Neuropeptides: Methods and Protocols (Methods in Molecular Biology, v789)

METHODS IN MOLECULAR BIOLOGY™ Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfi...

Author: Adalberto Merighi

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METHODS

IN

MOLECULAR BIOLOGY™

Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes: http://www.springer.com/series/7651

Neuropeptides Methods and Protocols Edited by

Adalberto Merighi Dipartimento di Morfofisiologia Veterinaria, Università degli Studi di Torino, Grugliasco, TO, Italy; Istituto Nazionale di Neuroscienze (INN), Università degli Studi di Torino, Grugliasco, TO, Italy

Editor Adalberto Merighi Dipartimento di Morfofisiologia Veterinaria Università degli Studi di Torino and Istituto Nazionale di Neuroscienze (INN) Università degli Studi di Torino Grugliasco, TO, Italy [email protected]

Please note that additional material for this book can be downloaded from http://extras.springer.com ISSN 1064-3745 e-ISSN 1940-6029 ISBN 978-1-61779-309-7 e-ISBN 978-1-61779-310-3 DOI 10.1007/978-1-61779-310-3 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2011936011 © Springer Science+Business Media, LLC 2011 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com)

Preface The term neuropeptide was originally coined to indicate small protein molecules that are contained in neurons. In the late 1970s and the 1980s of the last century, several tens of neuropeptides were localized by immunocytochemistry to discrete cell populations of the central and peripheral nervous system, and the concept of chemical neuroanatomy, originally developed by Tomas Hökfelt and coworkers, entered the scene of neurobiology. Since then, the field of neuropeptide biology has dramatically widened, and today the ultimate frontiers in neuropeptide research lie in the development of pharmacologically active compounds that are capable of crossing the blood–brain barrier to exert their biological role(s) in vivo and in the construction of genetic vectors to be employed in gene therapy. This book represents a readily reproducible collection of established and emerging techniques for neuropeptide research. Such a collection is preceded by a general introductory chapter (Chapter 1) that discusses a series of new concepts leading to a broader neuropeptide definition in light of the huge amount of data accumulated after more than half a century of neuropeptide research. The methods presented include immunocytochemical localization, biochemical characterization, functional analysis, development and production of genetic probes, and the design of neuropeptide derivatives for cellular neurobiology as well as the potential therapeutic applications. As a general indication to the readers, Chapters 2–10 are focused on a series of techniques for localization studies. They cover a broad range of protocols, such as the immunocytochemical detection of neuropeptides in nonmammalian vertebrates together with a detailed description of procedures for anesthesia and tissue preparations in these species (Chapter 2); the combined neuropeptide/receptor localization at the light and transmission electron microscope for connectivity studies (Chapter 3); the analysis of neuropeptide genes’ transcription by localization of pre-mRNA (Chapter 6) or mRNA/microRNA with in situ hybridization (Chapter 4), in situ PCR (Chapter 5), and laser capture/microdissection (Chapter 7); the visualization in vivo of neuropeptide secretion (Chapter 8) and translocation across the plasma membrane (Chapter 9); and the functional analysis of neuropeptide interactions in vitro with cells of the immune system (Chapter 10). Chapter 11 describes a series of electrophysiological protocols for functional studies in vitro and in vivo. Chapters 12–19 are devoted to biochemical/molecular biology techniques, ranging from radioimmunoassay (Chapter 12) to neuropeptidomics employing reverse-phase HPLC (Chapter 13) or mass spectrometry (Chapter 14), RNA analysis by suppression subtractive hybridization (Chapter 15), determination of neuropeptide release in vivo by microdialysis (Chapter 16) or antibody microprobes (Chapter 17), and measurement of neuropeptidases (Chapter 18) and neuropeptide autoantibody levels (Chapter 19) in biological fluids. Chapters 20–24 deal with a number of techniques developed to optimize neuropeptide administration to central neurons or to interfere with biological effects in vivo. These procedures include the intranasal delivery of neuropeptides (Chapter 20), the development of

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neuropeptide pro-drugs (Chapter 21), the use of phosphorothioate oligodeoxynucleotides that are capable of crossing the blood–brain barrier to knock down neuropeptides in the CNS (Chapter 22), the development of liposome-encapsulated neuropeptides for assessing the chronic actions of physiologically short-lived molecules (Chapter 23), the construction of recombinant adeno-associated viral vectors that can be used to locally or systemically enhance or silence neuropeptide gene expression (Chapter 24). Finally, Chapter 25 describes a calcium mobilization assay in mammalian cells to identify novel G-protein-coupled receptor family members that transduce the neuropeptide signals. All scientists who have excellently contributed to this book have a direct experience in one or more fields of neuropeptide research. I am very much indebted to all of them for their successful effort in emphasizing the description of the more common pitfalls in the techniques that they have described and of the hints to reduce the possibility of failure for beginners. The collection of protocols that forms this book is surely not exhaustive of the wide range of approaches that today can be employed in top level neuropeptide research. Yet it is intended for a large audience of scientists, including histologists, biochemists, cellular and molecular biologists, and electrophysiologists that are currently active in the field or are willing to enter such an exciting and still expanding area of neurobiology. Grugliasco, TO, Italy

Adalberto Merighi

Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1 What Are Neuropeptides? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J. Peter H. Burbach 2 Neuropeptide Localization in Nonmammalian Vertebrates . . . . . . . . . . . . . . . . . . . Paolo de Girolamo and Carla Lucini 3 Combined Light and Electron Microscopic Visualization of Neuropeptides and Their Receptors in Central Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chiara Salio, Laura Lossi, and Adalberto Merighi 4 Neuropeptide RNA Localization in Tissue Sections. . . . . . . . . . . . . . . . . . . . . . . . . Marc Landry, Shérine Abdel Salam, and Marie Moftah 5 Intron-Specific Neuropeptide Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Harold Gainer, Todd A. Ponzio, Chunmei Yue, and Makoto Kawasaki 6 Direct In Situ RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Laura Lossi, Graziana Gambino, Chiara Salio, and Adalberto Merighi 7 Laser Capture Microdissection and Quantitative-PCR Analysis . . . . . . . . . . . . . . . . Sarah J. Paulsen and Leif K. Larsen 8 Visualization of Peptide Secretory Vesicles in Living Nerve Cells . . . . . . . . . . . . . . . Joshua J. Park and Y. Peng Loh 9 Fluorescence Imaging with Single-Molecule Sensitivity and Fluorescence Correlation Spectroscopy of Cell-Penetrating Neuropeptides . . . . . . . . . . . . . . . . . Vladana Vukojevic´, Astrid Gräslund, and Georgy Bakalkin 10 Analysis of Neuroimmune Interactions by an In Vitro Coculture Approach . . . . . . . Tadahide Furuno and Mamoru Nakanishi 11 Electrophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Zhi-Qing David Xu 12 Localization of Neuropeptides by Radioimmunoassay . . . . . . . . . . . . . . . . . . . . . . . Fred Nyberg and Mathias Hallberg 13 Reversed-Phase HPLC and Hyphenated Analytical Strategies for Peptidomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anne-Marie Hesse, Sega Ndiaye, and Joelle Vinh 14 Neuropeptidomics: Mass Spectrometry-Based Qualitative and Quantitative Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ping Yin, Xiaowen Hou, Elena V. Romanova, and Jonathan V. Sweedler 15 Suppression Subtractive Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mohamed T. Ghorbel and David Murphy

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16 Neuropeptide Microdialysis in Free-Moving Animals . . . . . . . . . . . . . . . . . . . . . . . Tetsuya Kushikata and Kazuyoshi Hirota 17 Antibody Microprobes for Detecting Neuropeptide Release . . . . . . . . . . . . . . . . . . Rebecca J. Steagall, Carole A. Williams, and Arthur W. Duggan 18 Neuropeptidases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Manuel Ramírez, Isabel Prieto, Inmaculada Banegas, Ana B. Segarra, and Francisco Alba 19 Neuropeptide Autoantibodies Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sergueï O. Fetissov 20 Intranasal Delivery of Neuropeptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Michael C. Veronesi, Daniel J. Kubek, and Michael J. Kubek 21 Prodrug Design for Brain Delivery of Small- and Medium-Sized Neuropeptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Katalin Prokai-Tatrai and Laszlo Prokai 22 Measurement of Phosphorothioate Oligodeoxynucleotide Antisense Transport Across the Blood–Brain Barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . William A. Banks 23 Liposome-Encapsulated Neuropeptides for Site-Specific Microinjection . . . . . . . . . Frédéric Frézard, Robson A.S. dos Santos, and Marco A.P. Fontes 24 Recombinant Adeno-Associated Viral Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Marijke W.A. de Backer, Keith M. Garner, Mieneke C.M. Luijendijk, and Roger A.H. Adan 25 Deorphanizing G Protein-Coupled Receptors by a Calcium Mobilization Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Isabel Beets, Marleen Lindemans, Tom Janssen, and Peter Verleyen Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors SHÉRINE ABDEL SALAM s Department of Zoology, University of Alexandria, Alexandria, Egypt ROGER A.H. ADAN s Department of Neuroscience and Pharmacology, Rudolf Magnus Institute of Neuroscience, Utrecht University Medical Centre Utrecht, Utrecht, The Netherlands FRANCISCO ALBA s Department of Biochemistry and Molecular Biology III and Immunology, University of Granada Medical School, Granada, Spain MARIJKE W.A. DE BACKER s Department of Neuroscience and Pharmacology, Rudolf Magnus Institute of Neuroscience, Utrecht University Medical Centre Utrecht, Utrecht, The Netherlands GEORGY BAKALKIN s Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden INMACULADA BANEGAS s Unit of Physiology, Department of Health Sciences, University of Jaén, Jaén, Spain WILLIAM A. BANKS s Geriatrics Research, Education, and Clinical Center, Puget Sound Health Care System, Seattle, WA, USA; Division of Gerontology and Geriatric Medicine, Department of Internal Medicine, University of Washington, Seattle, WA, USA J. PETER H. BURBACH s Rudolf Magnus Institute of Neuroscience, Department of Neuroscience and Pharmacology, University Medical Center Utrecht, Utrecht, The Netherlands ISABEL BEETS s Research Group of Functional Genomics and Proteomics, K.U. Leuven, Leuven, Belgium ARTHUR W. DUGGAN s Department of Preclinical Sciences, Royal Dick School of Veterinary Medicine, Edinburgh University, Edinburgh, Scotland, UK SERGUEÏ O. FETISSOV s Digestive System and Nutrition Laboratory (ADEN EA4311), Rouen University, Rouen, France MARCO A.P. FONTES s Departamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil FRÉDÉRIC FRÉZARD s Departamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil TADAHIDE FURUNO s School of Pharmacy, Aichi Gakuin University, Nagoya, Japan HAROLD GAINER s Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA GRAZIANA GAMBINO s Dipartimento di Morfofisiologia Veterinaria, Università degli Studi di Torino, Grugliasco, TO, Italy

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KEITH M. GARNER s Department of Neuroscience and Pharmacology, Rudolf Magnus Institute of Neuroscience, Utrecht University Medical Centre Utrecht, Utrecht, The Netherlands MOHAMED T. GHORBEL s Bristol Heart Institute, University of Bristol, Bristol, UK PAOLO DE GIROLAMO s Department of Biological Structures, Functions and Technology, University of Naples Federico II, Naples, Italy ASTRID GRÄSLUND s Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden MATHIAS HALLBERG s Division of Biological Research on Drug Dependence, Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden ANNE-MARIE HESSE s Laboratory of Biological Mass Spectrometry and Proteomics (SMBP), CNRS USR3149, ESPCI ParisTech, Paris, France KAZUYOSHI HIROTA s Department of Anesthesiology, Hirosaki Graduate School of Medicine, Hirosaki, Japan XIAOWEN HOU s Center for Biophysics and Computational Biology, University of Illinois, Urbana, IL, USA; Beckman Institute, University of Illinois, Urbana, IL, USA TOM JANSSEN s Research Group of Functional Genomics and Proteomics, K.U. Leuven, Leuven, Belgium MAKOTO KAWASAKI s Department of Orthopaedics, School of Medicine, University of Occupational and Environmental Health, Kitakyushu-City, Fukuoka, Japan DANIEL J. KUBEK s Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN, USA MICHAEL J. KUBEK s Department of Anatomy and Cell Biology and Program in Medical Neuroscience, Indiana University School of Medicine, Indianapolis, IN, USA TETSUYA KUSHIKATA s Department of Anesthesiology, Hirosaki University Hospital, Hirosaki, Japan MARC LANDRY s INSERM U862, University of Bordeaux, Bordeaux, France LEIF K. LARSEN s Molecular Biology, Vipergen, Copenhagen, Denmark MARLEEN LINDEMANS s Research Group of Functional Genomics and Proteomics, K.U. Leuven, Leuven, Belgium LAURA LOSSI s Dipartimento di Morfofisiologia Veterinaria, Università degli Studi di Torino, Grugliasco, TO, Italy; Istituto Nazionale di Neuroscienze (INN), Università degli Studi di Torino, Grugliasco, TO, Italy CARLA LUCINI s Department of Biological Structures, Functions and Technology, University of Naples Federico II, Naples, Italy MIENEKE C.M. LUIJENDIJK s Department of Neuroscience and Pharmacology, Rudolf Magnus Institute of Neuroscience, Utrecht University Medical Centre Utrecht, Utrecht, The Netherlands ADALBERTO MERIGHI s Dipartimento di Morfofisiologia Veterinaria, Università degli Studi di Torino, Grugliasco, TO, Italy; Istituto Nazionale di Neuroscienze (INN), Università degli Studi di Torino, Grugliasco, TO, Italy MARIE MOFTAH s Department of Zoology, University of Alexandria, Alexandria, Egypt DAVID MURPHY s Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Bristol, UK MAMORU NAKANISHI s School of Pharmacy, Aichi Gakuin University, Nagoya, Japan

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SEGA NDIAYE s Laboratory of Biological Mass Spectrometry and Proteomics (SMBP), CNRS USR3149, ESPCI ParisTech, Paris, France FRED NYBERG s Division of Biological Research on Drug Dependence, Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden JOSHUA J. PARK s Neurosciences, University of Toledo College of Medicine, Toledo, OH, USA SARAH J. PAULSEN s Gubra, Hørsholm, Denmark Y. PENG LOH s Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA TODD A. PONZIO s Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA ISABEL PRIETO s Unit of Physiology, Department of Health Sciences, University of Jaén, Jaén, Spain LASZLO PROKAI s Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, TX, USA KATALIN PROKAI-TATRAI s Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, TX, USA MANUEL RAMÍREZ s Unit of Physiology, Department of Health Sciences, University of Jaén, Jaén, Spain ELENA V. ROMANOVA s Department of Chemistry, University of Illinois, Urbana, IL, USA; Beckman Institute, University of Illinois, Urbana, IL, USA CHIARA SALIO s Dipartimento di Morfofisiologia Veterinaria, Università degli Studi di Torino, Grugliasco, TO, Italy ROBSON A.S. DOS SANTOS s Departamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil ANA B. SEGARRA s Unit of Physiology, Department of Health Sciences, University of Jaén, Jaén, Spain JONATHAN V. SWEEDLER s Department of Chemistry, University of Illinois, Urbana, IL, USA; Beckman Institute, University of Illinois, Urbana, IL, USA REBECCA J. STEAGALL s Department of Physiology, Quillen College of Medicine, East Tennessee State University, Johnson City, TN PETER VERLEYEN s Research Group of Functional Genomics and Proteomics, K.U. Leuven, Leuven, Belgium MICHAEL C. VERONESI s Program in Medical Neuroscience, Indiana University School of Medicine, Indianapolis, IN, USA JOELLE VINH s Laboratory of Biological Mass Spectrometry and Proteomics (SMBP), CNRS USR3149, ESPCI ParisTech, Paris, France VLADANA VUKOJEVIC´ s Department of Clinical Neuroscience, Karolinska Institute, Stockholm, Sweden CAROLE A. WILLIAMS† s Department of Physiology, Quillen College of Medicine, East Tennessee State University, Johnson City, TN, USA ZHI-QING DAVID XU s Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden PING YIN s Department of Chemistry, University of Illinois, Urbana, IL, USA; Beckman Institute, University of Illinois, Urbana, IL, USA CHUNMEI YUE s Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

Chapter 1 What Are Neuropeptides? J. Peter H. Burbach Abstract We know neuropeptides now for over 40 years as chemical signals in the brain. The discovery of neuropeptides is founded on groundbreaking research in physiology, endocrinology, and biochemistry during the last century and has been built on three seminal notions: (1) peptide hormones are chemical signals in the endocrine system; (2) neurosecretion of peptides is a general principle in the nervous system; and (3) the nervous system is responsive to peptide signals. These historical lines have contributed to how neuropeptides can be defined today: “Neuropeptides are small proteinaceous substances produced and released by neurons through the regulated secretory route and acting on neural substrates.” Thus, neuropeptides are the most diverse class of signaling molecules in the brain engaged in many physiological functions. According to this definition almost 70 genes can be distinguished in the mammalian genome, encoding neuropeptide precursors and a multitude of bioactive neuropeptides. In addition, among cytokines, peptide hormones, and growth factors there are several subfamilies of peptides displaying most of the hallmarks of neuropeptides, for example neural chemokines, cerebellins, neurexophilins, and granins. All classical neuropeptides as well as putative neuropeptides from the latter families are presented as a resource. Key words: Cerebellins, Chemokines, Granins, Adipose peptides, Neuropeptide synthesis

1. Introduction Nerve cells communicate with each other by virtue of chemical signals that are released by one cell and received by another. All vertebrate and invertebrate species engage a multitude of signal molecules to meet the complexity of their nervous systems. The chemical nature of signal molecules ranges from simple gaseous molecules, such as NO2, to aminergic and fatty molecules, amino acids, and proteins. Each of them has its own biochemical pathways of synthesis and degradation, and cell biological characteristics allowing it to participate in chemical communication. Neuropeptides are by far the largest and most diverse class of signaling molecules in the brain. They can act as neurotransmitters

Adalberto Merighi (ed.), Neuropeptides: Methods and Protocols, Methods in Molecular Biology, vol. 789, DOI 10.1007/978-1-61779-310-3_1, © Springer Science+Business Media, LLC 2011

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directly, as modulators of ongoing neurotransmission by other transmitters, as autocrine or paracrine regulators in a close cellular environment, and as hormones on long range. The modes of action can be recognized in many physiological processes and functions in which neuropeptides participate. But what are neuropeptides precisely? When is a signal molecule a neuropeptide? There are no simple answers for all signal molecules. Here, an attempt is made toward criteria to define neuropeptides. These are based on chemical nature, pathways of biosynthesis, cell biological characteristics, and modes of action. We know neuropeptides now for over 40 years as a term, but the historical lines that lead to their recognition have contributed to these criteria. These lines are outlined below as a brief history. A comprehensive historical account has been eloquently presented by Klavdieva (1–4).

2. How Were Neuropeptides Discovered?

Neuropeptides were encountered before they were designated as such, even before chemical signaling was a precipitated concept in physiology. In ancient times, it was thought that organs of animals, including those of man, could affect the body and the mind when eaten. In the nineteenth century, when experimental life sciences developed, extracts of organs became systematically investigated for their effects on physiological systems. During these times the fundaments of endocrinology were laid. Substances such as secretin, insulin, vasopressin, and oxytocin were encountered as biological activities in crude extracts, detected by bioassays in whole animals or organ systems (5–7). In 1905, such substances were named “hormones,” a term introduced by the English physiologist Ernest Starling (1866–1927). Starling coined the term, derived from the Greek verb “ormao” (to arouse or excite), during a Croonian Lecture at the Royal College of Physicians in London on the Chemical Control of the Functions of the Body (8–12). He defined hormones in terms of chemical messengers produced recurrently to answer the physiological needs of the organism, and carried from the organ where they are produced to the organ which they affect by means of the bloodstream. Among hormonally active substances were molecules that we now call neuropeptides, such as vasopressin, oxytocin, and substance P (6, 7, 13). Only in the 1950s and beyond, the chemical identities of hormones were elucidated: almost all of them appeared to be short chains of amino acids, i.e., peptides (14). Large-scale purification of releasing factors from hypothalamic extracts at immense scale was undertaken, resulting in the elucidation of the structures of releasing factors in the late 1960s, and award of the Nobel Prize to Guillemin and Schally in 1977 (15). From the late 1950s onward, Victor Mutt

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(1923–1998) identified many new peptide hormones from gut organs, and later also from the brain based on chemical properties (16). Guided by biological activity, specifically opioid activity, John Hughes and Hans Kosterlitz (1903–1996) identified the enkephalins in 1975 (17). It appeared a seminal moment, bringing neuropeptides definitely from endocrinology into the neurosciences. While one historical line leading to neuropeptides is the conception of peptide hormones, the second is that of neurosecretion. Preceded by early studies of Carl Speidel in 1917 (18), Ernst Scharrer (1905–1965) proposed the concept of neurosecretion in 1934 (19). Early on, it was recognized by physiologists and histologists that nerve cells in the brain contained substances that were stored in the cell and could be secreted not into the blood stream, but internally in the nervous system. For example, by chemical staining of disulfide bridges, peptides could be detected in the hypothalamus and ventral brain with fibers to the brain separate from the posterior pituitary gland, the canonical site of release for peripheral hormonal actions (20). These peptides were vasopressin, oxytocin, and their neurophysins, later demonstrated by immunocytochemistry (21). Neurosecretion of peptidergic substances is now a fundamental aspect of neuropeptides (22). A third line in the history of neuropeptides is the discovery of biological actions of peptides on the nervous system, and particularly of cognitive actions in the brain. In parallel to the discovery of hormones, bioassays were instrumental in the discovery of neuropeptides. Bioassays for activity on the enteric nervous system existed already for a long time, but for the central nervous system this was new in the 1960s. David de Wied (1925–2004) pioneered on the activity of peptide hormones on rat behavior from the 1950s onward and discovered that ACTH, MSH, and vasopressin acted on the brain and affected learning and memory processes (23–25). In the 1970s he coined the term “neuropeptides” to designate neuroactive peptide hormones and fragments thereof. At that time receptors were still undiscovered mediators of the biological effects. With the development of radioligand binding assays and molecular technologies, receptors for all neuropeptides were found, now explaining the central effects. Since then, the term “neuropeptides” has been adopted and has marked an exciting field in the neurosciences.

3. Criteria for Neuropeptides The history of neuropeptides illuminates three scientific milestones (1) peptide hormones as regulators in the endocrine system, (2) neurosecretion of peptidergic substances, and (3) responsiveness of nerve cells to peptides. Along these lines, an answer to the question “What are neuropeptides?” is proposed here. The three historical

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lines leading to neuropeptides have not always been able to explain uncertainties like the following: “Are all peptide hormones neuropeptides?”, “Are all proteineous substances secreted by neurons neuropeptides?”, “Are all peptides with effects when injected in the brain, neuropeptides?” The basic question “What are neuropeptides?” requires a definition to pinpoint the essence of a neuropeptide. The hallmarks of neuropeptides are (1) gene expression and biosynthesis by neurons, (2) storage and regulated release upon demand, and (3) the ability to modulate or mediate neural functioning directly through neural receptors. These hallmarks are commented below. 3.1. Gene Expression and Biosynthesis by Neurons

Neuropeptides are used by neurons to signal to other cells. A priori gene expression and biosynthesis occur in neurons. For most “classical” neuropeptides, studies using in situ hybridization and immunocytochemistry have unambiguously shown that the transcript and peptide products are produced by neurons. In a few cases, biosynthesis of peptides could actually be demonstrated by incorporation of radioactive amino acids in pulse-chase experiments (26). An issue arises in cases where the peptide is produced in the nervous system by other cells than neurons. Formally, these are not neuropeptides, but are growth factors and cytokines produced by glial cells (see Subheading 4). Glial cells were believed to have only a constitutive secretory pathway, thus releasing intact or partly processed precursors in a nonregulated fashion. However, emerging data from astrocytes and glial cell lines show that these cells can also have a regulated secretory pathway (27, 28). Therefore, putative neuropeptides may be recognized in peptide families expressed by glial cells.

3.2. Regulated Release

Controlled secretion is a basis for regulation of chemical communication. Neurons expressing classical neuropeptides use the regulated secretory pathway, allowing storage of biosynthesized peptides in large dense-cored vesicles and controlled release upon a stimulus (29–31). The constitutive and regulated secretory pathways are schematically compared in Fig. 1. A signal peptide sequence is key for entry into secretion routes. Generally, this is a short 20–25 amino acid extension at the N-terminal of the precursor, the prepro-peptide. It is directly removed from the nascent precursor during protein synthesis and thus never found on the precursor or in the peptide pool. The regulated secretory route imposes specific structural characteristics to the neuropeptide precursor to be sorted away from the default constitutive route. These structural prerequisites for sorting are situated in the 3D structure of the precursor and have remained largely uncovered (32). Furthermore, the proteolytic processing of the precursor into active peptides is directed by short motifs of basic amino acids. Thus, neuropeptide precursors contain salient structural details, illustrated in Fig. 1.


5

Fig. 1. Secretory pathways and the biosynthesis of neuropeptides. The flow of newly biosynthesized secretory proteins through the cell is schematically depicted with emphasis on the subcompartments that are passed and the processing steps that occur. The default pathway is the constitutive pathway with small clear-cored secretory vesicles that immediately fuse for secretion. Neuropeptides are generally released through the constitutive pathway. Neuropeptide precursors are sorted in the trans-Golgi network into vesicles of the secretory pathway. These vesicles acidify, condense the protein content, and activate proteolytic enzymes resulting in processing of the precursor protein into neuropeptides. Mature granules (dense-cored vesicles) are stored and await release upon a stimulus. Posttranslational modifications and their cellular sites are indicated. The figure was made available by Gerard Martens, Nijmegen.

1. The signal peptide. This 20–25 amino-acid long N-terminal portion is required for the entry of the newly synthesized gene product into the lumen of the endoplasmic reticulum (ER). It is cleaved off during passage of the ER membrane, leaving the proneuropeptide in the ER-Golgi for further sorting into the regulated secretory pathway. 2. The basic motifs. Pairs of the basic amino acids lysine (Lys, K) and arginine (Arg, R), or, more rarely, a single Arg in the appropriate structural environment of the precursor serve as recognition sites and substrates of prohormone convertases (PCs). These proteolytic enzymes belong to a small family of subtilisin proteases with cell specific expression. Cleavage of the neuropeptide precursor leads to the generation of specific peptides, and cell-specific differences in PCs lead to different sets of peptides from the same precursor.

6

J.P.H. Burbach

3. C-terminal amidation. PC-generated peptides can be subject to further modification by peptidyl-aminotransferase (PAM). This enzyme uses a C-terminal glycine (Gly, G) as amide donor for the preceding amino acid. This results in a peptide with an amidated C-terminus. This amide occurs in many neuropeptides and is often essential for biological activity. This structural characteristic has been elegantly exploited by Mutt to isolated novel neuropeptides and peptide hormones on the bases of a chemical assay for C-terminal amides, such as neuropeptide Y, peptide YY, and peptide HI (all named after the C-terminal amidated amino acid) (16). 4. Post-translational modifications. Chemical modifications include N- and O-glycosylation, phosphorylation, sulfation, and acetylation. These are found on stored and secreted peptides and can contribute to biological properties. 3.3. Modulation of Neural Functions and Receptors

Neuropeptides have biological activities in neural systems that can be observed at all levels of neural functioning: effects of neuropeptides have been established at the genetic, biochemical, cellular, behavioral, and organismal levels. In this respect, the biological activities of many neuropeptides have been indicated as “pleiotropic,” i.e., acting on a variety of cell types and tissues. Such effects are not always distinguishable from that of biologically active peptides indicated as growth factors or cytokines. Typical for the cellular action of neuropeptides are the relatively slow responses and actions as compared to “fast neurotransmitters” such as excitatory amino acids and amines. Neuropeptides often coexist with amino acid and amine neurotransmitters in nerve terminals, but are released only after intense or prolonged stimulation (22). This delayed response of neuropeptide secretion is due to storage in dense-cored vesicles that are not docked at the cellular release site, e.g., the synapse or synaptic button, requiring recruitment first, unlike the “fast neurotransmitters,” which are ready for release. This happens at elevated Ca2+ levels which require enhanced or repeated stimulation. The relatively slow response to released neuropeptides is due to the type of receptor that almost all neuropeptides act on: G-protein coupled receptors (GPCRs). These receptors trigger an intracellular cascade of molecular enzymatic events that result in cellular responses. The time span over which such response is in effect (seconds or longer) is considerably longer than that of neurotransmitters that modulate ion fluxes directly through action on ion channels (milliseconds). Databases of neuropeptide receptors are available: http:// www.gpcr.org/7tm/ and http://www.iuphar-db.org. There are more neuropeptides known than there are receptors. Several peptides may still have unknown receptors. Moreover, receptors are often responsive to multiple related neuropeptides.


7

4. Neuropeptides Defined Based on the above considerations, a strict definition of a neuropeptide would be as follows: “A neuropeptide is a small proteinaceous substance produced and released by neurons through the regulated secretory route and acting on neural substrates.” About 70 genes encoding for such neuropeptides have been recognized. These can be considered as “classical neuropeptides” and are listed in Table 1. The genes encoding these classical neuropeptides can be grouped in 18 subfamilies according to precursor and peptide structure or according to function. Examples of the former are the CRH-related gene family and the glucagon/secretin gene family; the latter is illustrated by the opioid gene family. Apart from these, a group of miscellaneous peptide genes still remains (Table 1). Cerebellins are complicated with respect to fitting such a definition (Table 2). But how about peptides that are known as peptide hormones of the endocrine system, chemokines of the immune system, or growth factors, if they are also synthesized and active in the nervous system? To appreciate this issue, these classes of proteinaceous signaling molecules are briefly addressed below, in view of the characteristics of neuropeptides. 4.1. Peptide Hormones

A hormone is a substance secreted by one organ and acting on another after transport by the bloodstream, as already proposed by Starling in 1904 (8–12). In this way, cells at different locations in the organism communicate in an endocrine fashion. Many peptide hormones are also synthesized by neurons and thus belong no disputably to the classical neuropeptides (see Table 1). Reversely, many classical neuropeptides are also synthesized by endocrine glands and function peripherally as hormones. However, this definition excludes hormones as being neuropeptides if they are not synthesized by neurons, even if they signal to the brain. Leptin and insulin are examples, and there is an emerging class of regulatory peptides originally found in adipose tissues (Table 3). Sometimes the relationships between the nervous system and peripheral endocrine glands are complicated by alternative processing and splicing. One example of alternative processing is the peptides derived from POMC. ACTH and b-lipotropin are the prime peptide hormones derived from POMC in the periphery, while a-MSH and b-endorphin are the major POMC neuropeptides in the brain. This is due to differences in processing between adrenocorticotropes of the anterior pituitary gland, secreting ACTH for peripheral action, and hypothalamic POMC neurons, secreting a-MSH and b-endorphin from neuronal processes (33). The different sets of biologically active POMC products are accommodated by differences between peripheral and central melanocortin receptors. Furthermore, differential expression of two paralogous

2q12.2

20p13

Pro-dynorphin gene (PDYN)

8q12.1

Chromosomal localization

Pro-opiomelanocortin gene (POMC)

Opioid gene family Pro-enkephalin gene (PENK)

Gene (gene symbol)

Gene expression

Table 1 Neuropeptide gene families: classical neuropeptides

Prepro-dynorphin

POMC

Prepro-enkephalin

Precursor

Dynorphin A, dynorphin B, a-neoendorphin, b-neo-endorphin, dynorphin-32, leu-morphin

a-Melanocyte-stimulating hormone (a-MSH), g-melanocyte-stimulating hormone (g-MSH), b-melanocytestimulating hormone (b-MSH), adrenocorticotropic hormone (ACTH), b-endorphin, a-endorphin, g-endorphin, b-lipoprotein (b-LPH), g-lipoprotein (g-LPH), corticotropinlike intermediate peptide (CLIP)

Leu-enkephalin, Met-enkephalin, amidorphin, adrenorphin, peptide B, peptide E, peptide F, BAM22P

Active peptide(s)

8 J.P.H. Burbach

8p21.1


20p13

17q21.2

Oxytocin gene (OXT)

CCK/gastrin gene family Gastrin gene (GAST)

Vasopressin/oxytocin gene family Vasopressin gene (AVP) 20p13

Orphanin gene, prepro-nociceptin gene (PNOC)

Gene (gene symbol)

Gene expression

Prepro-gastrin

Prepro-oxytocinneurophysin I

Prepro-vasopressinneurophysin II

Prepro-nociceptin, Prepro-orphanin

Precursor

(continued)

Gastrin-34, gastrin-17, gastrin-4

Oxytocin (OT), neurophysin I (NP 1)

Vasopressin (VP), neurophysin II (NP II), C-terminal glycopeptide CPP

Nociceptin (orphanin FQ), neuropeptide 1, neuropeptide 2

Active peptide(s)

1 What Are Neuropeptides? 9

3q27.3

1p36.22

Cortistatin gene (CST)

3p22.1


Somastostatin gene family Somastostatin gene (SST)

Cholecystokinin gene (CCK)

Gene (gene symbol)

Table 1 (continued)

Gene expression

SS-12, SS-14, SS-28, antrin

cortistatin-29, cortistatin-17

Prepro-cortistatin

CCK-8, CCK-33, CCK-58

Active peptide(s)

Prepro-somatostatin

Prepro-CCK

Precursor

10 J.P.H. Burbach


12q13.13

7p15.3

17q21.31

17q21.31

Neuropeptide FF gene (NPFF)

Neuropeptide Y gene (NPY)

Pancreatic polypeptide gene (PPY)

Peptide YY gene (PYY)

F- and Y-amide gene family 7p15.2 Gonadotropin inhibitory hormone gene, RF-amide related peptide gene (RFRP)

Gene (gene symbol)

Gene expression

PPY

PYY, PYY-(3-36)

Prepro-PYY

(continued)

NPY, C-flanking peptide CPON

Neuropeptide FF, neuropeptide AF, neuropeptide SF

QRF-amide (neuropeptide RF-amide, GnIH, p518, RF-related peptide-2), RF-related peptide-1, RF-related peptide-3, neuropeptide VF

Active peptide(s)

Prepro-PPY

Prepro-NPY

Prepro-NPFF

Prepro-NPRF

Precursor



11p15.2

12p12.1

Islet amyloid polypeptide gene (IAPP), Amylin gene

11p15.2

Calcitonin II gene (CALCB)

Calcitonin gene family Calcitonin I gene (CALCA)

Prolactin-releasing peptide 2q37.3 (PRLH)

Gene (gene symbol)

Table 1 (continued)

Not available

Gene expression

Prepro-IAPP

IAPP (amylin, amyloid polypeptide)

Calcitonin gene related peptide I (a-CGRP) Calcitonin gene related peptide II (b-CGRP)

Prepro-CGRP-alpha Prepro-CGRP-beta

Calcitonin, katacalcin

PrRP-31, PrRP-20

Active peptide(s)

Prepro-CALC

Prepro-PrRP

Precursor

12 J.P.H. Burbach

22q13.33

Adrenomedullin-2 gene (ADM2)

1p36.22

Natriuretic peptide precur- 2q37.1 sor C gene (NPPC)

Brain natriuretic factor gene (NPPB)

Natriuretic factor gene family Atrial natriuretic factor 1p36.22 gene (NPPA)

11p15.4


Adrenomedullin gene (ADM)

Gene (gene symbol)

Gene expression

Prepro-CNP

Prepro-BNP

(continued)

C-type natriuretic peptide (CNP-23), CNP-29, CNP-53

Brain natriuretic factor (natriuretic peptide B, BNF, BNP)

Atrial natriuretic factor (natriuretic peptide A, ANF, ANP, natriodilatine, cardiodilatine-related peptide)

Adrenomedullin-2, intermedin-long (IMDL), intermedin-short (IMDS)

Prepro-adrenomedullin-2

Prepro-ANP

Adrenomedullin, AM, PAMP

Active peptide(s)

Prepro-adrenomedullin

Precursor



15q25.2-q25.3

Endothelin gene family Endothelin 1 gene (EDN1) 6p24.1

Neuromedin B gene (NMB)

Bombesin-like peptide gene family Gastrin-releasing peptide 18q21.32 gene (GRP)

Gene (gene symbol)

Table 1 (continued) Gene expression

Prepro-endothelin 1 (PPET1)

Prepro-neuromedin B2

Prepro-neuromedin B1

Prepro-GRP-3

Prepro-GRP-2

Prero-GRP-1

Precursor

Endothelin 1 (ET-1)

Neuromedin B (Ranatensin-like peptide, RLP) Neuromedin B (Ranatensin-like peptide, RLP)

GRP-27, GRP-14, GRP-10 (neuromedin C) GRP-27, GRP-14, GRP-10 (neuromedin C) GRP-27, GRP-14, GRP-10 (neuromedin C)

Active peptide(s)

14 J.P.H. Burbach

6q25.2

Vasoactive intestinal peptide gene (VIP)

Prepro-VIP-1 Prepro-VIP-2

Prepro-secretin

11p15.5

Secretin gene (SCT)

Endothelin 3 (ET-3)

Prepro-endothelin 3 (PPET3)

(continued)

VIP, PHM-27/PHI-27, PHV-42 VIP, PHM-27/PHI-27, PHV-42

Secretin

Glicentin; Glicentin-related polypeptide (GRPP); Oxyntomodulin (OXY) (OXM); Glucagon; Glucagon-like peptide 1 (GLP-1); Glucagon-like peptide 1 (7-37) (GLP-1 (7-37)); Glucagon-like peptide 1 (7-36) (GLP-1 (7-36)); Glucagon-like peptide 2 (GLP-2)

Endothelin 2 (ET-2)

Active peptide(s)

Prepro-endothelin 2 (PPET2)2

Precursor

Prepro-glucagon

20q13.32

Endothelin 3 gene (EDN3)

Gene expression

Glucagon/secretin gene family Glucagon gene (CGC) 2q24.2

1p34.2


Endothelin 2 gene (EDN2)

Gene (gene symbol)


CRH-related gene family Corticotropin-releasing hormone gene (CRH)

8q13.1

Prepro-CRH

CRH

GIP (gastric inhibitory peptide, glucosedependent insulinotropic polypeptide)

Prepro-GIP

Gastric inhibitory peptide gene (GIP)

17q21.32

GHRH (somatoliberin,GRF, somatocrinin, somatorelin, sermorelin)

Prepro-GHRH

Active peptide(s)

Growth hormone releasing 20q11.23 hormone gene (GHRH)

Precursor

PACAP-38, PACAP-27, PRP-48

Gene expression Prepro-PACAP


18p11.32

Pituitary adenyl cyclaseactivated peptide gene (ADCYAP1)

Gene (gene symbol)

Table 1 (continued)

16 J.P.H. Burbach

2p23.3

Urocortin gene (UCN)

10p15.1

1p36.23

3q28

Urocortin III gene (UCN3)

Urotensin-II (UTS2)

Urotensin-II domain containing (UTS2D)

Urocortin II gene (UCN2) 3p21.31


Gene (gene symbol)

Not available

Not available

Gene expression

Prepro-urotensin-2B

Prepro-urotensin-2, isoform a Prepro-urotensin-2, isoform b

Prepro-UNC III

Prepro-UNC II

Prepro-UNC I

Precursor

Urotensin-2-related peptide,urotensin-2B

Urotensin-2

Urotensin-2

UNC III, stresscopin

(continued)

UNC II, stresscopin-related peptide

UNC I

Active peptide(s)



Neuromedin U gene (NMU)

Neuromedins Neuromedin S gene (NMS)

Preprotachykinin B gene (TAC3)

4q12

2q11.2

12q13.3

Kinin and tensin gene family Preprotachykinin A gene 7q21.3 (TAC1)

Gene (gene symbol)

Table 1 (continued)

Not available

Gene expression

Neuromedin S Neuromedin U

Prepro-neuromedin U, multiple isoforms

Neuromedin K, neurokinin B Neuromedin K, neurokinin B

Substance P, neurokinin A (NKA, substance K, neuromedin L), neuropeptide K, neuropeptide gamma Substance P, neuropeptide K, neurokinin A Substance P, neurokinin A, neuropeptide g Substance P, neuropeptide K, neurokinin A

Active peptide(s)

Prepro-neuromedin S

PPTB, isoform 1 PPTB, isoform 2

d-PPTA

g-PPTA

b-PPTA

a-PPTA

Precursor

18 J.P.H. Burbach


1q42.2

12q21.31

6p21.31

3p25.3

Angiotensin gene (AGT)

Neurotensin gene (NTS)

Motilin family Motilin gene (MLN)

Ghrelin gene (GHRL)

Tensins and Kinins Kininogen-1 gene (KNG1) 3q27.3

Gene (gene symbol)

Not available

Gene expression

Neurotensin (NT), neuromedin N

Prepro-neurotensin

Prepro-ghrelin isoform 1 Prepro-ghrelin isoform 2 Prepro-ghrelin isoform 3, Pro-obestatin Prepro-ghrelin isoform 4, Pro-obestatin Prepro-ghrelin isoform 5

Obestatin

Obestatin

Ghrelin, obestatin Ghrelin, obestatin Obestatin

(continued)

Motilin, motilin-associated peptide Motilin, motillin-associated peptide

Angiotensin I, angiotensin II, angiotensin-(1–7)

Angiotensinogen preprotein

Prepro-motilin isoform 1 Prepro-motilin isofrom 2

Bradykinin, kallidin, LMW-K-kinin, HMW-K-kinin Bradykinin, kallidin, LMW-K-kinin, HMW-K-kinin

Active peptide(s)

Kininogen-1 precursor, isoform 1 Kininogen-1 precursor, isoform 2

Precursor


Gonadotropin-releasing 20p13 hormone gene (GnRH2)

GnRH family Gonadotropin-releasing 8p21.2 hormone gene (GnRH1)

19q13.42

11q13.2

Galanin family Galanin gene (GAL)

Galanin-like peptide precursor gene (GALP)


Gene (gene symbol)

Table 1 (continued)

Not in mouse

Not in mouse

Not in mouse

Gene expression

Prepro-GNRH2, Isoform-a Prepro-GNRH2, Isoform-b Prepro-GNRH2, Isoform-c

GnRH2 (LHRH II, gonadoliberin II)



GnRH (LHRH, gonadoliberin)

Galanin-like peptide (GALP)

Galanin-like peptide precursor

Prepro-GnRH1

Galanin, galanin message associated peptide (GMAP)

Active peptide(s)

Prepro-galanin

Precursor

20 J.P.H. Burbach

9p24.1

9p24.1

19p13.12

Relaxin-2 gene (RLN2)

Relaxin-3 gene (RLN3)

10q26.2

Neuropeptides S gene (NPS)

Insulin/relaxins Relaxin-1 gene (RLN1)

16p13.3

17q25.3


Neuropeptide W gene (NPW)

Neuropeptide B/W family Neuropeptide B gene (NPB)

Gene (gene symbol)

Not in mouse

Not available

Gene expression

Prepro-relaxin-3

Prepro-relaxin-2, isoform 1 Prepro-relaxin-2, Isoform 2

Prepro-relaxin-1

Relaxin-3

Relaxin-2

Relaxin-2

Relaxin-1

Neuropeptide S

(continued)

Neuropeptide W-23 (peptide L8), neuropeptide W-30

Prepro-neuropeptide W, PPL8

Prepro-neuropeptide S

Neuropeptide B-23 (peptide L7), neuropeptide B-29

Active peptide(s)

Prepro-neuropeptide B, PPL7

Precursor


Prepro-MCH

Prepro-hypocretin

Melanin-concentrating 12q23.2 hormone gene (PMCH)

Hypocretin gene (HCRT) 17q21.2

Hypocretin-1 (orexin A), hypocretin-2 (orexin B)

MCH, neuropeptide Glu-Ile (NEI), neuropeptide Gly-Glu (NGE)

PTHrP-(1-36), PTHrP-(38-94), PTHrP-(107-139) (osteostatin)

Active peptide(s)

Prepro-PTH-like hormone, isoform CRA_a Prepro-PTH-like hormone, isoform CRA_b

Precursor

Parathyroid hormone-like 12p11.22 hormone gene (PTHLH)

Gene expression TRH (thyroliberin)

3q21.3

Chromosomal localization Prepro-TRH

No-family neuropeptides Thyrotropin-releasing hormone gene (TRH)

Gene (gene symbol)

Table 1 (continued)

22 J.P.H. Burbach

16q22.1

6p22.3

Xq25

Agouti-related protein homolog gene (AGRP)

Prolactin (PRL)

Apelin gene (APLN)

Metastasis-suppressor KiSS 1q32.1 (KISS1)

5q13.2


Cocaine- and amphetamine-regulated transcript gene (CART)

Gene (gene symbol)

Gene expression CART-(1-39), CART-(42-89)

Active peptide(s)

Kiss-1

Prepro-apelin

Prolactin precursor

(continued)

Metastin (kisspeptin-54), (golgi transport 1 homolog A,golt1a), kisspeptin-14, kisspeptin-13, kisspeptin-10

Apelin-13, apelin-17, apelin-36 (APJ ligand, AGTRL1 ligand)

Prolactin

AGRP precursor isoform 1 AGRP Prepro-AGRP isoform 2 AGRP

Prepro-CART

Precursor


2q14.2

1p13.3

3p13

2q12.2

12p12.1

Prokineticin-1 (PROK1)


Augurin (C2orf40)

Spexin (C12orf39)


Diazepam-binding inhibitor (DBI)

Gene (gene symbol)

Table 1 (continued) Gene expression

Spexin precursor

Augurin precursor

Prokineticin-2 precursor isoform 1 Prokineticin-2 precursor isoform 2

Prokineticin-1 precursor

DBI isoform 1 DBI isoform 2 DBI isoform 3

Precursor

Spexin

Augurin (esophageal cancer related gene 4, ECRG-4)

Prokineticin-2 (PK2)

Prokineticin-2 (PK2)

Prokineticin-1, (PK1)endocrine glandderived VEGF (EGVEGF)

Diazepam-binding inhibitory peptide Diazepam-binding inhibitory peptide Diazepam-binding inhibitory peptide

Active peptide(s)

24 J.P.H. Burbach

13q13.3

3q28

Periostin (POSTN)

Osteocrin (OSTN)

Gene expression

Osteocrin precursor

Periostin precursor, isoform 1 Periostin precursor isoform 2 Periostin precursor isoform 3 Periostin precursor isoform 4

Precursor

Osteocrin (musclin)

Periostin (Osteoblast-specific factor 2 (OSF-2), fasciclin I-like) Periostin (Osteoblast-specific factor 2 (OSF-2), fasciclin I-like) Periostin (Osteoblast-specific factor 2 (OSF-2), fasciclin I-like) Periostin (Osteoblast-specific factor 2 (OSF-2), fasciclin I-like)

Active peptide(s)

Data have been obtained from http://www.neuropeptides.nl with permission and updated. Genes are abbreviated according to the official gene nomenclature (http://www. gene.ucl.ac.uk/nomenclature/). The localization on the human genome is given. neuropeptides are presented as the processed, biologically active products of the neuropeptide precursors (prepro-peptide) and the encoding gene. Brain expression data represent in situ hybridization data of the mouse brain obtained from the Allen Brain Atlas (http://www.brain-map.org). See http://extras.springer.com/ for the color version of this figure.


Gene (gene symbol)


26

J.P.H. Burbach

Table 2 Putative neuropeptides: cerebellins Gene (gene symbol)


Cerebellin-1 gene (CBLN1)

Precursor

Active peptide(s)

Mouse brain expression

16q12.1

Cerebellin-1 precursor

Cerebellin-1 (Cbln1)

Not available


18q22.3

Cerebellin-2precursor



14q12




20q13.2


Cerebellin-4 (Cbln4, cerebellin-like glycoprotein-1)

Putative neuropeptides are presented as the processed, biologically active products of the neuropeptide precursors (prepro-peptide) and the encoding gene. The cerebellin family members do not all fulfill the criteria of the neuropeptide definition. Particularly, regulated release has not been established for all members. Genes are abbreviated according to the official gene nomenclature. (http://www.gene.ucl.ac.uk/nomenclature/), and the localization on the human genome is given. Brain expression data represent in situ hybridization data of the mouse brain obtained from the Allen Brain Atlas (http://www.brain-map.org). Data have been obtained from http://www.neuropeptides.nl with permission and updated for this chapter. See http://extras.springer.com/ for the color version of this figure.

genes, and differential splicing of one gene transcript can result in a nervous-system-specific population of neuropeptides which is distinct from the related peripheral peptide hormones. Examples are found in the calcitonin/CGRP and tachykinin families of genes and peptides (34, 35). A number of peptide hormones have not been found to be expressed in the brain or have not been specifically investigated, such as the pituitary hormones, growth hormone, prolactin, LH, FSH, and others. Formally, they cannot be considered neuropeptides and have not been included in the tables. However, with ongoing examination of brain-expressed genes, several of these may still appear to be synthesized by certain neurons. Therefore, the list of classical neuropeptides may still grow. 4.2. Granins

Granins form a family of peptides that do not fully fulfill all criteria of neuropeptides (Table 4). Members of this family, chromogranins and secretogranins, display the structural features of neuropeptide

3q27.3

7q22.2

19p13.2

Visfatin gene (PBEF1)

Resistin gene (RETN)

7q32.1

Adipose neuroepeptides Leptin/ob gene (LEP)

Adiponectin gene (ADIPOQ)


Gene (gene symbol)

Resistin-delta2 precursor

Resistin precursor

Visfatin precursor

Adiponectin precursor

Prepro-leptin

Precursor

Resistin (Cysteine-rich secreted protein FIZZ3, Adipose tissue-specific secretory factor, Cysteine-rich secreted protein A12-alpha-like 2, ADSF, Xcp4) Resistin-delta2

Visfatin-1 (pre-B cell colony enhancing factor-1 (PBEF1), nicotinamide phosphoribosyltransferase)

Adiponectin (Acpr30, adipocyte complement related protein; adipocyte, C1Q and collagen domain containing)

Leptin (obesin)

Active peptide(s)

(continued)


Table 3 Putative neuropeptides: regulatory peptides from adipose tissues and structural relatives expressed in the brain


Not in human

3q13.13

Not in human

11p15.1

Resistin-like beta gene (RETLB)

Resistin-like gamma gene (RETLG)

Nucleobindin-2/NEFA gene (NUCB2)


Resistin-like alpha gene (RETLA)

Gene (gene symbol)

Table 3 (continued)

Nucleobindin-2

Resistin-like molecule gamma precursor (RELMgamma)

Resistin-like molecule beta precursor (RELMbeta)

Resistin-like molecule alpha precursor (RELMalpha)

Precursor

Nesfatin-1

Resistin-like molecule gamma (Cysteine-rich secreted protein FIZZ3, Xcp1)

Resistin-like molecule beta (Cysteine-rich secreted protein FIZZ2, Colon and small intestine-specific cysteine-rich protein, Cysteine-rich secreted protein A12-alphalike 1, Colon carcinoma-related gene protein, Xcp3)

Resistin-like molecule alpha (found in inflammatory zone 1; FIZZ1, hypoxiainduced mitogenic factor, Xcp2)

Active peptide(s)

not available


28 J.P.H. Burbach

19p13.2

1p36.12

3p13

Ubiquitin-like 5 (UBL5)



Prokineticin-2 isoform a precursor Prokineticin-2 isoform b precursor

Prokineticin-1 precursor

Beacon precursor

Precursor

Prokineticin-2 isoform b

Prokineticin-2 isoform a

Prokineticin-1 (endocrine gland-derived VEGF, EG-VEGF)

Beacon

Active peptide(s)


Data have been obtained from http://www.neuropeptides.nl with permission and updated. Genes are abbreviated according to the official gene nomenclature (http://www. gene.ucl.ac.uk/nomenclature/). The localization on the human genome is given. Putative neuropeptides are presented as the processed, biologically active products of the neuropeptide precursors (prepro-peptide) and the encoding gene. These regulatory peptides originally found in adipose tissues have brain expression or have structural relatives expressed in the brain. A role as neuropeptide has not been established for all criteria. Brain expression data represent in situ hybridization data of the mouse brain obtained from the Allen Brain Atlas (http://www.brain-map.org). See http://extras.springer.com/ for the color version of this figure.


Gene (gene symbol)


14q32.12

20p12.3

2q36.1

15q21.2

Chromogranin B gene (CHGB)

Secretogranin II gene (SCG2)

Secretogranin III gene (SCG3)


Chromogranin A gene (CHGA)

Gene (gene symbol)

Secretogranin III precursor

Secretogranin II precursor, chromogranin C precursor

Chromogranin B precursor variant

Chromogranin B precursor

Chromogranin A precursor

Precursor

Secretogranin III

Secretogranin II (chromogranin C), EM66, secretoneurin

Chromogranin B (secretogranin I), CCB peptide, GAWK peptide Chromogranin B (secretogranin I)

Chromogranin A, beta-granin, vasostatin

Active peptide(s)


Table 4 Putative neuropeptides: the granin family, neuropeptide-like proteins with ambiguous biological activities

30 J.P.H. Burbach

7q22.1

VGF nerve growth factor inducible protein (VGF)

VGF-precursor

Secretory granule neuroendocrine precursor

Precursor

VGF (NGF-inducible protein, neurosectretory protein), TLPQ-62, TLPQ-21, AQEE-30, LQEQ-19

Secretory granule neuroendocrine protein-1 (7B2, secretogranin 5)

Active peptide(s)


Neuropeptides are presented as the processed, biologically active products of the neuropeptide precursors (prepro-peptide) and the encoding gene. The granin family members do not all fulfill the criteria of the neuropeptide definition. Particularly, granins and derived peptides do not display unequivocal biological activities and receptors have not been defined. Genes are abbreviated according to the official gene nomenclature. (http://www.gene.ucl.ac.uk/nomenclature/). The localization on the human genome is given. Brain expression data represent in situ hybridization data of the mouse brain obtained from the Allen Brain Atlas (http://www.brain-map.org). Data have been obtained from http://www. neuropeptides.nl with permission and updated. See http://extras.springer.com/ for the color version of this figure.

15q13.3


Secretory granule neuroendocrine protein 1, 7B2 gene (SGNE1)

Gene (gene symbol)


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J.P.H. Burbach

precursors, and they are synthesized by neurons. Granins have been identified as peptides cosynthesized, costored, or coreleased with neuropeptides. Many reports on biological activities of some processed forms of granins exist, for instance for vasostatin, catestatin, pancreastatin, secretoneurin, EL35, EM66, and WE14 (36). However, a clear-cut biological function as signaling molecules as well as defined receptors lacks for these peptides. An alternative view on these peptides is that they are chaperones of the regulated secretory pathway, assisting neuropeptides with sorting and transport (37). 4.3. Chemokines

Chemokines, from “chemotactic cytokines,” are originally known as secreted factors that mediate leukocyte migration. They form a family of about 50 proteins of 8–14 kDa and are divided by structural similarities that classify them in the C, CC, CXC, and CXXXC subfamilies, according to the cysteine configuration (38). The nomenclature of individual chemokines follows this configuration, for example XCL1, CCL1, CXCL1, and CX3CL1, respectively. An online database is available at http://cytokine.medic.kumamotou.ac.jp/. There is growing evidence for neuronal expression of chemokines, for chemokine receptor expression by neurons, and for electrophysiological effects of chemokines on neurons (38–40). Neural activities of chemokines have been implicated in neuronal migration, neuroinflammation, and neuronal excitability. Chemokines, at least a number of them, may thus be considered as potential neuropeptides. The ones with proven neuronal origin have been listed in Table 5.

4.4. Growth Factors

Growth factors are proteinaceous signaling molecules that are produced and secreted by cells. They provide extracellular support to proliferation, differentiation and maintenance of other or the same cells in a paracrine or autocrine fashion, respectively. Many growth factors are expressed in the brain, where they act during embryonic development, and during neuronal adaptation and maintenance states of the adult nervous system. Growth factors come in many families, often with a large and diverse family tree. Examples are the neurotrophins, fibroblast growth factors (FGFs), Wnts, bone morphogenic factors (BMPs), epidermal growth factor (EGF), transforming growth factors alpha and beta (TGFs), and others. Although growth factors are expressed, synthesized, and released by neurons, they are generally not considered as neuropeptides, most growth factors are released by the constitutive secretory pathway and regulated at the level of gene expression. They act through receptor protein kinases. Still, in some cases growth factors may fulfill the criteria of neuropeptides. One example is BDNF, one of the five neurotrophins (41, 42). The primary gene product is prepro-BDNF, a precursor having a


33

Table 5 Putative neuropeptides: neurally expressed chemokine family members Chromosomal localization Precursor

Active peptide(s)

17q25.3

CCL2 precursor

CCL2

C–C motif chemokine 17q12 ligand 3 (CCL3)

CCL3 precursor

CCL3

C–C motif 17q12 chemokine ligand 4 (CCL4)

CCL4 precursor

CCL4

C–C motif 17q12 chemokine ligand 5 (CCL5)

CCL5 precursor

CCL5

C–C motif chemokine ligand 20 (CCL20)

CCL20 precursor isoform 1 CCL20 precursor isoform 2

CCL20

Gene (gene symbol) C–C motif chemokine ligand 2 (CCL2)

2q36.3

CCL 20

C–C motif chemokine 9p13.3 ligand 21 (CCL21)

CCL21 precursor

CCL21

CXC motif chemokine 10 (CXCL10)

CXCL10 precursor

CXCL10 (small inducible cytokine B10)

4q21.1


(continued)

34

J.P.H. Burbach

Table 5 (continued) Gene (gene symbol) CXC motif chemokine ligand 12 (CXCL12)

Chromosomal localization Precursor

Active peptide(s)

10q11.21

CXCL12 (stromal cell-derived factor 1, SDF-1a) Stromal cell-derived factor 1, SDF-1b Stromal cell-derived factor 1, SDF-1c Stromal cell-derived factor 1, SDF-1d

CXCL12 precursor, SDF-1 alpha SDF-1 beta

SDF-1 gamma

SDF-1 delta

CX3C motif 16q21 chemokine ligand 1 (CX3CL1)

Fractalkine precursor, CX3CL1 precursor


CX3CL1 (fractalkine)

Putative neuropeptides are presented as the processed, biologically active products of the neuropeptide precursors (prepro-peptide) and the encoding gene. The chemokine family members do not all fulfill the criteria of the neuropeptide definition. Particularly, expression by neurons and/or regulated release has not been established for all members. A database of all chemokines is available at dbCFC (http://cytokine.medic.kumamoto-u.ac.jp/) Genes are abbreviated according to the official gene nomenclature (http://www.gene.ucl.ac.uk/nomenclature/), and the localization on the human genome is given. Brain expression data represent in situ hybridization data of the mouse brain obtained from the Allen Brain Atlas (http://www.brain-map.org). Data have been obtained from http://www. neuropeptides.nl with permission and updated. See http://extras.springer.com/ for the color version of this figure.

signal peptide and cleavage sites for typical prohormone convertases. The BDNF gene is widely expressed in neurons, and BDNF is generated by processing and present in dense-cored vesicles, and is subject to stimulated release (43, 44). There is a separate role for pro-BDNF because it acts on a different receptor other than mature BDNF (45). Thus, BDNF fulfills all criteria to be a neuropeptide, albeit it is historically considered to be a growth factor. These properties may also appear for other neurotrophins, namely, NGF, NT-3, NT-4/5 (46), and even GDNF (28).


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5. Conclusion Neuropeptides have emerged as a distinct class of chemical signaling molecules in the 1960s and 1970s, mostly initiated by studies on the endocrine system and central nervous system effects. Over 60 genes exist in the mammalian genome, encoding classical neuropeptides. Classical neuropeptides fulfill the criteria of synthesis and regulated release by neurons and action on brain receptors. In a broader perspective, peptides usually considered as peptide hormones, growth factors, and cytokines may also be considered neuropeptides. Together, these peptides help to fine-tune numerous functions of the nervous system. References 1. Klavdieva, M.M. (1995) The history of neuropeptides 1. Front. Neuroendocrinol. 16, 293–321. 2. Klavdieva, M.M. (1996) The history of neuropeptides II. Front. Neuroendocrinol. 17, 126–153. 3. Klavdieva, M.M. (1996) The history of neuropeptides III. Front. Neuroendocrinol. 17, 155–179. 4. Klavdieva, M.M. (1996) The history of neuropeptides IV. Front. Neuroendocrinol. 17, 247–280. 5. Bayliss, W.M., and Starling, E.H. (1902) The mechanism of pancreatic secretion. J. Physiol. 28, 325–353. 6. Oliver, C., and Shäfer, E.A. (1895) On the physiological actions of extracts of the pituitary body and certain other glandular organs. J. Physiol. 18, 277–279. 7. Von den Velden, R. (1913) Die Nierenwirkung von Hypophysenextrakten beim Menschen. Klin. Wochschr. (Berlin) 50, 2083–2086. 8. Starling, E.H. (1904) The chemical regulation of the secretory process (Croonian Lecture to the Royal Society). Proc. Royal Soc. 73B, 310–322. 9. Starling, E.H. (1905) Croonian Lecture: On the chemical correlation of the functions of the body I. Lancet 2, 339–341. 10. Starling, E.H. (1905) Croonian Lecture: On the chemical correlation of the functions of the body II. Lancet 2, 423–425. 11. Starling, E.H. (1905) Croonian Lecture: On the chemical correlation of the functions of the body III. Lancet 2, 501–503. 12. Starling, E.H. (1905) Croonian Lecture: On the chemical correlation of the functions of the body IV. Lancet 2, 579–583.

13. Von Euler, U.S., and Gaddum J.H. (1931) An unidentified depressor substance in certain tissue extracts. J. Physiol. 72, 74–87. 14. Du Vigneaud, V., Lawler, H.C., and Popenoe, E.A. (1953) Enzymatic cleavage of glycinamide from vasopressin and a proposed structure for this pressor-antidiuretic hormone of the posterior pituitary. J. Am. Chem. Soc. 75, 4880–4881. 15. Wade, N. (1981) The Nobel Duel. Doubleday, Garden City, New York. 16. Tatemoto, K., and Mutt, V. (1980) Isolation of two novel candidate hormones using a chemical method for finding naturally occurring polypeptides. Nature 285, 417–418. 17. Hughes, J., Smith, T.W., Kosterlitz, H.W. et al (1975) Identification of two related pentapeptides from the brain with potent opiate agonist activity. Nature 258, 577–580. 18. Speidel, C.C. (1919) Gland-cells of internal secretion in the spinal cord of the skaes. Carengie Institute Washington Publications 13, 1–31 19. Scharrer, E., and Scharrer, B. (1937) Über Drüsen-Nervenzellen und neurosekretorische Organe bei Wirbellosen und Wirbeltieren. Biol. Rev. 12, 185–216. 20. Sterba, G. (1964) Principles of histochemical and biochemical demonstration of neurosecretion (carrier protein of oxytocin) with pseudoisocyanine. Acta Histochem. 17, 268–92. 21. Bargmann W, Scharrer E (1951) The site of origin of the hormones of the posterior pituitary. Am. Sci. 39, 255–259. 22. Hökfelt, T., Johansson, O., Ljungdahl, A., Lundberg, J.M., and Schultzberg, M. (1980) Peptidergic neurones. Nature 284, 515–521. 23. Bohus, B., and De Wied, D. (1966) Inhibitory and facilitatory effect of two related peptides on

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extinction of avoidance behavior. Science 153, 318–320. 24. De Wied, D. (1969) Effects of peptide hormones on behavior. In: Ganong, W.F., and Martini, L. (eds), Frontiers in neuroendocrinology. Oxford University Press, New York, pp. 97–140. 25. De Wied, D. (1971) Long term effect of vasopressin on the maintenance of a conditioned avoidance response in rats. Nature 232, 58–60. 26. Brownstein, M.J. (1977) Studies of the distribution of biologically active peptides in the brain. Adv. Exp. Med. Biol. 87, 41–48. 27. Hur, Y.S., Kim, K.D., Paek, S.H. et al (2010) Evidence for the existence of secretory granule (dense-core vesicle)-based inositol 1,4,5-trisphosphate-dependent Ca2+ signaling system in astrocytes. PLoS One 5, e11973. 28. Lonka-Nevalaita, L., Lume, M., Leppanen, S. et al (2010) Characterization of the intracellular localization, processing, and secretion of two glial cell line-derived neurotrophic factor splice isoforms. J. Neurosci. 30, 11403–11413. 29. Brownstein, M.J., Russell, J.T., and Gainer, H. (1980) Synthesis, transport, and release of posterior pituitary hormones. Science 207, 373–378. 30. Tooze, S.A., and Huttner, W.B. (1990) Cell-free protein sorting to the regulated and constitutive secretory pathways. Cell 60, 837–847. 31. Lang, T., Wacker, I., Steyer, J. et al (1997) Ca2+−triggered peptide secretion in single cells imaged with green fluorescent protein and evanescent-wave microscopy. Neuron 18, 857–863. 32. Tooze, S.A., Martens, G.J., and Huttner, W.B. (2001) Secretory granule biogenesis: rafting to the SNARE. Trends Cell Biol. 11, 116–122. 33. Burbach, J.P.H., and Wiegant, V.M. (1990) Gene expression, biosynthesis and processing of proopiomelanocortin peptides and vasopressin. In: De Wied, D. (ed), Neuropeptides, basics and perspectives. Elsevier, Amsterdam, pp 45–103. 34. , S.G., Jonas, V., Rosenfeld, M.G. et al (1982) Alternative RNA processing in calcitonin gene expression generates mRNAs encoding different polypeptide products. Nature 298, 240–244.

35. Nawa, H., Kotani, H., Nakanishi, S. (1984) Tissue-specific generation of two preprotachykinin mRNAs from one gene by alternative RNA splicing. Nature 312, 729–734. 36. Zhao, E., Zhang, D., Basak, A. et al (2009) New insights into granin-derived peptides: evolution and endocrine roles. Gen. Comp. Endocrinol. 164, 161–174. 37. Braks, J.A., and Martens, G.J. (1994) 7B2 is a neuroendocrine chaperone that transiently interacts with prohormone convertase PC2 in the secretory pathway. Cell 78, 263–273. 38. Ubogu, E.E., Cossoy, M.B., and Ransohoff, R.M. (2006) The expression and function of chemokines involved in CNS inflammation. Trends Pharmacol. Sci. 27, 48–55. 39. de Haas, A.H., van Weering, H.R., de Jong, E.K. et al (2007) Neuronal chemokines:versatile messengers in central nervous system cell interaction. Mol Neurobiol. 36, 137–151. 40. Miller, R.J., Rostene, W., Apartis, E. et al (2008) Chemokine action in the nervous system. J. Neurosci. 28, 11792–11795. 41. Huang, E.J., and Reichardt, L.F. (2001) Neurotrophins:roles in neuronal development and function. Annu. Rev. Neurosci. 24, 677–736. 42. Thomas, K., and Davies, A. (2005) Neurotrophins:a ticket to ride for BDNF. Curr. Biol. 15, R262-R264. 43. Salio, C., Averill, S., Priestley, J.V. et al (2007) Costorage of BDNF and neuropeptides within individual dense-core vesicles in central and peripheral neurons. Dev. Neurobiol. 67, 326–338. 44. Yang, J., Siao, C.J., Nagappan, G. et al (2009) Neuronal release of pro-BDNF. Nat. Neurosci. 12, 113–115. 45. Teng, H.K., Teng, K.K., Lee, R. et al (2005) Pro-BDNF induces neuronal apoptosis via activation of a receptor complex of p75NTR and sortilin. J. Neurosci. 25, 5455–5463. 46. Dicou, E, (2007) Peptides other than the neurotrophins that can be cleaved from proneurotrophins:a neglected story. Arch. Physiol. Biochem. 113, 228–233.

Chapter 2 Neuropeptide Localization in Nonmammalian Vertebrates Paolo de Girolamo and Carla Lucini Abstract Neuropeptides are particularly suited to comparative and evolutionary studies, since they have been highly conserved during evolution. Based on primary amino-acid structure, neuropeptides can be arranged into families and synthesized as multiple molecular variants. They may play different functional roles in different organs or tissues of the same species, but also among species and classes. Immunohistochemistry (IHC) is powerful technique for localizing the molecular expression of proteins in tissues and cells of different classes of vertebrates and has been fully exploited in the study of the mammalian brain. The present chapter provides a detailed description of the protocols routinely used in our laboratory to analyze the presence and distribution of neuropeptides in nonmammalian vertebrate tissues. Single labeling protocols performed by both light and fluorescein IHC, and double labeling protocols using primary antisera raised in different species or in the same species are described. Antibody and method specificity are also discussed in detail. Key words: Neuropeptides, Immunohistochemistry, PAP, ABC, HRP, Immunofluorescence, Fish, Amphibians, Reptiles, Birds

1. Introduction Currently, the broader definition of a neuropeptide adopted by The International Neuropeptide Society includes those peptides, independently of whether they are secreted by neurons or nonneural cells, which express the same genetic information and undergo identical processes of synthesis and transport, and bind to similar families of receptors (1). Neuropeptides are expressed in the central and peripheral nervous system and often coexist with other transmitters, either a classical transmitter or one or several other neuropeptides (2). Furthermore, neuropeptides or closely related molecules can be also expressed and released by endocrine cells to act as circulating of tissutal hormones (3).


37

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P. de Girolamo and C. Lucini

Based on their primary amino acid structure, neuropeptides can be grouped into families, which are represented along the evolutionary scale, from the most simple of coelenterates (4–7). Nevertheless, they are often synthesized as multiple molecular variants and may play different roles in different regions, species and classes. In some cases, these molecules even appear to be identical in structure: e.g. the head activator peptide of Hydra occurs in identical form in mammals (8), and the enkephalins, originally extracted from mammals, were also shown to be present in the nervous system of the mollusks Mytilus edulis and Lymnaea stagnalis (9). On the contrary, as an example of neuropeptides that are not fully identical, but structurally closely related, the “oxytocins–vasopressins” of the vertebrate classes (10) can be mentioned. These phenomena can be explained by considering the possible mutagenesis (gene duplication, gene conversion, point mutations) of genes coding for neuropeptides precursors (11). During this process, the neuropeptides diverge structurally, but they may be expected to still show similarities. With regard to the diversity, tissue-specific expression of related neuropeptide genes and differences in neuropeptide precursor processing can be also considered. For this reason, the study of the distribution of neuropeptides in vertebrates and their phenotypic plasticity leads to an understanding of the basic cytochemical organization, gives an appreciation of the wide diversity among animal classes, and represents a field particularly suited to comparative and evolutionary studies (12–14). In numerous investigations where antibodies to vertebrate or invertebrates neuropeptides were used, immunohistochemical relations were reported in animals unrelated to the donor species of the neuropeptides that served as immunogens (15–18). So, immunohistochemistry (IHC) has proven to be a valuable technique for the accurate mapping of the cellular distribution of neuropeptides in nonmammalian vertebrate tissues. However, when IHC is applied to lower vertebrates’ tissues some additional difficulties with respect to mammalian studies are encountered. The major limitations of the immunohistochemical approach are associated with (1) that neuropeptides often exist in closely related isoforms in a given species and antisera raised against specific synthetic holopeptides tend to recognize all of the isoforms, (2) cross-reactivity with undefined tissue antigens, and (3) the “detection limit” of the assay, namely, the possibility to discriminate between signal and background in the tissue. For these reasons, in most cases without adequate positive controls (sites within tissue of mammalian species where known proteins are stored), there is a fat chance to arrive at a determination of the lower detection limit for an immunohistochemical assay of nonmammalian tissues. Furthermore, because a neuropeptide may be present at a specific location, but in amounts below the detection limit, it is not appropriate to draw definitive comments on where a neuropeptide is not located in a tissue of a given species.

2 Neuropeptides in Lower Vertebrates

39

In order to provide a starting point for peptide IHC, and try out changeable protocol points, it is fundamental to remember that virtually any laboratory that employs IHC for histological studies has modified the protocols, since every antiserum or tissue requires specific modifications according to specific experimental conditions. Thus, it is important to consult the data sheet describing initial characterization of the antiserum and its application to specific tissues and animal species. For commercially available antisera, standard protocols are commonly supplied. Nonetheless, information on nonmammalian species is often missing. The present chapter provides a detailed description of the immunohistochemical protocols, including double labeling techniques, routinely used in our laboratory to analyze the presence and distribution of neuropeptides in nonmammalian vertebrate tissues.

2. Materials 2.1. Anesthesia/ Euthanasia

1. MS-222: Commercially available as Tricaine Methane Sulfonate (TMS; 3-amino benzoic acid ethyl ester) for use in fishes (19) and amphibians (20) (see Note 1). 2. Ketamine: For use in reptiles (21) (see Note 2). 3. Isoflurane: Inhalant anesthetic of choice for lizards (see Note 3) and birds (22).

2.2. Fixation and Tissue Processing

1. Bouin’s Fixative: Mix 75 mL saturated aqueous solution of picric acid (see Note 4), 25 mL formalin (~40% aqueous solution of formaldehyde – see Note 5), 5 mL glacial acetic acid (see Note 6). 2. Buffered formalin 10%: Add 100 mL formalin (~40% aqueous solution of formaldehyde) (see Note 5) and 900 mL distilled water. Buffer the mixture, for precise buffering to neutrality (pH 7.0), with 4 g NaH2PO4⋅H2O and 6.5 g Na2HPO4. 3. Paraformaldehyde 4% in 0.1 M phosphate buffer: Heat 400 mL distilled H2O to 50–60°C. Add 40°g paraformaldehyde (see Note 5) and ten drops of 5°M NaOH until the solution is clear (to completely dissolve the aldehyde). Cool and add 500°mL 0.2°M phosphate buffer stock solution and enough H2O to 1°L. This fixative can be aliquoted and stored frozen in vials. After thawing, mix the solution well and check the pH. 4. 100, 95, 85, 70, and 50% ethanol. 5. Benzene (see Note 7). 6. Methyl benzoate (see Note 8). 7. Celloidin (see Note 9). 8. Paraffin with a melting point of 58–60°C (see Note 10).

40


2.3. Immunohistochemistry

1. 0.01°M sodium citrate buffer, pH 6: Prepare the solution immediately before use. Mix 9°mL of solution A (2.1°g citric acid in 100°mL distilled water) and 41°mL of solution B (2.941°g sodium citrate in 100°mL distilled water) and bring to 500°mL with distilled water. Solutions A and B can be stored at 4°C for many weeks. 2. 0.01°M phosphate-buffered saline (PBS), pH 7.2–7.4: Prepare the solution immediately before use. Mix 31.5°mL solution A (8.89°g Na2HPO4 × 2H2O in 100°mL distilled water) and 8.5°mL of solution B (6.9°g NaH2PO4 × 2H2O in 100 mL distilled water) and add 16 g NaCl. Bring the volume to 2 L with distilled water. Adjust pH with NaOH or HCl. Solutions A and B can be stored at 4°C for many weeks. Stir and heat solution A before use to dissolve crystals. 3. Hydrogen peroxide 3%: Can be easily purchased in prediluted solutions. Hydrogen peroxide should be stored at 4°C and, since it breaks down quickly when exposed to light, in an opaque container. 4. Damp chamber: It can easily obtained using a Petri dish with a wet paper and five slides at bottom where put the slides with sections. 5. Antibodies: Lyophilized antibodies are stable for two or even more years when stored from −20 to −70°C. Reconstitute with distilled water and store in concentrated form (up to 1/20) in small aliquots at −20°C in a manual defrost freezer for 1 year and more. Avoid freeze–thawing. Store small volumes of working dilutions of the antibody at 4°C, for a maximum of 1 week. Liquid antibodies containing sodium azide as preservative are stable at 4°C for 6 months. Dilute antibodies according to the suppliers’ information. However, it is often useful to determine the optimal concentration by carrying out a series of dilutions. 6. DAB solution: Before use, dissolve 10 mg 3,3¢-diaminobenzidine (DAB) in 15 mL Tris–HCl, filter and add 1.5 mL H2O2 (see Note 11). DAB is commercially available in prediluted forms, powder, or 10-mg tablets. The latter are the most suitable because often prediluted DAB is at a higher concentration than needed, and can increase background staining, whereas DAB powder is more harmful. 7. 0.05 M Tris–HCl, pH 7.6: Dissolve 6.1 g tris-(hydroxymethyl) aminomethane in 50 mL distilled water, add 37 mL HCl 1 N, and bring to 1 L. Store the solution at 4°C for at maximum 1 week. 8. Fluorochrome-conjugated secondary antibodies: It can be stored in the dark at 4°C. If they are supplied in lyophilized form, they can be rehydrated with distilled water and centrifuged


41

if the solution is not clear. Then, add an equal volume of glycerol for a final concentration of 50% and store at −20°C. After the addition of glycerol, the concentration of protein and buffer salts is one half of the original. 9. Neuropeptides (antigens): Lyophilized neuropeptides are stable for 2 years or else more when stored from −20 to −70°C. Reconstitute with distilled water and store at 4°C for at maximum 1 week.

3. Methods 3.1. Anesthesia/ Euthanasia (see Note 12)

1. Fish: Place animals in MS-222 anesthetic solution, which is then absorbed through the gills. Animals should be left in the solution for at least 10 min following cessation of respiratory movement. MS-222, like all fish anesthetic agents, has a dosedependent effect, which varies with species as well as individuals. A 0.03% solution of MS-222 is effective in zebrafish, although stronger solutions may be required in other species. MS-222 is acidic, and the solution should be kept at pH 7.0–7.5 by adding sodium bicarbonate (typically 2 g per g of MS-222). 2. Amphibians: Place animals in a dish containing 2–3 mm of MS-222 anesthetic solution at the bottom. The anesthetic is thus absorbed through the ventral skin. A 0.5–2.0 mg/L solution is the MS-222 effective dose in frog. For other amphibian species, inject 50–200 mg/kg MS-222 solution intramuscularly or subcutaneously (20, 21). 3. Reptiles: Injectable and inhalant anesthesia are commonly employed for treatment procedures in reptiles (21). For injectable anesthesia, in lizards administrate intramuscularly or subcutaneously 50–150 mg/kg ketamine. For inhalant anesthesia, in lizards administrate, by means of a cone constructed of an appropriate size syringe casing covered by a rubber glove, a 3–4% isoflurane solution for induction and a 1–2% isoflurane solution for maintenance when exposed to the annoying odor. Adequate induction can take anywhere from several minutes to hours. 4. Birds: Induce inhalant anesthesia in chickens by a 0.5–4% isoflurane solution and maintain by 1–3% isoflurane solution. The bird’s head is placed into the glass anesthetic mask with a latex rubber forming a firm seal around the feathers. The latex is cut from a surgical glove and secured to the mask using a rubber band. The mask should be placed on an electric blanket heated to 40°C in a draft-free environment (not a fume hood) to minimize loss of body heat from conduction and convection. Feather removal should be kept to a minimum to maintain

42


thermal insulation. Control of heat loss is particularly critical during anesthesia of very young chicks (12, 13). 3.2. Fixation and Tissue Processing

1. Fixation: Since all fixatives can influence tissue antigenicity, a selection of the fixatives listed in Subheading 2.2 may need to be determined by trial and error. Adequate fixation is obtained by simple immersion of small tissue pieces into the fixative solution. This is the only mode of fixation possible for many tissues of lower vertebrates and when it is necessary to process samples in multiple fixatives (see Note 13). 2. Place tissue samples into an appropriate volume of fixative immediately upon removal or as soon as possible after death. The optimal size for tissues to be fixed for light microscopy is about 2 cm2 and about 3–4 mm in thickness, but these figures vary with tissue density. For example, compared with a compact tissue such as a liver, much larger pieces of spongy tissues such as fish heart can be adequately fixed (see Note 14). 3. Immerse samples in fixative for 2–4 up to 48 h at room temperature, depending on sample size, tissue, and subsequent treatment (see Note 15). 4. Wash specimens fixed in Bouin’s solution in 70% alcohol to precipitate soluble picrates and until all the yellow color is removed from the tissue (see Note 16). Rinse specimens fixed in buffered formalin 10% and paraformaldehyde must be in PBS (several changes) at room temperature for 2 h overnight at 4°C. 5. Rinse the tissue for 2 × 20 min in 0.137 M NaPO4 buffer, keeping the vials at 4°C during rinses. 6. Dehydrate in a graded ethanol–water series: 50, 70, 85, and 95% for 10–20 min each. Then, place in 100% ethanol for two changes from 20 min to 1 h each. All solutions should be at 4°C. 7. Clear samples in methyl benzoate–celloidin solution (celloidin 1% in methyl benzoate) for 24 h (see Notes 17 and 18). 8. Immerse in pure benzene for 1–2 h. 9. Transfer to a mixture containing of equal parts of paraffin wax and pure benzene in the vacuum for 1 h. 10. Immerse in two changes of pure paraffin wax from 15 min to 3 h each, depending upon the thickness and the nature of the tissue (see Note 19). Orientate and embed tissue in fresh wax using warm molds and embedding cassettes. 11. Cut thin sections (3–10 mm, 5 mm is commonly used) by using a sliding microtome. Trim the block until the cutting surface is optimal, transfer paraffin ribbons with a wet paintbrush to egg-albumen-coated slides.


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12. Place the slides on a warming tray and add distilled water to float the paraffin sections and allow then to expand and straighten out. Excess water is thus removed and sections will adhere to the slides. 13. Allow the slides to dry in an oven at 35–37°C overnight. 3.3. Immunohistochemistry 3.3.1. PAP, ABC, and HRP Protocols: Single Labeling

Single labeling (Fig. 1) can be performed by both light and fluorescence IHC. In nonmammalian vertebrates single neuropeptide labeling is preferably detected by light IHC, by using peroxidase– anti-peroxidase (PAP), avidin-biotin (ABC), and polymer horseradish-peroxidase (HRP) methods (see Note 20) that offer higher sensitivity than immunofluorescence. 1. Remove wax in xylene and bring the sections to water through graded alcohols. 2. Retrieve antigens by microwave oven treatment (23) on the sections immersed in 0.01 M sodium citrate buffer (see Note 21). 3. Rinse in PBS for 5 min at room temperature. 4. Block endogenous peroxidase activity by immersion of the sections at room temperature for 30 min in a solution of 3% hydrogen peroxide (for PAP and polymer-HRP methods). Block endogenous biotin by immersion of the sections in 0.05% avidin solution for 15 min followed by brief rinse in PBS and then incubate the sections in 0.005% biotin solution for 15 min (for ABC method). All these procedure are carried out at room temperature. 5. Rinse in PBS for 15 min. Dry the slides except for the area of section and place in a damp chamber. 6. Incubate for 30 min with 1:5 normal serum belonging to the species in which the secondary antibody was raised to block aspecific background staining. 7. Do not rinse. Draw off serum with a tissue, except for the area of the section and place in a damp chamber. 8. Dilute primary antibody in PBS to the appropriate concentration (see Note 22). Apply the primary antibody in a volume of 50–100 mL (depending on the area of sections). 9. Incubate the sections overnight at 4°C or for shorter periods at room temperature or at 37°C (see Notes 23 and 24). 10. Rinse three times in PBS, 5 min each. Dry the slides except for the area of section and place in a damp chamber. 11. Dilute the secondary antibody in PBS to appropriate concentration (1:50–1:200). This antibody is unconjugated for PAP, conjugated to biotin for ABC and to a polymer for HRP. 12. Incubate the sections for 30 min up to 2 h at room temperature or for a shorter time at 37°C (see Note 25).

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Fig. 1. Light microscopic ICC of peptides in nonmammalian vertebrates. (a–c) Beacon immunoreactivity within the Gallus domesticus hypothalamus – PAP method: (a) Immunoreactive neurons (arrows) in the dorsal (d), medial (m) and ventral (v) part of nucleus magnocellularis preopticus (MPO); (b) Innervation of the internal zone (i.z.) and external zone (e.z.) of the median eminence; (c) Dense immunoreactive fiber (arrows ) arrangement in the median eminence (3V, third ventricle);


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13. Rinse three times in PBS, 5 min each. Dry the slides except for the area of section. 14. Dilute the PAP or ABC complex in PBS to appropriate concentration. Incubate the sections for 30 min up to 2 h at room temperature or for a shorter time at 37°C. For the sections treated with the method HRP skip this step and proceed directly to step 16. 15. Rinse three times in PBS, 5 min each. Dry the slides except for the area of section. 16. Put a drop of DAB solution on the section in a dark chamber at room temperature. Monitor the reaction at low magnification under a microscope. The reaction should be complete in 10–20 min, but it can be prolonged up to 1 h (see Note 26). 17. Dehydrate the slides into progressive alcohols and mount with a commercially available medium (see Note 27). The sections stained with DAB are very stable and can be stored for many years. 3.3.2. Immunofluorescence Protocols: Double Labeling Using Primary Antisera Raised in Different Species

To perform this method (Fig. 2), both primary antisera against the two target neuropeptides, the related secondary antisera and normal sera can be mixed (see Note 28). All steps are carried out at room temperature unless otherwise stated. 1. Remove wax in xylene and bring the sections to water through graded alcohols. 2. Retrieve antigen by microwave oven treatment on the sections immersed in 0.01 M sodium citrate buffer (see Note 21). 3. Rinse in PBS for 5 min. Block background by incubation for 30 min with 1:5 normal sera belonging to the species in which the secondary antibodies were raised (see Note 29). 4. Do not rinse. Draw off the sera with a tissue, except for the area of the section and place in a damp chamber. 5. Apply a mixture of primary antibodies diluted in PBS to the appropriate concentration in a volume of 50–100 mL (depending on the section area). Incubate the sections for 24–48 h.

Fig. 1. (continued) (d) Neurotensin immunoreactivity in endocrine cells (arrows) of the domestic duck antrum, PAP method; (e) Ghrelin-immunostained cells (arrows) in the mucosal layer at the base of glandular lobuli in the Gallus domesticus proventriculus, PAP method; (f–g) Vasoactive Intestinal Peptide in neurons (arrows) of the muscular stomach of a 7-day-old duck embryo at low (f ) and high (g) magnification, PAP method; (h) Vasoactive Intestinal Peptide positive neurons and fibers in the myenteric plexus ganglion of the muscular stomach of the domestic duck at hatching, PAP method; (i) Gastrin/ CCK immunoreactive cells (arrows) in the antrum of the domestic duck at hatching, PAP method; (l) Cells displaying apical Orexin A immunoreactivity (arrows) in the stomach of Xenopus laevis, ABC method; (m) Orexin A immunoreactive cell (arrow ) in the proximal region of the Lacerta viridis small intestine, ABC method; (n) Orexin B immunoreactive cells (arrows) in the proximal region of the Lacerta viridis small intestine, HRP method; (o) Immunohistochemical detection of Vasoactive Intestinal Peptide in the taste buds of Carassius auratus gill arch, PAP method. Scale bars: (c, f ) = 50 mm; (e, h, i, l) = 25 mm; (a, b, d, g, m, n) = 10 mm; (o) = 5 mm.

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Fig. 2. Single fluorescence immunohistochemistry. (a, b) Somatostatin immunoreactivity in mixed (a) and large (b) p ancreatic islet cells of the domestic duck, at low (a) and high (b) magnification. In (b), somatostatin cells are lining large islet capillaries; (c) An individual PACAP immunoreactive nerve cell body with a prominent process is present at the base of the raker cushion in the oral side of the gill arch of Carassius auratus; (d) Vasoactive Intestinal Peptide immunoreactive nerve cell bodies with long processes in the connective tissue of the oral side of the gill arch of Carassius auratus. Scale bars: (a) = 20 mm; (b, c, d) = 10 mm.

6. Rinse three times in PBS, 5 min each. Dry the slides except for the area of section and place in a damp chamber. 7. Apply a mixture of secondary antibodies (against the IgGs of the species in which the primary antibodies were raised) diluted in PBS to appropriate concentration (1:25–1:50) (see Note 30). These antibodies are conjugated to fluorescein isothiocyanate (FITC) and to tetramethyl rhodamine isothiocyanate (TRITC) (24) (see Notes 31 and 32). Incubate the sections for 2 h in the dark. Incubation can be carried out also at 37°C. 8. Rinse three times in PBS, 5 min each. Dry the slides except for the area of section. 9. Mount the slides with 1:1 glycerin and PBS or a commercially available aqueous medium, fixing coverslips with four glue drops at angles. 10. Observe and photograph slides as soon as possible or store in the dark at 4°C for few weeks or at −20°C for few months (see Note 33).

2 Neuropeptides in Lower Vertebrates 3.3.3. Immunofluorescence Protocols: Double Immunofluorescence Labeling Using Primary Antisera Raised in the Same Species

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The simultaneous visualization of two antigens is also difficult for problems related to the availability of commercial antibodies raised in different species. This problem is particularly true in nonmammalian vertebrates because, usually, there are not mouse monoclonal antibodies available against nonmammalian antigens, thus eliminating the mouse from the panels of antibody donor species. Double immunofluorescence labeling using primary antisera raised in the same species consists of two sequential labeling techniques, using monovalent F(ab)-fluorochrome conjugates as secondary reagents (see Note 34) to avoid cross talk of subsequent antibody probes. 1. Repeat steps 1–4 of Subheading 3.3.2. 2. Apply the first primary antibody diluted in PBS to the appropriate concentration in a volume of 50–100 mL (depending on the section area). Incubate the sections for 24–48 h (see Notes 30 and 35). 3. Rinse three times in PBS, 5 min each. Dry the slides except for the area of section and place in a damp chamber. 4. Apply the TRITC-conjugated F(ab) secondary antibody (against the species in which the primary antibodies of the first labeling was raised) diluted in PBS to appropriate concentration (1:25–1:50) (see Notes 36 and 37). Incubate the sections for 2 h in the dark. Incubation can also be carried out at 37°C. 5. Rinse three times in PBS, 5 min each. Dry slides except for area of section. 6. Block background by incubation for 30 min with 1:5 normal serum belonging to the species in which the secondary antibody of the second labeling was raised. 7. Do not rinse. Draw off the mixture with a tissue, except for the area of the section and place in a damp chamber. 8. Apply the second primary antibody diluted in PBS to the appropriate concentration in a volume of 50–100 mL (depending on the section area). Incubate sections for 24–48 h (see Note 35). 9. Rinse three times in PBS, 5 min each. Dry slides except for area of section and place in a damp chamber. 10. Apply FITC-conjugated IgG secondary antibody (against the species in which was raised the primary antibody of the second labeling) diluted in PBS to appropriate concentration (1:25–1:50) (see Note 32). Incubate the sections for 2 h in the dark. Incubation can also be carried out at 37°C. 11. Repeat steps 9–10 of Subheading 3.3.2 (see Note 38).

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3.3.4. Control Experiments for Single Immunolabeling

Each single experiment should be run positive as well as negative controls to test that the detection system works and that the primary antibody used is responsible for generation of the immunolabeling ((25, 26) – see Note 39). 1. Positive controls using known tissues: Carry on parallel experiments by using tissue(s) in which the molecule(s) of interest is(are) known to be expressed. These controls test a protocol or procedure and make sure that it works. If positive control tissue(s) display(s) negative staining, the procedure needs to be checked until a good positive staining is obtained (see Note 40). 2. Negative controls – Antibody replacement: Substitute the primary antibody, the secondary antibody or the PAP/ABC complex in repeated trials with buffer (PBS) or normal serum to check for specificity of the immunoreactivity. During these controls, all the other steps must be kept the same. If any staining results, then the antibodies are binding nonspecifically to different sites on the tissue. These controls are easy to achieve and can be used routinely. 3. Negative controls – Absorption of the primary antibody: Incubate (prior to its use) the primary antibody with an excess of its corresponding or a related purified antigen at the same temperature and for the same time of the tested primary antibody reaction on experimental tissue (see Notes 41 and 42). In order to minimize methodological variations, process at least two adjacent sections, using absorbed and unabsorbed antibodies respectively.

3.3.5. Control Experiments for Double Immunolabeling

Double immunofluorescence labeling needs, besides the controls described in Subheading 3.3.4, additional specific controls to exclude cross-reactions of the secondary antibodies. 1. Controls for double labeling experiments using primary antisera raised in different species: Incubate the sections with one of the two primary antisera and with the mismatched secondary antibody. This control should give a negative reaction, if the secondary antibody of one line is specific and does not recognize the primary antiserum of the other line. 2. Controls for double labeling experiments using primary antisera raised in the same species: Test the specificity of the immunoreactivity in double labeling with F(ab) fragments by omitting the primary antibody in the second staining, and by the two assays described below (27). 3. Assay 1: After the first staining, incubate the sections for 48 h at room temperature with a PAP rabbit complex, which serves as the second-staining primary antibody to prove the monovalence of the F(ab) fragment. After DAB treatment


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(see Subheading 3.3.1, step 16), no immunolabeling should be observed at the light microscope. 4. Assay 2: After the first training, incubate the sections for 24 h at 4°C with a peroxidase-coupled donkey anti-rabbit F(ab) fragment, which serves as the second-staining secondary antibody, to prove the complete F(ab) saturation of rabbit IgG epitopes. After DAB treatment (see Subheading 3.3.1, step 16), no light immunolabeling should be observed.

4. Notes 1. Tricaine is a benzoic acid derivative. Therefore, a solution of MS-222 that is not buffered to neutral pH is acidic and poorly absorbed, resulting in a prolonged induction time. The stock solution should be stored in a dark brown bottle, and refrigerated or frozen (if possible). The solution should be replaced monthly and any time a brown color is observed. All bottles should be labeled and be provided the expiration date. Warning: This compound is a potent carcinogen, so precautions must be taken when handling. 2. Perhaps it is the most popular reptile anesthetic agent. As a dissociative psychotropic agent, this drug is effective and has a high level of safety in the reptile patient. 3. It is widely used in reptiles and birds. Isoflurane tends to have a high margin of safety and displays extremely rapid induction and recovery times. Since it is very lipid soluble, increasing the temperature of the animal may release more isoflurane and result in a very prolonged recovery. This anesthetic can be used for box induction if system has calibrated vaporizer, and has been used in bell jars following brief exposure. Of all inhalants, this is the least metabolized and is preferable for birds and high-risk patients. Isoflurane should only be handled in fume hood. If used in bell jar, use as small of diameter jar as possible and ensure a tight-fitting lid. Keep exposure times extremely brief, as death can occur rapidly. 4. Picric acid crystals are highly explosive when dry. Therefore, picric acid is best handled as a commercially prepared saturated aqueous solution. 5. Formaldehyde is a colorless gas with a strong, pungent odor. It is commonly used in liquid form as a 40% aqueous solution known as formalin and in solid form as a white powder called paraformaldehyde. Because of its volatility, both formalin and paraformaldehyde will readily give off irritating formaldehyde vapor with a strong odor. Warning: Formaldehyde is a sensitizer

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by skin and respiratory contact, and toxic by ingestion and inhalation. Its main targets are organs of the respiratory system. Formaldehyde is a corrosive and carcinogenic agent and must be handled in appropriate fume hoods. 6. This dry salt mixture can be prepared in advance and carried into the field in plastic containers. Warning: Glacial acetic acid is corrosive and must be handled with appropriate care. It causes skin burns, permanent eye damage, and irritation to mucous membranes. 7. Warning: Benzene is a very flammable cancer-causing agent. The fluid, either pure or in solution, constitutes a fire risk. If benzene must be used in an experiment, it should be handled at all stages in a fume cupboard. Wear safety glasses and use protective gloves. 8. Methyl benzoate is an aromatic substance with good dehydrating and alcohol detracting capacities. It is a colorless flammable solvent miscible with most organic solvents and with paraffin wax. 9. Warning: This product is handled safely when wet with water and is classified as an explosive when dry. If ignited after drying, it will burn very rapidly producing great amounts of heat and toxic smoke. Store under wet conditions until ready for use. Store in a cool, ventilated area, away from heat sources. 10. Warning: Keep paraffin away from heat and from sources of ignition. Empty containers pose a fire risk, evaporate the residue under a fume hood. Ground all equipment containing material. Keep away from incompatibles such as oxidizing agents. Keep container tightly closed, in a cool, well-ventilated area. 11. Warning: DAB is suspected carcinogen. Appropriate care should be exercised when using this reagent including gloves, eye protection, lab coats, and good laboratory procedures. Care for tips on handling and disposing of DAB solution and plasticware that has been in contact with DAB. 12. This is a reference of suggested and possible doses of anesthetics for a variety of lower vertebrate species. However, there is no claim made that this information is complete, original, or unique. When using analgesic agents, the concept of preemptive analgesia should be followed. That is, relieving the potential pain before the pain is felt. To do so will result in a quicker, less stressful recovery of the patient. For other potential sources of information on anesthetic/analgesic or euthanasic doses, agents, and techniques, consult the veterinary and clinical animal care staff at facility, for advice during protocol preparation and during the conduct of the study.


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13. Fixation is one of the most critical steps in immunostaining of lower vertebrates’ tissues. An ideal fixative should yield the following: a good preservation of the cells and tissues in a life-like manner and, at the same time, preserve antigenicity, allow antibody access to the antigen, harden naturally soft tissue which allow for easy manipulation during subsequent processing and sectioning. Fixation is also necessary to protect the sample from the deleterious effects of the immunostaining process. Chemical fixatives preserve tissue by artifactual diffusion of cell components, denaturing proteins through coagulation, cross-linking (e.g., formaldehyde), or both (e.g., mercuric formalin). 14. It is possible to evaluate several histology protocols for whole small fish specimens (e.g. Danio rerio, Nothobranchius furzeri, and Oryzias latipes). High-quality sections can be difficult to produce because small fish are composed of a variety of tissues that range in consistency from soft visceral organs to hard bones and scales. In this case, the best fixative is the Bouin’s solution that balances the hardening and minor shrinkage properties of formaldehyde by the softening and shrinking action of picric acid. 15. Storage in fixative for too long period generally causes loss of antigenicity, whereas overly short fixation times lead to inadequate retention of antigen and suboptimal tissue preservation. If the tissues are fixed longer than 24 h, they tend to become brittle and difficult to section. If long fixation times are required, it is advisable to replace with fresh fixative each day. 16. Failure to remove the Bouin’s solution can result in inadequate staining. 17. Since paraffin is immiscible with water, the main constituent of tissue, samples need to be dehydrated by progressively more concentrated ethanol baths. This is followed by a clearing agent to remove the ethanol. Finally, molten paraffin wax infiltrates the sample and replaces the clearing agent. 18. IHC can be performed on cryostatic sections of differently embedded tissues and also on whole organs or embryos. However, in our experience, sections of routinely paraffin embedded tissues give no problems for neuropeptide detection in lower vertebrates and, on the contrary, give an excellent definition of tissue morphology. 19. Tissues should be kept in the vacuum evaporator just long enough for the paraffin wax to fully infiltrate them. Prolonged heating in the oven causes shrinking and hardening of the tissue, rendering sections difficult to cut. Furthermore, many antigens are easily destroyed by high temperature and, therefore, prolonged exposure to molten wax. Infiltrate tissue with wax at the lowest possible temperature to keep it molten. Specimens

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of brain and spinal cord about 5–10 mm thick, skin such as embryos need at least three changes of pure paraffin wax for a total time of about 2–6 h, whereas organs such as spleen, containing a large amount of blood, muscle fibrous tissue, require no longer than a total of 2–3 h in the paraffin baths. 20. PAP method, the more ancient of the three methods, is somewhat less sensible of ABC, but it does not recognize biotin which is widely diffused and can give false positive or background staining. The polymer-HRP method has highest sensitivity, but is more expensive. In nonmammalian vertebrate tissues, the distribution of endogenous peroxidases, biotin and the concentration of investigated antigen are generally not known; thus, it is always preferable to compare all the three methods. 21. Fixation can influence tissue antigenicity, but several studies have suggested that increased heating may reverse the effects of longer fixation (23). Because studies on nonmammalian vertebrates often involve many different specimens, it may be difficult to standardize the optimal time of fixation. Therefore, different heating times and pHs of sodium citrate buffer could be tested. However, using the solution at pH 6.0 and the microwave oven treatment for 10 min at 750 W is a good standard to start with for most specimens. 22. The primary antibody concentration must be carefully determined because it is an essential requisite to achieve a good immunoreaction. The concentrations suggested by suppliers are often too much higher and have to be gradually tested at progressive dilutions. Their final dilution depends on neuropeptide concentrations in tissues and the sensitivity of the method. 23. Temperature and time of incubation are, after the dilution of the antibody, two other essential requisites for a good immunoreaction. Temperature and time are in inverse relationship to each other. Usually, the best reactions are achieved by incubating slides overnight at 4°C because low temperatures decrease background. 24. Commercially available antibodies are generally directed against human or, at most, mouse antigens. Theoretically, these antibodies in nonmammalian vertebrate tissues could not recognize their target neuropeptides and might cross-react with other unknown peptides. This contingency is quite infrequent because neuropeptides are usually well conserved through vertebrates in their aminoacidic sequence. However, to obtain a good reaction and minimal cross-reaction staining, it is best practice to choose those primary antibodies which are directed against epitopes showing the most conservative sequence.


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Many free programs are suitable for comparing peptides of different species. To this purpose we often employ Mega program (http://www.megasoftware.net/). Another control to check for the actual presence of a neuropeptide in nonmammalian tissue highly similar to a mammalian neuropeptide is to treat the experimental nonmammalian sections with several different antibodies that are directed against the same neuropeptide to show that the same structure(s) is(are) labeled. 25. Secondary antibody concentration, time, and temperature of incubation have to be tested for each primary antibody and tissue employed. For PAP method, the secondary antibody has to be clearly in excess to leave an antigenic site free to bind to PAP complex. 26. Time incubation in DAB solution and concentration of primary antibody are in inverse proportion. To avoid background and then misunderstanding of positive stainings, it is better diluting the primary antibody to obtain a slow reaction of DAB solution. Warning: DAB is suspected carcinogen (see Note 11). 27. Warning: Histological mounting medium is flammable and hazardous to health when inhaled. Thus, mounting the slides must be conducted in a fume hood. 28. The visualization of two antigens is quite difficult for problems related to antibody penetration. Thus, the fluorescence technique, even if less sensitive of light techniques, is more suitable because the secondary fluorescent dye-liked antibody is a complex sterically smaller than secondary and tertiary layer molecules of light techniques. 29. To obtain the final optimal dilution for the antibody mixture, remember that each antibody solution represents a dilution buffer for the other antibody. 30. Fluorescence needs more concentrated primary antibodies than light immunohistochemistry (the ratio is about one to ten). To improve the sensitivity of immunofluorescence, it might be better to increase the time and the temperature of incubation with primary antibody. 31. FITC is the form of fluorescein used for conjugation to antibodies. Fluorescein conjugates absorb light maximally at 492 nm and fluoresce maximally at 520 nm. FITC is a widely used fluorochrome due to its long history. The major disadvantage of fluorescein is its rapid photobleaching (fading). TRITC conjugates absorb light maximally at 550 nm and fluoresce at 570 nm. Better color separation could be achieved by using Texas Red, but it has been reported that use of Texas Red may lead to higher background staining (24).

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32. Secondary antibodies for multiple labeling must be affinity-purified and it is mandatory that they do not recognize one another, or other primary antibodies used in the assay system or the endogenous immunoglobulins that are present in tissues under investigation. 33. Attention: Steps 7–10 in Subheading 3.3.2 must be performed in the dark to avoid any bleaching of fluorochromes. 34. Monovalent F(ab) fragments of affinity-purified, secondary antibodies cover the surface of immunoglobulins for double labeling primary antibodies from the same host species. Thus, it is necessary to employ a high concentration of conjugated F(ab) to achieve effective blocking of the first primary antibody. 35. Best results are obtained when the primary antibody with the highest affinity is used in the second stage. When both primary antibodies have approximately the same affinity, better results are obtained when the primary antibody raised against the less abundant antigen is applied first. 36. Because monovalent F(ab) fragment must be applied in excess (see Note 34), the right concentrations have to be deducted by a series of controls (see Subheading 3.3.5). 37. In the first labeling, it is better to avoid using a FITC-conjugated F(ab) secondary antibody because this fluorochrome could fade during the second labeling reaction. 38. Attention: Steps 2–11 in Subheading 3.3.3 must be performed in the dark to avoid any bleaching of fluorochromes. 39. Specificity is the most important and difficult criterion of validity to define in immunocytochemical stainings of nonmammalian vertebrate tissues and requires two independent sets of validation: specificity of the method and specificity of the antibody. Thus, the requirements for a valid immunohistochemical method are as follows: (1) it should include a careful design of experiments with preliminary standardization of all steps of the control tests (e.g., incubation time, antibody dilution, the second and following antibodies used), which should be identical to those used for the immunohistochemical localization procedure; (2) handling of the antigens in the test system should be identical to those in the tissue section (e.g., specificity is often dependent on the fixative used); (3) it should include the use of other significant and appropriate information to support the validity of the localization (e.g., reliable positive and negative controls) and the correct interpretation of the results. 40. If the positive control tissue gives positive staining and the nonmammalian tissue gives negative reaction, it does not necessarily mean that in the latter the searched neuropeptide is lacking, since it may be only present in lower concentration.


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Thus, in these occurrences further reactions employing more concentrated antibodies or more sensitive detection systems (see Note 20) are needed. 41. The absorption of primary antibody with its corresponding antigen should give a negative staining if the tested primary antiserum is specific and its antigenic sites were saturated by a sufficient amount of antigen. The absorption of primary antiserum with related antigens should not influence the positive reaction if the tested primary antiserum is specific and does not cross-react with related antigens. 42. The amounts of antibody and antigen that have to be mixed will vary depending upon the titer and avidity of the antibody and upon the stability of the antigen. However, it is better to absorb the primary antibody at its maximum dilution that gives a good staining. The amount of related antigens (generally up to 100 mg/mL) is much more than the specific antigen (generally up to 50 mg/mL). References 1. Kastin, A. J. (2000) What is a neuropeptide? Trends Neurosci. 23, 113–114. 2. Salio, C., Lossi, L., Ferrini, F., and Merighi, A. (2006) Neuropeptides as synaptic transmitters. Cell Tissue Res. 326, 583–598. 3. Solcia, E., Usellini, L., Buffa, R., et al (1998) Endocrine cells producing regulatory peptides. In: Polak JM, (ed.) Regulatory peptides. Experientia supplementum, vol. 56. Basel/ Boston/Berlin: Birkha user Verlag; 220–246. 4. Holmgren, S. and Jensen, J. (1994) Comparative aspects on the biochemical identity of neurotransmitters of autonomic neurons. In: Burnstock G, series ed. The autonomic nervous system. Nilsson S, Holmgren S, volume eds. Comparative physiology and evolution of the autonomic nervous system. London: Gordon and Breach Science Publishers; 69–95. 5. Thorndyke, M. C. (1986) Immunocytochemistry and evolutionary studies with particular reference to peptides. In: Polak JM and Noorden S Van (eds.) Immunocytochemistry, J. Wright and Sons, Bristol; 300–327. 6. Thorndyke, M. C. and Goldsworthy, G.J. (1988) Neurohormones in invertebrates. Cambridge: Cambridge University Press. 7. Joosse, J. What is special about peptides as neuronal messengers? (1988) In: Thorndyke MC; Goldsworthy GJ, eds. Neurohormones in inver tebrates. Cambridge: Cambridge University Press; 1–3.

8. Bodenmüller, H., and Schaller, H. C. (1981) Conserved amino acids sequence of a neuropeptide, the head activator, from the coelenterates to humans. Nature 293, 579–580. 9. Leung, M. K., Boer, H. H., van Minnen, J., lundy, J. and Stefano, G. B. (1990) Evidence for an enkephalinergic system in the nervous system of the pond snail Lymnea stagnalis. Brain Res. 531, 66–71. 10. Hadley, M. E. (1988) Endocrinology, PrenticeHall, Inglewood Cliffs, NJ, USA. 11. Wilson, A. C. (1985) The molecular basis of evolution. Sci. Am. 253, 164–173. 12. Fasolo, A., and Vaudry, H. (1992) Neuropeptides in evolution. Ann. Sci. Nat. 12, 124–136. 13. Holmgren, S. and Jensen, J. (2001) Evolution of vertebrate neuropeptides. Brain Res. Bull. 55, 723–735. 14. Conlon, J.M. and Larhammar, D. (2005) The evolution of neuroendocrine peptides. Gen. Comp. Endocrinol. 142, 53–59. 15. Bjenning, C. and Holmgren, S. (1988) Neuropeptides in the fish gut. Histochemistry 88, 155–163. 16. Conway, K. M. and Gainer, H. (1987) Immunocytochemical studies of vasotocin, mesotocin, and the neurophysins in the Xenopus hypothalamo-neurohypophysial system. J. Comp. Neurol. 264, 494–508.

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17. Schot, L. P. C., Boer, H. H. and MontagneWajer, C. (1984) Characterization of multiple immunoreactive neurons in the central nervous system of the pond snail Lymnea stagnalis. Neuroscience 81, 373–378. 18. Veenstra, J. A., Romberg-Privee, H. M., Schooneveld, H. and Polak, J. M. (1985) Immunocytochemical localization of peptidergic neurons and neurosecretory cells in the neuroendocrine system of the Colorado potato beetle with antisera to vertebrate regulatory peptides. Histochemistry 82, 9–18. 19. Nusslein-Volhard, C. and Dahm, R. (2002) Zebrafish: a practical approach. Oxford University Press. 20. Wright, K. M. and Whitaker, B. R. (2001) Amphibian medicine and captive husbandry. Krieger Publishing Company, Malabar, USA. 21. Fish, R. E., Danneman, P.J., Brown, M., and Karas, A. (2008) Anesthesia and analgesia in laboratory animals. Academic Press. 22. Abou-Madi, N. (2001) Avian anesthesia. Vet. Clinics of North America: Exotic Animal Practice 4, 147–167.

23. Taylor, C. R., Shi, S. R., Chen, C., Young, L., Yang, C., and Cote, R, J,. (1996) Comparative study of antigen retrieval heating methods: microwave, microwave and pressure cooker, autoclave, and steamer. Biotech. Histochem. 71, 263–270. 24. Wessendorf, M. W., and Brelje, T. C. (1992) Which fluorophore is brightest? A comparison of the staining obtained using fluorescein, tetramethylrhodamine, lissamine rhodamine, Texas red, and cyanine 3.18. Histochemistry. 98, 81–85. 25. Burrt, R. W. (2000) Specificity controls for immunocytochemical methods. J. Histochem. Cytochem. 48, 163–165. 26. Petrusz, P. (1983) Essential requirements for the validity of immunocytochemical staining procedures J. Histochem. Cytochem. 31, 177–179. 27. Negoescu, A., Labat-Moleur, F., Lorimer, P., Lamarcq, L., Guillermet, C., Chambaz, E., and Brambilla, E. (1994) F(ab) secondary antibodies: a general method for double immunolabeling with primary antisera from the same species. Efficiency control by chemiluminescence. J. Histochem. Cytochem. 42, 433–437.

Chapter 3 Combined Light and Electron Microscopic Visualization of Neuropeptides and Their Receptors in Central Neurons Chiara Salio, Laura Lossi, and Adalberto Merighi Abstract The study of neuronal connections and neuron to neuron (or neuron to glia) communication is of fundamental importance in understanding brain structure and function. Therefore, ultrastructural investigation by the use of immunocytochemical techniques is a really precious tool to obtain an exact map of the localization of neurotransmitters (neuropeptides) and their receptors at different types of synapses. However, in immunocytochemical procedures one has always to search for the optimal compromise between structural preservation and retention of antigenicity. This is often made difficult by the need to localize not only small transmitter molecules, as in the case of transmitter amino acids and neuropeptides, but also their specific receptors that are usually large proteins very sensitive to fixation procedures. We describe here a preembedding procedure employing the Fluoronanogold™ reagent, a probe consisting of fluorescein-tagged antibodies conjugated with ultrasmall gold particles that can be made visible under the electron microscope by a gold intensification procedure. This technique permits correlative fluorescence and electron microscopy observations, providing a very useful tool for the study of neuronal connectivity. Moreover, the Fluoronanogold™ procedure can be combined with conventional postembedding immunogold techniques in multiple labeling studies. Key words: Neurons, Electron microscopy, Fluoronanogold™, Neuropeptides, Somatostatin, SSTR2a receptors, GDNF, GFRa1 receptors, BDNF, fl-trkB receptors

1. Introduction The ultrastructural localization of neuropeptides and their receptors at synapses (and nonsynaptic sites) is of great importance to understand the mechanism(s) of action of this family of neurotransmitter molecules and the organization of neuronal networks. Nevertheless, our knowledge of the role of neuropeptides in different areas of the brain and spinal cord is still largely incomplete, since a thorough


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anatomical and functional description of neuronal circuitry is often lacking, such is the precise localization of messenger molecules and their receptors at synapses. Different immunocytochemistry (ICC) labeling methods have been developed starting from the seventies of the last century to detect neuropeptides (and other neurotransmitters) at the transmission electron microscope (TEM) level (see for example (1–4)). Initial work in the field was mainly carried out by a preembedding approach with adaptation of the widely employed peroxidase– anti-peroxidase (PAP) and/or avidin-biotin complex (ABC) methods for light microscopy for use in ultrastructural studies. In parallel, the development of colloidal gold probes opened the way to the use of postembedding procedures that allowed a better preservation of ultrastructure in labeled samples but were associated with an often too drastic reduction of antigenicity that seriously limited their suitability for detection of large receptor molecules (5). More recently, the preembedding Fluoronanogold™ technique (Fig. 1) that we describe here has emerged as a new approach for the detection of a wide number of neurotransmitters and receptors resulting as an excellent method that combines several advantages of the pre and postembedding procedures and allows for a precise correlation of anatomical distributional observations at the light microscope with subcellular localization TEM studies (6–10). As an example of the possibilities that have been opened to combined histological/ultrastructural investigations of central neurons by the use of the Fluoronanogold™ technique, we describe here the localization of the somatostatin (SST) receptor 2 (SSTR2) and the glial-derived neurotrophic factor (GDNF)-family receptor a1 (GFRa1) at central synapses. Moreover, the Fluoronanogold™ procedure can be combined with conventional postembedding immunogold (4) in multiple labeling studies, as shown here by the concurrent visualization of the high-affinity brain-derived neurotrophic factor (BDNF) tyrosine kinase receptor (trkB) with neuropeptides such as calcitonin-gene related peptide (CGRP) or substance P (SP). All these peptide molecules have multiple roles in sensory pathways, acting via specific high-affinity receptors (11–18). Namely, SSTR2, one of the five SST G-protein coupled receptors (SSTR1-SSTR5) (19, 20), is expressed in spinal cord (21) in two different isoforms, SSTR2a and SSTR2b (22, 23), and seems to mediate the effect of SST in the control of nociceptive information. GFRa1 and RET are the two receptors forming the multireceptor complex that mediates the biological effects of GDNF. GFRa1 is the high-affinity ligand-binding component of the complex, anchored to the outer plasma membrane by a glycosyl phosphatidylinositol link (24–27). RET is the transmembrane tyrosine kinase receptor, acting as the signal transducing component (24–30).

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Fig. 1. Fluoronanogold™ immunostaining technique. (A) Structure of Alexa Fluor 488, Streptavidin and the 1.4 nm Nanogold™ particle, covalently conjugated to form the complex Alexa Fluor 488-Fluoronanogold™-Streptavidin. (B) Enlargement of 1.4 nm gold particle with the Gold Enhancement kit. The time of incubation in the Gold Enhancement kit is from 5 to 15 min, during which gold ions are catalytically deposited all around the 1.4 nm gold particle to give rise to an enlarged electrondense particle of 25–30 nm in size. (C) Fluorescent immunostaining of GFRa1 receptors in the mouse spinal cord dorsal horn obtained using a goat anti-GRFa1 primary antibody (Neuromics, Minneapolis, USA), a secondary biotinylated anti-goat antibody (Vector Laboratories, Burlingame, CA, USA) and the Alexa Fluor-488-Fluoronanogold-Streptavidin complex (Nanoprobes, Yaphank, NY). GFRa1-immunoreactive fibers are mainly distributed in laminae I and II of the dorsal horn. In the inset, a dense mesh of varicose processes is shown. (D) Electron micrograph of the same area of the dorsal horn shown in (C) after the gold-intensification procedure. A GFRa1-immunoreactive axon terminal is labeled by irregular electron-dense gold particles of 25–30 nm in size distributed along the plasma membrane. Scale bar: (C): 250 mm; inset in (C): 20 mm; (D): 200 nm. See http://extras.springer.com/ for the color version of this figure.

TrkB is the high-affinity tyrosine kinase receptor mediating BDNF cellular actions (31, 32). Three different trkB receptor isoforms are generated by alternative splicing of trkB mRNA (33, 34): the full-length trkB (fl-trkB) receptor and two truncated trkB receptors (tr-trkB; (33, 35)). All trkB isoforms share a common extracellular domain, whereas the fl-trkB receptor is the only one with the intracellular tyrosine kinase domain and is thus able to trigger the signal transduction pathways utilized by BDNF to exert its biological functions.

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2. Materials 2.1. Tissue Preparation for Preembedding Electron Microscopy with Fluoronanogold™

1. Sodium pentobarbital (30 mg/kg). 2. Phosphate buffer (PB): Prepare a stock solution “A” with 0.2 M NaH2PO4∙H2O and a stock solution “B” with 0.2 M Na2HPO4. Prepare working solution by adding 19 mL “A” and 81 mL “B” solutions to 100 mL of distilled water. Adjust pH to 7.4 if necessary. Store at room temperature. 3. Phosphate buffered saline (PBS): Prepare a 9‰ NaCl solution in 900 mL distilled water and 100 mL 0.2 M PB. Adjust pH to 7.4 if necessary. Store at 4°C. 4. Ringer solution: Dissolve 2 g NaCl, 0.0625 g KCl, and 0.125 g NaHCO3 in 237.5 mL distilled water. Add 14 mL PB 0.2 M. Adjust pH to 7.4. Store at 4°C. 5. Paraformaldehyde/glutaraldehyde fixative: prepare a 4% (w/v) paraformaldehyde solution (available from Sigma, St. Louis, MO), in PB. Dissolve the solution by carefully heating it with a stirring hot plate in a fume hood, then cool at room temperature and finally store at 4°C. Immediately before use, add 0.01–0.5% glutaraldehyde (from a 25% glutaraldehyde stock solution, available from Sigma). 6. Surgical instrumentation and peristaltic pump for perfusion. 7. Vibrating microtome (Leica Microsystems, Weitzlar, Germany).

2.2. Fluorescence Microscopy Labeling with Fluoronanogold™

1. 50 mM glycine solution in PBS. 2. 0.1%(v/v) Triton X-100 (TX) in PBS (PBS-0.1%TX). Store at 4°C. 3. 5% (v/v) Normal Serum (NS, available from Vector Laboratories, Burlingame, CA), made in the same species of the secondary antibody, diluted in PBS (PBS-5%NS) or PBS0.1%TX (PBS-5%NS-0.1%TX). As an alternative, use 6% (w/v) Bovine Serum Albumin, Fraction V (BSA, Sigma) in PBS (PBS-6%BSA) or PBS-0.1%TX (PBS-6%BSA-0.1%TX). 4. Primary antibodies: goat anti-SSTR2a (Santa Cruz, Biotech nology, Santa Cruz, CA); goat anti-GRFa1 (Neuromics, Minneapolis, MN), chicken anti-fl-trkB (Promega Corpora tion, Madison, WI), chicken anti-human BDNF (Promega Corporation, Madison, WI), rabbit anti-CGRP (obtained from JM Polak, Imperial College, UK), and rat anti-SP (BD Pharmingen, Franklin Lakes, NJ). 5. Secondary biotinylated anti-species antibody (available from Vector Laboratories) plus Alexa Fluor 488 (or 594)FluoroNanogold™-Streptavidin (Nanoprobes, Yaphank, NY) (see Note 1) or directly secondary FluoroNanogold™-anti-species Fab’-Alexa Fluor 488 (or 594) (Nanoprobes) (see Note 2).


2.3. Gold Enhancement, Tissue Processing and Ultrathin Sectioning for TEM

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1. Gold Enhancement kit (Nanoprobes): Prepare the mixture immediately before use. Store at 4°C. 2. Osmium tetroxide (available from Electron Microscopy Sciences, Hatfield, PA): Prepare an aqueous stock solution of 2% osmium tetroxide at least 24 h before use. Store at 4°C (see Note 3). 3. Osmium ferrocyanide: mix equal volumes of 2% aqueous osmium tetroxide and 3% potassium ferrocyanide in distilled water (see Note 4). 4. Maleate buffer stock solution: Dissolve 23.2 g. maleic acid in 200 mL distilled water and 8 g NaOH in 200 mL distilled water to obtain a 1 N solution. Mix the two solutions and dilute to 1000 mL with distilled water. 5. Maleate buffer pH 5.2: To 100 mL of stock solution, add approximately 14.4 mL 0.2 N NaOH and then dilute to 400 mL with distilled water. 6. Uranyl acetate (available from Electron Microscopy Sciences) in maleate buffer: Prepare a 1% (w/v) solution of uranyl acetate (available from Electron Microscopy Sciences) in maleate buffer pH 6. 100 mL of maleate buffer stock solution plus approximately 53.8 mL 0.2 N NaOH. Dilute to 400 mL with distilled water (see Note 5). 7. Ethanol 70%, Ethanol 90%, Absolute Ethanol. 8. Propylene oxide (available from Sigma Chemicals – see Note 6). 9. Base resin: Araldite M, Araldite M Hardener 964, and dibutylphthalate (all these reagents are available from Sigma Chemicals). Mix together 10 mL of Araldite M, 10 mL of Araldite M Hardener 964 and 0.25 mL of dibutylphthalate to obtain base resin. Prepare just before using. 10. Base resin plus accelerator: Prepare a solution of 2% Accelerator (available from Sigma Chemicals) in base resin. Put resin plus Accelerator at 37°C for 30 min before use. 11. Acetate foils (blank EM photographic negatives) or polyethylene capsules for section embedding (see Note 7). 12. Glass knife maker (Leica, Germany) for making glass knives starting from glass knife strips. 13. Glass knife strips (available from Electron Microscopy Sciences). 14. Diatome Diamond knife (Electron Microscopy Sciences) for ultrathin sections (optional). 15. Grid Coating Pen (Electron Microscopy Sciences). 16. Chloroform. 17. 200 mesh nickel grids (Electron Microscopy Sciences).

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18. Syringe filters – pore size 0.22 mm (available from Whatman, Maidstone, England). 19. Aqueous uranyl acetate solution: Prepare a saturated uranyl acetate (available from Electron Microscopy Sciences) solution in double-distilled water. Filter the solution with syringe filters – pore size 0.22 mm (see Note 5). 20. Lead citrate (Reynold’s): Dissolve 1.33 g Pb(NO3)2 in 30 mL double-distilled water, then add 1.76 g C6H5Na3O7∙2H2O. Mix for 30 min, and then add 8 mL NaOH 4 N to clarify the solution. Add double-distilled water to 50 mL final volume. Store the solution at 4°C, in the dark. Filter the solution with syringe filters – pore size before use. 21. Parafilm M. 22. Glass Petri dishes.

3. Methods The choice of the immunolabeling procedure for the ultrastructural localization of neuropeptides and their receptors is critical and should be carefully related to the structure, size, and molecular weight of the molecules that one wants to detect under the TEM. The preembedding immunoperoxidase method (PAP or ABC) that has been often used for the subcellular visualization of receptors, presents several disadvantages, primarily because the product of the reaction is often larger than the organelle(s) of interest, commonly masks the underlying cellular membranes, and thus does not lead to reliable identification of subcellular storage sites. An alternative technique is the preembedding Nanogold™ immunostaining procedure with ultrasmall gold clusters. In this case colloidal gold particles or gold clusters are silver-intensified before observation with TEM (5). Although this technique allows a good subcellular antigen localization, its main disadvantage is the difficulty in correlating light and electron microscopic observations. The Fluoronanogold™ preembedding technique is a rather new approach that, by using fluorescein-tagged antibodies conjugated with intensified small gold particles, allows a precise correlation between the fluorescence immunostaining observed at the light level and the gold-enhanced signal at the electron microscope, providing a useful tool for high-resolution correlative microscopy. The Fluoronanogold™ probes (Fig. 1) have different advantageous properties: (1) penetration into aldehyde-fixed tissues greater than colloidal gold particles (36), (2) penetration into tissues as readily as conventional immunofluorescent probes (7), (3) a covalent link between the fluorochrome, the antibody, and 1.4-nm gold particles


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that avoids the dissociation of antibodies from gold particles, as reported for conventional colloidal gold immunoprobes (37), (4) good fluorescent labeling that is not reduced from the proximity to the gold cluster (36), and (5) visualization of the labeling at both the fluorescence and electron microscope. By the use of these probes, receptor molecules are clearly localized over the plasma membrane of different neuronal processes that can be easily identified as being axonal or dendritic, pre or postsynaptic, etc. The content of labeled profiles (i.e., synaptic vesicles, mitochondria, etc.) is not obscured by immunostaining that takes the form of very electron-dense particles of relatively large sizes (25–30 nm) and irregular shapes. Gold-intensified particles are easily spotted under the TEM (Fig. 2) and immediately distinguishable from colloidal gold in multiple labeling procedures (Fig. 3). 3.1. Tissue Preparation for Preembedding Electron Microscopy with Fluoronanogold™

1. Under deep pentobarbital anesthesia (30 mg/kg), perfuse animals through the descending aorta with Ringer solution followed by cold fixative solution (see Note 8). 2. After perfusion, carefully dissect out areas of interest, cut them into small blocks (not exceeding 5 mm in thickness), and postfix for 2–4 additional hours in the same aldehyde mixture at 4°C. 3. Thoroughly rinse tissue blocks in PBS and cut sections with a vibratome at a thickness of 50–100 mm. 4. Store the sections in PBS and further process them as freefloating.

3.2. Florescence Microscopy Labeling with Fluoronanogold™

1. Incubate the sections for 5 min in 50 mM glycine in PBS (see Note 9). 2. Transfer the sections in PBS-5%NS or PBS-5%NS-0.1%TX (see Note 10) or alternatively in PBS-6%BSA or PBS-6%BSA0.1%TX for 30 min at room temperature in continuous agitation (see Note 11). 3. Incubate the sections with the primary antibody of interest (goat anti-SSTR2a diluted 1:100; goat anti-GRFa1, diluted 1:300; chicken anti-fl-trkB diluted 1:500) in the same buffer of step 2 overnight at 4°C under continuous agitation (see Note 12). 4. Wash in PBS three times for 10 min each. 5. Incubate for 1 h with an anti-species biotinylated secondary antibody at the right concentration in PBS at room temperature and then for 1 h with Alexa Fluor 488 (or 594)Fluoronanogold™-Streptavidin at the right dilution, or directly with a secondary Fluoronanogold™-anti-species Fab¢-Alexa Fluor 488 (or 594) antibody (see Note 13).

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Fig. 2. Ultrastructural localization of GFRa1 and SSTR2a receptors in mouse spinal dorsal horn using the Fluoronanogold™ labeling technique. (A) A GFRa1-immunoreactive type I glomerular terminal (GIa) is surrounded by several unlabeled dendrites (d). (B) A GFRa1-immunoreactive central terminal, in a characteristic type II glomerular arrangement (GII), is surrounded by unlabeled dendrites (d). Both in GIa and GII glomerular terminals, GFRa1 gold-intensified labeling is associated with the plasma membrane. Note in the insets that GFRa1-labeling is characterized by irregular electron-dense gold particles. The gold particle intensification was made directly on nickel uncoated grids, after the electron microscopy embedding procedure. The grids were rinsed in double-distilled water and then gold particles were intensified by the Gold Enhancement kit for 15 min, reaching a diameter of 25–30 nm. (C) An SSTR2a-immunoreactive nonglomerular axon terminal (At) contacts an unlabeled dendrite (d). (D) Two SSTR2a-immunolabeled dendrites (d1 and d2) are postsynaptic to an unlabeled axon terminal. SSTR2a immunostaining appears in the form of electron-dense particles of relatively large size and irregular shape, distributed along the plasma membrane. The gold particle intensification was made on free-floating sections before the electron microscopy embedding procedure. Sections were rinsed in PBS and then incubated in the Gold Enhancement kit for 15 min to intensify gold particles until a size of 25–30 nm. Scale bars: 300 nm; insets: 30 nm.


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Fig. 3. Ultrastructural localization of fl-trkB receptors, BDNF, CGRP and/or SP in mouse spinal dorsal horn using the Fluoronanogold™ labeling technique combined with conventional postembedding immunogold procedure. (A) A BDNF + CGRPimmunoreactive type Ib terminal (GIb) is surrounded by several dendrites (d), one of which is fl-trkB immunolabeled. BDNF (10-nm gold particles; see inset) and CGRP (20-nm gold particles; see inset) are costored in large granular vesicles (LGVs) of the type Ib glomerulus, while trkB is distributed along the dendrite plasma membrane. (B) A BDNF + CGRPimmunoreactive nonglomerular axon terminal (At) makes a synapse (arrowheads) with a fl-trkB-labeled dendrite (d). BDNF (10-nm gold particles) and CGRP (20-nm gold particles) are costored in LGVs of the axon terminal, while trkB is distributed along the intracellular aspect of the dendrite plasma membrane. Note in the inset that trkB-labeling is characterized by irregular electron-dense gold particles. (C) A fl-trkB + BDNF + SP-immunopositive GIb central terminal is surrounded by several unlabeled dendrites (d). BDNF (10-nm gold particles) and SP (20-nm gold particles) show a selective localization in LGVs, while trkB displays intracellular plasma membrane labeling. The two labels are easily distinguishable. BDNF and SP postembedding immunolabeling is associated with the presence of gold particles of very regular round shapes and constant size (10 and 20 nm respectively; arrows in the inset), while trkB preembedding immunostaining is in the form of very electron-dense particles of relatively large sizes (25–30 nm) and irregular shapes (arrowhead in the inset). Scale bars: 300 nm; 20 nm (inset in A); 30 nm (inset in B, C).

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6. Thoroughly wash the sections in PBS. 7. Check the specificity of immunostaining with a fluorescence microscope (see Note 14). An example of the fluorescence immunolabeling for GFRa1 receptors in mouse spinal cord lamina II is shown in Fig. 1C. 3.3. Gold Enhancement, Tissue Preparation, and Ultrathin Sectioning for TEM

Two alternative protocols are given since the gold enhancement step can be carried out either before embedding in resin, or directly on ultrathin sections on grids. In the preembedding protocol skip step 11. In the on-grid protocol skip steps 1 and 2. Both are equally suitable for analysis of CNS connectivity in term of morphological preservation and retention of antigenicity. Examples of the goldintensified probes before resin embedding are shown in Fig. 2C, D. Examples of the gold-intensified probes directly on grid after electron microscopy embedding are shown in Figs. 2A, B and 3. 1. Rinse free-floating sections in PBS and incubate in the Gold Enhancement kit for 5–15 min to intensify gold particles (Figs. 1B, D and 2C, D). The kit consists of four solutions that should be prepared immediately before use. Wait for 5 min after mixing solutions A (enhancer) and B (activator) and then add solutions C (initiator) and D (buffer) (see Note 15). The development is not highly light sensitive, so it may be conducted under normal room lighting. 2. Thoroughly wash gold-enhanced sections in PBS. 3. Postfix in osmium ferrocyanide for 1 h at 4°C. Remember to prepare the solution immediately before use. 4. Wash four times for 5 min each in maleate buffer pH 5. 5. Stain with 1% uranyl acetate in maleate buffer pH 6 for 1 h at 4°C. 6. Wash four times for 5 min each in maleate buffer pH 5. 7. Dehydrate in increasing concentrations of ethanol, starting from 70% ethanol for 5 min, followed by 95% ethanol for 5 min, and then 100% ethanol, three times for 10 min each. 8. Transfer sections in propylene oxide 2× 10 min each and then in propylene oxide-base resin 1:1 overnight at 37°C. 9. Flat-embed in base resin plus accelerator for 24 h at 60°C (see Note 6). 10. Cut ultrathin sections (70–80 nm) and collect them on precleaned uncoated nickel grids (see Note 16). 11. Rinse the grids in double-distilled water and intensify the gold particles with the Gold Enhancement kit for 5–15 min (Fig. 1B, D). The kit consists of four solutions that should be prepared immediately before use. Wait for 5 min after mixing solutions A (enhancer) and B (activator) and then add solutions


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C (initiator) and D (buffer). After mixing, a drop of the solution is placed on a sheet of parafilm in a glass Petri dish and the grids are floated on individual drops for the required time (see Note 15). The development is not highly light sensitive, so it may be conducted under normal room lighting. Once intensification is terminated thoroughly wash in double-distilled water. 12. Counterstain sections on grids with aqueous uranyl acetate for 1 min, wash in double-distilled water, counterstain with lead citrate for an additional minute, and wash again in doubledistilled water. 13. Allow grids to dry on filter paper and observe at TEM.

4. Notes 1. Alexa Fluor-FluoroNanogold™-Streptavidin contains a fluorochrome molecule, a 1.4-nm diameter gold particle and streptavidin, covalently bound together (see Fig. 1A). 2. Alexa Fluor-FluoroNanogold™-anti-species Fab¢ consists of a 1.4-nm diameter gold particle conjugated with a specific Fab¢ fragment and a fluorochrome. 3. Osmium tetroxide is highly poisonous, even at low exposure levels, in particular upon inhalation, and must be handled with precautions. Wear appropriate protective clothing, always work under a fume hood and discharge it in an apposite disposal container. 4. The osmium ferrocyanide solution must have a brown colour. If solution becomes black, discharge it. It is advisable to prepare fresh for each experiment. 5. Uranyl acetate is a nuclear fuel derivative, and thus its use and possession are sanctioned by international law. It is very toxic by ingestion and if inhaled as dust or by skin contact. Keep it in a special metal container, wear appropriate protective clothing when using, and carefully transfer it to an apposite disposal container and arrange its removal by a disposal company. 6. Propylene oxide is a probable human carcinogen; it is harmful by ingestion, inhalation and through skin contact. Wear appropriate protective clothing when using, always work under a fume hood, and discharge it in an apposite disposal container. 7. Different methods of section embedding are available according to specific needs. Acetate foils (blank EM photographic negatives) are used to flat embed large sections from which it is possible to select the area(s) of interest to be cut at the ultramicrotome. This method is really useful to preserve the

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right spatial orientation of the section for further TEM observations. Polyethylene capsules are used for embedding small pieces of tissue and when the orientation is not of primarily importance. In this case, tissue specimens tend to shift inside the capsule during polymerization, changing the initial orientation. It is convenient to place the surface of the specimen from which sections are desired next to the tip of the capsule. Different types of polyethylene capsules are available, depending on the diameter size, length of the capsule, shape of the tip (conical or pyramid) or the bottom (flat). 8. Fixation is carried out with minimal concentrations of glutaraldehyde to minimize the reduction of tissue antigenicity. The percentage of glutaraldehyde also depends on the primary antibody used (some antibodies need high concentrations of glutaraldehyde, whereas others do not work in presence of this aldehyde molecule). 9. Glycine solution in PBS is a useful step to inactivate residual aldehyde groups, which may still be present after aldehyde fixation without detrimental effects on tissue morphology. 10. To prevent nonspecific background staining (due to interactions between the primary antibody and the cell surface or intracellular structures of the specimen), it is important to use a nonimmune serum from the same animal species that produces the secondary antibody or a high concentration high-molecularweight protein solution, such as BSA. This avoids nonspecific positive staining due to binding of excess secondary antibody to components in the protein solution. 11. Triton X-100 has some detrimental effects on tissue ultrastructure, so use only if necessary and at very low concentration (do not exceed 0.1%). 12. The length and temperature of incubation should change depending on the primary antibody(ies) employed. Some antibodies work well only at room temperature; others need to stay at least for 24–48 h at 4°C. 13. Owing to some quenching of fluorescence by the gold particle, slightly higher concentrations (1:50–1:100) of Streptavidin/ secondary Fluoronanogold™-anti-species Fab’-antibody are recommended for incubations. Optimal antibody dilution should be determined by titration. 14. Alexa Fluor-488 has the maximum absorption near to 494 nm, and the maximum emission near to 519 nm. These values are very similar to those of fluorescein (green), so a fluorescein filter set is recommended for fluorescence observation. Alexa Fluor-594 has the maximum absorption near to 590 nm, and the maximum emission near to 619 nm. These values are very


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similar to those of rhodamine (red) or Texas Red, and so one of these filter sets is recommended for observation. 15. The enlargement of particles with the GoldEnhance kit depends on the length of application: 5–15 min will give rise to particles from 5 to 30 nm in size, so the intensification time should be adjusted time to time according to specific needs. 16. In order to enhance specimen adhesion to the grid, it is recommended to clean grids before using. Different cleaning protocols are available depending on the type of grids (nickel, copper, gold). In particular, for copper and nickel grids, such a treatment is effective for long periods in preventing the oxidation of the metal surface that tend to interfere with specimen adhesion to the grid. For nickel grids, the following cleaning procedure is recommended: wash grids with chloroform for 5 min in a sonicator, then in 1% glacial acetic acid in double-distilled water in a sonicator, followed by ethanol 95% for 1–2 min, and finally wash three times in double-distilled water. The use of nickel grids is recommended because they are chemically inert and avoid the risk of unwanted chemical reactions during the labeling procedures.

Acknowledgments This work was supported by grants of the Compagnia di San Paolo (Torino – Italy) and the Italian MiUR (Fondi PRIN 2008). References 1. Faulk, W. P. and Taylor, G. M. (1971) An immunocolloid method for the electron microscope. Immunochemistry 8, 1081–1083. 2. Priestley, J.V. (1984) Pre-embedding ultrastructural immunocytochemistry: Immunoenzyme techniques, in Immunolabelling for electron Microscopy (Eds J.M. Polak and I.M. Varndell), pp. 37–52, Elsevier, Amsterdam. 3. Merighi, A. (1992) Post-embedding electron microscopic immunocytochemistry, p. 51–87. In J. M. Polak and J. V. Priestley (Eds.), Electron Microscopic Immunocytochemistry. Oxford University Press, London. 4. Merighi, A. and Polak, J. M. (1993) Postembedding immunogold staining. p. 229–264. In A. Cuello (Ed.), Immunohistochemistry II. John Wiley & Sons, London. 5. Aimar, P., Lossi, L., and Merighi, A. (2002) Immunocytochemical labeling methods and related techniques for ultrastructural analysis

on neuronal connectivity, p. 161–180. In A. Merighi and G. Carmignoto (Eds.), Cellular and Molecular Methods in Neuroscience Research. Springer-Verlag, New York. 6. Powell, R. D., Halsey, C. M., Spector, D. L., Kaurin, S. L., McCann, J. and Hainfeld, J. F. (1997) A covalent fluorescent-gold immunoprobe: simultaneous detection of a pre-mRNA splicing factor by light and electron microscopy. J. Histochem. Cytochem. 45, 947–956. 7. Robinson, J. M. and Vandré, D. D. (1997) Efficient Immunocytochemical Labeling of Leukocyte Microtubules with FluoroNanogold: An Important Tool for Correlative Microscopy. J. Histochem. Cytochem. 45, 631–642. 8. Takizawa, T. and Robinson, J. M. (2000) FluoroNanogold is a bifunctional immunoprobe for correlative fluorescence and electron microscopy. J. Histochem. Cytochem. 48, 481–486. 9. Robinson, J. M., Takizawa, T., Pombo, A. and Cook, P. R. (2001) Correlative fluorescence

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and electron microscopy on ultrathin cryosections: bridging the resolution gap. J. Histochem. Cytochem. 49, 803–808. 10. Takizawa, T. and Robinson, J. M. (2003) Ultrathin Cryosections: An Important Tool for Immunofluorescence and Correlative Microscopy. J. Histochem. Cytochem. 51, 707–714. 11. Proudlock, F., Spike, R. C. and Todd, A.J. (1993) Immunocytochemical study of somatostatin, neurotensin, GABA, and glycine in rat spinal dorsal horn. J. Comp. Neurol. 327, 289–297. 12. Jiang, N., Furue, H., Katafuchi, F., and Yoshimura, M. (2003) Somatostatin directly inhibits substantia gelatinosa neurons in adult rat spinal dorsal horn in vitro. Neuroscience Research 47, 97–107. 13. Holstege, J. C., Jongen, J. L., Kennis, J. H., van Rooven-Boot, A. A., and Vecht, C. J. (1998) Immunocytochemical localization of GDNF in primary afferents of the lumbar dorsal horn. Neuroreport 9, 2893–2897. 14. Jongen, J. L., Dalm, E., Vecht, C. J., and Holstege, J. C. (1999) Depletion of GDNF from primary afferents in adult dorsal horn following peripheral axotomy. Neuroreport 10, 867–871. 15. Pezet, S., Malcangio, M., and McMahon, S.B. (2002) BDNF: a neuromodulator in nociceptive pathways? Brain Res. Brain Res. Rev. 40, 240–249. 16. Malcangio, M. and Lessmann, V. (2003) A common thread for pain and memory synapses? Brain-derived neurotrophic factor and trkB receptors. Trends Pharmacol. Sci. 24, 116–121. 17. Merighi, A., Carmignoto, G., Gobbo, S., Lossi, L., Salio, C., Vergnano, A.M., and Zonta, M. (2004) Neurotrophins in spinal cord nociceptive pathways. Prog. Brain Res. 146, 291–321. 18. Merighi, A., Salio, C., Ghirri, A., Lossi, L., Ferrini, F., Betelli, C., and Bardoni, R. (2008) BDNF as a pain modulator. Prog. Neurobiol. 85, 297–317. 19. Patel, Y. C., and Srikant, C. B. (1997) Somatostatin receptors. Trends Endocrinol. Metab. 8, 398–405. 20. Olias, G., Viollet, C., Kusserow, H., Epelbaum, J. and Meyerhof, W. (2004) Regulation and function of somatostatin receptors. J. Neurochem. 89, 1057–1091. 21. Segond Von Banchet, G., Schindler, M., Hervieu, G. H., Beckmann, B., Emson, P. C., and Heppelmann, B. (1999) Distribution of somatostatin receptor subtypes in rat lumbar spinal cord examined with gold-labelled soma-

tostatin and anti-receptor antibodies. Brain Research 816, 254–257. 22. Schulz, S., Schreff, M., Schmidt, H., Handel, M., Przewlocki, R. and Hollt, V. (1998a) Immunocytochemical localization of somatostatin receptor sst2A in the rat spinal cord and dorsal root ganglia. Eur. J. Neurosci. 10, 3700–3708. 23. Schulz, S., Schmidt, H., Handel, M., Schreff, M. and Hollt, V. (1998b) Differential distribution of alternatively spliced somatostatin receptor 2 isoforms (sst2A and sst2B) in rat spinal cord. Neurosci. Lett. 257, 37–40. 24. Jing, S., Wen, D., Yu, Y., Holst, P. L., Luo, Y., Fang, M., Tamir, R., Antonio, L., Hu, Z., Cupples, R., Louis, J. C., Hu, S., Altrock, B. W. and Fox, G. M. (1996) GDNF-induced activation of the ret protein tyrosine kinase is mediated by GDNFR-alpha, a novel receptor for GDNF. Cell 85, 1113–1124. 25. Treanor, J. J., Goodman, L., de Sauvage, F., Stone, D. M., Poulsen, K. T., Beck, C. D., Gray, C., Armanini, M. P., Pollock, R. A., Hefti, F., Phillips, H. S., Goddard, A., Moore, M. W., Buj-Bello, A., Davies, A. M., Asai, N., Takahashi, M., Vandlen, R., Henderson, C. E. and Rosenthal, A. (1996) Characterization of a multicomponent receptor for GDNF. Nature 382, 80–83. 26. Naveilhan, P., ElShamy, W. M. and Ernfors, P. (1997) Differential regulation of mRNAs for GDNF and its receptors Ret and GDNFR alpha after sciatic nerve lesion in the mouse. Eur. J. Neurosci. 9, 1450–1460. 27. Sanicola, M., Hession, C., Worley, D., Carmillo, P., Ehrenfels, C., Walus, L., Robinson, S., Kaworski, G., Wei, H., Tizard, R., Whitty, A., Pepinsky, R.B. and Cate, R. L. (1997) Glial cell line-derived neurotrophic factor-dependent RET activation can be mediated by two different cell-surface accessory proteins. Proc. Natl. Acad. Sci. USA 94, 6238–6243. 28. Durbec, P., Marcos-Gutierrez, C. V., Kilkenny, C., Grigoriou, M., Wartiowaara, K., Suvanto, P., Smtih, D., Ponder, B., Costantini, F., Saarma, M., Sariola, H. and Pachnis V. (1996) GDNF signaling through the Ret receptor tyrosine kinase. Nature 381, 789–793. 29. Trupp, M., Arenas, E., Fainzilber, M., Nilsson, A. S., Sieber, B. A., Grigoriu, M., Kilkenny, C., Salazar-Grueso, E., Pachnis, V. and Arumae, U. (1996) Functional receptor for GDNF encoded by the c-ret proto-oncogene. Nature 381, 785–789. 30. Worby, C. A., Vega, Q. C., Zhao, Y., Chao, H. H., Seasholtz, A. F. and Dixon, J. E. (1996) Glial cell line-derived neurotrophic factor signals through the RET receptor and activates

3 Fluoronanogold™ Labeling of Neuropeptides and Their Receptors mitogen-activated protein kinase. J. Biol. Chem. 271, 23619–23622. 31. Kaplan, D.R. and Stephens, R.M. (1994) Neurotrophin signal transduction by the Trk receptor. J. Neurobiol. 25, 1404–1417. 32. Kaplan, D.R. and Miller, F.D. (1997) Signal transduction by the neurotrophin receptors. Curr. Opin. Cell Biol. 9, 213–221. 33. Middlemas, D.S., Lindberg, R.A., and Hunter, T. (1991) trkB, a neural receptor protein-tyrosine kinase: evidence for a full-length and two truncated receptors. Mol. Cell Biol. 11, 143–153. 34. Barbacid, M. (1994) The Trk family of neurotrophin receptors. J. Neurobiol. 25, 1386–1403.

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35. Klein, R., Conway, D., Parada, L.F., and Barbacid, M. (1990) The trkB tyrosine protein kinase gene codes for a second neurogenic receptor that lacks the catalytic kinase domain. Cell 61, 647–656. 36. Takizawa, T., Suzuki, K. and Robinson, J. M. (1998) Correlative Microscopy Using FluoroNanogold on Ultrathin Cryosections: Proof of Principle. J. Histochem. Cytochem. 46, 1097–1102. 37. Kramarcy, N. R. and Sealock, R. (1991) Commercial preparations of colloidal gold-antibody complexes frequently contain free active antibody. J. Histochem. Cytochem. 39, 37–39.

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Chapter 4 Neuropeptide RNA Localization in Tissue Sections Marc Landry, Shérine Abdel Salam, and Marie Moftah Abstract In situ hybridization has become a routine technique to provide insights into RNA localization. However, different protocols exist for multiple purposes, and it is, therefore, important to clearly define specific needs to choose the most suitable one(s). For instance, in situ hybridization can target different types of RNA, including mRNA or small noncoding RNA such as micro RNA (miRNA). Detection protocols are developed for light or electron microscopy and can be combined with immunocytochemistry to study RNA coexpression with proteins or peptides. In this chapter, we present some protocols to illustrate the diversity of in situ hybridization methods. We focus on the detection of mRNA or miRNA and show that the protocols are quite similar but use dedicated probe types, namely, oligo- or riboprobes and locked nucleic-acid probes. Key words: mRNA, miRNA, In situ hybridization, Oligoprobes, Riboprobes, Locked nucleic-acid probes

1. Introduction The birth of new concepts in chemical neurotransmission such as cotransmission, volume transmission, or neuronal versatility frequently occurred thanks to the advances in neurocytochemical technologies. First, the identification and in situ localization of neurotransmitters, related enzymes, and receptors greatly benefited from the development of more and more specific and sensitive cytochemical techniques: after histochemistry of acetylcholinesterase, the chemical neuroanatomy of monoamine neurons originated from histofluorescence methods. But it is the development of immunocytochemistry (ICC) at optic and electron microscope levels that has given a universal and versatile tool for such cartographies, first of neuropeptides, then of enzymes of neurotransmitter


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metabolism, and finally of the neurotransmitters themselves after the pioneering work of Steinbusch for serotonin [1). However, these mappings, completed by that of receptors through ligand binding or ICC, do not allow, for example, to assume that the protein molecules identified are truly synthesized within the sites where they are detected. The in situ hybridization (ISH) of mRNAs leads to the detection and cellular localization of another step of the protein and peptide synthesis. However, ISH is not restricted to the detection of coding RNAs and can also be applied to the visualization of small noncoding RNAs that have essential and diverse cellular functions. They may function as catalysts, adapters, and guides in processes such as pre-mRNA splicing, translation, and amino-acid transfer. Other categories of noncoding RNAs act as regulators of gene expression, such as micro RNAs (miRNA). MiRNA are implicated in the pathogenesis of many human diseases, including cancers and neurological affections (2). It is thus of the utmost importance to detail the tissue and cellular distribution of miRNA to provide new insights into their regulatory roles in pathological conditions. As for coding RNAs, ISH is the most suitable tool for the localization of noncoding RNAs, including miRNA. We describe and comment here some protocols for the detection of mRNA and miRNA with light and electron microscopy. We also give some clues for the combination of ISH with other histochemical approaches, i.e., ICC.

2. Materials 2.1. Preparation of Biological Samples

1. Cryostat or paraffin embedding station and microtome (see Note 1). 2. Super Frost Plus Gold slides (Menzel-Glaser, Braunschweig, Germany). 3. Water bath (only if using paraffin sections). 4. Slide oven (only if using paraffin sections).

2.2. Probes (see Note 2)

1. DNA oligoprobes (25–30 nucleotides). 2. RNA riboprobes. 3. Locked nucleic acid (LNA™) probes prelabeled with digoxigenin or fluorochromes (Exiqon, Vedbaek, Denmark).

2.2.1. Probe Labeling and Purification (see Note 3)

1. dNTP (Roche Diagnostics, Mannheim, Germany). 2. Terminal transferase or RNA polymerase (available from Roche Diagnostics).

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3. Labeling buffer (see below). 4. Ethanol (molecular biology grade). 5. LiCl. 6. EDTA or DNase I (available from Roche Diagnostics). 7. SP6/T7 RNA polymerase labeling kit (Roche Diagnostics) for digoxigenin labeling of riboprobes. 8. Digoxigenin-11-dUTP (Roche Diagnostic) for digoxigenin labeling of oligoprobes. 9. Brightstar Psoralen Kit (BioRad, Hercules, CA) for biotin labeling of oligo- or riboprobes. 2.3. Reagents and Buffers 2.3.1. Tissue Pretreatments

1. 0.1 M and 0.01 M phosphate-buffered saline (PBS – available from Sigma Chemicals, St. Louis, MO). 2. Phosphate buffer (PB): Prepare a stock solution “A” with 0.2 M NaH2PO4∙H2O and a stock solution “B” with 0.2 M Na2HPO4. Prepare working solution by adding 19 mL “A” and 81 mL “B” solutions to 100 mL of distilled water. Adjust pH to 7.4 if necessary. Store at room temperature. 3. Proteinase K stock solution: 1 mg/mL in water. 4. Acetylation solution: Add 1.165 mL triethanolamine to 100 mL distilled water and then add 250 mL acetic anhydride. 5. Ethanol. 6. Chloroform. 7. 4% paraformaldehyde in 0.1 M phosphate buffer.

2.3.2. Hybridization

1. Standard saline citrate (SSC 1×): 0.15 M NaCl, 0.015 M Na citrate, pH 7.2 (available from Sigma Chemicals). 2. Denhardt’s solution: 0.02% bovine serum albumin (BSA – available from Sigma Chemicals), 0.02% Ficoll, 0.02% polyvinylpyrrolidone. 3. Hybridization buffer for oligoprobes and LNA probes: 50% deionized formamide, 4× SSC, 1× Denhardt’s solution, 0.02 M NaPO4, pH 7.0, 1% N-lauroylsarcosine, 10% dextran sulfate (to be omitted when using free-floating sections), 500 mg/L denatured salmon testis DNA. 4. Hybridization buffer for riboprobes: 50% deionized formamide, 5× SSC, 50 mg/mL yeast RNA, 1% sodium dodecyl sulfate (SDS). 5. RNAse A (available from Roche Diagnostics): 100 mg/mL in 1× SSC.

2.3.3. Posthybridization Rinsing

1. Posthybridization rinsing solution for oligo- and riboprobes: 1× SSC.

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2. Posthybridization rinsing solution 1 for LNA probes: SSC 1×, 50% formamide, 0.1% Tween. 3. Posthybridization rinsing solution 2 for LNA probes: SSC 0.2×. 2.3.4. Detection

1. TNT: 0.1 M Tris–HCl, 0.15 M NaCl, 0.5% blocking reagent (provided in Perkin Elmer Tyramide system amplification kit). 2. TNB: 0.1 M Tris–HCl, 0.15 M NaCl, 0.05% Tween. 3. BSA. 4. Buffer A: 1 M NaCl, 200 mM MgCl2, 0.1 M Tris–HCl, pH 7.5. 5. Buffer B: 1 M NaCl, 5 mM MgCl2, 0.1 M Tris–HCl, pH 9.5. 6. Alkaline phosphatase substrate: 5-bromo-4-chloro-3- indolylphosphate (0.45%, BCIP, Roche Diagnostics) and Nitro-blue tetrazolium chloride (0.35%, NBT, Roche Diagnostics) in buffer B. 7. 0.05 M Tris–HCl, pH 7.6. 8. 0.025% 3,3¢ diaminobenzidine in 0.05 M Tris–HCl, pH 7.6.

2.3.5. Mounting or Embedding

1. Antifading mounting medium (Dako, Glostrup, Denmark). 2. Osmium tetroxide. 3. Epon resin. 4. Uranyl acetate.

2.4. Antibodies and Detection Systems

1. HRP- or AP-conjugated streptavidin (Roche Diagnostics). 2. HRP-conjugated anti-DIG (Roche Diagnostics). 3. Avidin/biotin blocking kit (Vector laboratories, Burlingame, CA). 4. Tyramide system amplification kit (TSA): FITC- or biotin conjugated tyramide (TSA-Plus Fluorescein (or Biotin) system-tyramide signal amplification Kit – Perkin Elmer, Waltham, MA). 5. Ultrasmall gold particles-conjugated anti-DIG antibody (Nanoprobes, Yaphank, NY, USA). 6. Silver enhancement kit for electron microscopy (Nanoprobes). 7. Anti-synaptoporin antibody (Synaptic systems, Goettingen, Germany). 8. AlexaFluor 568-anti-mouse secondary antibody (Invitrogen, Carlsbad, CA).

2.5. Disposables

1. Eppendorf tubes. 2. Plastic cuvettes. 3. 12-well tissue culture plates. 4. 24-well tissue culture plates.

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3. Methods Various protocols are available to perform ISH with different probe types, as well as with different detection systems. Important criteria drive the choice of the method. In particular, the nature (mRNA or miRNA) and relative abundance of target RNA will help defining the type of probe (oligoprobe, riboprobe, or LNA probe) as well as the detection system to be applied. Oligoprobes are most convenient, as they do not need so much precaution such as RNAse-free medium for riboprobes (Fig. 1). However, riboprobes remain known to be more sensitive because of their RNA nature, and the high Tm of the hybrids made with riboprobes. As described above, LNA probes are most suitable for short targets such as miRNA (Fig. 2). Another element that guides the choice of the method is the possible requirement for multiple labeling. A double labeling experiment needs that the two reactions are compatible in terms of fixation, pretreatments, temperature of reaction, and detection. There is no major hindrance to the detection of two mRNAs if the probes are of the same type. By contrast, a good preservation of antigenicity during the ISH reaction is a major condition for coupling ICC and ISH. In addition, the signals given by each technique should be readily distinguishable. Moreover, the detectable molecule, i.e., the reporter molecule, introduced chemically or enzymatically should not interfere with the hybridization reaction or the stability of the resulting hybrid. Also, it should remain accessible to the detection system used later on. If two enzymatic labelings have to be performed, the reaction products must be of sharp different colors (e.g., purple and brown) (3). New protocols have arisen that allow multiple fluorescent detections (4, 5). To improve the sensitivity of detection systems, it is often required to amplify the signal. One of the most effective procedures uses tyramide signal amplification (TSA). TSA may actually provide up to a 100-fold increase in signal as compared with conventional detection systems. This detection system is based on the reaction of peroxidase with hydrogen peroxide and the phenolic part of tyramide, which produces a quinone-like structure bearing a radical on the C2 group. This activated tyramide then covalently binds to tyrosine in close vicinity to the peroxidase (6). Such a reaction can be applied either to ICC or ISH detection with a similar protocol after peroxidase is bound to the target via an antibody. The tyramide can be conjugated to a fluorochrome or a hapten. Hence, direct or indirect detection of enzymatically deposited tyramides is possible and can be coupled to another labeling (3).

Fig. 1. (a, b) Nonradioactive ISH with oligoprobes was applied to the detection of mRNA coding for the GABAB receptor subunit B1. TSA detection provides a high signal-to-noise ratio that allows identifying GABAB-containing neurons (arrowheads) throughout the dorsal horn of the rat spinal cord (a). At a higher magnification (b), subcellular distribution of B1 mRNA can be detailed and ISH shows the presence of transcripts in neuronal processes (double arrows). (c) ISH with oligoprobes was used to detect mRNA coding for a neuropeptide, galanin, with electron microscopy. The digoxigenin reporter molecule was visualized with silver-enhanced ultrasmall gold particles. High magnification shows the location of galanin mRNA (arrows) on the cytoplasmic side of the endoplasmic reticulum membrane in neurons of the rat locus coeruleus. B1: GABAB1 subunit of the GABAB receptor; AP-Dig: alkaline phosphatase detection of the digoxigenin reporter molecule; USgold-Dig: silver enhancement of ultrasmall gold particles coupled to anti-digoxigenin antibody. Bar: 50 mm (a, b); 200 nm (c).

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Fig. 2. (a, b): ISH with LNA probes was applied to the detection of miRNA miR-134 in the rat spinal cord. Hybrids were visualized with a fluorescent TSA method. Signal was intense on the ventral horn of the spinal cord, and especially in presumed motoneurons (arrowheads). At higher magnification, labeling appears punctuate both in cell bodies and neuronal processes (double arrows). (c) ISH with LNA probes was coupled to ICVC for synaptoporin, a marker of nociceptive sensory fibers, in the dorsal horn of the spinal cord. No colocalization can be seen while some apposition between immunopositive nerve endings and miR-134 containing cell bodies are detected (arrows). (d) ISH with LNA probes was detected with silver enhanced ultrasmall gold particles in the rat spinal cord. Hybrids are observed in lysosomes, corresponding to degrading compartments where miRNA/mRNA hybrids are likely to be addressed. LNA Locked nucleic acid, TSA Detection with Tyramide System Amplification of digoxigenin-labeled LNA probe, USgold ultrasmall gold detection of digoxigenin-labeled LNA probe, L Lysosome. Bar: (a) 50 mm; (b) 25 mm; (c) 10 mm. See http://extras.springer.com/ for the color version of this figure.

At the electron microscope level, an important feature is the resolution of the reporter molecules. Autoradiographic studies require the use of 3H instead of 35S to limit the diffusion of the signal. Nevertheless, beside the difficulties of such a technique, the resolution remains poor. Also, enzymatic products are known to diffuse throughout the cytoplasm. To date, the use of silver-enhanced gold particles represents the marker ensuring the best resolution for preembedding ICC and ISH (7) studies, provided that the duration and temperature of the silver-enhancement procedure are well optimized.

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3.1. Direct Chromogenic Detection of Oligoprobes (mRNA)

1. Air-dry sections at room temperature (RT) for 2 h.

3.1.1. Prehybridization

5. Quickly wash the sections in RNase-free water.

2. Fix with 4% paraformaldehyde for 20 min (see Note 4). 3. Incubate with proteinase K diluted 1:2,000 for 15 min in PB (see Note 5). 4. Stop reaction with 1 M Tris–HCl, 0.5 M EDTA (v/v). 6. Dehydrate through graded ethanols, once in 50, 70, 95% ethanol, and twice in 100% ethanol, 1 min each. 7. Incubate in chloroform for 5 min (for lipid removing). 8. Wash twice in 100% ethanol and once in ethanol 95%. 9. Air-dry the slides for at least 2 h.

3.1.2. Hybridization

1. Before hybridization, incubate the hybridization buffer at 42°C for 1 h. 2. Incubate the sections at 42°C overnight in hybridization buffer containing the oligoprobe at 2 pmol/mL (labeled probe from a stock at 1 pmol/mL – see Notes 6 and 7).

3.1.3. Posthybridization and Detection

1. Wash the sections 4 × 15 min each in SSC 1× at 55°C, then for 30 min in SSC 1× at RT. 2. Incubate in buffer A/BSA (1%) pH 7.5 for 30 min at RT. 3. Incubate with the alkaline phosphatase-conjugated anti-digoxigenin antibody 1:5,000 in buffer A/BSA for 30 min at RT. 4. Wash for 10 min in buffer A and then for 10 min in buffer B pH 9.5 at RT. 5. Detect alkaline phosphatase activity with BCIP/NBT in 5 mL developing solution, pH 9.5 for few hours (see Note 8). 6. Wash thoroughly in water. 7. Air-dry the sections for at least 2 h. 8. Mount in PBS–glycerol (v/v) solution.

3.2. Chromogenic Detection of Riboprobes with a TSA Enhancement Protocol (mRNA) 3.2.1. Preparation of Labeled RNA Probes 3.2.2. Single-Labeling In Situ Hybridization

1. Synthesize the riboprobe using SP6 (T7 for sense riboprobe) RNA polymerase. Reporter molecules (digoxigenin or biotin) are incorporated, respectively, by adding 11 digoxigenin-dUTP into the medium or by using a Brightstar Psoralen Kit (BioRad, Hercules, CA) according to the manufacturer’s instructions, for 45 min under 365 nm UV light (3).

1. Equilibrate the sections to RT. 2. Fix in 4% paraformaldehyde for 20 min (see Note 4). 3. Deproteinize with proteinase K 10 mg/mL for 15 min (see Note 5). Stop reaction with 0.5 M EDTA in 1 M Tris–HCl, pH 8 (v/v).

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4. Dehydrate through Subheading 3.1.1).

graded

ethanols

(see

step

6

in

5. Delipidate the sections in chloroform for 5 min. 6. Rehydrate in 70% ethanol and air-dry. 7. Hybridize the sections overnight at 65°C with 10 ng of digoxigenin- or biotin-labeled probes diluted in hybridization buffer. 8. Rinse slides 2 × 5 min each in 1× SSC. 9. Treat with RNAse A for 30 min at 37°C. 10. Wash for 5 min in SSC 1×, 5 min in SSC 0.5× at RT, 30 min in SSC 0.1× at 65°C two times each, and 15 min in SSC 0.1× at RT (see Note 9). 11. Transfer the sections to buffer A containing 0.5% BSA for 30 min. 3.2.3. Detection

1. Incubate the sections overnight at 4°C with sheep HRPconjugated anti-digoxigenin, or anti-biotin antibody at (1/1,1000) dilution. 2. Rinse in TNT containing 0.5% blocking reagent (TNB). 3. Incubate in biotinylated tyramide diluted 1:500 in the provided diluent for 10 min at 37°C (see Note 10). 4. Rinse in TNT. 5. Incubate in TNB with alkaline phosphatase-conjugated streptavidin diluted 1:5,000 for 30 min. 6. Rinse 3 × 10 min each in buffer A and then for 10 min in buffer B. 7. Detect alkaline phosphatase with NBT/BCIP (see Note 8). 8. Rinse and mount the sections as described above (steps 6–8 in Subheading 3.1.3).

3.3. Double Chromogenic Detection of Riboprobes (mRNA)

1. Synthesize digoxigenin and biotin labeled probes (see Subheading 3.2.1). 2. Perform section pretreatment as in the single labeling protocol (steps 1–6 in Subheading 3.2.2). 3. Hybridize the sections with a mixture of both probes (see step 7 in Subheading 3.2.2). 4. Carry out posthybridization rinsing (see steps 8–11 in Subheading 3.2.2). 5. First detect the biotin-labeled probe was first detected using HRP-conjugated streptavidin (1/200, 2 h in TNB). 6. Visualize the biotin-labeled probe with diaminobenzimide. 7. Block unreacted biotin with the avidin/biotin blocking kit prior to the second detection.

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8. Incubate the sections overnight at 4°C with sheep HRP conjugated anti-digoxigenin, or anti-biotin antibody diluted 1,000. 9. Rinse in TNT containing 0.5% blocking reagent (TNB). 10. Incubate in biotinylated tyramide diluted 1:500 in the provided diluent for 10 min at 37°C. 11. Rinse in TNT. 12. Incubated in TNB with alkaline phosphatase-conjugated streptavidin diluted 1:5,000 for 30 min. 13. Rinse 3 × 10 min each in buffer A and then for 10 min in buffer B. 14. Perform colorimetric detection with NBT/BCIP. This double detection results in brown (for TSA) and purple (for alkaline phosphatase) staining, respectively. 15. Rinse and mount the sections as described above (steps 6–8 in Subheading 3.1.3). 3.4. Fluorescent Detection of LNA Probes with a TSA Enhancement Protocol (miRNA)

1. Thaw the Eppendorf tubes containing frozen sections for ISH (see Note 11). Pour thawed floating sections in a 12-well tissue culture plate containing a plastic cuvette with a perforated bottom. 2. Rinse the sections with 0.01 M PBS twice. 3. Prepare the acetylation medium. Care should be taken not to add the acetic anhydride until the last minute before incubation. 4. Incubate the sections in the acetylation medium for 10 min at RT. 5. Rinse 3 × 5 min each with 0.01 M PBS. 6. Add 2 mM LNA probe to the hybridization buffer and incubate the mixture for 5 min at 70°C. 7. Transfer the tubes on ice for 3 min to unwind the probe. 8. Transfer the floating sections into a sterile 24-well tissue culture plate and incubate with the probe hybridization buffer overnight at 55°C. 9. Rinse the sections in a 12-well plate 2 × 20 min each with rinsing solution 1 at 65°C. 10. Rinse for 10 additional minutes with rinsing solution 2 at 65°C. 11. Rinse again 3 × 10 min with TNT at RT. 12. Block sections for 30 min in the TNB blocking solution in a 24-well culture plate. 13. Incubate the floating sections with the anti-digoxigenin HRPconjugated antibody diluted 1:5,000 in TNB.

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14. Rinse in a 12-well plate 3 × 10 min each with TNT. 15. Incubate in a 24-well culture plate with tyramide FITC diluted 1:50 in its amplification diluent for 10 min at RT. 16. Rinse 3 × 10 min each in a 12-well plate with TNT at RT. 17. Transfer the sections on slides and let them dry for 30 min in a 37°C incubator. 18. Mount them with coverslips using an antifading agent. 19. Store the slides at −20°C in dark until examination. 3.5. Double Fluorescent Labeling Combining ICC and ISH with LNA Probes (miRNA)

1. Repeat steps 1–11 in Subheading 3.4. 2. Dilute primary antibody at optimal titer in TNT + 1% BSA. 3. Incubate the sections overnight with the primary antibody in a 24-well plate at 4°C. 4. Rinse 3 × 10 min each with TNT in a 12-well culture plate. 5. Block the sections for 30 min in the TNB blocking solution in a 24-well culture plate. 6. Incubate floating sections with the anti-digoxygenin HRPconjugated antibody diluted 1:100 and Alexa Fluor 568 antispecies diluted 1:500 in TNB for 3 h at RT (in dark). 7. Rinse in a 12-well plate 3 × 10 min each with TNT. 8. Incubate in a 24-well culture plate with Tyramide FITC diluted 1:50 in its amplification buffer for 10 min at RT. 9. Rinse 3 × 10 min each in a 12-well plate with TNT at RT. 10. Transfer the sections on slides and let them dry for 30 min in a 37°C incubator. 11. Mount them with coverslips using an antifading agent. 12. Store the slides at −20°C in dark until examination.

3.6. ISH at the Electron Microscope Level with Silver-Enhanced Gold Particles (mRNA and miRNA)

1. Use cryoprotected and cryocut chemically fixed cells or tissue sections. 2. Hybridize digoxigenin labeled oligoprobes or LNA probes according to the above methods. 3. Carry out posthybridization washings. 4. Preincubate in a blocking solution (PBS-BSA, gelatin, normal goat serum) 2 h at RT. 5. Incubate with 1 nm gold-conjugated anti-digoxigenin antibody diluted in the blocking solution for 30 min at RT. 6. Rinse in PBS. 7. Postfix in paraformaldehyde 4% in PBS for 10 min at RT. 8. Rinse in PBS. 9. Perform silver enhancement according to the manufacturer instructions, rinse in water and PB (see Note 12).

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10. Postfix in OsO4 1% diluted in PB for 10 min at RT. 11. Embed in resin. 12. Cut ultrathin sections and counterstain with uranyl acetate. 13. Observe under a TEM.

4. Notes 1. Most commonly, ISH is carried out on tissue sections from paraffin-embedded material or on frozen sections. Paraffin embedding leads to unsurpassed histological preservation. It also allows storage at room temperature at low cost. The main disadvantage is a reduced sensitivity as compared with frozen sections. The latter are suitable for chemically unfixed material, lack of fixation offering an increase in sensitivity. Moreover, frozen tissue provides better probe access to target RNA that further increases when using oligoprobes (8). Altogether, frozen sections make ISH more sensitive than with any other type of sections. It is of course possible to use frozen sections of fixed material. In this case, animals are perfused with 4% paraformaldehyde, tissues are dissected out, postfixed, cryoprotected with 10% glycerol and 25% saccharose, and embedded in Tissue-Tek. Use a cryostat to cut sections (14 mm thickness), thaw-mount them on Super Frost Plus Gold slides, and, if necessary, store at −80°C. 2. Either DNA or RNA probes can be used. The former type is made of oligoprobes usually ranging between 25 and 50 nucleotides. The latter corresponds to riboprobes whose length depends on the cDNA that has been subcloned. The detection of miRNA relies on shorter probes, between 17 and 25 nucleotides. The short length of miRNA makes traditional oligoprobes unsuitable for ISH because of the low melting temperature (Tm) of formed hybrids. LNA is a modification that dramatically increases the Tm of duplexes, allowing their use even in stringent conditions. To increase sensitivity, LNA probes are double labeled at both 5¢ and 3¢ ends, thus improving the detection of low abundance miRNA. The signal-tonoise ration benefits from a cooperative effect of the two digoxigenin labeled and makes the detection of lowly expressed targets possible. 3. To label oligoprobes, use twenty-five 100 pmoles of probe and nonradioactive (digoxigenin, biotin, fluorescein) dNTP (Roche Diagnostics). For riboprobes 1 g template is transcribed, and reporter molecule can be inserted by using nonradioactive NTP or by performing chemical addition.

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4. Fixation is the first and a crucial step for ISH and ICC, since it must allow both a good morphological preservation and a good efficiency of the histochemical reactions, two criteria being most often contradictory. Two kinds of fixative can be considered: chemical and physical fixatives. Among the former, precipitating fixatives give a nice morphology but are generally not well suited for RNA. Nonprecipitating aldehydic fixatives have been introduced in ISH studies in the eighties, and 4% paraformaldehyde is now the most common fixative for both ICC and ISH and their combination. The fixation by the formaldehyde or its derivatives is largely reversible. Thus, the chemical properties of nucleic acids are not modified by the fixation and hybrids Tm remains unchanged upon aldehyde fixation. Despite these favorable properties, it is preferable to use slight concentrations of formaldehyde on nervous tissue to allow a better accessibility of the probes to the target. The glutaraldehyde is known to be of a poor interest in ISH studies since it creates a dense network of double bonds making difficult the probe penetration. Finally, the use of picric acid should be banned since it modifies chemical properties of the probe and can alter it. Physical fixations have also been used and proved to be of a great interest at least for double ISH. Thus, drying is a very easy way to fix nervous tissues and air-dried cryostat sections (8) or cell cultures (9) are more sensitive to mRNA detection than fixed material. Also, microwave heating has been reported to improve hybridization efficiency without altering tissue morphology (10). 5. Tissue section pretreatments are performed to facilitate probe penetration and access to the target. Actually, RNA can be associated to many RNA-binding proteins that make the access difficult. Protease treatment allows exposing cellular RNAs to the probe. However, overdigestion makes morphology suboptimal and hinder cellular, and of course subcellular, localization. 6. To increase the sensitivity of ISH with oligoprobes, a cocktail of several oligoprobes, all complementary to the same TNA species, can be applied. Every probe hybridizes to a specific sequence of the target and contributes to the signal (11). 7. Various controls should be conducted when performing ISH to test the specificity of probes, antibodies and detection systems. Positive controls should monitor the quality of sample preparation and tissue; they also confirm that all reagents are properly prepared. A positive control could be a probe or antibody that is highly expressed in the cell type considered. Probes against ribosomal RNA are good positive controls for ISH. Negative controls are used to assess the level of nonspecific background. The best control for ISH is to use probes that are known to work but not expressed in the tissue being analyzed.

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For LNA probes against miRNA, control probes include probes with few mismatches, probes with scrambled sequence, and pretreatment of the samples with RNase to ensure that probes hybridize to RNAs. 8. Incubation time in NBT/BCIP must be adjusted according to the probe used and the RNA targeted. Typically, incubation time ranges between 2 and 3 h, but it can increase up to36 h. One of the major problems of long incubation time is a strong background. To reduce background, and therefore to increase signal-to-noise ratio, levamisole is used to block endogenous alkaline phosphatases before applying digoxigenin-conjugated reagents (levamisole solution: 1 mM levamisole-containing buffer A). 9. Posthybridization rinsing is important, especially when using riboprobes or LNA probes, and rinsing that are described in protocol are only indicative. An alternative washing protocol for riboprobes can be as follows: two times 30¢ at 70°C (50% formamide; 5× SSC, 1% SDS), two times 30¢ at 65°C (50% formamide; 2× SSC). 10. It is important for reducing background when using TSA to block endogenous peroxidase by preincubating the sections in H2O2 at 2% in PB or Tris buffer. 11. When using chemically fixed tissue, ISH is preferably performed on free-floating sections rather on slide attached sections, to further increase sensitivity. The protocols described here are intended for free-floating sections, but they can be easily adapted to thaw-mounted sections. In this case, incubations are done by applying drops of solution (typically 200 mL per slide) on sections surrounded by a repellant medium. ISH can be also applied on adherent cell types including primary cell cultures (9) with no or minimal modifications as compared to tissue sections. 12. Overamplification is another source of nonspecific staining when using the silver enhancement procedure. Silver enhancement time should then be adapted to every experiment. References 1. Steinbusch, H.W., Verhofstad, A.A., and Joosten, H.W. (1978) Localization of serotonin in the central nervous system by immunohistochemistry: description of a specific and sensitive technique and some applications. Neuroscience 3, 811–819. 2. Liu, X., Fortin, K., and Mourelatos, Z. (2008) MicroRNAs: biogenesis and molecular functions. Brain Pathol. 18,113–121. 3. Landry, M., Bouali-Benazzouz, R., El Mestikawy, S., Ravassard, P., and Nagy, F. (2004)

Expression of vesicular glutamate transporters in rat lumbar spinal cord, with a note on dorsal root ganglia. J. Comp. Neurol. 468, 380–394. 4. Landry, M., Vila-Porcile, E., Hökfelt, T., and Calas, A. (2003) Differential routing of coexisting neuropeptides in vasopressin neurons. Eur. J. Neurosci. 17, 579–589. 5. de Planell-Saguer, M., Rodicio, M.C., and Mourelatos, Z. (2010) Rapid in situ codetection of noncoding RNAs and proteins in cells and formalin-fixed paraffin-embedded tissue

4 RNA Localization sections without protease treatment. Nat. Protoc. 5, 1061–1073. 6. van Gijlswijk, R.P., Zijlmans, H.J., Wiegant, J., Bobrow, M.N., Erickson, T.J., Adler, K.E., Tanke, H.J., and Raap, A.K. (1997) Fluorochrome-labeled tyramides: use in immunocytochemistry and fluorescence in situ hybridization. J. Histochem. Cytochem. 45, 375–382. 7. Vila-Porcile, E., Xu, Z.Q., Mailly, P., Nagy, F., Calas, A., Hökfelt, T., and Landry, M. (2009) Dendritic synthesis and release of the neuropeptide galanin: morphological evidence from studies on rat locus coeruleus neurons. J. Comp. Neurol. 516, 199–212. 8. Dagerlind, A., Friberg ,K., Bean, A.J., and Hökfelt, T. (1992) Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry. Histochemistry 98, 39–49.

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9. Kerekes, N., Landry, M., and Hökfelt, T. (1999) Leukemia inhibitory factor regulates galanin/galanin message-associated peptide expression in cultured mouse dorsal root ganglia; with a note on in situ hybridization methodology. Neuroscience 89, 1123–1134. 10. Lan, H.Y., Hutchinson, P., Tesch, G.H., Mu, W., and Atkins, R.C. (1996) A novel method of microwave treatment for detection of cytoplasmic and nuclear antigens by flow cytometry. J. Immunol. Methods. 190, 1–10. 11. Trembleau, A., Roche, D. and Calas, A. (1993) Combination of non-radioactive and radioactive in situ hybridization with immunohistochemistry: a new method allowing the simultaneous detection of two mRNAs and one antigen in the same brain tissue section. J. Histochem. Cytochem. 41, 489–498.

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