loss of orexin/hypocretin neurons in the human brain in all cases of narcolepsy patients examined (Peyron et al., 2000). Thannikal et al. separately reported that post-mortem brains of human narcoleptics contain almost no orexin neurons. All these observations implicate orexin system in the sleep disorder narcolepsy and, potentially, in the regulation of normal sleep processes (Thannkikal et al., 2000).

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3. OREXIN RECEPTORS

3.1. STRUCTURES The actions of orexins are mediated via two GPCRs named the orexin-1 (OX1R) and orexin-2 receptor (OX2R) (Sakurai et al., 1998). Among various classes of G-protein-coupled receptors, OX1R is structurally more similar to certain neuropeptide receptors, most notably to the Y2 Neuropeptide Y (NPY) receptor (26% similarity), followed by the thyrotropinreleasing hormone (TRH) receptor, cholecystokinin type-A receptor and NK2 neurokinin receptor (25%, 23% and 20% similarity, respectively). The amino acid identity between the deduced full-length human OX1R and OX2R sequences is 64%. Thus, these receptors are much more similar to each other than they are to other GPCRs. Amino acid identities between the human and rat homologues of each of these receptors are 94% for OX1R and 95% for OX2R, indicating that both receptor genes are highly conserved between the species (Sakurai et al., 1998). 3.2. CHROMOSOMAL LOCALIZATION In radiation hybrid mapping, the MIT markers showing tightest linkage to the human OX1R and OX2R genes are the STS markers D1S195 and D1S443, and WI-5448 and CHLC.GATA74F07, respectively. The inferred cytogenetic locations between these markers are lp33 for OX1R, and 6 p l l - 6 q l l for OX2R (accurate cytogenetic locations are often difficult to interpret from radiation hybrid maps in which the gene lies near the centromere). 3.3. PHARMACOLOGY Competitive radioligand binding assays using CHO cells expressing OX1R (CHO/OX1R cells) suggested that orexin-A is a high-affinity agonist for OX1R. The concentration of cold orexin-A required to displace 50% of specific radioligand binding (ICs0) was 20 nM. Human orexin-B also acted as a specific ligand on CHO/OX1R cells (Fig. 4). However, human orexin-B has significantly lower affinity compared to human OX1R: the calculated ICs0 in competitive binding assay was 250 nM for human orexin-B, indicating two orders of magnitude lower affinity as compared with orexin-A. Binding experiments using CHO cells expressing the human OX2R cDNA (CHO/OX2R) demonstrated that OX2R is a high affinity receptor for human orexin-B with ICs0 of 20 nM. Orexin-A also had high affinity for this receptor with ICs0 of 20 nM, which is similar to the value for orexin-B, suggesting that OX2R is a non-selective receptor for both orexin-A and -B, while OX1R is significantly selective for orexin-A over orexin-B (Fig. 4). 3.4. SIGNALING Orexin-A induced a transient increase in [Ca2+]i in CHO/OX1R cells in a dose-dependent manner (Sakurai et al., 1998). This calcium mobilization is likely caused at least in part by the activation of the Gq class of heterotrimeric G-proteins. The calculated concentration of orexin-A required to induce half-maximum response (ECs0) from the results of experiments using FLIPR (Molecular Devices) was 0.06 nM (Asahi et al., 1999). Synthetic human orexinB also acted as a specific agonist on CHO/OX1R cells in a parallel set of experiments. In accordance with the results of binding experiment, human orexin-B has significantly lower 252

Orexin receptors

120

.~.

Ch. V

i

OX1R

OX2R 120

10X-A

I

OX-B

OX-A

~

100.

OX-B

100

< .=-

x

80

? u')

c,,I

60

60

"o t.:3 o 133

40

40

! 20

...........................................

10

9

8

20

7

Concentration (-log[M])

6

5

...........................................

10

9

8

7

6

5

Concentration (-log[M])

Fig. 4. Displacement of [125I-Tyr17]orexin-Abinding to cells expressing human OX1R (left) and OX2R (right) by

increasing concentrations of cold orexin-A and human orexin-B, determined in quadruplicate. Level of non-specific binding was approximately 20% of the binding in the absence of competitor. affinity for the human OX1R as compared with orexin-A: the ECs0 in the [Ca2+]i transient assay measured by FLIPR was 1.5 nM for human orexin-B, indicating two to three orders of magnitude lower affinities as compared with orexin-A. In accordance with the results of competitive binding study, [Ca2+]i transient doseresponse studies using stably transfected CHO cells expressing the human OX2R cDNA demonstrated that OX2R is a high affinity receptor for both orexins. ECs0 values 0.06 nM and 0.13 nM for orexin-A and -B respectively. Thus, OX2R is a non-selective receptor for both orexin-A and -B, while OX1R is selective for orexin-A. 3.5. LIGAND-RECEPTOR STRUCTURE-ACTIVITY RELATIONSHIPS Activities of synthetic orexin-B analogs in cells transfected with either OX1R or OX2R were examined to define the structural requirements for activity of orexins (Asahi et al., 1999). The ability of N- or C-terminally truncated analogs of orexin-B to increase cytoplasmic Ca 2+ levels in the cells showed that the absence of N-terminal residues had little or no effect on the biological activity and selectivity of both receptors. Truncation from the N-terminus to the middle part of orexin-B resulted in moderate loss of activity, in the order of peptide length (Table 1). In particular, deletion of the conserved sequence between orexin-A and -B caused a profound loss of biological activity, and the C-terminally truncated peptides were also inactive for both receptors. These results suggest that the consensus region between orexin-A and -B is important for the activity of both receptors. Substitution of each amino acid of the natural sequence of orexin-B by L-alanine revealed that the residues in the N-terminal region could be substituted by L-alanine without loss of activity of both receptors. However, substitution in the C-terminal region (especially at positions 24-28) decreased the activity, just as C-terminal truncation did (Table 2). Substitution of each amino acid of orexin-B by the corresponding D-amino acid also showed that the C-terminal region is highly important for the activity of orexin-B (Asahi et al., 1999). 253

TABLE 1 . Biological activities of N-truncated analogs of human orexin-B on OXlR or OX2R expressing CHO cells

2

P

Peptide

OXB OXB(2-28) OXB(3-28) OXB(4-2 8) OXB(5-28) OXB(G28) OXB(7-28) OXB(8-28) OXB(9-28) OXB(10-28) OXB(l1-28) OXB( 12-28) OXB( 13-28) OXB(14-28) OXB( 15-28) OXB( 1 6 2 8 ) OXB( 17-28) OXB( 18-28) OXB( 19-28) OXB(20-28) OXB(21-28)

Sequence

RSGPPGLQGRLQRLLQASGNHAAGILTM-amide SGPPGLQGRLQRLLQASGNHAAGILTM-amide GPPGLQGRLQRLLQASGNHAAGILTM-amide PPGLQGRLQRLLQASGNHAAGILTM-amide PGLQGRLQRLLQASGNHAAGILTM-amide GLQGRLQRLLQASGNHAAGILTM-amide LQGRLQRLLQASGNHAAGILTM-amide

QGRLQRLLQASGNHAAGILTM-amide GRLQRLLQASGNHAAGILTM-amide RLQRLLQASGNHAAGILTM-amide LQRLLQASGNHAAGILTM-aide QRLLQASGNHAAGILTM-amide RLLQASGNHAAGILTM-amide LLQASGNHAAGILTM-amide LQASGNHAAGILTM-amide QASGNHAAGILTM-amide ASGNHAAGILTM-amide SGNHAAGILTM-amide GNHAAGILTM-amide NHAAGILTM-amide HAAGILTM-amide

ECSO(nM)

PECSO(peptide) - PEGO(OXW

PEG0

OXlR

OX2R

OXlR

OX2R

1.428 3.850 2.650 3.400 3.900 4.500 15.500 26.000 29.500 280.000 2,050.000 7,850.000 11,400.000 41,000.000 66,000.000 > 100,000 > 100,000 > 100,000 > 100,000 > 100,000 > 100,000

0.131 0.220 0.210 0.265 0.335 0.790 1.155 1.400 1.720 6.850 29.000 43.000 76.000 300.000 745.000 1,303.333 710.000 1,833.333 1,333.333 1,466.667 37,666.667

8.845 8.415 8.577 8.469 8.409 8.347 7.810 7.585 7.530 6.553 5.688 5.105 4.943 4.387 4.180

9.884 9.658 9.678 9.577 9.475 9.102 8.937 8.854 8.764 8.164 7.538 7.367 7.119 6.523 6.128 5.885 6.149 5.737 5.875 5.834 4.424

-

Biological activities were determined as their ability to increase cytoplasmic Ca2+ levels in cells monitored by FLIPR.

OXlR

0x2r

-0.430 -0.268 -0.376 -0.436 -0.498 - 1.035 - 1.260 -1.315 -2.292 -3.157 -3.740 -3.902 -4.458 -4.665

-0.226 -0.206 -0.307 -0.409 -0.782 -0.947 -1.030 -1.120 - 1.720 -2.346 -2.517 -2.765 -3.361 -3.756 -3.999 -3.735 -4.147 -4.009 -4.050 -5.460

q

TABLE 2. Biological activities Peptide OXB IAla-OXB 2Ala-OXB 3Ala-OXB 4Ala-OXB 5Ala-OXB 6Ala-OXB 7Ala-OXB 8Ala-OXB 9Ala-OXB 1OAla-OXB 1 1Ala-OXB 12Ala-OXB I3Ala-OXB 14Ala-OXB 15Ala-OXB 16Ala-OXB 17Gly-OXB 18Ala-OXB 19Ala-OXB 20Ala-OXB 21Ala-OXB 22Gly-OXB 23Gly-OXB 24Ala-OXB 25Ala-OXB 26Ala-OXB 27Ala-OXB 28Ala-OXB

of

L-alanine-substituted analogs of human orexin-B on OXlR or OX2R expressing CHO cells

Sequence

RSGPPGLQGRLQRLLQASGNHAAGILTM-amide

ASGPPGLQGRLQRLLQ ASGNHAAGILTM-amide

RAGPPGLQGRLQRLLQASGNHAAGILTM-amide RSAPPGLQGRLQRLLQASGNHAAGILTM-amide RSGAPGLQGRLQRLLQASGNHAAGILTM-amide RSGPAGLQGRLQRLLQASGNHAAGILTM-amide RSGPPALQGRLQRLLQASGNHAAGILTM-amide RSGPPGAQGRLQRLLQASGNHAAGILTM-amide RSGPPGLAGRLQRLLQASGNHAAGILTM-amide RSGPPGLQARLQRLLQASGNHAAGILTM-amide RSGPPGLQGALQRLLQASGNHAAGILTM-amide

RSGPPGLQGRAQRLLQASGNHAAGILTM-amide RSGPPGLQGRLARLLQASGNHAAGILTM-amide RSGPPGLQGRLQ ALLQASGNHAAGILTM-amide

RSGPPGLQGRLQRALQASGNHAAGILTM-amide RSGPPGLQGRLQRLAQASGNHAAGILTM-amide RSGPPGLQGRLQRLLAASGNHA AGILTM-amide RSGPPGLQGRLQRLLQGSGNHAAGILTM-amide RSGPPGLQGRLQRLLQAAGNHAAGILTM-amide RSGPPGLQGRLQRLLQASANHAAGILTM-amide RSGPPGLQGRLQRLLQASGAHAAGILTM-amide RSGPPGLQGRLQRLLQASGNAAAGILTM-amide RSGPPGLQGRLQRLLQASGNHGAGILTM-amide RSGPPGLQGRLQRLLQASGNHAGGILTM-amide RSGPPGLQGRLQRLLQASGNHAAAILTM-amide RSGPPGLQGRLQRLLQASGNHAAGALTM-amide RSGPPGLQGRLQRLLQASGNHAAGIATM-amide RSGPPGLQGRLQRLLQASGNHAAGILAM-amide RSGPPGLQGRLQRLLQASGNHAAGILTA-amide

EC50 (nM)

0

PEGO

OXlR

OX2R

OXlR

OX2R

1.428 2.333 2.433 2.333 3.200 3.067 1.667 1.238 3.000 1.058 8.900 10.183 3.400 1.700 0.817 38.878 1.510 0.41 3 6.133 14.275 32.000 5.700 7.500 22.000 682.500 41666.667 790.000 140.333 263.333

0.131 0.195 0.220 0.183 0.227 0.290 0.117 0.127 0.463 0.197 0.773 0.094 0.230 0.134 0.066 2.186 0.127 0.091 1.563 1.090 2.303 0.7 13 0.273 0.827 286.000 390.000 150.750 10.900 16.200

8.845 8.632 8.614 8.632 8.495 8.513 8.778 8.907 8.523 8.976 8.05 1 7.992 8.469 8.770 9.088 7.410 8.821 9.384 8.212 7.845 7.495 8.244 8.125 7.658 6.166 4.380 6.102 6.853 6.579

9.884 9.710 9.658 9.737 9.645 9.538 9.933 9.897 9.334 9.706 9.112 10.027 9.638 9.873 10.180 8.660 9.895 10.043 8.806 8.963 8.638 9.147 9.563 9.083 6.544 6.409 6.822 7.963 7.790

~~

~~

__

Biological activities were determined as their ability to increase cytoplasmic Ca2+ levels in cells monitored by F'LIPR

~

pEC5~(peptide) - pEC5o (OXB)

$.

OXlR

OX2R

2 CI

-0.213 -0.231 -0.21 3 -0.350 -0.332 -0.067 0.062 -0.322 0.131 -0.794 -0.853 -0.376 -0.075 0.243 -1.435 -0.024 0.539 -0.633 - 1.000 -1.350 -0.601 -0.720 -1.187 -2.679 -4.465 -2.743 -1.992 -2.266

-0.174 -0.227 -0.148 -0.240 -0.347 0.049 0.013 -0.550 -0.178 -0.773 0.143 -0.246 -0.01 1 0.296 -1.224 0.01 1 0.158 -1.078 -0.922 -1.247 -0.738 -0.321 -0.802 -3.341 -3.475 -3.063 - 1.922 -2.094

20 rd

2

Ch. V

T. Sakurai et al.

TABLE 3. Distribution of OX1R and OX2R in the adult rat brain Region

OX1R-ir

OX1R mRNA

OX2R m R N A

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Telencephalon Olfactory system Anterior olfactory nucleus Piriform cortex Tenia tecta Neocortex Agranular insular cortex Neocortex layer 6 Neocortex layer 5 Neocortex layer 2 Claustrum Metacortex Cingulate/retrosplenial cortex Basal ganglia Caudate putamen Globus pallidus Substantia nigra, pars compacta Subthalamic nucleus Nucleus accumbens, rostral Hippocampal formation CA1 region CA2 region CA3 region Dentate gyrus Amygdala Amygdaloid nuclei Substantia innominata Septal and basal magnocellular nuclei Bed nucleus of the stria terminalis Lateral septal nucleus, dorsal part Medial septal nucleus Nucleus of the horizontal limb of the diagonal band Nucleus of the vertical limb of the diagonal band Thalamus Anteromedial thalamic nucleus, dorsal Centrolateral thalamic nucleus Centromedial thalamic nucleus Paracentral thalamic nucleus Paraventricular thalamic nucleus Reticular thalamic nucleus Zona incerta Lateral and medial geniculate nuclei Subthalamus Subthalamic nucleus Hypothalamic preoptic nuclei Anteroventral preoptic area Magnocellular preoptic area Medial preoptic nucleus Median preoptic nucleus Supraoptic nucleus Ventrolateral preoptic area Ventromedial preoptic area

256

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Orexin receptors

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OX 1R-ir

OX 1R mRNA

OX2R mRNA

Hypothalamus Anterior hypothalamic area Arcuate hypothalamic nucleus Dorsomedial hypothalamic nucleus Lateral hypothalamic area Magnocellular preoptic nucleus Medial mammillary nucleus Paraventricular hypothalamic nucleus Posterior hypothalamic area Premammillary nucleus Supraoptic nucleus Suprachiasmatic nucleus Ventromedial hypothalamic nucleus

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Mesencephalon Dorsal tegmental nucleus Inferior colliculus Interpeduncular nuclei Periaqueductal gray Principal sensory trigeminal nucleus Raphe nuclei Substantia nigra, pars compacta Superior colliculus

Rhombencephalon Facial nucleus Locus coeruleus Pontine reticular nucleus Spinal trigeminal nucleus

Cerebellum Cerebellar cortex Deep cerebellar nuclei

D

++

Spinal cord and dorsal root ganglia Spinal cord (gray matter; dorsal and ventral horn) DRG

+++ +++

Orexin'A(15-33), the C-terminal half of orexin-A, and orexin-B(10-28) have similar sequences, however, their selectivity to OX1R and OX2R is different (Asahi et al., 1999). This finding indicates that not only the activity but also the ligand/receptor selectivity is closely related to the C-terminal half of the orexin sequence.

4. DISTRIBUTION OF OREXIN RECEPTOR mRNA AND PROTEIN IN MAMMALIAN CENTRAL NERVOUS SYSTEM

4.1. OVERALL DISTRIBUTION OF OREXIN RECEPTOR mRNA IN RAT CENTRAL NERVOUS SYSTEM OX1R and OX2R exhibit marked differential distribution (Table 3). The pattern of OX1R and OX2R mRNA tend to be complementary. For instance, within the hypothalamus, a low level 257

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of OX1R mRNA expression is observed in the dorsomedial hypothalamus (DMH), while a higher level of OX2R mRNA expression is observed in this region. Other areas of OX2R prominence are the arcuate nucleus and paraventricular nucleus (PVN), lateral hypothalamic area, and most significantly, the tuberomammillary nucleus (Trivedi et al., 1998; Marcus et al., 2001). In these regions, there was little or no OX1R signal. OX1R mRNA is abundant in anterior hypothalamic area and VMH. Outside the hypothalamus, high levels of OX1R mRNA expression are also detected in the tenia tecta, hippocampal formation, dorsal raphe nucleus, and most prominently, the locus coeruleus. OX2R mRNA is mainly expressed in the cerebral cortex, nucleus accumbens, subthalamic nucleus (PVT), and paraventricular thalamic nuclei, anterior pretectal nucleus (Trivedi et al., 1998; Marcus et al., 2001).

4.2. DISTRIBUTION OF OREXIN RECEPTORS IN THE RAT CENTRAL NERVOUS SYSTEM Unfortunately, reliable information about the protein distribution of OXRs is currently not available in the literature, so we will mainly discuss the distribution of receptor mRNAs here. However, we will describe our recent observation regarding OX1R protein distribution. The description of nuclear and subnuclear patterns corresponds to the nomenclature of the rat brain of Paxinos and Watson (1998).

4.2.1. Telencephalon 4.2.1.1. Isocortex

Both OX1R and OX2R mRNAs were observed in the cerebral cortex (Trivedi et al., 1998; Marcus et al., 2001). A low density diffuse hybridization signal for OX1R mRNA was observed in the prelimbic, infralimbic, and dorsal peduncular cortices in layers 2, 5 and 6 (Marcus et al., 2001). OX2R mRNA was diffusely observed throughout all layers of the cerebral cortex. In addition, moderate to densely labeled cells were observed in layer 5, extending into layer 6 of the cortex. Immunohistochemical study of OX1R using anti-OX1R anti-serum demonstrated OX1Rlike immunoreactivity in many isocortical areas, including the primary and secondary motor areas, claustrum, primary and secondary somatosensory areas, gustatory area, anterior cingulate area, visceral area, agranular insular area (dorsal, ventral and posterior parts), retrosplenial cortex, posterior-parietal region association area, dorsal auditory area, primary auditory area, ventral auditory area, primary auditory area, anterolateral, primary, rostrolateral and anteromedial visual areas. Immunostaining was particularly concentrated in layers Ill/IV. Most stained cells resembled principal pyramidal neurons (Hervieu et al., unpublished result). 4.2.1.2. Olfactory cortex

In situ hybridization study showed that OX2R mRNA was abundant in the olfactory tubercle, while OX1R mRNA was not observed in this region. However, the olfactory tubercles were heavily immunostained with the OX1R antiserum (Hervieu et al., unpublished result). OX1Rlike immunoreactivity was also seen in the olfactory nuclei. Dense signals for OX1R-like 258

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immunoreactivity were also observed in the dorsal part of the tenia tecta and in the piriform cortex.

4.2.1.3. Hippocampal formation CA2 regions of the hippocampus, and amygdalohippocampal area displayed high levels of OX1R mRNA expression. CA1 field displayed hybridization only slightly above the background. A dense signal for OX2R mRNA was observed in the CA3 region and dentate gyrus layer (Trivedi et al., 1998; Marcus et al., 2001). A weak signal for OX2R mRNA was observed in the CA2 region. In the immunohistochemical study for OX1R, CA2 and CA3 were more densely labeled than CA1. OX1R-like immunoreactivity was mainly located in the stratum pyramidale in Ammon's horn. The granule cell layer of the dentate gyrus was lightly stained. Interneuronlike cells were immunostained in the hilus (Hervieu et al., unpublished result).

4.2.1.4. Amygdala The OX1R gene was relatively highly expressed in the amygdaloid regions, mRNA and protein signals were seen in the medial and basomedial nuclei (Trivedi et al., 1998; Hervieu et al., unpublished result). A weak diffuse signal for OX2R was observed in the anterior cortical nucleus and medial nucleus of the amygdala. The posterior cortical nucleus displayed a moderate signal for OX2R mRNA (Marcus et al., 2001).

4.2.1.5. Septal regions A high level of OX1R mRNA expression was observed in the tenia tecta, induseum griseum, septohipocampal nucleus and bed nucleus of the stria terminalis (Trivedi et al., 1998). Within the medial septal nucleus and nucleus of the diagonal band, small numbers of densely labeled cells were observed (Marcus et al., 2001). Immunohistochemical study demonstrated OX1R in the medial septal nucleus, and nucleus of the diagonal band of Broca as well (Hervieu et al., unpublished result). Moderately labeled cells for OX2R mRNA were observed in the medial septal nucleus, and diagonal band of Broca (Trivedi et al., 1998). Signals for OX2R mRNA were also found over neurons in the substantia innominata in a pattern consistent with the location of basal forebrain cholinergic neurons. Light diffuse labeling for OX2R mRNA was present in the lateral septum (Marcus et al., 2001). A moderately strong signal for OX2R mRNA was observed in the preoptic and medial divisions of the bed nucleus of the stria terminalis (Marcus et al., 2001).

4.2.1.6. Corpus striatum A weak signal for OX2R mRNA was observed in the globus pallidus, while no signal for OX1R mRNA was observed in this area. However, both lateral and medial segment of the globus pallidus were positive for OX1R-like immunohistochemistry (Hervieu et al., unpublished result). OX1R-like immunoreactivity was also observed in the caudate-putamen, substantia innominata and magnocellular preoptic nucleus.

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4.2.2. Diencephalon 4.2.2.1. Thalamus

The thalamus contained high levels of OXR signals. Both OX1R and OX2R mRNAs were observed in the paraventricular thalamic nucleus (PVT). OX1R mRNA, but not OX2R mRNA was observed in the ventral anterior thalamic nucleus. Moderately labeled cells were also found in the anteromedial thalamic nucleus and intergeniculate leaflet. A low density signal was also detected in other nuclei including interanteromedial and reuniens nuclei. On the other hand, OX2R mRNA, but not OX1R mRNA, was observed in the central medial thalamic nucleus (Trivedi et al., 1998). A dense signal for OX2R mRNA was observed in the rhomboid nucleus. OX1R-like immunoreactivity was detected in the reticular nucleus, PVT, paratenial nucleus, anterodorsal nucleus, anteroventral nucleus, lateral dorsal nucleus, ventral anterolateral complex, central medial nucleus, central lateral nucleus, paracentral nucleus, ventral posterior lateral and the medial nucleus. The subthalamic nucleus and zona incerta were also positive for OX1R-like immunoreactivity (Hervieu et al., unpublished result). Both the medial geniculate and lateral geniculate nucleus contained OX1R-like immunoreactivity (Hervieu et al., unpublished result). 4.2.2.2. Hypothalamus

Both orexin receptor subtypes were very abundantly expressed in the hypothalamus. A dense signal for OX1R mRNA was observed in the VMH, especially in the dorsomedial portion (Trivedi et al., 1998; Marcus et al., 2001). Moderately dense signals for OX1R mRNA were observed in the lateroanterior nucleus as in the DMH and posterior hypothalamus (PH) (Trivedi et al., 1998; Marcus et al., 2001). Marcus et al. reported that weak hybridization signals for OX1R mRNA were spread diffusely across the LHA. Weak signals for OX1R mRNA were present in the supraoptic nucleus (SON) (Marcus et al., 2001). Moderately densely labeled cells were observed in the ventral premammillary nucleus, subthalamic nucleus, and zona incerta (Marcus et al., 2001). Although Trivedi et al. (1998) and Marcus et al. (2001) reported that signals for OX1R mRNA were not observed in the paraventricular nucleus (PVH), OX1R-like immunoreactivity was observed in this region (Hervieu et al., unpublished result) (Fig. 5). The suprachiasmatic nucleus, supraoptic nucleus, arcuate nucleus, VMH, DMH and overlying zona incerta were also heavily immunostained. In the PVH, immunostaining was seen in the lateral zone of the posterior magnocellular region, the dorsal parvocellular, medioparvocellular and the dorsal zone of the medial parvocellular region. More rostrally, labeling was recorded in

Fig. 5. Microscopic demonstration of OX1R immunoreactivity in hypothalamus. A dense population of OX1R receptor immunostained cells was detected in the supraoptic nucleus (SO in A), paraventricular nucleus (PVH in B), suprachiasmatic nuclei (SCH in C), arcuate nucleus (AN in D), ventromedial nucleus (VMH in E and E'), dorsomedial nucleus (DMH in F), perifornical area of the lateral hypothalamus (LHA in G) and tuberomammillary nucleus (TMv in H). In the paraventricular nucleus (PVH), staining was present in the lateral zone of the posterior magnocellular region (PVHpml), dorsal parvocellular, medio-parvocellular and the dorsal zone of the medial parvocellular region (resp. PVHdp, PVHmpv and PVHmpd in B). In the ventromedial nucleus, all sub-regions were immunostained; i.e. the ventrolateral, dorsomedial and the central parts (vl, dm and c in E'). Calibration bars: A-C,F, 270 Ixm;D,E, 130 Ixm;E', 1000 Ixm; G,H: 540 Ixm.

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the PH as well as the dorsal part of the posterior mammillary, the medial mammillary and tuberomammillary nuclei (Fig. 5). The magnocellular preoptic nucleus contained many densely labeled cells for OX2R mRNA, as did the subparaventricular zone. Signals for OX2R mRNA were also evident in the anteroventral periventricular nucleus. Very dense signals for OX2R mRNA were observed in the PVH and tuberomammillary nucleus. Moderate signals for OX2R mRNA were observed in the arcuate nucleus and VMH. Light signals for OX2R mRNA were observed in the DMH as was LHA and PH (Trivedi et al., 1998; Marcus et al., 2001). The strongest signals for OX2R mRNA were present in the tuberomammillary nucleus, which contained many cells displaying densely hybridized cells. A moderately dense signal was found in the ventral premammillary, dorsal premammillary and lateral mammillary nuclei (Marcus et al., 2001).

4.2.3. Mesencephalon and rhombencephalon (midbrain and hindbrain) The periaqueductal gray matter and substantia nigra pars compacta showed moderately dense signals for OX1R mRNA in a moderate number of cells. The pedunculopontine and laterodorsal tegmental nuclei contained many cells with moderate to highly dense labeling for OX1R mRNA. The ventral tegmental area also contained many cells with moderate to highly densely labeling for OX1R mRNA. Very dense signals for OX1R mRNA were observed in the locus coeruleus (Trivedi et al., 1998; Marcus et al., 2001), where the densest signals for orexin-positive nerve fibers were observed (Peyron et al., 1998; Nambu et al., 1999; Date et al., 1999). In addition, A4, A5, and A7 adrenergic cell groups also contained signals for OX1R mRNA, while, OX2R mRNA was not observed in these regions. The nucleus of the solitary tract displayed a weak signal for OX1R mRNA in an A2 cell pattern. Possible A1 or C1 cells in the ventral lateral medulla were also moderately labeled. A moderately dense signal for OX1R mRNA was also observed in the dorsal motor nucleus of the vagus. Moderately signals for OX1R mRNA were observed in both the dorsal and median raphe nuclei. Dense signals for OX2R mRNA were also observed in the dorsal raphe nucleus, and median raphe nucleus (Marcus et al., 2001). A moderate dense signal for OX2R mRNA was also observed in the pontine raphe nucleus. In immunohistochemical study, the locus coeruleus (LC) was densely stained with antiOX1R antiserum (Fig. 6). OX1R-like immunoreactivity was also detected in the periaqueductal gray, dorsal tegmental nucleus, pontine central gray, suprageniculate nucleus and interpeduncular nucleus. Weak staining for OX1R-like immunoreactivity was observed in the caudal pontine reticular nucleus and in the gigantocellular reticular nucleus (Hervieu et al., unpublished result). A moderately dense signal for OX2R mRNA was observed in the VTA and ventral periaqueductal gray. The midbrain reticular formation also displayed a moderate signal for OX2R mRNA. In the pontine nuclei, a weak diffuse signal for OX2R mRNA was observed. A moderate number of cells in the laterodorsal and pedunculopontine tegmental nuclei displayed moderate labeling for OX2R mRNA. Several areas in the pons and medulla showed expression of OX2R mRNA, including the trigeminal nuclei, ventral lateral medulla, and dorsal motor nucleus of the vagus. Less dense signals for OX2R mRNA were seen in the nucleus of the solitary tract, facial motor nucleus, hypoglossal nucleus, nucleus ambiguous, and in the external cuneate and gracile nuclei (Marcus et al., 2001). No signal for either receptor was seen in the cerebellar cortex, but the deep cerebellar 262

Orexin receptors

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Fig. 6. Microscopic demonstration of the OX1R immunoreactivityin locus coeruleus. Calibration bar: 270 Ixm.

nucleus, interpositus cerebellar nucleus, and medial dorsolateral cerebellar nucleus were moderately immunostained with anti-OX1R antibody (Hervieu et al., unpublished result).

4.2.4. Spinal cord There were dense signals for both mRNA and immunoreactivity for OX1R in the lumbar part of the spinal cord (Hervieu et al., unpublished result). All subdivisions of the gray matter (dorsal and ventral horns) were stained (ventromedial, dorsomedial, interrnediolateral, central, ventrolateral, dorsolateral and retrodorsolateral). Dense immunostaining was seen in the dorsal root ganglia, with two types of cells being labeled based on morphometric characteristics (large and small cells) (Hervieu et al., unpublished result).

5. COMPARISON OF OX1R AND OX2R DISTRIBUTION The patterns of OX1R and OX2R mRNA were largely distinct and complementary in several regions. For example, OX1R mRNA was most dense in the CA2 region of the hippocampus and less dense in the dentate gyrus. In contrast, OX2R mRNA was most dense in the CA3 region. The tenia tecta and induseum griseum displayed dense OX1R signal, and lower levels of OX2R mRNA. The hypothalamus also displayed contrasting areas of both receptors. While the level of OX1R mRNA expression in the DMH was low, OX2R expression in this region was high. 263

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Hierachies [Part Ii Of Ii]

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