Peptide Receptors, Part II (Handbook of Chemical Neuroanatomy)

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PEPTIDE RECEPTORS PART II

This Page Intentionally Left Blank

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

Volume 20

PEPTIDE RECEPTORS PART II Editors:

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

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

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

2003

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

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

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Notice No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drugs dosages should be made. First edition 2003 Library of Congress Cataloging in Publication Data A catalog record from the Library of Congress has been applied for. British Library Cataloguingin Publication Data Peptide receptors Part 2 editors, R. Q u i r i o n , A. B j o r k l u n d , T. ( H a n d b o o k of c h e m i c a l n e u r o a n a t o m y ; v. 20) l.Neuropeptides - Receptors I . Q u i r i o n , Remi, 1955- I I . B j o r k l u n d , A n d e r s , I I I . H o k f e l t , T. (Tomas) 573.8'6465

ISBN: ISBN: ISSN:

Hokfelt.

-

1945-

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

O The paper used in this publication meets the requirements of ANSI/NISO Z39.48-1992 (Permanence of Paper). Printed in The Netherlands.

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

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

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

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

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

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

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

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

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

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

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

vi

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

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

vii

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Contents

List of Contributors P refa c e

I.

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

4.

5. 6. 7. 8. II.

vii

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

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

1 1

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

31

31 31 32 33 34 ix

.

.

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

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

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

76

5.8.

,

7. 8. 9. III.

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

2.

,

4. 5.

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

76 78 85 86 92 92

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

6.

7.

xii

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

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

8.

IV.

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

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

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

195

1. 2.

3.

4.

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

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

V.

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

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

OREXIN RECEPTORS M. YANAGIS AWA

245

.

xiv

-

T. SAKURAI, G. HERVIEU AND

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

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

3.

4.

5. 6. 7. 8. 9.

10. 11. 12. 13. VI.

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

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

3. 4.

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

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

323 323 323 323 325 325 326 327 327 XV

5.

6. 7. 8. 9.

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

Subject Index

xvi

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

CHAPTER I

Opioid receptors CATHERINE ABBADIE AND GAVRIL W. PASTERNAK

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

2. OPIOIDS

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

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

Ch. I

C. Abbadie and G.W. Pasternak

CH3

CH3

/__~N

I

I

N8

? ~? 6 ~ . )--/-~

o~ N - C ~

CH3CH2C[~

7

HO

CH3CO

OH

(,9

Morphine

OCCH3

Heroin

Fentanyl

H3

CH3 I

N

H ~ HO

"~-

OH -"'<'COOH

CH30

Morphine 613-Glucuronide

CH3

/ HO/

HO/

Levorphanol

CH3CH2CII

--CH3

Meperidine

Codeine

CH3\c--c CH3/ HxCH2 I

I

~

<5)--

J

CH 3

Pentazocine

II CH3 CH3CH 2~C-- C--CH2--CH --N/ I CH3 \CH3

Methadone Fig. 1.

OH

Structures of opioids.

NHO~O

Naloxone

~ ~--C--CH3 HO/L-~ O//~----~OCH?H

Buprenorphine

Opioid receptors

Ch. I

TABLE 1. Structure of some common opioid peptides Natural opioid peptides [LeuS]Enkephalin [MetS]Enkephalin Peptide E (amidorphin)

Endomorphin- 1 Endomorphin-2 Orphanin FQ/Nociceptin Orphanin FQ2 Nocistatin

Tyr-Gly-Gly-Phe-Leu Tyr-Gly-Gly-Phe-Met Tyr-Gly-Gly-Phe-Met-Lys-Lys-Met-Asp-Glu-Leu-Tyr-Pro-Leu-Glu-Val-GluGlu-Glu-Ala-Asn-Gly-Gly-Glu-Val-Leu Tyr-Gly-Gly-Phe-Met-Lys-Lys-Met-Asp-Glu-Leu-Tyr-Pro-Leu-Glu-Val-GluGlu-Glu-Ala-Asn-Gly-Gly Tyr-Gly-Gly-Phe-Met-Lys-Lys-Met-Asp-Glu-Leu-Tyr-Pro-Leu-Glu-Val-GluGlu-Glu-Ala-Asn Tyr-Gly-Gly-Phe-Met-Lys-Lys-Met-Asp-Glu-Leu-Tyr-Pro-Leu-Glu-Val-GluGlu-Glu Tyr-Gly-Gly-Phe-Met-Lys-Lys-Met-Asp-Glu-Leu-Tyr Tyr-Gly-Gly-Phe-Met-Arg-Val Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg--Pro-Lys--Leu-Lys-Trp-Asp-Asn-Gln Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Pro-Lys Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Pro Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-LeuPhe-Lys-Asn-Ala-Ile-Ile-Lys-Asn-Ala-Tyr-Lys-Lys-Gly-Glu Tyr-Pro-Trp-Phe-NH2 Tyr-Pro-Phe-Phe-NH2 Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-S er-Ala-Arg-Lys-Leu-Ala-Asp-Glu Phe-Ser-Glu-Phe-Met-Arg-Gln-Tyr-Leu-Val-Leu-S er-Met-Gln-Ser-Ser-Gln Thr-Glu-Pro-Gly-Leu-Glu-Glu-Val-Gly-Glu-Ile-Glu-Gln-Lys-Gln-Leu-Gln

Synthetic opioid peptides DPDPE DADLE DALDA DAMGO DSLET Deltorphin II CTOP

[D-Pen2,D-Pen5]enkephalin [D-Ala2,D-Leu5]enkephalin Tyr-D-Arg-Phe-Lys-NH2 [D-Ala2,MePhe4,Gly(ol)5]enkephalin [D-Ser2,Leu5]enkephalin-Thr6 Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2 D-Phe-c[Cys-Tyr-D-Trp-Orn-Thr-Pen]-Thr-NH2

BAM 22 BAM 20 BAM 18 BAM 12 Metorphamide Dynorphin A Dynorphin B 0t-Neoendorphin ~-Neoendorphin ~h-Endorphin

actions, such as their ability to modulate the release of a variety of hormones. There also is evidence of additional effects upon the immune system and other peripheral organ systems. However, these remain to be more fully defined. Opiates such as morphine act by mimicking endogenous peptides within the CNS, the endorphins (Table 1) (Evans et al., 1988). There are three major classes of opioid peptides, each generated from a precursor peptide with its own gene. ~-Endorphin, a 31 amino acid, is derived from pro-opiomelanotropin that also generates ACTH and 0~-MSH. The enkephalins are pentapeptides (Tyr-Gly-Gly-Phe-Met and Tyr-Gly-Gly-Phe-Leu) derived from pre-proenkephalin, which contains multiple copies of the two pentapeptides, as well as a number of additional extended enkephalins. The processing of the enkephalins is quite complex. Although the extended enkephalins are generated, along with the enkephalins, the physiological importance of the longer peptides is still not clear. The precursor for the third major family of opioid peptides, pre-prodynorphin, generates dynorphin A, as well as a number of other putative opioid peptides such as dynorphin B and ot-neoendorphin. Most recently, peptides with morphine-like activities have been reported, termed the endorphins (Zadina et al., 1997). They have been isolated from the brain, but their precursor has not yet been identified. Without additional information, their potential significance remains tentative,

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but their unique receptor selectivity for Ix receptors suggests that they may be the endogenous morphine-like ligand. The complexity of the opioid peptides and their processing is beyond the scope of this review, but this complexity gives insights into the potential complexity of the opioid receptors for which they serve as ligands.

3. OPIOID RECEPTOR SUBTYPES The three major classes of opioid receptors were originally defined pharmacologically, with tx receptors mediating the actions of morphine, • receptors responsible for activity of the benzomorphan ketocyclazocine and ~ receptors selective for the enkephalins (Table 2) (Hughes et al., 1975; Martin et al., 1976; Lord et al., 1977; Pasternak, 1993; Reisine and Pasternak, 1996). As noted above, the endomorphins are likely the endogenous Ix ligands and dynorphin A is the endogenous ~: ligand. Classical pharmacological approaches have implied subtypes within these classifications. The identification and classification of these receptors evolved over several decades and a full review is beyond the scope of this chapter. All the opioid receptors share the ability to mediate analgesia. Since most opiate and opioid peptides show only modest selectivity among the receptors and all the receptors can elicit a similar pharmacological response, it has been difficult to dissect the actions of each alone. This has been helped in recent years with the cloning of the various receptors and both knockout and antisense models, although each has uncovered surprising results.

TABLE 2. Opioid receptor classification and localization of analgesic actions Receptor

Clone

Analgesia

Other

tx IX1

MOR-1 Supraspinal

IX2

Spinal

Prolactin release Acetylcholine release in the hippocampus Feeding Respiratory depression Gastrointestinal transit Dopamine release by nigrostriatal neurons Guinea pig ileum bioassay Feeding

M6G

Spinal and supraspinal

1 K2

KOR-1

Spinal and supraspinal

KOR-1/DOR- 1

Unknown Supraspinal

~:3 DOR-1 31 32

DOR-1

Psychotomimesis Sedation Diuresis Feeding Unknown Mouse vas deferens bioassay Dopamine turnover in the striatum Feeding

Supraspinal Spinal and supraspinal

Some of the actions attributed to a general family of receptor have not yet been associated with a specific subtype. All the correlations in this table are based upon animal studies, which can show species differences.

Opioid receptors

Ch. I

3.1. Ix OPIOID RECEPTORS The first binding studies identified morphine-like, or Ix, receptors (Pert and Snyder, 1973; Simon et al., 1973; Terenius, 1973). These sites have high affinity for traditional Ix opioids, such as morphine, dihydromorphine and the antagonist naloxone. Soon after these initial descriptions, the possibility of Ix receptor binding subtypes was raised (Pasternak and Snyder, 1975; Pasternak et al., 1980a,b), leading to the proposal of Ixl and Ix2 receptors (Wolozin and Pasternak, 1981). This classification was based, in large part, upon the antagonists naloxazone and naloxonazine and attributed different pharmacological activities to these two Ix subtypes, with the Ix1 receptors mediating supraspinal analgesia and Ix2 receptors responsible for respiratory depression and the inhibition of gastrointestinal transit, a major contributor to the constipation seen with opioids clinically (Table 2). The pharmacology of the morphine metabolite morphine-6~-glucuronide (M6G) implied the presence of yet another subtype of Ix receptor distinct from the Ix1 and Ix2 classification (Rossi et al., 1995a, 1996, 1997a). This subtype was particularly intriguing since it appeared to be important in the actions of heroin. The pharmacological classification of Ix receptor subtypes has been reviewed (Pasternak, 1993, 2001; Pasternak and Standifer, 1995). The Ix opioid receptor was cloned shortly after the 3 receptor. A member of the large G-protein coupled receptor family, MOR-1 (also termed MOP1) initially was reported to contain four exons (exons 1-4) (Fig. 2) and has been associated with morphine analgesia both through antisense mapping (Chen et al., 1995; Lai et al., 1995; Rossi et al., 1994, 1995a, 1997a; Pasternak, 2001) and gene disruption strategies (Matthes et al., 1996; Sora et al., 1997; Loh et al., 1998; Schuller et al., 1999). A major question remained, however. How could a single cloned receptor be reconciled with the pharmacological studies implying multiple Ix opioid receptors? A possible explanation came soon afterwards with reports of alternative splicing of MOR-1 (Fig. 2) (Bare et al., 1994; Zimprich et al., 1995). Both MOR-1A and MOR-1B displayed alternative splicing at the intracellular carboxy tail of the receptor, having little effect on the overall binding characteristics (Fig. 2B). These reports were then followed by a number of additional splice variants, as well as the identification of both MOR-1A and MOR-1B in the mouse (Pan et al., 1999, 2000). Like the earlier ones, they also involved splicing at the carboxy terminus. Their binding characteristics show the expected Ix ligand selectivity, but they have different regional distributions immunohistochemically, as discussed below. Ix Subtypes also have been suggested from molecular studies. Antisense mapping reveals differences between morphine and M6G analgesia and even between supraspinal and spinal morphine analgesia, as well as other functions (Rossi et al., 1994, 1995a,b, 1996, 1997a; Leventhal et al., 1996, 1997, 1998). Furthermore, interesting results were observed with an MOR-1 knockout mouse (Schuller et al., 1999). As with other MOR-1 knockout mice, morphine analgesia was lost in these exon 1 knockout animals. Yet, heroin and M6G analgesia remained intact, although with a slight decrease in potency. This observation provides clear evidence for differences between morphine and both heroin and M6G at the molecular level. 3.2. 30PIOID RECEPTORS 3 Receptors were initially defined pharmacologically by their high affinity and selectively for the enkephalins in the mouse vas deferens bioassay, followed soon afterwards by binding studies (Lord et al., 1977; Chang et al., 1979; Reisine and Pasternak, 1996). Like tx drugs, ligands are effective analgesics. Although Ix and ~ receptors share the ability to induce

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C. Abbadie and G.W. Pasternak

A. Oprm Gene Structure 1 Intron (kb)

2 ~28

3

5

0.8 ~11

4 ~8.5

10 10

6 16

7 ,~7.8

8 >30

9 >40

B. Alternatively spliced MOR-1 variants Amino acid sequence following exon 3

Exon composition

111213141 1112131 111213151 ] 1

2 I 3 1718191

MOR-1

- LENLEAETAPLP

MOR-1A

- VRSL

MOR-1B

- KIDLF

MOR-1C

- PTLAVSVAQIFTGYPSPTHVEKPCKSCM DRGMRNLLPDDGPRQESGEGQLGR

1 ] 2 I 3 18191

MOR-1D

" RNEEPSS

11213

MOR-1E

- KKKLDSQRGCVQHPV

MOR-1F

- APCACVPGANRGQTKASDLLDLELETVG SHQADAETNPGPYEGSKCAEPLAISLVPLY

161718191

I 1 I 2 I 3 ]10161718[91

-

Splice site

Fig. 2. Structure of MOR-1 gene. (A) Gene structure. The gene structure of the tx opioid receptor (Oprm) is presented. Note that the original gene comprised exons 1-4. Since then, additional exons have been reported and the gene now encompasses approximately 250 kb. The number of the exons was based upon the order of their discovery. (B) Schematic of protein structure. The Oprm gene undergoes extensive splicing, as demonstrated in this schematic. Note that these variants all differ at the carboxy terminus within the cell. Exon 1 encodes the first transmembrane domain, while exons 2 and 3 each encode another three transmembrane domains. Thus, all the variants presented above contain identical structures throughout the entire membrane spanning regions, differing only at the carboxy tips of the proteins. Also note that although several of these variants share exons, the amino acid sequence of each is distinct due to either frameshifts and/or early termination.

analgesia, the remainder of the pharmacology differs. Unlike ~ drugs, ~ compounds lack respiratory depressant and constipating effects. There have been enormous efforts made to establish the structure-activity relationships of the enkephalins and thousands of analogs have been synthesized, yielding a number of highly selective ~ ligands as well as others selective for ~ receptors (Table 1). Many of these ligands, such as [D-PenZ,B-PenS]enkephalin have been radiolabeled and widely used in binding studies and autoradiographically. As additional ligands were developed, particularly antagonists, investigators proposed two subtypes of 3 receptors, termed 3l and ~2 receptors (Table 2) (Jiang et al., 1991; Negri et al., 1991; Takemori and Portoghese, 1993; Xu et al., 1993). Like the 1~ subtypes, the binding differences between the two 3 subtypes are subtle and few ligands can definitively distinguish between them. This has hampered the interpretation of many of the autoradiographic studies, which were likely labeling various proportions of multiple subtypes. The 3 receptor was the first opioid receptor cloned (Evans et al., 1992; Kieffer et al., 1992). Like MOR-1, the cloned 3 receptor DOR-1 (also termed DOP1), is a member of the G-protein coupled receptor family. It has high homology to MOR-1, particularly in the transmembrane regions, but differs in that DOR-1 contains only three exons. Like MOR-1, the splice site between exons 1 and 2 is located just after the first transmembrane domain and the splice site

Opioid receptors

Ch. I

between exons 2 and 3 is within the third extracellular loop. Antisense mapping (Standifer et al., 1994) and knockout models (Kieffer, 1999; Zhu et al., 1999) confirm the role of this receptor behaviorally. Splice variants of the 8 receptor have been suggested from antisense mapping (Rossi et al., 1997b) and several have been cloned (Gav6riaux-Ruff et al., 1997). However, the cloned variants are truncated and do not appear to encode functional receptors. Thus, the suggestion of 8 receptor subtypes provided by both pharmacological and antisense approaches has not yet been confirmed at the molecular level. There is some evidence supporting more than one 8 receptor. Antisense mapping differentiated between several 8 drugs (Rossi et al., 1997b). Disruption of the DOR-1 receptor eliminated spinal 8 analgesia, as expected, but supraspinal analgesia remained intact (Zhu et al., 1999). The supraspinal 8 analgesia observed in the wild-type controls differed in several respects. Although still sensitive to the 8 antagonist naltrindole, the residual analgesia was less sensitive. In addition, the non-peptide ~ drugs were actually far more potent in the knockout animals when compared to wild-type controls. Finally, antisense oligodeoxynucleotides that effectively blocked supraspinal in the wild-type mice were ineffective in the knockout mice. Thus, disruption of DOR-1 eliminated spinal analgesia, but left intact, if not more prominent, a novel form of supraspinal 8 analgesia. The relationship of this residual response to the 81 and 82 receptors previously proposed is unknown, but it does support the possibility of 8 receptor heterogeneity at the molecular level. 3.3. ~: OPIOID RECEPTORS Receptors were first proposed from classical pharmacological studies exploring a series of benzomorphans (Brown et al., 1976) long before the isolation of dynorphin A, its endogenous ligand (Goldstein et al., 1979). Over time, a number of Kreceptor subtypes have been proposed (Zukin et al., 1988; Clark et al., 1989; Rothman et al., 1990). K1 Receptors are commonly defined by their high affinity for U50,488H and U69,593 and are the receptors for dynorphin A. However, even among U50,488H-sensitive sites, studies have implied two populations of binding sites (Clark et al., 1989; Rothman et al., 1990). K2 Receptors are U50,488Hinsensitive sites with a unique pharmacological selectivity (Zukin et al., 1988). Initially defined as a separate receptor, recent evidence suggests that it actually represents a dimer of KOR-1 and DOR-1 receptors (Jordan and Devi, 1999). Finally, another U50,488H-insensitive site has been identified, termed ~:3 (Clark et al., 1989). It has a unique pharmacology, both in binding studies and in vivo (Price et al., 1989; Gistrak et al., 1990; Paul et al., 1990). Although it has been classified within the K receptor family, it is quite distinct from the other K subtypes. Like the others, K drugs are potent analgesics and several have been used widely clinically, including pentazocine, nalbuphine and levorphanol (Reisine and Pasternak, 1996). However, the widespread use of a number of other K drugs has been limited by their potential to elicit unpleasant psychomimetic effects and dysphoria (Pfeiffer et al., 1986). K Drugs will impede gastrointestinal transit, although not as completely as Ix drugs, and they are thought not to have appreciable effects on respiration. The K1 receptor, termed KOR-1 or KOP1, was cloned in 1993 (Minami et al., 1993; Reisine and Bell, 1993; Yasuda et al., 1993). The KOR-1 is comprised of three exons that encode a typical G-protein coupled receptor with seven transmembrane domains. It is highly homologous to the other opioid receptors, particularly within the transmembrane domains. When expressed, KOR-1 is pharmacologically indistinguishable from the K1 receptor and antisense mapping studies support the possibility that they are the same (Adams et al., 1994; Chien and Pasternak, 1995; Pasternak et al., 1999). As noted above, KOR-1 also is

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a component of the •2 receptor when dimerized with DOR-1. The molecular biology of the K3 receptor is less clear. Evidence strongly suggests an association with the orphanin FQ/nociceptin receptor, ORL-1/KOR-3 (Pan et al., 1994, 1995), but the two are not identical. Additional studies are needed to define K3 receptors at the molecular level. 4. DISTRIBUTION OF OPIOID RECEPTORS IN THE RAT BRAIN

4.1. AUTORADIOGRAPHIC LOCALIZATION OF OPIOID RECEPTORS The first autoradiographic distribution of opioid binding sites within the brain goes back almost three decades, before the classification of the three major classifications and long before the definition of subtypes (Kawabata et al., 1992; Raffa et al., 1993). Since then, a variety of radioligands have been used to define the distribution of these various classes of receptors (Atweh and Kuhar, 1977a,b,c; Goodman et al., 1980; Herkenham and Pert, 1980; Pearson et al., 1980; Bonnet et al., 1981; Bowen et al., 1981; Duka et al., 1981; Foote and Maurer, 1982; Goodman and Snyder, 1982a; Pfeiffer et al., 1982; Wamsley et al., 1982, Wise and Herkenham, 1982; Quirion et al., 1982, 1983; Kent et al., 1982; Lewis et al., 1983; Maurer et al., 1983; Slater and Patel, 1983; Edley and Herkenham, 1984; Tempel et al., 1984; Goodman and Pasternak, 1985; Morris and Herz, 1986; McLean et al., 1987; Mogil et al., 1994). However, as our understanding of the opioid system has progressed, it has become increasingly clear that these studies must be interpreted cautiously due to the limited selectivity of the tools available at the time. This is particularly relevant with regards to subtypes. 4.1.1. t~ Opioid receptors

The first extensive autoradiographic study of opiate receptors was done in rat brain (Atweh and Kuhar, 1977a,b,c). Although [3H]diprenorphine is not particularly selective, it labeled predominantly t~ sites in these studies. In the telencephalon, dense labeling was seen in the presubiculum and in most nuclei of the amygdala, patchy areas in the caudate-putamen and accumbens nucleus, the subfornical organ, the interstriatal nucleus of the stria terminalis and the external part of the anterior olfactory nucleus. Lower densities were found in the hippocampal formation (stratum oriens, stratum lacunosum-moleculare), the deeper layers of the cerebral cortex, the entopeduncular nucleus, globus pallidus, septum and paratenial thalamic nucleus. In the diencephalon, low binding densities were found in most nuclei of the hypothalamus. In the thalamus, the density varied from low in the lateral nuclei to intense in medial nuclei (centromedian and periventricular). In the brainstem, areas showing very dense or dense localizations of receptors included the parabrachial nuclei, the superior colliculus, the ventral median raphe nucleus, components of the accessory optic system, portions of the habenulo-interpeduncular complex, the pretectal nuclei and the ventral lateral geniculate and the infundibulum. In the lower medulla, the area postrema, in components of the vagal system, including the vagus nerve, nucleus tractus solitarius, nucleus commissuralis, nucleus intercalatus, nucleus ambiguus and nucleus originis dorsalis vagus, and in layers I and II (substantia gelatinosa) of the dorsal horn of the spinal cord and of the spinal trigeminal nucleus all had very high receptor densities. The agonist [3H]etorphine revealed similar labeling as the antagonist [3H] diprenorphine. In these early studies, the radioligand was administered in vivo, followed by sectioning of

Opioid receptors

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the brain. Similar results were later seen by incubating [3H] diprenorphine with lightly fixed brain sections mounted on microscope slides (Young III and Kuhar, 1979). Later studies using more selective IX ligands, such as [3H]DAMGO reported similar distributions of tx receptors (Fig. 3) (Goodman et al., 1980). Distinguishing between IXl and IX2 binding sites required the development of computerized pixel-by-pixel subtraction technique, an innovative approach at that time (Goodman and Pasternak, 1985). This approach demonstrated that the general distributions of the two IXreceptor subtypes were similar, but their ratios within regions varied significantly.

4.1.2. 80pioid receptors The distribution of 3 receptors within the brain (Fig. 4) is quite distinct from that of IX sites labeled with [3H]DAMGO (Goodman et al., 1980; Lewis et al., 1983; Quirion et al., 1983; Mansour et al., 1987; Renda et al., 1993; Hiller et al., 1996). The earlier studies used moderately selective agents, such as [3H]DADLE. However, DADLE still retains affinity for at least one subpopulation of IX binding sites, complicating the interpretation of these studies (Goodman et al., 1985). 3 Sites were found in all laminae of the neocortex, olfactory tubercle, diffusely throughout the striatum, and in the basal, lateral, and cortical nuclei of the amygdala. Using a more selective ligand, [3H]DPDPE, Sharif and Hughes revealed high 3 receptor concentrations in the olfactory bulb (external plexiform layer), striatum, nucleus accumbens, amygdala and cortex (layers I-II and V-VI) (Sharif and Hughes, 1989). Mansour and colleagues reported similar distributions, except in the central amygdaloid nucleus where they observed very little binding (Mansour et al., 1993). High levels of [3H]DPDPE binding also were seen in the external plexiform layer of the olfactory bulb, the nucleus accumbens and the olfactory tubercule. Moderate concentrations were observed in the granular layer of the olfactory bulb, all layers of the cerebral cortex, the striatum, the medial, cortical, basolateral and lateral nuclei of the amygdala. Low concentrations are found in the glomerular layer of the olfactory bulb, accessory olfactory bulb, pyramidal layer of the hippocampus, superior and inferior colliculi, habenula, globus pallidus, ventromedial nucleus of the hypothalamus and the central medial, medial dorsal and ventromedial thalamic nuclei, stria terminalis, periaqueductal gray, substantia nigra, nucleus of the solitary tract and the spinal cord.

4.1.3. K Opioid receptors K 1 Receptors have high affinity for U50,488H, U69,593 and the endogenous opioid peptide dynorphin A (Reisine and Pasternak, 1996). The abundance of K1 receptors in mice and rats are relatively low, making it difficult to examine them. However, the guinea pig has high levels of these receptors. Early studies looking at K receptors in the rat utilized [3H] ethylketocyclazocine or [3H]bremazocine in the presence of unlabeled DAMGO and DPDPE to block IX and 3 sites, leaving only K receptors (Goodman and Snyder, 1982a,b; Slater and Patel, 1983; Mansour et al., 1987). There was dense labeling in the amygdala, olfactory tubercle, nucleus accumbens, caudate putamen, medial preoptic area, hypothalamus, median eminence, periventricular thalamus, and interpeduncular nucleus. However, both ethylketocyclazocine and bremazocine are not very selective and it is likely that these early studies were looking at more than simply K1 binding sites, although in one study all the labeling was effectively displaced by U50,488H (Morris and Herz, 1986). Subsequent studies used [3H]U69,593, a highly selective ~:1 ligand (Quirion et al., 1987). The labeling of [3H]U69,593 is more restricted than that of either [3H] ethylketocyclazocine

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Fig. 3. Schematic sagittal representations of tx opioid receptors. Schematic distributions of the Ix receptors are presented using autoradiography, in situ hybridization and immunohistochemistry. Autoradiographic data (Goodman et al., 1980) using [3H]DAMGO, in situ hybridization (Delfs et al., 1994a; Mansour et al., 1994a) and protein distributions (Arvidsson et al., 1995b; Ding et al., 1996) are from the literature.

10

Opioid receptors

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Fig. 4. Schematic sagittal representations of ~ opioid receptors. Schematic distributions of the 3 receptors are presented using autoradiography, in situ hybridization and immunohistochemistry. Autoradiographic data (Tempel and Zukin, 1987; Sharif and Hughes, 1989; Mansour et al., 1993) using [3H]DPDPE, in situ hybridization (Mansour et al., 1994a) and protein distribution (Arvidsson et al., 1995a; Bausch et al., 1995) are from the literature.

11

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or [3H]bremazocine in the presence of an excess of the Ix and 3 blockers DAMGO and DPDPE. This is evident in the olfactory bulb, dorsal hippocampus, dentate gyrus, medial habenula, ventromedial and ventrolateral thalamus, pontine nuclei and the cerebellum, where [3H]bremazocine binding is readily detected while [3H]U69,593 binding is undetectable (Mansour et al., 1994d). It is important to note that the correspondence between localization of ~:1 mRNA and the distribution of [3H]U69,593 was better than that of the less selective radioligands (Mansour et al., 1994d). There are significant species differences between the guinea pig and other rodents, with the density of ~:1 receptors in the guinea pig far exceeding levels in the others. Furthermore, these differences extended beyond simple differences in absolute receptor density and involved relative distributions among regions as well. For example, major differences were observed in the hippocampus, with very low binding in the pyramidal cell layer, the dentate granular cell layer and the commissural-associational zone of the dentate molecular layer of the guinea pig compared to moderately dense labeling of these structures in the rat. In addition, the terminal field of the mossy fiber system in the hilus was enriched in the guinea pig and absent in the rat (Foote and Maurer, 1986). In the telencephalon, Mansour (Mansour et al., 1987) and Unterwald (Unterwald et al., 1991) reported little k: binding in the olfactory bulb, although ~: sites are seen in the olfactory tubercule (Fig. 5). K Sites are present at low levels in all layers of the neocortex. Dense binding is present in the nucleus accumbens, especially in its medial part and in patches along the ventromedial edge. In the basal ganglia, ~: sites are present in the nucleus accumbens, the claustrum and the endopiriform nucleus. The globus pallidus contains low level of ~c sites, whereas the medial septum and bed nucleus of the stria terminalis exhibit moderate levels of ~: binding. There is a low density of ~: sites in the hippocampus. In the diencephalon, ~: binding is dense in the midline nuclei of the thalamus (paraventricular, centromedian, rhomboid and anteromedial nuclei), the habenula, the hypothalamus (periventricular, ventromedial and dorsomedial nuclei), median eminence and lateral hypothalamic area. In the midbrain and the pons, ~: binding is found in the superior colliculus, the periaqueductal gray, the interpeduncular nucleus, the substantia nigra (both pars comparta and reticulata), the inferior colliculus, the raphe nuclei, the locus coeruleus and the parabrachial nuclei. A very low density of K sites is found in the cerebellum. In the spinal cord, ~: sites are more abundant in the superficial laminae. 4.2. OPIOID RECEPTOR mRNAs Following the cloning of the opioid receptors, their distributions within the brain were evaluated using in situ hybridization. Unlike traditional receptor autoradiography, the selectivity of labeling with in situ hybridization is far more selective. However, the question of mismatches remains a potential problem. Hybridization to the mRNA localizes the cell bodies responsible for synthesizing the receptors, but the receptors themselves might be transported long distances along axons, leading to a potential mismatch between the distribution of the mRNA and the protein. Despite this potential problem, in situ hybridization has proven a valuable approach to define the anatomy of opioid receptors.

4.2.1. IX Opioid receptors The reported distributions of tx opioid receptor (MOR-1) mRNA in the rat brain are very similar among laboratory groups (Fig. 3) (Delfs et al., 1994b; Mansour et al., 1994a,e; Minami 12

Opioid receptors

Ch. I

Fig. 5. Schematic sagittal representations of • opioid receptors. Schematic distributions of the ~ receptors are presented using autoradiography, in situ hybridization and immunohistochemistry. Autoradiographic data (Mansour et al., 1987; Unterwald et al., 1991) using U69593, in situ hybridization (Mansour et al., 1994a,d) and protein distributions (Arvidsson et al., 1995c; Mansour et al., 1996) are from the literature.

13

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et al., 1994). The original MOR-1 variant remains the most abundant of all the variants, and these studies have defined its distribution. However, these studies do not distinguish among the various splice variants.

4.2.1.1. Telencephalon Cells of the internal granular, mitral and glomerular layers of the olfactory bulb express tx opioid receptor mRNA. However, these internal layers have low levels of [3H]DAMGO binding. High levels of MOR-1 mRNA can be found in the cells of the olfactory bulb that also display high binding levels. In the frontal, parietal, occipital and temporal cortex, cells expressing mRNA are scattered in layers II and III, and in layer VI, whereas binding has been reported in all layers. Patchy signals of MOR mRNA have been detected in the caudate-putamen and in the nucleus accumbens that correspond to the patchy areas of binding. In the globus pallidus and the ventral pallidus, mRNA expression is moderate to intense. In the septum, cells expressing mRNA are found in the medial and lateral part. Cells in the diagonal band of Broca express low to moderate level of mRNA. In the amygdaloid complex, MOR mRNA is moderate to intense and is found in the basolateral, medial, cortical and central amygdaloid nuclei. No detectable binding has been detected in the central nucleus despite moderate mRNA expression. In the hippocampal formation, cells expressing mRNA are disseminated and are predominantly found in the stratum pyramidale, with few cells in the stratum oriens and radiatum. In the dentate gyrus, the cells of the granular layer express mRNA for the MOR, whereas binding is prominent in the molecular layer. Cells expressing mRNA can also been found in the presubiculum and subiculum, in the endopiriform nucleus and claustrum where binding is also present.

4.2.1.2. Diencephalon The distribution of cells expressing mRNA is heterogeneous. Intense levels are found in most thalamic nuclei, including the paraventral, paratenial, mediodorsal, paracentral, centromedial, centrolateral, ventrolateral, ventromedial, rhomboid and posterior. Moderate levels are seen in the lateroposterior nucleus. In the ventral posterolateral and posteromedial nuclei, mRNA levels are weak in the anterior part, but intense in the posterior part. In the anterior nuclei, such as the anterior ventral and anterior dorsal nuclei, mRNA expression is not detectable. In the lateral and medial geniculate nuclei, mRNA expression is intense in the dorsal part and moderate in the ventral part. High levels of expression are present in the medial, but not the lateral habenula. In the hypothalamus, moderate mRNA expression is found in the lateral, anterior, arcuate, supramammillary and medial mammillary nuclei.

4.2.1.3. Mesencephalon Moderate MOR-1 mRNA is found in the superior colliculus, but labeling in the inferior colliculus is intense, as is the -labeling in the interpeduncular nucleus. In the raphe nuclei, MOR-1 mRNA is intense in the medial nucleus and weak in the dorsal nucleus. In the periaqueductal gray, cells expressing MOR-1 mRNA are predominantly found in the ventral and lateral parts, whereas binding is mainly observed in the dorsal part. In the substantia nigra, only cells located in the pars compacta, but not the pars reticulata express moderate levels of mRNA. The rostral part of the ventral tegmental area exhibits low to moderate levels of MOR mRNA, whereas the caudal part has higher expression. 14

Opioid receptors

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4.2.1.4. M e d u l l a

Scattered cells within the reticular nuclei express high mRNA levels. High levels of MOR-1 mRNA are found in the locus coeruleus, in ~the lateral, medial and ventral division of the parabrachial and in the ambiguus nucleus. Similarly, high mRNA expression is present in the area postrema, the nucleus of the solitary tract, the cuneatus and gracilis nuclei and the dorsal motor nucleus of the vagus. No cells expressing mRNA are detectable in the lobules of the cerebellum, but cells expressing moderate levels are found in the deep cerebellar nuclei. 4.2.1.5. Spinal cord

Intense labeling of MOR-1 mRNA is present only in the superficial lamina of the dorsal horn and low in all other laminae.

4.2.2. 80pioid receptors A number of laboratories have examined the anatomical distribution of DOR-1 mRNA (Fig. 4) (George et al., 1994; Grimm et al., 1994; Le Moine et al., 1994; Mansour et al., 1994a). 4.2.2.1. Telencephalon

According to Mansour and colleagues, high levels of DOR-1 mRNA are observed in the cells of the intemal granular and mitral cell layers of the olfactory bulb. In the anterior olfactory nucleus, 8 receptor expression is high in the dorsal, medial, lateral, ventral and posterior subdivisions. High levels of expression are found in the agranular insular, lateral orbital, prefrontal, cingulated, piriform, entorhinal, frontal, parietal, occipital and temporal cortices with the highest levels in layers II-III, and V-VI. In the hippocampal formation, cells expressing ~ receptor mRNA are localized in the pyramidal cell layer, with occasional cells observed in the stratum oriens. In the subiculum, ~ mRNA expression is high, and is low in the parasubiculum and presubiculum. In the amygdala, the highest level of mRNA are found in the lateral, basolateral, medial and basomedial nuclei, with moderate levels in the posterior medial and cortical nuclei. 3 Expression is also found in the bed nucleus of the stria terminalis. Cells expressing 3 receptors have a, homogenous distribution in the caudate-putamen as compared to the tx expressing cells. The shell of the accumbens nucleus and the olfactory tubercule contain cells expressing 3 mRNA. Very few cells express 3 mRNA in the globus pallidus and ventral pallidum, and in the lateral and medial septum. 4.2.2.2. D i e n c e p h a l o n

,", ~. . :J"

Cells expressing 3 receptor mRNA have a limited distribution in the hypothalamus except in the ventromedial nucleus. Similarly, expression in the thalamus is low in the laterodorsal, reticular and parafascicular nuclei and in the zona incerta and the lateral geniculate body. No mRNA was detectable in the mammillary nuclei. In the mesencephalon, cells expressing ~ mRNA are localized to the intermediate and deep layers of the superior colliculus and the dorsal, central and external cortex of the inferior colliculus. A very low level of ~ receptor mRNA is present in the periaqueductal gray, substantia nigra and ventral tegmental area. In the interpeduncular complex, ~ receptor mRNA is primarily localized in the rostral part. 15

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C. Abbadie and G.W. Pasternak

In the mesencephalon, the pontine nuclei express very high levels of 3 receptor mRNA, but the median raphe and the raphe magnus express low levels and the raphe pallidus moderate levels. No expression is seen in the locus coeruleus and low levels are detected in the parabrachial nucleus. In the trigeminal nucleus, expression is high in the cells of the motor and spinal trigeminal and moderate in the sensory nucleus. Low levels are detected in the olivary complex and moderate levels in the cochlear and vestibular nuclei. In the brainstem, the gigantocellular cells express high levels of mRNA, but very low levels are detected in the nucleus of the solitary tract. No expression is seen in the ambiguous nucleus and in the dorsal motor nucleus of the vagus. In the cuneatus nucleus, receptor expression is high. Moderate to high levels are detected in the trapezoid nucleus, hypoglossal nucleus, lateral reticular nucleus. In the cerebellum, cells in the granular cell layer show expression as well as cells in the interposed and medial cerebellar nuclei. In the spinal cord, cells expressing 3 receptors were found in all laminae.

4.2.3. K Opioid receptors Mansour and colleagues (Mansour et al., 1994a,b) reported KOR-1 mRNA in the internal granular cell layers of the olfactory bulb, high levels in the dorsal and lateral divisions of the anterior olfactory nucleus. High levels of • receptor mRNA are observed in the lateral orbital cortex and in the entorhinal cortex (Fig. 5). Cells expressing mRNA are localized in layers V-VI of the parietal, temporal and occipital cortices. No mRNA is detectable in the hippocampal formation. In the parasubiculum, KOR-1 mRNA expression is high, but low in the subiculum and presubiculum. In the amygdala, the highest mRNA levels are found in the central lateral, basolateral and medial nuclei, with moderate levels in the centromedial and basomedial nuclei. KOR-1 mRNA expression is particularly high in the medial posterior division of the bed nucleus of the stria terminalis. In the dorsal striatum, cells expressing K receptor mRNA are localized in the medial and ventral part. In the nucleus accumbens, K receptor expression is organized into clusters. High expression is also observed in the olfactory tubercule. In the globus pallidus and ventral pallidum, scattered large cells express ~: mRNA. Moderate level of expression is detected in the medial and lateral septum. In the diencephalon, ~: receptor expression predominates in the hypothalamus. Rostrally, K receptors are observed in the medial preoptic area. More caudally, the paraventricular, periventricular, supraoptic, suprachiasmatic dorsomedial, ventromedial and arcuate nuclei, the median eminence and the infundibular stem express high mRNA levels. In the mammillary complex, KOR-1 mRNA expression is limited to the ventral premammillary and supramammillary nuclei. With the thalamus, high expression levels are limited to the paraventricular, centromedial, paracentral and parafascicular nuclei, with more moderate levels in the lateral habenula, laterodorsal nucleus, rhomboid, zona incerta and the ventrolateral geniculate body. Within the mesencephalon, superficial gray, optic nerve, intermediate gray and deep gray layers of the superior colliculus display moderate expression of KOR-1 mRNA. Cells expressing KOR-1 mRNA are widely distributed in the periaqueductal gray with moderate levels of expression in the dorsal and ventrolateral parts. In the substantia nigra, the medial portion of the pars compacta shows high levels and the pars reticulata moderate levels. The ventral tegmental area also has dense labeling. Low levels are found in the rostral, caudal and intermediate subdivisions of the interpeduncular nucleus. In the mesencephalon, the pontine nuclei do not express KOR-1 mRNA, in contrast to the higher levels in the dorsal raphe and raphe obscurus and moderate levels in the median raphe. High levels are also seen in the locus coeruleus and in the lateral part of the parabrachial 16

Opioid receptors

Ch. I

nucleus. In the trigeminal nucleus, expression is low in the motor and sensory nuclei, but is high in the spinal trigeminal nucleus. Low levels are detected in the olivary complex and vestibular nuclei and moderate in the cochlear nucleus. In the brainstem, the gigantocellular cells express high levels of mRNA, but low levels are detected in the nucleus of the solitary tract. No expression is seen in the ambiguous nucleus, but a high level is detected in the dorsal motor nucleus of the vagus. In the cuneatus and gracilis nuclei, receptor expression is very low. Very low levels also are seen in the trapezoid nucleus, hypoglossal nucleus and lateral reticular nucleus. In the cerebellum, cells in the interposed and medial cerebellar nuclei show low levels. In the spinal cord, cells in all laminae expressed KOR- 1 mRNA. 4.3. IMMUNOHISTOCHEMICAL DISTRIBUTION OF THE OPIOID RECEPTORS With the sequence of the cloned opioid receptors, it was possible to map them immunohistochemically. The selected epitopes were unique and, overall, these studies have provided valuable insights into the distribution of the cloned receptors. However, these studies do not address the issue of splice variants, as discussed below with the MOR-1 receptor variants. Thus, even these studies must be interpreted cautiously, taking into account the location of the epitopes and the possibility that a specific epitope might be contained within more than one variant.

4.3.1. I~ Opioid receptors MOR-l-like immunoreactivity (MOR-1-LI) was initially defined by antisera generated against the intracellular carboxy terminus (Fig. 3) (Mansour et al., 1994c, 1995; Arvidsson et al., 1995b; Ding et al., 1996; Wang and Wessendorf, 1999). Overall, these studies correspond quite well to the earlier autoradiography studies. Although the various immunohistochemical studies show remarkable agreement, there are several differences. Although Ding et al. (1996) reported weak labeling in the granule cell layer of the dentate gyrus, Arvidsson et al. (1995b) described the labeling as intense. The second difference was found in the dorsal root ganglia, where one study reported labeling only in small diameter cells and the other found that all cells exhibit MOR-LI. In the olfactory bulb, MOR-1-LI is weak to moderate in the glomerular cell layer and in the mitral cell layer. The external part of the anterior olfactory nucleus shows moderate to intense MOR-1-LI, but other regions exhibit weak or no detectable labeling. Throughout the cerebral cortex, weak labeling was found within cell bodies in layers II-IV. In the hippocampal formation, MOR-1-LI is distributed in cell bodies of the pyramidal cell layer and in the stratum oriens and radiatum. In the dentate gyrus, MOR-LI can be found in the molecular layer, granular cell layer and polymorph layer. MOR-1-LI is weak to moderate in the septum. In the caudate-putamen and in the nucleus accumbens MOR-1-LI shows a patchy distribution. Moderate MOR-LI can be seen in the globus pallidus and weak labeling is found in the ventral pallidum and the entopeduncular nucleus. In the amygdala, MOR-1-LI is distributed in the intercalated, lateral, anterior, basolateral, and central amygdaloid nuclei. MOR-1 is weak in the cortical, medial, lateral and basomedial nuclei of the amygdala. Very weak MOR-1-LI is detected in the preoptic region. In the hypothalamus, weak labeling is detected in all nuclei, except the supraoptic nucleus which lacks detectable MOR-1-LI. No MOR-1-LI is found in the mammillary complex. In the habenula, MOR labeling is intense. Most of the thalamic nuclei exhibit moderate MOR-1-LI: paratenial, paraventricular, reunions, 17

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C. Abbadie and G.W. Pasternak

lateroposterior, anterodorsal, anteromedial, mediodorsal, centrolateral, centromedian, ventrolateral, gelatinosus, rhomboid, laterodorsal and posterior nuclei. However, no MOR-1-LI is detected in the anteroventral, parafascicular nuclei and caudal part of the reticular thalamic nucleus. Moderate to intense labeling is found in the lateral and medial geniculate nuclei. In the superior colliculus, MOR-1-LI is weak to moderate in all layers. MOR-1-LI is weak in the periaqueductal gray and the in the dorsal raphe nucleus. Some MOR-1-LI can be seen in the pars reticulata of the substantia nigra, but not in the pars compacta. In the inferior colliculus, moderate MOR-LI is found in the dorsal and external cortices, but not in the central nucleus. Intense labeling is seen in the median raphe nucleus, and weak in the dorsal raphe nucleus. Very intense MOR-LI is detected in the lateral and medial part of the parabrachial nucleus as well as in the locus coeruleus. Moderate to intense labeling is found in the spinal trigeminal nuclei, the nucleus of the solitary tract and the ambiguus nucleus. No MOR staining is seen in either the cerebellar cortex or the nuclei. In the spinal cord, very dense labeling in lamina II where cell bodies can be detected. Weak staining is seen in laminae III-VI and X, no MOR-LI in motoneurons. MOR-1 undergoes extensive alternative splicing at the T-end of exon 3 (Fig. 2). In MOR1A, exon 4 is absent and the predicted coding region extends beyond the normal splice site in exon 3, resulting in four amino acids instead of the 12 amino acids coded by exon 4 in MOR-1 (Bare et al., 1994). MOR-1B contains an alternatively spliced exon 5 instead of the original exon 4 (Zimprich et al., 1995). However, the splicing of MOR-1 is far more extensive than originally believed. We recently reported five additional exons in the MOR-1 gene (Pan et al., 1999, 2000). In these variants, combinations of the five exons are alternatively spliced at the T-end of exon 3 to replace the original exon 4 and thereby generate the new MOR-1 variants, all of which contain the same exons 1, 2 and 3 as originally reported in MOR-1 (Fig. 2B). In MOR-1C, the 12 amino acids encoded by exon 4 in MOR-1 are replaced by 52 amino acids derived from the combination of the new exons 7, 8 and 9. In MOR-1D, exon 4 is replaced by exons 8 and 9, producing a 7 amino acid sequence. The presence of frame shifts among the variants sharing exons downstream from exon 3 gives each splice variant a unique amino acid sequence that allows its distribution to be explored immunohistochemically (Fig. 2B). Since all these variants share exons 1, 2 and 3, in situ hybridization cannot distinguish among them unless special care is taken to label unique sequences, which is difficult since most of the exons are present in more than one variant. The antisera used by Arvidsson and coworkers (Arvidsson et al., 1995b) was generated against the amino acids almost exclusively encoded by exon 4 and was similar to that used by Ding (Ding et al., 1996). However, Mansour and colleagues generated their antisera from a longer sequence, including almost 50 additional amino acids encoded by exon 3 (Mansour et al., 1995). Preabsorption of their antisera with the 12 amino acids encoded by exon 4 of MOR-1 only partially blocked the immunohistochemical staining. These differences might be explained by alternative splicing. The exon 4 epitope is contained only within MOR-1 itself. However, epitopes within exon 3 would be present in virtually all the variants identified to date. Thus, it is interesting that Mansour and colleagues found intense staining in regions where we subsequently observed MOR-1C-LI and little labeling with the MOR-1 antisera (see below). These regions include the hypothalamic nuclei, such as the periventricular nucleus, the median eminence and the deep laminae of the spinal cord. Thus, the presence of multiple MOR-1 variants has introduced a complexity in the interpretation of these immunohistochemical results not appreciated previously and illustrates the need to document the exon encoding the epitope recognized by the antisera. 18

Opioid receptors

Ch. I

No data are available on the distribution of MOR-1A in the CNS. MOR-1B is mainly expressed in the olfactory system, with little expression elsewhere (Schulz et al., 1998). Using an antiserum against a unique 20 amino acid carboxy sequence of MOR-1C, we reported its distribution in the rat CNS (Abbadie et al., 2000a,b,c). The distribution of MOR-1C-LI differed from MOR-1-LI (Fig. 6). For example, in contrast to the intense MOR-1-LI patches seen in the striatum, we did not observe any MOR-1C-LI. Conversely, the intensity of MOR-1C-LI exceeded MOR-1-LI in the lateral septum, the deep laminae of the spinal cord and most hypothalamic nuclei. MOR-1D-LI was seen in the dentate gyrus and in the mossy fibers of the hippocampal formation, the nucleus of the solitary tract and the area postrema, the inferior olivary nucleus, the nucleus ambiguus, the spinal trigeminal nucleus and the spinal cord. MOR-1D-LI was not observed in some regions containing dense MOR-1-LI, such as the striatum or the locus coeruleus. In regions containing MOR-1, MOR-1C and MOR-1D, the pattern of each variant was unique (Fig. 6), with no colocalization found except in the lateral septum where MOR-1 and MOR-1C were found in a subset of neuronal cell bodies.

4.3.2. 80pioid receptors The distribution of DOR-1-LI in the CNS has been examined in the rat (Fig. 4) (Arvidsson et al., 1995a; Kalyuzhny et al., 1996) and primates (Honda and Arvidsson, 1995) using a C-terminus antiserum and in mouse using an N-terminus (Bausch et al., 1995), with similar results showing punctate staining that likely corresponds to axonal labeling. In the olfactory bulb, DOR-1-LI is reported in the mitral cell layer and sub-glomerular region. In the hippocampal formation, DOR-1-LI is found in the stratum radiatum, oriens and pyramidale and in stratum moleculare and granulosum. In the cerebral cortex, all layers exhibit DOR-1-LI. Very faint staining is reported in the caudate-putamen. In the brainstem, DOR-1-LI is present in the substantia nigra, red nucleus, interpeduncular nucleus, periaqueductal gray, locus coeruleus, parabrachial nuclei, in all raphe nuclei, pontine reticular nuclei, trigeminal motor nucleus, tegmental nuclei, facial motor nucleus, vestibular nucleus, ambiguous nucleus, inferior olivary complex, medullary reticular formation, dorsal vagal nucleus, hypoglossus nucleus, nucleus of the solitary tract, and the spinal trigeminal nucleus. In the spinal cord, staining can be found throughout all laminae, but is more intense in laminae I-II.

4.3.3. KOpioid receptors The distribution of KOR-1 is described using different antisera recognizing the C-terminus of KOR-1 (Fig. 5) (Arvidsson et al., 1995c; Mansour et al., 1996). As with the other receptors, there was reasonably close agreement with the immunohistochemical labeling and that seen autoradiographically. In the telencephalon, KOR-1-LI is localized throughout the cerebral cortex and predominantly in layers V-VI. In the telencephalon, KOR-1-LI is detected in the caudate-putamen, olfactory tubercule, nucleus accumbens, endopiriform nucleus and claustrum. The hippocampal formation of the rat did not show any labeling in the rat, but KOR-1-LI is observed in this region in the guinea pig. The amygdaloid complex contains dense KOR-1-LI as well as the bed nucleus of the stria terminalis. KOR-1-LI is also observed in the septum, globus pallidus and the ventral pallidum. In the diencephalon, dense KOR staining is seen in the preoptic area and in all hypothalamic 19

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20

C. Abbadie and G.W. Pasternak n

Fig. 6. Comparison of the distribution of three MOR-1 splice variants. The distribution of MOR-1 (exon 4-L1), MOR-1C (exons 7/8/9-L1) and MOR-ID (exons 8/Y-LI) was examined using selective antibodies directed against the C-terminal sequence of the receptor. In the dorsal horn of the spinal cord (first row), MOR- I is abundant in lamina I1 and to a lesser extent in lamina I; MOR-IC is mainly found in axons in laminae 1-11 as well as in lamina X and a few fibers can also be observed in laminae V-VI; MOR-ID is dense in lamina I and more diffuse in laminae 11-Ill. At the level of the striatum (second row), MOR-1 can be found in patches in the caudate-putamen and the accumbens whereas MOR-IC and MOR-ID are not present in the striatum. Ncuronal cell bodies expressing MOR-lC can be found in the ventral part of the lateral septum.

5 R

Q

3

e&b.+ 3

E-

Opioid receptors

Ch. I

nuclei. KOR-LI is more restricted in the thalamus to the paraventricular, zona incerta, centromedial, centrolateral, reunions and rhomboid nuclei. In the mesencephalon, the superior colliculus shows KOR-1-LI staining in the zonal, superficial gray and intermediate gray layers. In the inferior colliculus, KOR-1-LI is restricted to the external cortex. KOR-1-LI is also seen throughout the periaqueductal gray and the raphe. In the substantia nigra, KOR-1-LI is restricted to the pars reticulata. More caudally, KOR-1-LI is sparse. KOR is expressed in the lateral parabrachial, trigeminal nuclei, nucleus of the solitary tract, area postrema and dorsal motor of the vagus. In the spinal cord, dense immunoreactivity is observed in the superficial laminae and lighter expression is detected in laminae III-X. 4.4. ULTRASTRUCTURAL LOCALIZATION OF THE OPIOID RECEPTORS Electron microscopy studies have looked at the subcellular localization of the opioid receptors in various regions. There is no simple relationship between a given opioid receptor and its axonal or dendritic localization. For example, KOR-LI was initially reported to be postsynaptic (Arvidsson et al., 1995c), whereas later studies revealed that KOR-LI was mainly expressed in pre-synaptic sites in the posterior pituitary (Shuster et al., 1999) and the hippocampal formation (Drake et al., 1996). Detailed analysis of the ultrastructural distribution of the opioid receptors revealed that more than 80% of the total neuronal profiles containing MOR-LI are dendrites and dendritic spines are in the patches of the striatum (Wang et al., 1997). Similarly in the dorsal raphe nucleus 75% of MOR-1-LI profiles are dendrites (Wang and Pickel, 1998). However, in the hippocampal formation, a substantial amount of MOR-LI is pre-synaptic (Drake and Milner, 1999). At the level of the spinal cord, MOR-LI is equally distributed in pre- and postsynaptic sites (Aicher et al., 2000). In the caudate-putamen nucleus, DOR-LI is found within the cytoplasm of spiny and aspiny neurons. DOR-LI is preferentially localized to membranes of the smooth endoplasmic reticulum revealing a prevalent association of DOR with cytoplasmic organelles that is involved in intracellular trafficking of cell surface proteins (Wang and Pickel, 2001). In the nucleus accumbens, 60% of DOR-immunoreactive profiles are axon terminals and small unmyelinated axons, whereas the remainder are mainly dendrites and dendritic spines (Svingos et al., 1998). In the area postrema, most of DOR-LI profiles observed are axon terminals (67.4%), whereas 28.3% are dendrites (Guan et al., 1997). In the spinal cord, the majority of DOR-LI is found in axon and axon terminals (Cheng et al., 1995). In the guinea pig hippocampal formation, KOR-LI is mainly localized presynaptically. More precisely, in the stratum lacunosum-moleculare of the CA1 region and all layers of the CA3 region of the hippocampus, KOR-LI is restricted to unmyelinated axons and axon terminals, and is associated with plasma membranes, large dense-core vesicles, and cytoplasmic surfaces of small vesicles. In the dentate gyrus, KOR-LI terminals form asymmetric synapses with granule cell perikarya and large unlabeled dendrites (Drake et al., 1996). More functionally, in vasopressin magnocellular neurosecretory neurons in the posterior pituitary KOR-LI is associated with the membrane of peptide-containing large secretory vesicles, and is rarely associated with the plasma membrane in unstimulated nerve terminals. However after stimulation (salt-loading that elicits vasopressin release) KOR1-LI is translocated from these vesicles to the plasma membrane. This stimulus-dependent translocation of receptors to the presynaptic plasma membrane provides a novel mechanism for regulation of transmitter release (Shuster et al., 1999). 21

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5. CONCLUSIONS The distribution of the opioid receptors has been explored for decades. The early attempts utilized autoradiographical approaches that depended upon the binding of selective radioligands to the receptors. These provided a general understanding of the localizations of these receptors. However, as our understanding of opioid systems has increased, many of the agents used have been found to lack the specificity originally attributed to them. The cloning of the opioid receptors has provided more specific tools to define their localization, but even here there is a complexity that makes the interpretation of the results difficult. Alternative splicing, especially of MOR-1, has significant implications upon both the in situ and the immunohistochemical studies due to sharing of nucleotide sequences and epitopes among more than one variant. New approaches will be required to resolve these issues and to define, unambiguously, the various receptors within the brain.

6. ABBREVIATIONS

I-II V-VI Abl Acb Ace Aco AD Amb Ame AO Arc BST Cb CC ce

CIC CL CM CPu DMH DG DR DTg Ecx Epl Fcx G Gi G1 GP 22

laminae I and II of the spinal cord laminae V and VI of the spinal cord basolateral amygdaloid nucleus nucleus accumbens central amygdaloid nucleus cortical amygdaloid nucleus anterodorsal thalamic nucleus ambiguus nucleus medial amygdaloid nucleus olfactory nucleus arcuate nucleus bed nucleus of the stria terminalis cerebellum corpus callosum central canal of the spinal cord central nucleus of the inferior colliculus centrolateral thalamic nucleus centromedian thalamic nucleus caudate putamen dorsomedial hypothalamic area dentate gyrus dorsal raphe nucleus dorsal tegmental nucleus entorhinal cortex external plexiform layer of the olfactory bulb frontal cortex nucleus gelatinosus of the thalamus gigantocellular reticular nucleus glomerular layer of the olfactory bulb globus pallidus

Opioid receptors Gr

HPC Int IP LA LC LD LG LHb LRN LS MD ME Med MG MHb Mi MM MS MVE NTS PB Pcx

PAG Pn Po PO PrS Pv PVN Re RM Rme SCIC Sex

SuG Tcx

SON SN SpV Tu Tz VDB VH VM VMH VP VPL

Ch. I

intermediate granular cell layer of the olfactory bulb hippocampus interposed cerebellar nucleus interpeduncular nucleus lateral hypothalamic area locus coeruleus laterodorsal thalamic nucleus lateral geniculate nucleus lateral habenula lateral reticular nucleus lateral septum mediodorsal thalamic nucleus median eminence medial cerebellar nucleus medial geniculate nucleus medial habenula mitral cell layer of the olfactory bulb mamaillary nucleus medial septum medial vestibular nucleus nucleus of the solitary tract parabrachial nucleus parietal cortex periaqueduuctal gray pontine reticular nucleus posterior thalamic nucleus preoptic area presubiculum paraventricular thalamic nucleus paraventricular hypothalamic nucleus reuniens thalamic nucleus raphe magnus nucleus raphe median nucleus superior colliculus striate cortex superficial gray layer of the superior colliculus temporal cortex supraoptic nucleus substantia nigra spinal trigeminal nucleus olfactory tubercule nucleus of the trapezoid body nucleus of the vertical limb of the diagonal band ventral horn of the spinal cord ventromedian thalamic nucleus ventromedial hypothalamic area ventral pallidus ventroposterolateral thalamic nucleus 23

Ch. I VTA Zi

C. Abbadie and G.W. Pasternak ventral tegmental area zona incerta

7. ACKNOWLEDGEMENTS This work was supported by Research Grants (DA02615, DA06141 and DA07242) and a Senior Scientist Award (DA00220) from the National Institute on Drug Abuse to G.W.E, as well as by a core grant to MSKCC (CA08748) from the National Cancer Institute.

8. REFERENCES Abbadie C, Gultekin SH, Pasternak GW (2000a): Immunohistochemical localization of the carboxy terminus of the novel mu opioid receptor splice variant MOR-1C within the human spinal cord. NeuroReport 11:1-5. Abbadie C, Pan YX, Drake CT, Pasternak GW (2000b): Comparative immunohistochemical distributions of carboxy terminus epitopes from the mu opioid receptor splice variants MOR-1D, MOR-1 and MOR-1C in the mouse and rat central nervous systems. Neuroscience 100:141-153. Abbadie C, Pan YX, Pasternak GW (2000c): Differential distribution in rat brain of mu opioid receptor carboxy terminal splice variants MOR-1C and MOR-l-like immunoreactivity: evidence for region-specific processing. J Comp Neurol 419:244-256.

Adams JU, Chen X, Deriel JK, Adler MW, Liu-Chen L-Y (1994): Intracerebroventricular treatment with an antisense oligodeoxynucleotide to kappa-opioid receptors inhibited kappa-agonist-induced analgesia in rats. Brain Res 667:129-132.

Aicher SA, Sharma S, Cheng PY, Liu-Chen LY, Pickel VM (2000): Dual ultrastructural localization of II-opiate receptors and substance P in the dorsal horn. Synapse 36:12-20. Arvidsson U, Dado RJ, Riedl M, Lee J-H, Law PY, Lob HH, Elde R, Wessendorf MW (1995a): ~-Opioid receptor immunoreactivity: distribution in brainstem and spinal cord, and relationship to biogenic amines and enkephalin. J Neurosci 15:1215-1235. Arvidsson U, Riedl M, Chakrabarti S, Lee J-H, Nakano AH, Dado RJ, Loh HH, Law P-Y, Wessendorf MW, Elde R (1995b): Distribution and targeting of a ~-opioid receptor (MOR1) in brain and spinal cord. J Neurosci 15:3328-3341. Arvidsson U, Riedl M, Chakrabarti S, Vulchanova L, Lee J-H, Nakano AH, Lin X, Lob HH, Law P-Y, Wessendorf MW, Elde R (1995c): The kappa-opioid receptor is primarily postsynaptic: combined immunohistochemical localization of the receptor and endogenous opioids. Proc Natl Acad Sci USA 92:5062-5066. Atweh SF, Kuhar MJ (1977a): Autoradiographic localization of opiate receptors in rat brain. I. Spinal cord and lower medulla. Brain Res 124:53-67. Atweh SF, Kuhar MJ (1977b): Autoradiographic localization of opiate receptors in rat brain. II. The brain stem. Brain Res 129:1-12.

Atweh SF, Kuhar MJ (1977c): Autoradiographic localization of opiate receptors in rat brain. III. The telencephalon. Brain Res 134:393-405.

Bare LA, Mansson E, Yang D (1994): Expression of two variants of the human I~ opioid receptor mRNA in SK-N-SH cells and human brain. FEBS Lett 354:213-216. Bausch SB, Patterson TA, Appleyard SM, Chavkin C (1995): Immunocytochemical localization of delta opioid receptors in mouse brain. J Chem Neuroanat 8:175-189. Bonnet KA, Groth J, Gioannini T, Cortes M, Simon EJ (1981): Opiate receptor heterogeneity in human brain regions. Brain Res 221:437-440. Bowen WD, Gentleman S, Herkenham M, Pert CB (1981): Interconverting I~ and 3 forms of the opiate receptor in rat striatal patches. Proc Natl Acad Sci USA 78:4818-4822. Brown MJ, Martin JR, Asbury AK (1976): Painful diabetic neuropathy. Arch Neurol 33:164-171. Chang K-J, Cooper BR, Hazum E, Cuatrecasas P (1979): Multiple opiate receptors: different regional distribution in the brain and differential binding of opiates and opioid peptides. Mol Pharmacol 16:91-104. Chen XH, Adams JU, Geller EB, Deriel JK, Adler MW, Liu-Chen L-Y (1995): An antisense oligodeoxynucleotide to I~-opioid receptors inhibits t~-opioid receptor agonist-induced analgesia in rats. Eur J Pharmacol 275:105108.

24

Opioid receptors

Ch. I

Cheng PY, Svingos AL, Wang H, Clarke CL, Jenab S, Beczkowska IW, Inturrisi CE, Pickel VM (1995): Ultrastructural immunolabeling shows prominent presynaptic vesicular localization of 3-opioid receptor within both enkephalin- and nonenkephalin-containing axon terminals in the superficial layers of the rat cervical spinal cord. J Neurosci 15:5976-5988. Chien C-C, Pasternak GW (1995): (-)-Pentazocine analgesia in mice: interactions with a ~ receptor system. Eur J Pharmaco1294:303-308. Clark JA, Liu L, Price M, Hersh B, Edelson M, Pasternak GW (1989): Kappa opiate receptor multiplicity: evidence for two U50,488-sensitive kappal subtypes and a novel kappa3 subtype. J Pharmacol Exp Ther 251:461-468. Delfs JM, Kong H, Mestek A, Chen Y, Yu L, Reisine T, Chesselet M-F (1994a): Expression of mu opioid receptor mRNA in rat brain: an in situ hybridization study at the single cell level. J Comp Neurol 345:46-68. Delfs JM, Yu L, Reisine T, Chesselet M-F (1994b): The distribution and regulation of mu opioid receptor mRNA in rat basal ganglia. Regul Pept 54:79-80. Ding YQ, Kaneko T, Nomura S, Mizuno N (1996): Immunohistochemical localization of tx-opioid receptors in the central nervous system of the rat. J Comp Neurol 367:375-402. Drake CT, Milner TA (1999): Mu opioid receptors are in somatodendritic and axonal compartments of GABAergic neurons in rat hippocampal formation. Brain Res 849:203-215. Drake CT, Patterson TA, Simmons ML, Chavkin C, Milner TA (1996): Kappa opioid receptor-like immunoreactivity in guinea pig brain: ultrastructural localization in presynaptic terminals in hippocampal formation. J Comp Neurol 370:377-395. Duka T, Wuster M, Schubert P, Stoiber R, Herz A (1981): Selective localization of different types of opiate receptors in hippocampus as revealed by in vitro autoradiography. Brain Res 205:181-186. Edley SM, Herkenham M (1984): Comparative development of striatal opiate receptors and dopamine revealed by autoradiography and histofluorescence. Brain Res 305:27-42. Evans CJ, Hammond DL, Frederickson RCA (1988): The opioid peptides. In: Pasternak GW (Ed), The Opiate Receptors. Clifton, NJ: Humana Press, pp. 23-74. Evans CJ, Keith Jr. DE, Morrison H, Magendzo K, Edwards RH (1992): Cloning of a delta opioid receptor by functional expression. Science 258:1952-1955. Foote RW, Maurer R (1982): Autoradiographic localization of opiate kappa-receptors in the guinea-pig brain. Eur J Pharmacol 85:99-103. Foote RW, Maurer R (1986): Distribution of opioid binding sites in the guinea pig hippocampus as compared to the rat: a quantitative analysis. Neuroscience 19:847-856. Gavrriaux-Ruff C, Peluso J, Befort K, Simonin F, Zilliox C, Kieffer BL (1997): Detection of opioid receptor mRNA by RT-PCR reveals alternative splicing for the 3- and kappa-opioid receptors. Mol Brain Res 48:298-304. George SR, Zastawny RL, Briones-Urbina R, Cheng R, Nguyen T, Heiber M, Kouvelas A, Chan AS, O'Dowd BF (1994): Distinct distributions of mu, delta and kappa opioid receptor mRNA in rat brain. Biochem Biophys Res Commun 205:1438-1444. Gistrak MA, Paul D, Hahn EF, Pasternak GW (1990): Pharmacological actions of a novel mixed opiate agonist/antagonist, naloxone benzoylhydrazone. J Pharmacol Exp Ther 251:469-476. Goldstein A, Tachibana S, Lowney LI, Hunkapiller M, Hood L (1979): Dynorphin-(1-13), an extraordinarily potent opioid peptide. Proc Natl Acad Sci USA 76:6666-6670. Goodman RR, Pasternak GW (1985): Visualization of mul opiate receptors in rat brain using a computerized autoradiographic subtraction technique. Proc Natl Acad Sci USA 82:6667-6671. Goodman RR, Snyder SH (1982a): Autoradiographic localization of kappa opiate receptors to deep layers of the cerebral cortex may explain unique sedative and analgesic effects. Life Sci 31:1291-1294. Goodman RR, Snyder SH (1982b): Kappa opiate receptors localized by autoradiography to deep layers of cerebral cortex: relation to sedative effects. Proc Natl Acad Sci USA 79:5703-5707. Goodman RR, Snyder SH, Kuhar MJ, Young WS (1980): Differentiation of delta and mu opiate receptor localizations by light microscopic autoradiography. Proc Natl Acad Sci USA 77:6239-6243. Goodman RR, Adler AA, Pasternak GW (1985): Regional differences in mul binding of 3-DADL-enkephalin: comparisons of thalamus and cortex. Neurosci Lett 59:155-158. Grimm LJ, Clock BJ, Cox BM (1994): Comparison of the distribution of mu and delta opiate receptor mRNAs in rat and mouse brain by in situ hybridization. Regul Pept 54:111-112. Guan JL, Wang QR Nakai Y (1997): Electron microscopic observation of delta-opioid receptor-1 in the rat area postrema. Peptides 18:1623-1628. Herkenham M, Pert CB (1980): In vitro autoradiography of opiate receptors in rat brain suggests loci of 'opiatergic' pathways. Proc Natl Acad Sci USA 77:5532-5536.

25

Ch. I

C. Abbadie and G.W. Pasternak

Hiller JM, Fan LQ, Simon EJ (1996): Autoradiographic comparison of [3H]DPDPE and [3H]DSLET binding: evidence for distinct 81 and 82 opioid receptor populations in rat brain. Brain Res 719:85-95. Honda CN, Arvidsson U (1995): Immunohistochemical localization of delta- and mu-opioid receptors in primate spinal cord. NeuroReport 6:1025-1028. Hughes J, Smith TW, Kosterlitz HW, Fothergill LA, Morgan BA, Morris HR (1975): Identification of two related pentapeptides from the brain with potent opiate agonist activity. Nature 258:577-579. Jiang Q, Takemori AE, Sultana M, Portoghese PS, Bowen WD, Mosberg HI, Porreca F (1991): Differential antagonism of opiate delta antinociception by [D-AlaZ,Cys6]enkephalin and naltrindole-5'-iosothiocyanate: evidence for subtypes. J Pharmacol Exp Ther 257:1069-1075. Jordan BA, Devi LA (1999): G-protein-coupled receptor heterodimerization modulates receptor function. Nature 399:697-700.

Kalyuzhny AE, Arvidsson U, Wu W, Wessendorf MW (1996): Ix-Opioid and 8-opioid receptors are expressed in brainstem antinociceptive circuits: studies using immunocytochemistry and retrograde tract-tracing. J Neurosci 16:6490-6503. Kawabata A, Nishimura Y, Takagi H (1992): L-Leucyl-L-arginine, naltrindole and D-arginine block antinociception elicited by L-arginine in mice with carrageenin-induced hyperalgesia. Br J Pharmacol 107:1096-1101. Kent JL, Pert CB, Herkenham M (1982): Ontogeny of opiate receptors in rat forebrain: visualization by in vitro autoradiography. Dev Brain Res 2:487-504. Kieffer BL (1999): Opioids: first lessons from knockout mice. Trends Pharmacol Sci 20:19-26. Kieffer BL, Befort K, Gaveriaux-Ruff C, Hirth CG (1992): The 8-opioid receptor: isolation of a cDNA by expression cloning and pharmacological characterization. Proc Natl Acad Sci USA 89:12048-12052. Lai J, Bilsky EJ, Porreca F (1995): Treatment with antisense oligodeoxynucleotide to a conserved sequence of opioid receptors inhibits antinociceptive effects of delta subtype selective ligands. J Recept Res 15:643-650. Le Moine C, Kieffer B, Gaveriaux-Ruff C, Befort K, Bloch B (1994): Delta-opioid receptor gene expression in the mouse forebrain: localization in cholinergic neurons of the striatum. Neuroscience 62:635-640. Leventhal L, Bodnar RJ, Cole JL, Rossi GC, Pan YX, Pasternak GW (1996): Antisense oligodeoxynucleotides against the MOR-1 clone alter weight and ingestive responses in rats. Brain Res 719:78-84. Leventhal L, Stevens LB, Rossi GC, Pasternak GW, Bodnar RJ (1997): Antisense mapping of the MOR-1 opioid receptor clone: modulation of hyperphagia induced by DAMGO. J Pharmacol Exp Ther 282:1402-1407. Leventhal L, Silva RM, Rossi GC, Pasternak GW, Bodnar RJ (1998): Morphine-6~-glucuronide-induced hyperphagia: characterization of opioid action by selective antagonists and antisense mapping in rats. J Pharmacol Exp Ther 287:538-544.

Lewis ME, Pert A, Pert CB, Herkenham M (1983): Opiate receptor localization in rat cerebral cortex. J Comp Neurol 216:339-358.

Loh HH, Liu HC, Cavalli A, Yang WL, Chen YF, Wei LN (1998): Ix Opioid receptor knockout in mice: effects on ligand-induced analgesia and morphine lethality. Mol Brain Res 54:321-326. Lord JAH, Waterfield AA, Hughes J, Kosterlitz HW (1977): Endogenous opioid peptides: multiple agonists and receptors. Nature 267:495-499. Mansour A, Khachaturian H, Lewis ME, Akil H, Watson SJ (1987): Autoradiographic differentiation of mu, delta, and kappa opioid receptors in the rat forebrain and midbrain. J Neurosci 7:2445-2464. Mansour A, Thompson RC, Akil H, Watson SJ (1993): Delta opioid receptor mRNA distribution in the brain: comparison to delta receptor binding and proenkephalin mRNA. J Chem Neuroanat 6:351-362. Mansour A, Fox CA, Burke S, Meng F, Thompson RC, Akil H, Watson SJ (1994a): Mu, delta, and kappa opioid receptor mRNA expression in the rat CNS: an in situ hybridization study. J Comp Neurol 350:412-438. Mansour A, Fox CA, Burke S, Watson SJ (1994b): Immunohistochemical localization of the kappal opioid receptors. Regul Pept 54:177-178. Mansour A, Fox CA, Burke S, Watson SJ (1994c): Immunohistochemical localization of the mu opioid receptors. Regul Pept 54:179-180.

Mansour A, Fox CA, Meng F, Akil H, Watson SJ (1994d): Kappa 1 receptor mRNA distribution in the rat CNS: comparison to kappa receptor binding and prodynorphin mRNA. Mol Cell Neurosci 5:124-144. Mansour A, Fox CA, Thompson RC, Akil H, Watson SJ (1994e): Ix-Opioid receptor mRNA expression in the rat CNS: comparison to Ix-receptor binding. Brain Res 643:245-265. Mansour A, Fox CA, Burke S, Akil H, Watson SJ (1995): Immunohistochemical localization of the cloned Ix opioid receptor in the rat CNS. J Chem Neuroanat 8:283-305. Mansour A, Burke S, Pavlic RJ, Akil H, Watson SJ (1996): Immunohistochemical localization of the cloned kappa 1 receptor in the rat CNS and pituitary. Neuroscience 71:671-690.

26

Opioid receptors

Ch. I

Martin WR, Eades CG, Thompson JA, Huppler RE, Gilbert PE (1976): The effects of morphine and nalorphine-like drugs in the nondependent and morphine-dependent chronic spinal dog. J Pharmacol Exp Ther 197:517-532. Matthes HWD, Maldonado R, Simonin F, Valverde O, Slowe S, Kitchen I, Befort K, Dierich A, Le Meur M, Doll6 P, Tzavara E, Hanoune J, Roques BP, Kieffer BL (1996): Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the Ix-opioid-receptor gene. Nature 383:819-823. Maurer R, Cortes R, Probst A, Palacios JM (1983): Multiple opiate receptor in human brain: an autoradiographic investigation. Life Sci 33:231-234. McLean S, Rothman RB, Jacobson AE, Rice KC, Herkenham M (1987): Distribution of opiate receptor subtypes and enkephalin and dynorphin immunoreactivity in the hippocampus of squirrel, guinea pig, rat and hamster. J Comp Neuro1255:497-510.

Minami M, Toya T, Katao Y, Maekawa K, Nakamura S, Onogi T, Kaneko S, Satoh M (1993): Cloning and expression of a cDNA for the rat kappa-opioid receptor. FEBS Lett 329:291-295. Minami M, Onogi T, Toya T, Katao Y, Hosoi Y, Maekawa K, Katsumata S, Yabuuchi K, Satoh M (1994): Molecular cloning and in situ hybridization histochemistry for rat Ix-opioid receptor. Neurosci Res 18:315-322. Mogil JS, Marek P, O'Toole LA, Helms ML, Sadowski B, Liebeskind JC, Belknap JK (1994): Ix-Opiate receptor binding is up-regulated in mice selectively bred for high stress-induced analgesia. Brain Res 653:16-22. Morris BJ, Herz A (1986): Autoradiographic localization in rat brain of k opiate binding sites labelled by [3H]bremazocine. Neuroscience 19:839-846. Negri L, Potenza RL, Corsi R, Melchirri P (1991): Evidence for two subtypes of delta opioid receptors in rat brain. Eur J Pharmacol 196:335-336.

Pan YX, Cheng J, Xu J, Pasternak GW (1994): Cloning, expression and classification of a kappa3-related opioid receptor using antisense oligodeoxynucleotides. Regul Pept 54:217-218. Pan YX, Cheng J, Xu J, Rossi GC, Jacobson E, Ryan-Moro J, Brooks AI, Dean GE, Standifer KM, Pasternak GW (1995): Cloning and functional characterization through antisense mapping of a kappa3-related opioid receptor. Mol Pharmacol 47:1180-1188.

Pan YX, Xu J, Bolan EA, Abbadie C, Chang A, Zuckerman A, Rossi GC, Pasternak GW (1999): Identification and characterization of three new alternatively spliced mu opioid receptor isoforms. Mol Pharmacol 56:396-403. Pan YX, Xu J, Bolan EA, Chang A, Mahurter L, Rossi GC, Pasternak GW (2000): Isolation and expression of a novel alternatively spliced mu opioid receptor isoform, MOR-1E FEBS Lett 466:337-340. Pasternak GW (1993): Pharmacological mechanisms of opioid analgesics. Clin Neuorpharmacol 16:1-18. Pasternak GW, Snyder SH (1975): Identification of a novel high affinity opiate receptor binding in rat brain. Nature 253:563-565.

Pasternak GW, Standifer KM (1995): Mapping of opioid receptors using antisense oligodeoxynucleotides: correlating their molecular biology and pharmacology. Trends Pharmacol Sci 16:344-350. Pasternak GW, Childers SR, Snyder SH (1980a): Naloxazone, long-acting opiate antagonist: effects in intact animals and on opiate receptor binding in vitro. J Pharmacol Exp Ther 214:455-462. Pasternak GW, Childers SR, Snyder SH (1980b): Opiate analgesia: evidence for mediation by a subpopulation of opiate receptors. Science 208:514-516. Pasternak GW (2001): Incomplete cross tolerance and multiple mu opioid peptide receptors. Trends Pharmacol Sci 22:67-70. Pasternak KR, Rossi GC, Zuckerman A, Pasternak GW (1999): Antisense mapping KOR-I: evidence for multiple kappa analgesic mechanisms. Brain Res 826:289-292. Paul D, Levison JA, Howard DH, Pick CG, Hahn EF, Pasternak GW (1990): Naloxone benzoylhydrazone (NalBzoH) analgesia. J Pharmacol Exp Ther 255:769-774. Pearson J, Brandeis L, Simon EJ, Hiller J (1980): Radioautography of binding of tritiated diprenorphine to opiate receptors in the rat. Life Sci 26:1047-1052. Pert CB, Snyder SH (1973): Opiate receptor: demonstration in nervous tissue. Science 179:1011-1014. Pfeiffer A, Pasi A, Mehraein P, Herz A (1982): Opiate receptor binding sites in human brain. Brain Res 248:87-96. Pfeiffer A, Branti V, Herz A, Emrich HM (1986): Psychotomimesis mediated by • opiate receptors. Science 233:774-776.

Price M, Gistrak MA, Itzhak Y, Hahn EF, Pasternak GW (1989): Receptor binding of 3H-naloxone benzoylhydrazone: a reversible kappa and slowly dissociable IX opiate. Mol Pharmacol 35:67-74. Quirion R, Bowen WD, Herkenham M, Pert CB (1982): Visualization and solubilization of rat brain opiate receptors with a 'k' ligand selectivity pattern. Cell Mol Neurobiol 2:333-346. Quirion R, Zajac JM, Morgat JL, Roques BP (1983): Autoradiographic distribution of mu and delta opiate receptors in rat brain using highly selective ligands. Life Sci 33:227-230. 27

Ch. I

C. Abbadie and G.W. Pasternak

Quirion R, Pilapil C, Magnan J (1987): Localization of kappa opioid receptor binding sites in human forebrain using [3H]U69,593: comparison with [3H]bremazocine. Cell Mol Neurobiol 7:303-307. Raffa RB, Mathiasen JR, Kimball ES, Vaught JL (1993): The combined immunological and antinociceptive defects of beige-J mice: the possible existence of a 'ix-repressin'. Life Sci 52:1-8. Reisine T, Bell GI (1993): Molecular biology of opioid receptors. Trends Neurosci 16:506-510. Reisine T, Pasternak GW (1996): opioid analgesics and antagonists. In: Hardman JG, Limbird LE (Eds), Goodman and Gilman's: The Pharmacological Basis of Therapeutics. New York: McGraw-Hill, pp. 521-556. Renda T, Negri L, Tooyama I, Casu C, Melchiorri P (1993): Autoradiographic study on [3H]-[D-Ala2]-deltorphin-I binding sites in the rat brain. NeuroReport 4:1143-1146. Rossi GC, Pan YX, Cheng J, Pasternak GW (1994): Blockade of morphine analgesia by an antisense oligodeoxynucleotide against the mu receptor. Life Sci 54:PL375-PL379. Rossi GC, Pan YX, Brown GP, Pasternak GW (1995a): Antisense mapping the MOR-1 opioid receptor: evidence for alternative splicing and a novel morphine-6~-glucuronide receptor. FEBS Lett 369:192-196. Rossi GC, Standifer KM, Pasternak GW (1995b): Differential blockade of morphine and morphine-6~-glucuronide analgesia by antisense oligodeoxynucleotides directed against MOR-1 and G-protein ~ subunits in rats. Neurosci Lett 198:99-102.

Rossi GC, Brown GP, Leventhal L, Yang K, Pasternak GW (1996): Novel receptor mechanisms for heroin and morphine-6~ -glucuronide analgesia. Neurosci Lett 216:1-4. Rossi GC, Leventhal L, Pan YX, Cole J, Su W, Bodnar RJ, Pasternak GW (1997a): Antisense mapping of MOR-1 in the rat: distinguishing between morphine and morphine-6~-glucuronide antinociception. J Pharmacol Exp Ther 281:109-114.

Rossi GC, Su W, Leventhal L, Su H, Pasternak GW (1997b): Antisense mapping DOR-1 in mice: further support for 3 receptor subtypes. Brain Res 753:176-179. Rothman RB, Bykov V, DeCosta BR, Jacobson AE, Rice KC, Brady LS (1990): Interaction of endogenous opioid peptides and other drugs with four kappa opioid binding sites in guinea pig brain. Peptides 11:311-317. Schuller AG, King MA, Zhang J, Bolan E, Pan YX, Morgan DJ, Chang A, Czick ME, Unterwald EM, Pasternak GW, Pintar JE (1999): Retention of heroin and morphine-6 beta-glucuronide analgesia in a new line of mice lacking exon 1 of MOR-1. Nat Neurosci 2:151-156. Schulz S, Schreff M, Koch T, Zimprich A, Gramsch C, Elde R, H611t V (1998): Immunolocalization of two mu-opioid receptor isoforms (MOR1 and MOR1B) in the rat central nervous system. Neuroscience 82:613-622. Sharif NA, Hughes J (1989): Discrete mapping of brain Mu and delta opioid receptors using selective peptides: quantitative autoradiography, species differences and comparison with kappa receptors. Peptides 10:499-522. Shuster SJ, Riedl M, Li XR, Vulchanova L, Elde R (1999): Stimulus-dependent translocation of kappa opioid receptors to the plasma membrane. J Neurosci 19:2658-2664. Simon EJ, Hiller JM, Edelman I (1973): Stereospecific binding of the potent narcotic analgesic [3H]Etorphine to rat-brain homogenate. Proc Natl Acad Sci USA 70:1947-1949. Slater P, Patel S (1983): Autoradiographic localization of opiate kappa receptors in the rat spinal cord. Eur J Pharmacol 92:159-160. Sora I, Takahashi N, Funada M, Ujike H, Revay RS, Donovan DM, Miner LL, Uhl GR (1997): Opiate receptor knockout mice define Ix receptor roles in endogenous nociceptive responses and morphine-induced analgesia. Proc Natl Acad Sci USA 94:1544-1549. Standifer KM, Chien C-C, Wahlestedt C, Brown GP, Pasternak GW (1994): Selective loss of ~ opioid analgesia and binding by antisense oligodeoxynucleotides to a 3 opioid receptor. Neuron 12:805-810. Svingos AL, Clarke CL, Pickel VM (1998): Cellular sites for activation of 3-opioid receptors in the rat nucleus accumbens shell: relationship with MetS-enkephalin. J Neurosci 18:1923-1933. Takemori AE, Portoghese PS (1993): Enkephalin antinociception in mice is mediated by 81- and 32-opioid receptors in the brain and spinal cord, respectively. Eur J Pharmacol 242:145-150. Tempel A, Zukin RS (1987): Neuroanatomical patterns of the IX, 3 and k opioid receptors of rats brain as determined by quantitative in vitro autoradiography. Proc Natl Acad Sci USA 84:4308-4312. Tempel A, Gardner EL, Zukin RS (1984): Visualization of opiate receptor upregulation by light microscopy autoradiography. Proc Natl Acad Sci USA 81:3893-3897. Terenius L (1973): stereospecific interaction between narcotic analgesics and a synaptic plasma membrane fraction of rat cerebral cortex. Acta Pharmacol Toxicol 32. Unterwald EM, Knapp C, Zukin RS (1991): Neuroanatomical localization of kappa1 and kappa2 opioid receptors in rat and guinea pig brain. Brain Res 52:57-65. Wamsley JK, Zarbin MA, Young WS, Kuhar MJ (1982): Distribution of opiate receptors in the monkey brain: an autoradiographic study. Neuroscience 7:595-613.

28

Opioid receptors

Ch. I

Wang H, Pickel VM (1998): Dendritic spines containing Ix-opioid receptors in rat striatal patches receive asymmetric synapses from prefrontal corticostriatal afferents. J Comp Neurol 396:223-237. Wang H, Wessendorf MW (1999): IX- and 8-opioid receptor mRNAs are expressed in spinally projecting serotonergic and nonserotonergic neurons of the rostral ventromedial medulla. J Comp Neurol 404:183-196. Wang H, Moriwaki A, Wang JB, Uhl GR, Pickel VM (1997): Ultrastructural immunocytochemical localization of Ix-opioid receptors in dendritic targets of dopaminergic terminals in the rat caudate-putamen nucleus. Neuroscience 81: 757-771. Wang H, Pickel VM (2001): Preferential cytoplasmic localization of delta-opioid receptors in rat striatal patches: comparison with plasmalemmal mu-opioid receptors. J Neurosci 21:3242-3250. Wise SP, Herkenham M (1982): Opiate receptor distribution in the cerebral cortex of the rhesus monkey. Science 218:387-389.

Wolozin BL, Pasternak GW (1981): Classification of multiple morphine and enkephalin binding sites in the central nervous system. Proc Natl Acad Sci USA 78:6181-6185. Xu H, Partilla JS, De Costa BR, Rice KC, Rothman RB (1993): Differential binding of opioid peptides and other drugs to two subtypes of opioid 3ncx binding sites in mouse brain: further evidence for 8 receptor heterogeneity. Peptides 14:893-907. Yasuda K, Raynor K, Kong H, Breder CD, Takeda J, Reisine T, Bell GI (1993): Cloning and functional comparison of kappa and ~ opioid receptors from mouse brain. Proc Natl Acad Sci USA 90:6736-6740. Young III WS, Kuhar MJ (1979): A new method for receptor autoradiography: [3H]opioid receptors in rat brain. Brain Res 179:255-270.

Zadina JE, Hackler L, Ge LJ, Kastin AJ (1997): A potent and selective endogenous agonist for the Ix-opiate receptor. Nature 386:499-502. Zhu YX, King MA, Schuller AGE Nitsche JF, Reidl M, Elde RP, Unterwald E, Pasternak GW, Pintar JE (1999): Retention of supraspinal delta-like analgesia and loss of morphine tolerance in 8 opioid receptor knockout mice. Neuron 24:243-252. Zimprich A, Simon T, Hollt V (1995): Cloning and expression of an isoform of the rat Ix opioid receptor (rMOR 1 B) which differs in agonist induced desensitization from rMOR1. FEBS Lett 359:142-146. Zukin RS, Eghbali M, Olive D, Unterwald E, Tempel A (1988): Characterization and visualization of rat and guinea pig brain kappa opioid receptors: evidence for kappa1 and kappa2 opioid receptors. Proc Natl Acad Sci USA 85:4061-4065.

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CHAPTER II

The melanin-concentrating hormone GUILLAUME J. HERVIEU, LAURENCE MAULON-FERAILLE, JONATHAN K. CHAMBERS, JANE E. CLUDERAY, SHELAGH WILSON, FRANCOISE PRESSE AND JEAN-LOUIS NAHON

The purpose of this review is to present the most recent findings on structural analysis and neurobiology of melanin-concentrating hormone (MCH), with special emphasis on the cloning, functional characterisation and neuronal distribution of at least two MCH receptors. It was a long-standing and important achievement in the field. First, the reader is proposed a survey of the milestones that marked the discovery of the peptide MCH in 1983 until the characterisation of two MCH receptors, named MCH-R1 and MCH-R2, in the late 1990s and the development of MCH-R1 antagonists that show potential anxiolytic, antidepressant and/or anorectic actions in 2002. Previous reviews have been already published on the functions and brain localisation of MCH in fishes and mammals (Eberle, 1988; Baker, 1991, 1994; Nahon et al., 1993; Nahon, 1994; Knigge et al., 1996; Griffond and Baker, 2002) and on the pairing of the MCH ligand with two subtype receptors so far (Saito et al., 2000; Boutin et al., 2002; Griffond and Baker, 2002). Recent neuroanatomical comments related to the feeding properties of MCH have been written by Sawchenko (1998); Tritos and Maratos-Flier (1999); Kilduff and de Lecea (2001). The effect of a pharmaceutically drug discovery molecule acting as an antagonist at one of the MCH receptor, MCH-R1, and affecting both affective and energy balances, is discussed by Schwartz and Gelling (2002).

1. A SURVEY OF THE MELANIN-CONCENTRATING SYSTEM: SEMINAL BACKGROUND STUDIES AND PHARMACEUTICAL INTEREST

1.1. MCH HAS A CONCERTED SET OF ACTIONS IN THE FISH MCH was first isolated from fish teleost pituitaries in 1983 by its role in melanin-concentration action (Kawauchi et al., 1983). The 'MCH enigma' was solved. For 50 years, ichthyologists and life scientists interested in pigmentary control had wondered if two substances with opposite actions existed to regulate the fish colour tegument for the component dark/light. It was already known that s-melanin-stimulating hormone (MSH), one of the very first neuropeptides ever biochemically isolated, had darkening properties by allowing melanin to spread with melasome suborganelles. Viewed from a pharmacological aspect, one was asking if the inhibition of the MSH darkening properties just happening by biological

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

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desensitisation/degradation or by active pharmacological/functional antagonism of another substance or both. MCH is classified as a melanotropin, i.e. a factor implicated in regulating colour tegument (with a potency ECs0 as low as pM range to evoke melanin concentration in certain teleost fishes). However, unlike the MSH and adrenocorticotropic hormone (ACTH) peptide family, all derived from the prepro-opio-melanocortin (POMC) precursor, MCH is not classified as a melanocortin because it lacks the consensus melanocortin sequence - H i s - P h e - A r g - T r p - . Consistent with that, MCH does not signal through the specific set of not less than six G-protein-coupled receptor (GPCR) proteins binding MSHs and ACTH, and termed from MC-1 to MC-6 binding (see Adan and Gipsen, 1997). Intriguingly, it may be possible that MCH and MSH are evolutionary related as suggested by Matsunaga et al. (1986). In the fish, few other functions than melanophore concentration were noted for MCH, i.e. an action of the peptide regulating the stress axis (see Baker, 1994) and more recently, stimulating the immune system (Harris and Bird, 2000). It may well illustrate a coordinated, goal-orientated function of the peptide in the vital neural activation related to a potentially hazardous situation perceived by the fish. This could be particularly pertinent in predatory situations, when camouflage can be achieved by matching tegument colour to background environment colour through a simple monocolour balance from dark to light, and when neurobehavioural mechanisms are triggered that should normally optimise the animal's response in dealing with dangerous and potentially fatal threatening stimuli. 1.2. MCH ALSO EXISTS IN MAMMALS Immunochemical studies indicated the very probable existence of an MCH-like factor in the rat, as an antiserum raised against salmon MCH resulted in a very strong and localised immunosignal within the hypothalamus in rats (Zamir et al., 1986a,b) (see Fig. 1). The rat orthologue peptide was purified in the laboratory of Wilie Vale at the Clayton Foundation for Peptide Studies, The Salk Institute in California in 1989 from 60,000 hypothalami using the same extracts that served to purify the growth hormone-releasing hormone (Vaughan et al., 1989). Rat and salmon MCH displayed strong homology mainly within the loop structure (Fig. 2A). Molecular studies allowed the identification of the genetic rat sequence encoding the prepro-MCH precursor and establish the possibility that two other

......... ~.

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Fig. 1. MCH-containing neurons and projections in the rat. MCH-producing cells and projections are noted, respectively, by dots and lines.

32

The melanin-concentrating hormone A

Rat MCH

Ch. H

H-~pmel~---]z~tz_eulz~~.c~ ~t]z~=~lGzy~ v~ ~ ~ ~-oc~ ~IG~F~-]-C~

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Fig. 2. (A) Comparison of the rat and salmon MCH. Identical amino acids are boxed. (B) Schematic representation of the MCH mRNA precursor and peptide sequences. Basic amino acid residues are noted in black in the precursor. Arrowheads indicate putative or overt cleavage sites.

peptides, namely neuropeptide-GE (NGE) and neuropeptide-EI (NEI), could be released upon proteolytic maturation of the prepro-MCH precursor (Breton et al., 1989; Nahon et al., 1989) (Fig. 2B). 1.3. MCH AS A 'GUT-BRAIN' PEPTIDE A new mammalian peptidergic system had thus emerged. It would fit within the general rules that govern neuropeptidergic expression: notably, the neuropeptide is in fact both expressed in the central nervous system (see Fig. 1; Bittencourt et al., 1992) and in the periphery, notably and not surprisingly in the enteric nervous system. By different techniques either related to mRNA studies (Northern blot, mRNA in situ hybridisation, RT-PCR) or peptide studies (immunohistochemistry, radio-immunoassays), MCH mRNA and peptides have been characterised in the peripheral tissues of the mouse (Breton et al., 1993b), rat (Hervieu and Nahon, 1995; Takahashi et al., 1995) and human (Takahashi et al., 1995; Viale et al., 1997). Expression level is much lower in the periphery than in the brain. There is a widespread expression of MCH in the gastrointestinal organs (stomach, duodenum, jejunum, ileum, colon), in the immune system (thymus, spleen), in the cardiopulmonary tissues (heart, lung) and in the reproductive organs (testis, ovary). Histological studies showed that in the testis, the MCH peptide is present in the Sertoli cells and is also strikingly localised to the germ cell nuclei (Hervieu and Nahon, 1995; Hervieu et al., 1996b) while in the gastrointestinal tract, the peptide is produced by white cells and is shown to regulate the hydro-ionic balance of the gastrointestinal tract in an in vivo model (Hervieu et al., 1996a). As alluded before, immunomodulatory effect of MCH have been reported on rainbow trout phagocytes and leucocytes (Harris and Bird, 2000). These characteristics are not at odds with general features of neuropeptides and the established concept of a dual and reciprocal communication between the nervous, humoral and immune system (Blalock, 1989). 33

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1.4. "A [MAMMALIAN] PEPTIDE STILL IN SEARCH OF FUNCTIONS" (Bittencourt et al., 1992) For 20 years, it is fair to say that it proved difficult to assign to the mammalian MCH a clear set of biological actions. First of all, there is no evidence for a role of MCH in the control of mammalian melanogenesis (reviewed by Baker, 1994; Nahon, 1994), nor melanoma incidence (see gene array study by Bittner et al., 2000). That MCH does not act directly on pigmentary control may be partially explained by a strikingly different neuroanatomical location of the fish and rodent brain MCH system. While in the fish MCH is a hypophysiotropic factor released from the pituitary with quasi-instant control over the pigmentory system, there is, on the contrary, a marked low peptide content at both the median eminence and pituitary levels. Rat MCH is neither synthesised in the neurohypophysis, nor in the hypothalamic nuclei with strong neural connections to the median eminence, part of the hypothalamo-anterior pituitary portal system that links hypothalamus to the adenohypophysis gland in the rat. Also, of particular importance, numerous, but unsuccessful, attempts by laboratories have been made to identify MCH receptor(s) for more than a decade. Undoubtedly, the absence of a simple MCH bioactivity assay (i.e. melanin concentration in fish skin bioassay) and its non-reliable characteristics, have certainly refrained attempts to characterise the receptor. Significant chemistry work was performed in Switzerland by Alex Eberle, a leader in the melanocortins field. Photoactivable and radio-active MCH peptide mimetics are used to demonstrate evidence of specific MCH receptor-like entities in a number of mouse melanomas (Drozdz et al., 1995). It is well known that peptides are difficult to experiment with, mostly because of their usually high molecular weight and other unfavourable generally hydrophobic physicochemical properties. They render peptides 'sticky' to the tube in vitro and when injected in vivo, peptides bind mostly to albumins in the peripheral blood and to brain ventricular component (see study by Bittencourt and Sawchenko, 2000, discussing if centrally administered neuropeptides access cognate receptors, using the CRF system as a case study). Peptides are also readily degradable by enzymes. Their usual short shelf-life in biological fluids and the sheer multiplicity of product degradation by-passes that may still retain pharmacological activity, add another degree of complexity for studying neuropeptidergic systems. Amongst other endocrine and neural systems, the brain contains probably the richest source of peptides, both in terms of diversity and abundance. Brain central effects of a peptide can very often be only evidenced through surgical intervention and intracerebral administration (intrathecal/intracerebroventricular). To demonstrate an in vivo central effect of any compound, it is most preferred to experiment with a compound that is not a peptide, is orally available and at least reasonably penetrant into the brain. It is perhaps why few out of the sheer plethora of naturally purified and in silico potential identified peptides, fulfil the criteria as neurotransmitter. Meanwhile, sporadic evidence attributed the peptide a role in sensory gating (Miller et al., 1993), epileptogenesis (Knigge et al., 1997), and, in the regulation of the hypothalamopituitary adrenal axis, though reported with somehow opposite actions (Jezova et al., 1992; Bluet-Pajot et al., 1995; Ludwig et al., 1998). It was possibly a lead in confirming an evolutionary conservation of the peptide function to intervene in the stress axis, both in the fish and in the rat.

34

The melanin-concentrating hormone

Ch. H

1.5. MCH REGULATES FOOD INTAKE IN RATS Strong and sudden impetus to research in the MCH field probably all came from a study published in Nature in 1996 showing that the peptide induces feeding behaviour (Qu et al., 1996). Numerous review works have been published looking at the possibility of developing drugs that would regulate the energy balance, notably to fight obesity, a leading cause of mortality in the 'Western world' (see Campfield, 1998; Bray and Tartaglia, 2000; Kopelman, 2000; Clapham et al., 2001; see also reviews on the neurocircuitries implicated in the regulation of feeding behaviour, e.g. Morley, 1987; Flier and Maratos-Flier, 1998; Elmquist et al., 1999; Schwartz et al., 2000). From that point, interest in MCH was no more purely academic. Major pharmaceuticals and biotechs entered a race to characterise the receptor (SmithKline Beecham, Glaxo-Wellcome, Schering-Plough, Synaptic, Merck, Sharp and Dohm, Takeda), and to develop peptide and non-peptide mimetics ligand of MCH (legacy SmithKline Beecham, GlaxoSmithKline, Schering-Plough, Servier, Synaptic, Takeda). Major academic laboratories studying the feeding behaviour entered the competition too. 1.6. SLC-1 AND ANOTHER ORPHAN GPCR ARE PARALOGUE RECEPTORS FOR MCH An orphan GPCR called SLC-1 (GPR24) was identified in 1996 by Kolakowski et al., 1996. SLC-1 has most homology with the somatostatin receptor family, but does not bind somatostatin-14, nor somatostatin-28, nor corticostatin. Kolakowski then commented that 'the abundance of SLC-1 mRNA expression in brain with regional localisation to discrete areas involved in functions such as emotion, memory and sensory perception, make the isolation of the endogenous ligand of this receptor an important priority. This heralds the exciting potential of identifying a novel peptidergic neurotransmitter signalling system in the brain' (Kolakowski et al., 1996). Darlinson and Richter (1999), while reviewing evolutionary aspects of paired peptidergic ligand/receptor systems as well as orphan ligands or receptors, also highlighted the importance in identifying the natural ligand of SLC-1. SLC-1 was later characterised as being a receptor for MCH (Chambers et al., 1999; Saito et al., 1999; Bachner et al., 1999; Lembo et al., 1999; Shimomura et al., 1999; reviewed in Saito et al., 2000). Some of the approaches used a method best described as 'reverse pharmacology approach' (RPMA) (e.g. Chambers et al., 1999; for review, Stadel et al., 1997) while others approaches relied on systematic agonist compound bank screening or biochemical purification of ligands that can activate the transfected receptor in heterologous cell lines. No more than a year later, a second receptor with low sequence identity to that of MCH-R1 was identified as a biologically relevant second MCH receptor subtype or a paralogue to MCH-R1 (reviewed in Boutin et al., 2002). 1.7. THE MCH SYSTEM APPEARS AS A COMPLEX EVOLUTIONARY MODEL The MCH and its receptor system, apart from its biological significance, seems to have had a very intriguing purely genetic evolution history. There are two very distinct loci for human MCH gene chromosomal localisation. A truncated version of the MCH gene was identified with a different chromosomal localisation to that of the 'authentic' MCH gene (Breton et al., 1993a,b; Pedeutour et al., 1994). It is only found in the Hominidae (Viale et al., 1998, 2000). That very example was published in the Science issue reporting the data on human genome sequencing by Craig Venter and colleagues as part of phylogenetic case study (Courseaux 35

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and Nahon, 2001). Interestingly, this gene may code for a putative nuclear protein, the signal peptide of the pro-MCH being replaced by a nuclear-localisation signal (NLS) in the new gene (Viale et al., 2000). Coincidentally perhaps, Hervieu et al. (1996a,b) localised MCHlike immunoreactivity in the nuclei of germ cells. Prepro-MCH antisense transcripts were characterised in the rat (Hervieu and Nahon, 1995), the human (Miller et al., 1998) and cell lines (Presse et al., 1997; Borsu et al., 2000). Toumaniantz et al. (1996) characterised a peptide called MGOP that could be derived from the alternative splicing of the prepro-MCH mRNA. Regarding MCH receptors, data seem firmly to establish that non-human species (rat, mouse, hamster, guinea, pig, and rabbit) do not have functional MCH-R2 receptors, or encode a non-functional MCH-R2 pseudogene while retaining MCH-R1 functional expression (see Tan et al., 2002). Such a late evolutionary process for both a variant putative peptide as well as a receptor subtype must be seen as a rare occurrence.

2. THE PRO-MCH GENE, REGULATION OF EXPRESSION AND PRECURSOR PROCESSING 2.1. STRUCTURE, CHROMOSOMAL MAPPING AND EVOLUTION OF THE PRO-MCH GENE AND LINKED GENES The organisation of the gene encoding the MCH precursor in the rat, mouse and human (reviewed in Nahon et al., 1993; Nahon, 1994) is clearly established. This gene comprises three exons and two introns coveting about 1.4 kb of genomic DNA (Fig. 3A). The first exon encodes the 5'-untranslated region of the mRNA and the N-terminal part of pro-MCH including the signal peptide that allows targeting to the secretory pathway. The second exon comprises the sequence corresponding to NGE, NEI and the first three amino acids of MCH. The last 15 amino acids of MCH and the T-untranslated amino acids are localised on exon III. Intron B splits, methionine codon with the nucleotide A on exon II and the bases TG on exon III. This intronic organisation is identical in the rat mouse and human MCH gene. This unusual intron position for a neuropeptide encoding gene is of primary importance to generate by alternative splicing a new protein named MCH-gene-overprinted-polypeptide (MGOP) (Toumaniantz et al., 1996) (Fig. 3B). In addition, dense MGOP projections were encountered in the suprachiasmatic, ventromedial and arcuate nuclei, as well as median eminence, suggesting neuroendocrine functions for MGOE Recently, comparison of MCH and MGOP distribution in the rat brain revealed a striking colocalisation in neurones of the zona incerta/lateral hypothalamus and unique expression of MGOP in neurones of the hypothalamic periventricular nucleus and of many brain areas (cortex, amygdala, caudate putamen, lateral septal nucleus) (Toumaniantz et al., 2000). Characterisation of high molecular weight MCH gene transcripts in PC12 cells and rat tissues revealed the existence of antisense RNAs complementary to the MCH gene (Presse et al., 1997; Borsu et al., 2000). This followed the discovery of shorter natural antisense transcript in the rat gut (Hervieu and Nahon, 1995). Human antisense prepro-MCH mRNAs were also reported by Miller et al. (1998) and Viale et al. (2000). In the pheochromocytoma cell line PC12, two classes of antisense RNAs were found: (1) non-coding unspliced RNAs overlapping exon II/exon III and flanking intronic sequences of the MCH gene; and (2) alternative spliced mRNAs coding for new RNA/DNA binding proteins. We named this gene AROM for Antisense-RNA-overlapping-MCH. This gene maps at the same locus than 36

The melanin-concentrating hormone

Ch. H

Fig. 3. The MCH gene. (A) Comparison of the human, mouse and rat MCH genes. Exons and introns are noted by boxes and lines, respectively. Percent identities are indicated. (B) Structure of the MCH and MGOP mRNA and deduced proteins. Alternative splicing is noted by dotted line.

MCH gene and appears highly conserved among rat, mouse and human (Borsu et al., in preparation). Because of the reciprocal regulation of MCH and AROM gene expression in PC12 cells (Borsu et al., 2000) and in vivo (Presse and Nahon, unpublished data) it is feasible that AROM mRNA and/or proteins may control MCH gene expression but this remains to be directly tested. The pro-MCH (PMCH) locus was localised to chromosome 12q23 in human (Pedeutour et al., 1994; Viale et al., 1997). We previously hypothesised that the MCH gene could be a candidate gene for the Darier's disease and spinocerebellar ataxia type 2, but further studies excluded MCH and identified the actual genes involved in these diseases (Sanpei et al., 1996; Sakuntabhai et al., 1999). A second MCH-like gene system was identified in the human genome. Indeed a truncated version (named variant MCH gene) of the authentic MCH was found duplicated on the long an short arms of the human chromosome 5 (reviewed in Nahon, 1994). Interestingly, this gene may code for a putative nuclear protein, the signal peptide of the pro-MCH being replaced by a nuclear-localisation signal (NLS) in the new gene (Viale et al., 2000). Phylogenetic analysis revealed that the variant MCH gene arose by very complex mechanisms only in the Hominidae lineage (Viale et al., 1998; Courseaux and Nahon, 2001). Thus, the MCH gene family provides a unique model to investigate the structural and functional switches of genes that diverged during late primate evolution. 2.2. REGULATION OF PREPRO-MCH GENE EXPRESSION The regulation of MCH gene expression has been most extensively investigated with in vivo models. In particular, MCH mRNA increases during postnatal development at the 37

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suckling-transition time in rodents (Presse et al., 1992; Breton et al., 1993b; Brischoux et al., 2001) and exhibits a marked circadian variation (Presse and Nahon, 1993; Bluet-Pajot et al., 1995), consistent with the important role of MCH in feeding behaviour, locomotor activity or higher integrative behaviours. Furthermore, low level of MCH mRNA expression was found associated with short-term footshock stress (Presse et al., 1992) and dehydration or hypertonic saline regimen (Presse and Nahon, 1993), suggesting an inhibitory function for the MCH system in the stress response and fluid homeostasis (see below). Furthermore, an increase in MCH mRNA was recently reported (Herv6 et al., 1998) following acute polyethylene glycol (PEG) injection that induced a reduction of the extracellular fluid volume. The apparent discrepancy between previous and recent data on the regulation of MCH mRNA following osmotic challenges most likely resulted from a differential effect of the experimental models on volaemia or osmolability as discussed by Herv6 et al. (1998). During recent years, a number of studies have accumulated with respect to variations of MCH mRNA expression, feeding behaviour and possibly obesity. First, activation of MCH neurones was observed following insulin and 2-deoxyglycose injections in the rat (BahjaouiBouhaddi et al., 1994; Presse et al., 1996) or lesions of the neuromedial hypothalamic nuclei (VMN) (Griffond et al., 1995). Then, direct involvement of the MCH neuronal system in the control of feeding behaviour was supported by the increase of MCH mRNA levels observed following food-deprivation in the rats (Presse et al., 1996; Herv6 and Fellmann, 1997) and mice (Qu et al., 1996). Finally, overexpression of MCH mRNA and/or pro-MCH derived peptides was found in various obese rodents, including ob/ob mice (Qu et al., 1996; Mondal et al., 2002); Huang et al., 1999), db/db mice (Mizuno et al., 1998; Mondal et al., 2002), fat~fat mice (Rovbre et al., 1996) and Ay/a (agouti) mice (Hanada et al., 2000). Alteration in adiposity may also have some influences on the MCH expression. This was exemplified by the decrease of MCH mRNA observed in brown adipose tissue-deficient mice which developed both obesity and hyperleptinaemia (Tritos et al., 1998a) and the higher levels of MCH mRNA found in thin ewes by comparison with the fat animals (Henry et al., 2000). Leptin either decreased MCH gene expression following acute injection in rats (Sahu, 1998) and ob/ob mice (Tritos et al., 2001) or, conversely, stimulated MCH mRNA peptide expression under chronic treatment in lean and ob/ob mice (Huang et al., 1999). Stricker-Krongrad et al. (2001) recently reported that in obese hyperphagic Zucker, the absence of leptin signaling in rats is associated with an increased hypothalamic expression and circulating release of MCH and that it probably contributed to their obesity syndrome. Using canine distemper virus (CDV) which can target hypothalamic nuclei, and lead to obesity syndrome in the late stages of infection, Verlaeten et al. (2001) showed a specific down-regulation of melanin-concentrating hormone precursor mRNA (ppMCH) in infected obese mice. The MCH gene expression is decreased in adult male rats treated with lipopolysaccharide (LPS), an inflammatory agent. Anorexia is often a consequence of inflammatory processes and the down-regulation of MCH gene expression may contribute to hypophagic behaviours (Sergeyev et al., 2001). Apart from regulation by stress, food or water deprivation, the MCH neuronal system is also affected by the steroid hormones status of the animals. Early studies (Parkes and Vale, 1992b; Presse et al., 1992) have suggested that endogenous glucocorticoids in adrenalectomised rats or addition of dexamethasone in vivo or in hypothalamic cells in culture, stimulated the synthesis of MCH mRNA and pro-MCH derived peptides (MCH and NEI). The effects of gonadal steroids on MCH mRNA or peptide production were recently examined in models of ovariectomised (OVX) female rats (Murray et al., 2000) or OVX female macaques (Viale et al., 1999a). The animals were exposed to oestradiol benzoate to suppress secretion of luteinising hormone (LH) in the first step, then a 'mid-cycle-like' LH-surge operated 38

The melanin-concentrating hormone

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naturally in the primates or after administration of progesterone in the rat model. In the rats, MCH mRNA content decreased in a subpopulation of cell bodies of the medial zona incerta following the oestradiol benzoate-treatment. Negative regulation by oestrogen on MCH expression was further confirmed in an oestrogen-cachexia-induced model. Oestrogeninduced anorexia in rodents is associated with decreased MCH gene expression (Mystkowski et al., 2000). Surprisingly, in view of the effect of MCH on the LH release (see below), the addition of progesterone provoked a LH surge but did not affect the reduction of MCH mRNA levels observed in the zona incerta. In the macaques, oestradiol treatment induced basic variations in MCH and NEI contents that paralleled those of gonadotrophin-releasing hormone (GnRH). This suggested that pro-MCH-derived peptides may indeed participate in the regulation of the LH secretion through interaction with the GnRH neural system localised in the medial preoptic area. Furthermore, in addition to mature MCH or NEI, other MCH-ir and NEI-ir products were identified in oestradiol-treated OVX monkeys, suggesting that post-translational regulation at the level of processing and/or degradation of pro-MCH may operate (Viale et al., 1999a). 2.3. PEPTIDE CHARACTERISATION AND PRECURSOR PROCESSING Regarding the structure of the mammalian pro-MCH derived peptides, it is worth noting that exclusively mature peptides, i.e. cyclic MCH and amidated NEI, were found in the rat and human brains (Hervieu et al., 1996a; Viale et al., 1997) (Fig. 4A). Conversely, neither MCH nor NEI were found in human peripheral organs and a larger peptidic form was identified in the colon, thymus or adipose tissues (Viale et al., 1997) (Fig. 4A). This large MCH-ir peptide contained, at least, a NEI sequence in its N-terminus and ends with a C-terminal MCH sequence (Viale et al., 1997). Similarly, pro-MCH processing intermediates are likely to exist in the rat gut (Hervieu et al., 1996a), testis (Hervieu et al., 1996b) and spleen (Hervieu and Nahon, unpublished data) but their structures remained elusive. More recently, a pro-MCH derivative similar to this detected in the human peripheral organs was characterised in the mouse spleen (Viale et al., 1999b) and fat tissues (Viale and Nahon, unpublished data). In this context, the processing of MCH precursor was examined using various cellular systems and an in vivo PC2 KO-mouse model (Viale et al., 1999b) (Fig. 4B). The main finding was that active pro-convertase (PC2) is necessary and sufficient to generate NEI in the brain and in PC12 cells. In contrast, several PC, including PC1/3, PC2 and PC5/6A may cleave at the appropriate site to produce MCH either in vaccinia expression systems or in the mouse brain. Incidently, co-localisation studies demonstrated simultaneous expression of MCH mRNA and PC2 in all MCH-expressing cell bodies of the lateral hypothalamus, whereas only 15-20% of these cells contained PC1. Therefore, PC2 is likely to be the key enzyme that cleaves MCH-precursor to generate MCH and NEI in the brain. The counterpart in peripheral organs remained unknown.

3. FEATURES OF THE MCH SYSTEM IN THE RAT CNS

Long before MCH was known in the rat brain, immunoreactive signals related to the POMCderived 0~-MSH were observed where immunosignals for the other POMC-derived peptides could not be seen. This suggested that the antiserum recognised an as yet unknown factor, bearing immunological resemblance with the c~-MSH. By retrograde tracing studies combined to immunohistochemistry, a system with lateral hypothalamic origin was identified and which 39

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A

Hypothalamus 15 600

NEI

400

200

200

100

15

5

Periphery 400

200]

25 (rain)

25

15

300

32

300

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45 (min)

35

32

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45 (min)

MCH

' or.’,

MC.

I

MCH

I

I

Fig. 4. MCH precursor processing and peptide production. (A) Schematic representation of RP-HPLC analysis of protein extracts from hypothalamus or peripheral organs. Mature NEI and MCH migrate at 15 and 32 min, respectively. At the periphery a larger product migrates at 45 min (Viale et al., 1997). (B) Role of pro-convertases in pro-MCH processing. Sequences at the cleavage sites are noted. The question marks indicate that PC involved at the putative cleavage site is unknown.

innervated the entire neuraxis. It was termed the 'et-2' system (for second ot-MSH system). That system was to be later identified as the MCH system. The antiserum was targeting the amidated motif of NEI (Nahon et al., 1989). The ~-MSH is amidated, as could also be the NEI, a peptide of the pro-MCH precursor protein, because of a consensus motive for amidation. At the same time, it brings support for the predicted amidated NEI to exist in the rat brain (see Eberle, 1988; Baker, 1994; Nahon, 1994; Sawchenko, 1998). The seminal paper by Bittencourt et al. (1992) is a very thorough description of the MCH system in the rat, and pertinently proposed some directions in which to investigate the role of the peptide. Other neuroanatomical studies have been done in the guinea pig (Knigge et al., 1996), in the monkey (Bittencourt et al., 1998) and the human (Elias et al., 1998, 2001).

40

The melanin-concentrating hormone

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3.1. A STRIKING HYPOTHALAMIC LOCALISATION OF THE MCH IMMUNOREACTIVE CELL BODIES The hypothalamic location of MCH neurones is striking: more than 95% of the MCH gene neurones in the CNS are hypothalamic with more than 90,000 cells there expressing the MCH gene (see Knigge et al., 1996). The MCH gene expression level here is just below those of oxytocin and vasopressin (Bittencourt et al., 1992). In fact, hypothalamic MCH peptide levels are amongst the highest in the brain (Nahon, 1994) and animals do not need to receive colchicine treatment (which inhibits axonal transport of prepro-MCH out of the cell body) in order to allow visualisation of MCH cell body immunosignals (Bittencourt et al., 1992). Interestingly, the high level of MCH gene expression was confirmed in substractive molecular biology experiments at the Salk Institute: two clones identified as both the MCH and the hypocretins/orexins were read-out as being particularly strongly enriched in rat hypothalamic cDNA libraries (Gautwik et al., 1996). MCH is an excellent peptidergic marker of the lateral hypothalamus and would stand as the only specific peptidergic marker of the lateral hypothalamus until the discovery of the orexins/hypocretins (Sakurai et al., 1998). Bittencourt, Presse and colleagues identified secondary sites of brain MCH expression in the olfactory tubercle and in a previously uncharacterised part of the paramedian pontine reticular formation in the rat (Bittencourt et al., 1992; Presse et al., 1992) but not in the monkey (Bittencourt et al., 1998). Of interest, lactation induces novel hypothalamic expression site of the female rat MCH system in the medial preoptic nucleus, preoptic periventricular nucleus and rostral area of the paraventricular hypothalamus (Knollema et al., 1992). Some differences are noted in the way MCH neurones are organised in the hypothalamus of different animals: the MCH cell group is always positioned in the lateral hypothalamus and zona incerta but with differences in the subdivisions of these regions: for instance, a tail of immunopositive cells extend in the guinea pig until reaching the posterior hypothalamus at the supramammillary commissure level (see Knigge et al., 1996). A reliable feature for all species studied so far (mouse, rat, hamster, guinea pig, rabbit, dog, monkey, human) is that the MCH cell group do not populate any of the traditional proper hypothalamic nuclei (see Knigge et al., 1996). In the human, no differences was reported regarding the number and position of MCH cell bodies in males versus females. The most prominent cluster of MCH cells ran along the entire rostrocaudal extension of the fornix. The MCH perikarya were observed extending back into the posterior hypothalamic area, just above the mammillary body and close to the third ventricle (Elias et al., 1998). MCH gene expression is noted at early stages of development: in the rat, MCH mRNA and peptide are detected at day 13 of gestation while in the human, MCH immunoreactivity is observed at the 7th week of foetal life (Bresson et al., 1987; Brischoux et al., 2001). The ontogeny of rat hypothalamic MCH neurones was carefully studied using the bromodeoxyuridine method, combined to immunochemical techniques, in order to determine the period of birth of these neurones. The maturation of the total MCH neuronal population occurred throughout prenatal stages and the early postnatal period. No subpopulations of MCH neurones could be identified based on ontogenic criteria, but it was found that the spatiotemporal pattern of MCH cell genesis had striking similarities with the one described for the hypothalamic parvicellular neuroendocrine neurones (Brischoux et al., 2001). Cytological features of the non-colchicinised MCH neuropeptidergic adult rat hypothalamic cell include: a medium-size, a shape ranging from multipolar to fusiform, giving rise to 2-5 41

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primary dendrites and common secondary dendritic branching. Varicose axons (0.1-0.2 ~m in diameter) arose from labelled perikarya or the proximal portion of primary dendrites. Large and bipolar neurones (12 x 40 ~m) are observed at the level of the internal capsula (Bittencourt et al., 1992; Knigge et al., 1996). Electron microscopy using an antiserum directed towards the rat MCH revealed the ultrastructural details of MCH neurones: densecored granules (75-200 nm in diameter) in a moderate number, distributed throughout the cytoplasm, with immunosignals located in a subset of Golgi saccules. Labelled cell bodies are often apposed to vascular elements such as venules, separated by fine interposed astrocytic processes. Axons are most commonly unmyelinated and terminals are characteristic of a neuropeptidergic neurone with small electron-lucent vesicles and large dense-cored vesicles. These vesicles were detected at the level of the external lamina of the median eminence (Bittencourt et al., 1992). 3.2. FEATURES OF THE MCH INNERVATION WITHIN THE MAMMALIAN BRAIN MCH nerve immunoreactivity is broadly distributed within the rat brain (neocortex, allocortex and hippocampal formation, basal ganglia, diencephalon, brainstem/pons/reticular formation, myelencephalon) (Figs. 1 and 9G) with very similar projections to these of orexins (Peyron et al., 1998; see also Chapter 5). The peptide was not detected in the anterior pituitary. Extremely dense terminal fields of MCH nerves are observed in parts of the extrapyramidal system, the reticular formation throughout the brainstem, the precerebellar nuclei and the spinal cord. In the neocortex, the MCH immunosignals distributes within laminae, a characteristic of a non-specific cortical afferent innervation, which appears to be a basis for generalised cortical arousal. In the diencephalon, MCH is not predicted to have major neuroendocrine effects because of a relatively low peptide content in key areas like the paraventricular and supraoptic nuclei and also, a relatively sparse innervation of median eminence. Other MCH-immunoreactive territories enriched with MCH are the autonomic-related structures of the brainstem, though the levels of peptide are much lower than in the forebrain. Signals are observed in all sorts of nuclei (motor nucleus of the V, parabrachial nuclei, locus coeruleus) and reach the spinal cord, particularly enriched around the central canal (Bittencourt et al., 1992; Bittencourt and Elias, 1998; Sawchenko, 1998). Some investigations have more especially focused on the MCH immunoreactive projections to the parabrachial nucleus and pontine gustatory area (Touzani et al., 1993), within the hypothalamus (Broberger et al., 1998; Elias et al., 1998; Broberger, 1999; Abrahamson et al., 2001; Abrahamson and Moore, 2001), to the peri-aqueductal grey matter (PAG; Elias and Bittencourt, 1997) and to the medial septum and thoracic spinal cord (Bittencourt and Elias, 1998). By combining retrograde tracing studies and immunochemistry, it was shown that projections to the periaqueduct area originated from two major sources: the zona incerta supplied afferents via a medial pathway that entered the PAG dorsally at rostral levels, and a pathway originating in the lateral hypothalamus that entered the PAG ventrally at more caudal levels. The medial subdivision of the PAG, which encompassed the Gerrits column I contained the greatest level of MCH-ir fibres (Elias and Bittencourt, 1997). In the spinal cord, MCH-ir fibres were concentrated primarily in lamina X (surrounding the central canal) and secondarily in layers I, III and IV. In the septal complex, MCH-ir fibres were enriched in the medial aspects and in the vertical and horizontal limbs of the medial septum and the nucleus of the diagonal band (Bittencourt and Elias, 1998). The medial area of the parabrachial nucleus, also termed the pontine taste area, receives many ascending gustatory afferents and is implicated 42

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in the palatability of nutrients. Fifty to 60% of the MCH-ir neurones from the juxtacapsular region and 20-30% of neurones in the perifornical area project to the taste area (Touzani et al., 1993). A recent study using neurotropic virus pseudorabies in conjunction with the immunocytochemical localisation of MCH to better define the pathways in the rat hypothalamus related to the reflex control of thermogenesis has shown that MCH neurones from the lateral hypothalamus project to brown adipose tissue (Oldfield et al., 2002). These data set the scene for the peptide to play a role in an extremely vast array of brain activities, ranging from neocortical to autonomic modulations. MCH appears as to be one out of the many other factors which contribute to the bridging of diverse biological systems, such as mood regulation, reward system, feeding and water intake, sexual behaviour, motor and somatosensory controls. It is presently uncertain to what extent the peptide is significantly implicated within each of them but evidences are accumulating that MCH is a key regulator in body homeostasis by its role on those diverse systems. 3.3. COLOCALISATION DATA It is a rule of thumb that neuropeptides usually colocalise with other neuromodulators within the cell that produces them. To date, investigations have mainly focused on feeding regulators. 3.3.1. Neurochemical colocalisation

As expected, MCH coexists with the NEI peptide (released from the prepro-MCH precursor protein) with a 96% overlap except in the interanterodorsal nucleus of the thalamus (Bittencourt et al., 1992). It has still not yet been proven that the NGE, the other peptide potentially released from the prepro-MCH precursor protein, also co-exists in MCH neurones. Also in the lateral hypothalamus, the MGOP-ir cells have a very similar location to that of MCH cells (Toumaniantz et al., 1996). Overt co-localisation of the MGOP-ir and MCH-ir was demonstrated in the LHA whereas only MGOP-ir was found in neurons of the periventricular area of the hypothalamus (Toumaniantz et al., 2000). MCH and orexin form distinct cell population groups in the lateral hypothalamus of the rodent (less than 1% overlap; Broberger et al., 1998; Elias et al., 1998) and the human (Elias et al., 1998). MCH co-exist with the anorectic neuropeptide cocaine- and amphetamine-regulated transcript (CART) (65% of the MCH population; Broberger, 1999; Elias et al., 2001; Brischoux et al., 2001) and glutamate (Abrahamson and Moore, 2001; Abrahamson et al., 2001). Comforting the role of MCH in energy balance, the MCH neurones receive a dense innervation by NPY, AgRP and ~-MSH neurones of the basal hypothalamus (Elias et al., 1998) and harbour on the cell membrane leptin receptor (Hakansson et al., 1998a) as well as the NK3 receptor (55% of the MCH population, Griffond et al., 1997) and contain within their nuclei STAT-3-1ike immunosignal (Hakansson et al., 1998). In a more recent study, Iqbal et al. (2001) showed that all MCH neurones harbour the long form of the leptin receptor, OB-Rb. MCH neurones innervating VIP and AVP fibres coming from the hypothalamic suprachiasmatic nucleus (Abrahamson et al., 2001) could implicate the MCH neurones as a component of the arousal system. Lastly, strong acetylcholine-esterase activity and immunoreactivity is present in the MCH neurones at reticular and nuclear envelope level, suggesting a possible cholinergic receptivity (see Griffond et al., 1998).

43

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3.3.2. Functional colocalisation

Energy metabolism is also thought to be critically controlled by the LHA, as it contains a population of neurones which is sensitive to glucose levels and is activated by hypoglycaemia. The identity of these glucostat cells has remained elusive, probably because the lateral hypothalamus is not as topologically organised as other major nuclei of the hypothalamus. Cells are scattered within the medial forebrain bundle, the most prominent brain fibre network bundle. This has added a great deal of complexity to the design of experiments to study the LHA that would not damage the medial forebrain bundle itself (see Bernardis and Bellinger, 1996). The principal path of MCH projections though the basal forebrain, hypothalamus and rostral midbrain is virtually superimposable upon a map of sites showing increased metabolic activity in response to rewarding electrical stimulations of the LHA (see Bittencourt et al., 1992; Sawchenko, 1998). This data would imply the MCH neuronal population be the brain glucose-sensor neuronal group. However, experimental data do not favour that hypothesis as shown by injection of non-metabolisable glucose analog such as goldthioglucose, which does not affect MCH gene expression (Grillon et al., 1997) and 2-deoxyglucose used on slice cultures of rat hypothalamus (Bayer et al., 1999a). Rather, it would seem that the orexin neurones are the anatomical substrate of the hypothalamic glucostat, as reported by an activation of the orexin neurons lateral hypothalamic for instance by insulin-induced acute hypoglycaemia (Moriguchi et al., 1999). 3.4. NEUROCHEMICAL ENVIRONMENT AND SURVIVAL OF MCH NEURONES 3.4.1. Neurochemical environment

The MCH neurones are strongly intermingled with orexin, GABA, dopamine, neurotensin and galanin neurones. MCH neurones are surrounded by a rich neuropil containing dopamine, serotonin, neurotensin, galanin, neurophysin, GABA, NPY, ~-MSH, AgRR somatostatin, VIR AVP and neurokinin B. Nodular figures and pericellular baskets suggest the occurrence of synapses between the different systems (see Griffond et al., 1998). 3.4.2. Survival of M C H neurones in culture

Compagnone et al. (1991) devised a serum-free medium culture system for growing hypothalamic neurones containing MCH immunoreactive cells. Using a co-culture model, it was shown that diffusible factors from the arcuate nucleus and the diagonal band increased the number and the size of the MCH neurones. The glia in the arcuate nucleus produced factors important for MCH neurite outgrowth and expressed inhibitory factors, preventing the adhesion of MCH cells on arcuate glial cells. Contacts between MCH and dopaminergic cells were increased (Compagnone et al., 1993). Hypothalamic slices prepared from 6- to 8-day-old rats and containing the MCH neurones, when treated with carbachol, a cholinergic agonist, resulted in an increase of MCH gene expression, whereas that regulation was abolished using atropine and hexamethonium, respectively, a muscarinic and nicotinic antagonist, respectively (Bayer et al., 1999b). That mice deleted for the muscarinic M3 receptor have reduced level of MCH (Yamada et al., 2001) comes as supportive evidence for a close interaction between the MCH and cholinergic systems. Also hypothalamic slices containing the MCH neurones prepared from 6- to 8-day-old rats, could be maintained for 1 month in culture with MCH 44

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neurones responsive to leptin at day 10 of growth (Bayer et al., 1999c). Organotypic cultures might thus represent an adequate model in which to investigate further the pharmacology and physiology of the MCH system. A study using synaptically coupled neurones from the rat embryonic hypothalamus in tissue culture has shown that MCH exerted profound inhibition of synaptic activity on those cells. Both glutamate and GABA synaptic transmission were inhibited by MCH (Gao and van den Pol, 2001). In a study on the hippocampus, Varas et al. (2002) observed a long-lasting potentiation on the hippocampal evoked response on dentate gyms induced by MCH. 3.5. PHYSIOLOGICAL SECRETION OF MCH To date, dexamethasone, cAMP analogues, phorbol esters, noradrenaline and glutamate/ NMDA have been shown to induce the release of MCH from neurones in culture while CRF inhibits MCH release (see Nahon, 1994). 3.6. PERIPHERAL PLASMATIC AND CENTRAL MCH While Takahashi et al. (1995) were unable to detect rat peripheral blood circulating levels of MCH, a recent study using a commercial radio-immunoassay has shown that MCH-like immunoreactive material was detectable with levels ranging from 54 to 400 pg/ml of plasma (Bradley et al., 2000). A recent topic in neuropeptidergy has been to propose transcytosis of peptides between the brain and the periphery (in both ways) (refer to: Strand et al., 1994; Crawley and McLean, 1996). Sensor-systems at the periphery-brain barrier level exist in order to transduce an humoral messenger into a brain signal, like for leptin. It implies a cooperation at the blood-brain barrier (BBB) level mostly while circumventricular organs (e.g. median eminence, choroid plexus, area postrema... ) vasculature, though devoid of BBB, are not a first-place of entry because part of the ependyme is joined by tight junctions. Peptides can cross the BBB due to their lipophilicity, but the existence of saturable transporters for peptides across the blood-brain barrier has been reported (refer to: Strand et al., 1994; Crawley and McLean, 1996; Gao et al., 2000). Such a system could exist for MCH. While almost all of the peptides and polypeptides tested so far cross the BBB at a faster rate than the vascular marker albumin, the MCH analog, [125I][Phe13,Tyr19]MCH, did not cross faster than Tc-99m-albumin. This is probably because of its binding to serum proteins (Kastin et al., 1999; see also Kastin et al., 2000). 3.7. DEGRADATION OF MCH BY PEPTIDASES The endopeptidase EC 3.4.24.11 (neutral endopeptidase, enkephalinase) in vitro attacked MCH at three sites of the molecule with an apparent affinity of about 12 txM and a kcat of 4 rain -1 . The first site of cleavage was at CysV-Met 8, i.e. within the peptide loop formed by the internal disulphide bridge, and necessary to bioactivity. NEP could therefore be considered as one of the main MCH-inactivating peptidases since the degradation products generated are probably devoid of biological activity (Checler et al., 1992; Maulon-Feraille et al., 2002). NEP is particularly abundant in choroid tissues and meninges which are the first sites sensing the i.c.v. MCH injection. Hence it is probable that MCH may have a short half-life in the blood and cerebrospinal fluid (CSF). However, it has to be noticed that the dipeptide H-NEIMCH-OH is fully resistant to degradation by both aminopeptidase M and endopeptidase 24.11 as well as by exo- and endo-proteases using brain extracts and purified proteases. It retains 45

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biological activity on MCH-R1 and has physiologically a more potent orexigenic effect than MCH itself (Maulon-Feraille et al., 2002). It may well be that the dipeptide is a physiological agonist at MCH-R1.

4. C E N T R A L E F F E C T S OF MCH We shall focus here on three cerebral functions supported by substantial, although sometimes controversial, experimental evidence. 4.1. MCH AND THE REGULATION OF THE HPA (Fig. 5) Historically, the first study dealing with the MCH role in mammals was to test the effect of salmon MCH on ACTH release from isolated rat pituitary cells (Baker et al., 1985). This initiated the long series of conflicting results since the direct inhibitory action of salmon MCH (Baker et al., 1985) could not be reproduced using the synthetic rat MCH (Navarra et al., 1990). In the same line, Jezova et al. (1992) reported a central stimulatory effect of rat MCH on basal ACTH release after intracerebroventricular (i.c.v.) administration in conscious rats. Another study, however, found that i.c.v, injection of rat MCH (and NEI) did not modify the basal secretion of ACTH at day or night time (Bluet-Pajot et al., 1995). On the contrary, MCH appears to inhibit ACTH secretion after an ether stress (Bluet-Pajot et al., 1995) or after a mild handling stress (Ludwig et al., 1998). Interestingly, NEI (Bluet-Pajot et al., 1995) or MSH (Ludwig et al., 1998) can prevent the inhibitory action of MCH on ACTH release. Since none of these peptides may antagonise the binding of MCH ligand or activation of the MCH receptor in transfected cells (see below), it is likely that they act through their own

Fig. 5. The hypothalamic-pituitary-adrenal axis and central neurotransmitter circuitry. During stress, stimuli are

integrated at the levels of the brainstem and hypothalamus. +, activator of the stress response; -, inhibitor of the stress response. Stress leads to secretion of glucocorticoids which in turn inhibit the activators at multiple levels in the brain and possibly induces putative activator (like MCH) in the hypothalamus. 46

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receptor either on the same cellular target or downstream of MCH in the stress-responseregulating pathway. Lastly, another work has reported that MCH administered intravenously (i.v.) induced a strong and significant dose-dependent increase in plasmatic corticosterone level (Ashmeade et al., 2000). It is thus presently a confusing situation where both activatory and inhibitory actions of MCH on the stress axis are reported and it is hoped that some light can be shed on these discrepancies in the near future. The anxiolytic and antidepressant properties of SNAP-7941, an MCH-R1 antagonist (Borowsky et al., 2002), may, however, favour the hypothesis that MCH is activatory within the stress axis. 4.2. MCH AND REPRODUCTIVE FUNCTIONS Based on neuroanatomical considerations, Gonzalez, Wilson and colleagues made a comprehensive study of MCH and sexual or reproductive behaviours in female rats. Infusion of rat MCH in the medial preoptic area (MPOA) or the ventromedial nucleus (VMN) of the hypothalamus stimulated lordosis of sexually non-receptive rats (Gonzalez et al., 1996). MCH administered into the MPOA or the median eminence has also a stimulatory effect on the LH release (Gonzalez et al., 1997a). Interestingly, this action seems of true physiological significance since MCH antiserum injected in the MPOA fully prevented the progesteroneinduced rise in LH release. Furthermore, MCH and MSH had opposite effects on LH release after injection in MPOA or ME (Gonzalez et al., 1997a). Later, the same group (Murray et al., 2000) confirmed the stimulatory action of MCH on LH secretion when injected into the MPOA and suggested that this peptide could be a weak agonist at the MC5 receptor. However, another laboratory (Tsukamura et al., 2000) found very recently that central injection of rat MCH decreased both plasma LH concentrations and LH pulse frequencies. There is no obvious explanation for these opposite results other than the fact that the stimulatory effect was noted when MCH was injected at low concentrations (100 ng/side) into discrete hypothalamic areas (MPOA, VMN) whereas the inhibitory effect was observed when highest doses (1-10 ~g/animal) were infused into the third cerebroventricle. Another group recently observed an effect of MCH on the stimulation of both LH and FSH gonadotropins from proestrous pituitaries similar to the effect produced by luteinising hormone-releasing hormone (LHRH). Simultaneous incubation of pituitaries with MCH and LHRH did not modify LH, but increased the FSH release induced by LHRH. This suggest that MCH could be involved in the regulation of preovulatory gonadotropin secretion (Chiocchio et al., 2001). 4.3. A ROLE FOR MCH IN REGULATING WATER BALANCE Given the particularly robustly strong (and quasi-uniquely localised) MCH gene expression in the lateral hypothalamus and zona incerta in the all mammals studied so far (rat, guinea pig, mouse, monkey, human; see Section 3.1), a role for MCH in the regulation of the hydric and energy balance was suggested on the basis of functional neuroanatomical considerations. The evidence for a role of MCH in water balance was known prior to the biochemical purification of the rat MCH: a 5-day salt loading triggered an elevation of MCH immunoreactivity in the hypothalamus and pituitary (Zamir et al., 1986a). Gene-expression studies showed that rat hypothalamic MCH gene expression was down-regulated by a 6-day salt-loading experiment. Females and males show differences and clusters of MCH cells within subdivisions of the lateral hypothalamus do not behave evenly (Presse and Nahon, 1993). Also, the MCH is a secretagogue of arginine-vasopressin (AVP) as demonstrated in vivo (Forsling and Zhou, 1997). 47

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Lactation induces novel hypothalamic expression of MCH in the preoptic area and paraventricular hypothalamus (Knollema et al., 1992). Also MCH evokes the release of oxytocin in a rat hypothalamo-pituitary explants (Parkes and Vale, 1992a). Lactation, which can be considered as an energy-deficient component, is an obvious link between feeding behaviour and internal fluid regulation. 4.4. MCH AND THE CONTROL OF FEEDING BEHAVIOUR It is now clearly established that MCH is a critical factor involved in feeding behaviour and energy regulation (Presse et al., 1996; Qu et al., 1996; Rossi and Bloom, 1997; Ludwig et al., 1998; Rossi et al., 1999; Edwards et al., 1999) (Fig. 6). The determining role of the LHA in controlling feeding behaviour made the MCH an early putative candidate for that role. One can grossly define the lateral hypothalamus as a 'feeding centre'. A lesioned or chemically damaged LHA makes the animals die by voluntary starvation (the so-called syndrome of aphagia). There was a long-term controversy dating back from the 1940s and 1950s, which led to the proposal of a dual centre hypothesis for feeding intake control where the LHA would be the feeding centre, while the ventromedial hypothalamic nucleus would be the satiety centre (Hetherington and Ranson, 1940; Anand and Brobeck, 1951). The mechanistic as well as the detailed anatomical observations would finally support that hypothesis and equally revealed a great hormonal/neural neurochemical diversity in natural agents regulating feeding behaviour (Morley, 1987; Elmquist et al., 1999). However, while clearly demonstrated for NPY, the implication of MCH within energy balance was a more frustrating area of investigation, as the lateral hypothalamus is extremely difficult to experiment with. Also feeding intake is an extremely complex behaviour to analyse. Leptin is produced by adipocytes in the periphery upon satiety and transmits to brain to induce food intake cessation. It acts within the hypothalamus where it regulates an increasingly reported intermingled circuitry of neuromodulators (mainly neuropeptides) and neurotransmitters (noradrenaline, glutamate, GABA, serotonin) (for reviews see Morley, 1987; Schwartz et al., 2000). A first report established that MCH was anorectic at specific times of the circadian rhythm (Presse et al., 1996). An orexigenic action of MCH was subsequently identified (Qu et al.,

Fig. 6. Effects of MCH on feeding behavior. Intracerebroventricular injection of MCH may inhibit or activate

food-intake according to the studies of Presse et al. (1996) or Qu et al. (1996). 48

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1996) and confirmed by others (Rossi and Bloom, 1997; Ludwig et al., 1998; Rossi et al., 1999; Edwards et al., 1999). The discrepancy may be due to the use of a low dose of peptide in the first study and the existence of a biphasic effect of MCH on appetite and food consumption. Indeed, the anorectic effect of MCH was characterised by i.c.v, injection of MCH in very low doses of peptides (1-100 ng/animal) in male Wistar rats with prior fasting for 2-24 h. This reduction in food intake was found also when 1 ng rat MCH was bilaterally infused into the central borders of the ZI-LHA in the morning, but not at the beginning of the feeding active period (Presse et al., 1996). Orexigenic effects of MCH was found with i.c.v. injection of 5 [xg MCH at different times of the dark phase consistently increased feeding for 2-4 h in Long Evans rats (Qu et al., 1996). The appetite-stimulating effect of MCH occurred both in the light and dark phases of the day (Rossi and Bloom, 1997) and is comparable to that of orexins and galanin (Edwards et al., 1999). Rat strain differences in terms of MCH sensitivity were identified (Della-Zuana et al., 2002). Confirmation of an orexigenic role for MCH came from studies showing that mice deleted for the prepro-MCH gene had a hypophagic and lean phenotype with virtually no fat deposit and an altered metabolism (Shimada et al., 1998). It was the first example that deletion of a gene encoding a single orexigenic peptide can result in leanness. Possible pathophysiological implication of MCH in feeding disorders was indicated by a three-fold increase of MCH mRNA and peptide in human obese as compared to lean subjects (Zhang et al., 1998). However, one would expect MCH to have a chronic orexigenic action that leads to weight gain to cause obesity, as for NPY. Twice daily administration of MCH only caused an transient increase in food intake for 5 consecutive days, after which time the effect was lost. Daily food intake and weight were not altered too (Rossi and Bloom, 1997). These data are in contrast to those obtained by Della-Zuana et al. (2002) who made a continuous infusion of MCH in Wistar or Sprague-Dawley rats (8 txg/animal/day) and +/+, +/ob or ob/ob mice (4 Ixg/animal/day) and reported both stimulation of feeding and enhanced body weight after 5 days in rat and 4 days in mice. Long-term infusion of the peptide still resulted in a feeding-promoting effect of MCH after 12 days of administration in both Wistar and Sprague-Dawley rats. Also in satiated C57B1/6J mice (both ob/ob and ob/+), feeding was still stimulated after more than a week. Also, MCH overexpression in transgenic mice leads to obesity as well as insulin resistance (Ludwig et al., 2001). So, while the role of MCH on short-term feeding behaviour seemed clearly established, its effect on long-term weight remained controversial. As alluded in Section 3.7, the dipeptide H-NEI-MCH-OH is resistant to degradation by exo- and endo-proteases to which MCH is highly sensitive and has been shown to have a more potent feeding-inducing effect than MCH itself (Maulon-Feraille et al., 2002). A cell-specific precursor processing may well naturally produce the dipeptide and one would expect a longer-lasting effect than MCH on activating food intake (see Section 2.3). Functional interactions of MCH with others peptidic systems involved in feeding control and energy balance homeostasis are now well documented. Indeed, MCH may act as a functional antagonist of MSH in food intake (Ludwig et al., 1998), consistent with the evolutionary conserved blocking action of either of these peptides under several paradigms (reviewed in Tritos and Maratos-Flier, 1999). Manipulation of these peptidergic systems with MCH agonists and ~-MSH antagonists quite reproducibly triggers a feeding intake effect. Both MCH and MSH peptides are part of a new feeding regulatory circuitry with the Agouti protein and its related protein, AgRP, and the newly discovered Mahogany protein (see Schwartz et al., 2000). Other anorectic peptides, such as glucagon-like peptide (GLP)-I or neurotensin, prevent also the appetite stimulating effect of MCH with differential action on 49

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the NPY neuronal system (Tritos et al., 1998b). The hypothesis that MCH and NPY act on feeding behaviour through independent pathways was recently sustained by Della-Zuana et al. (2002), using selective NPY receptor antagonists. However, it has also been found that two structurally different NPY Y-1-receptor antagonists, namely BIBO 3304 and GR231118, could inhibit MCH-induced feeding which may mean that the orexigenic action of MCH involves the Y-1-receptor (Chaffer and Morris, 2002). Galanin and MCH would control food-intake behaviour by using separate, parallel neuronal circuits (Rossi et al., 1999). The orexin-induced feeding effect is independent from that of MCH (Lopez et al., 2002). In summary, the MCH action on food consumption is independent from the galanin and melanocortin receptors (Rossi et al., 1999), possibly from the NPY pathway (Della-Zuana et al., 2002) but is abolished with ct-MSH (Ludwig et a1.,'1998) as well as neurotensin and GLP- 1 (Tritos et al., 1998b). Further evidence of a paramount role for MCH in regulating energy balance come from neuroendocrine studies on the influence of MCH on the hypothalamo-pituitary-thyroid (HPT) axis. The thyroid axis is important in energy homeostasis and starvation leads to profound suppression of the HPT axis. MCH suppresses TRH release from hypothalamic explants as well as decreasing plasmatic levels of TSH (Kennedy et al., 2001). In conclusion, there is now strong evidence supporting a crucial role for MCH in the hypothalamic control of feeding and long-term body weight maintenance. The additional effects on the HPA axis, reproduction and other higher integrative functions (reviewed in Knigge et al., 1996) could represent integrative mechanisms aimed at producing the most appropriate and coordinated response to food stimulation.

5. THE MCH RECEPTORS

For more than 15 years, advances in understanding of MCH biology have been hampered by the lack of information about the MCH receptor(s). This was mostly due to technical difficulties inherent in the cyclical nature of the MCH molecule and the absence of a simple assay to monitor MCH bioactivity. Using a reverse-pharmacology approach (see Stadel et al., 1997), Chambers et al. (1999) identified an MCH receptor. SLC-1, previously an orphan receptor (Kolakowski et al., 1996), was shown to be activated by MCH with high specificity and affinity. Another research group also identified the same MCH receptor by using a brain purification extract approach (Saito et al., 1999). Several other reports describing the identification of SLC-1 as being an MCH receptor followed (Bachner et al., 1999; Lembo et al., 1999; Shimomura et al., 1999; reviewed by Saito et al., 2000). A second human MCH receptor, named, MCH-2, MCH2, MCH-2R or MCH-R2 or SLT was subsequently characterised with little sequence identity to SLC-1 (An et al., 2001; Hill et al., 2001; Moil et al., 2001; Rodriguez et al., 2001; Sailer et al., 2001; Wang et al., 2001). Consequently to the discovery of a second MCH receptor subtype, SLC-1 is also called MCH-R1 or MCH-1 or MCH1 (reviewed by Saito et al., 2000; Boutin et al., 2002). 5.1. BIOASSAYS AVAILABLE FOR MELANOTROPINS Bioassays are based on the ability of MCH to induce pigment aggregation in melanophores of lower vertebrates (amphibians and teleost fish species; mostly Hyla, Anolis, Xenopus, Ctenopharyngodon, Synbranchus). The hormonal response is quantified by determination 50

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of the degree of skin darkening, either by naked eye estimation of colour change, by microscopic observation of melanophores, or by photoelectric measurements of reflection or absorption of light. Sensitivity of these assays are high (subpicomolar and subfemtomolar ranges, respectively, in in vivo and in vitro melanophore assay). The response of isolated melanophores to hormonal aggregation is complete within 20-40 min. However, the routine use of isolated melanophores is cumbersome given the need for large quantities of organelles. Consequently, isolated skin is a more practical tool for repetitive serial tests (see Eberle, 1988). The use of these assays has defined gross, but key, pharmacophores for MCH bioactivity (see Section 5.6). But a functional MCH receptor has not been characterised by cloning from the teleost melanosomes to date. 5.2. MCH-BINDING SITES A small number of studies have used radiolabelled forms of MCH to characterise high affinity binding sites in tissue and cell preparations. Qualitative studies with tritiated MCH provided the first evidence of MCH binding sites in mammalian tissues (Drozdz and Eberle, 1995a) but quantitative studies were greatly facilitated by the development of a biologically active, high specific activity radioiodinated ligand, [125I][Phe13,Tyr19]MCH (Drozdz and Eberle, 1995b), which has been used to characterise specific high affinity binding sites on SVK14 keratinocytes (Burgaud et al., 1997) and mouse melanoma cells (Drozdz et al., 1995). The use of MCH radioiodinated on Tyr 13 ([125I]MCH) was initially avoided as it was shown to lack biological activity (Eberle, 1988). However, studies with a cloned MCH receptor, SLC-1 (see below) have demonstrated the ability of this ligand to radiolabel MCH receptors with high affinity (Chambers et al., 1999). This radioligand was subsequently used to characterise MCH binding sites in membrane preparations from several areas of the human brain, with similar (subnanomolar) affinities for MCH to those reported at the cloned receptor (Sone et al., 2000). No autoradiographic studies of binding site distribution in tissue sections have been reported, most likely due to the technical problems of lipophilicity and sensitivity to oxidation associated with MCH. More recently, oxidatively stable (Hintermann et al., 1999) and photoreactive (Drozdz et al., 1999) analogues of radioiodinated MCH have been developed. Hintermann et al. (2001a) reported further evidence of MCH binding sites on the mouse B 16 melanoma cell line. In addition, Audinot et al. (2001b) reported the synthesis of [125I]$36057, a shortened, more stable and weakly hydrophobic peptide analogue of MCH, which proved to be a more potent and more stable radioligand than [125I][3-iodo-Tyr13]MCH and may represent a reliable tool for binding assays in the search of novel MCH ligand receptors as well as providing great help for autoradiographic studies of the MCH receptors. These new ligands may circumvent some of the technical difficulties of the past, and may provide the means to identify and clone novel subtypes of MCH receptor. The Synaptic compound SNAP-7941, an MCH-R1 antagonist, appears that it will be a valuable tool to that end (Borowsky et al., 2002). 5.3. MOLECULAR CLONING, CHROMOSOMAL LOCALISATION, AND PHYLOGENY 5.3.1. SLC-1 Diverse approaches were used by a number of groups to identify firstly the human (Chambers et al., 1999; Saito et al., 1999), and later the rat (Bachner et al., 1999; Lembo et al., 1999; Shimomura et al., 1999) and mouse (Kokkotou et al., 2001) orphan G-protein-coupled receptor designated SLC-1 as a receptor for MCH. Three forms of the SLC-1 protein have been char51

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acterised from human cDNA libraries: depending on the initiation codon used for translation, three proteins of 353, 417 and 422 amino acids could be potentially produced (Shimomura et al., 1999). Also, two splice variant forms of human SLC-1, differing in length and sequence at the N-terminus, have been cloned from cDNA libraries. The shorter version, a 353 amino acid sequence which arises from the excision of a single exon during processing (Lakaye et al., 1998), has high homology to equivalent expressed sequences in rat and mouse, and has been characterised as a functional MCH receptor. However, the putative protein product of the longer splice variant (Kolakowski et al., 1996), which arises from the failure to splice out the single intron, lacks glycosylation sites in the extracellular N-terminal tail region (Lakaye et al., 1998) and does not appear to be expressed efficiently on cell membranes in the heterologous systems used so far (Chambers et al., 1999). Consequently, there is no evidence yet that this longer form is a functional receptor. Also, rat and mouse equivalents of the longer form have not been successfully cloned from expression libraries, and the predicted protein products deduced from equivalent genomic sequences in rat, mouse and man would be predicted to exhibit low homology in the N-terminal region. For these reasons, it is likely that the longer form, although expressed, may represent an aberrant processing of SLC-1 primary transcripts, which appeared after the evolutionary divergence of primates and rodents (Fig. 7A). The resulting mRNA produced from the slc-1 gene locus is reported with a size of 2.4 kb (Kolakowski et al., 1996) in the rat and human or 2.0 kb (Saito et al., 1999) in the rat by Northern blot. The rat and human orthologue SLC-1 protein sequences are greatly conserved (Fig. 7B; accession number: U71092 for human sequence; U77953 for the rat sequence). In the human, the SLC- 1 gene is located on the chromosome 22ql 3.3 (Kolakowski et al., 1996). Highest sequence identity with other receptors than SLC-1 is observed with the somatostatin receptor (SSTR) gene family (Fig. 7C). SLC-1 encodes 40% protein sequence identity with the SSTRs in the transmembrane domains and an overall 29-32% with the SSTRs. Compared with other SSTRs, the SLC-1 protein has a large amino acid terminus and a short cytoplasmic tail. SLC-1 has also 26-30% protein sequence identity with the opioid receptors (see Darlinson and Richter, 1999). 5.3.2. MCH2 MCH2 was initially identified in a genomic survey sequence as being homologous to SLC-1 receptors (An et al., 2001; Hill et al., 2001; Mori et al., 2001; Sailer et al., 2001; Wang et al., m~

Fig. 7. SLC-1 dendrogram and schematic representation comparing human and rat SLC-1 sequences. (A) Schematic representation comparing the human 'long' and 'short' SLC-1 sequences. In the rat, normal expression of SLC-1 involves processing to excise a single intron, producing a 353-amino acid form of SLC-1. However, in the human, abberant processing results in the production of a longer form of SLC-1 protein (402 aa), in addition to the 353-amino acid receptor. The longer form, although found in cDNA libraries, lacks glycosylation signals in the N-terminal region, and has not been demonstrated to be a functional MCH receptor. Genomic sequences are indicated by thick bars, and protein products by thin bars. Dark arrows indicate areas of homology, and arrows with crosses indicate sequences lacking in homology. Large open arrows depict translation, splicing and transcription. Small triangles indicate consensus sites for asparagine-linked glycosylation. (B) Schematic representation comparing human and rat orthologue SLC-1 sequences. An alignment between the protein sequences of the human (HSllCBY) and the rat (RNllCBY) orthologue SLC-1 sequences. White-boxed residues indicate species amino-acid mismatch. (C) Dendrogram. A dendrogram indicating the sequence identity of SLC-1 with opioid/nociceptin (in grey), somatostatin and other GPCR receptors: SLC-1 as well as GPR7 and GPR8 sequences are classified as somatostatin-receptor-like/opioid-receptor-like sequences.

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2001; Rodriguez et al., 2002). Using this sequence, a full-length cDNA was generated from human foetal brain tissue with an open reading frame of 1023 bp, encoding a polypeptide of 340 amino acids, with 38% identity to SLC-1 and with many of the structural features conserved in G-protein-coupled receptors (Hill et al., 2001). Indeed the receptor contains a short N-terminus, seven distinct hydrophobic membrane-spanning domains and the highly conserved DRY motif located at the interface between the third transmembrane helix and the

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cytoplasm. The receptor has only one initiator methionine in the open reading frame, which contrasts with three such putative initiator methionines in SLC-1. There are two putative N-linked glycosylation sites and there is no characteristic signal peptide. BLAST analysis of public databases revealed SLC-1 to be its most homologous relative. The gene locus is located to the human chromosome 6q14,3-q15 and Northern-blot data showed a 4-kb transcript predominantly expressed in the brain (Rodriguez et al., 2002). The two GPCRs are 57% identical at the nucleotide level, 59% similar and 38% identical at the amino acid level. A short splice-variant of MCH2 (with a predicted protein sequence lacking the two last transmembrane domain) was identified by RACE-PCR but not Northern blot (An et al., 2001; Rodriguez et al., 2002). 5.4. SIGNALLING Intracellular signalling resulting from the activation of native MCH receptors has been reported in melanophores of the tilapia, Oreochromis niloticus (Oshima and Wannitikul, 1996). In these fish cells, MCH produced a clear reduction of cAMP levels and forskolin inhibited the aggregation response to MCH. In rat synaptically coupled lateral hypothalamic neurones in tissue culture, blockade of the Gi/o protein with pertussis toxin eliminated the actions of MCH (Gao and van den Pol, 2001). 5.4.1. SLC-1 The intracellular signalling events following SLC-1 MCH receptor activation have so far been largely explored in mammalian cell lines heterologously expressing recombinant SLC-1 (Chambers et al., 1999; Lembo et al., 1999; Saito et al., 1999; Shimomura et al., 1999) (Table 1). In these cells, MCH induced a pertussis toxin sensitive, dose-dependent inhibition of forskolin-elevated levels of intracellular cAMP, demonstrating that SLC-1 couples to Gproteins of the Gi/o class. At higher doses, MCH caused a SLC-1 mediated transient elevation of intracellular Ca 2+ concentrations in these cells, suggesting coupling to Gaq proteins. However, this response was inhibited by pertussis toxin (Lembo et al., 1999), suggesting that G~u subunits released from G~i/o proteins may be responsible via a direct activation of PLC~. Further evidence for coupling to G-proteins of the Gi/o class and activation of phospholipase C has been observed after heterologous expression of SLC-1 in Xenopus oocytes, where MCH

T A B L E 1. Reported potencies in functional assays of mammalian MCH at SLC-1 expressed stably in CHO and HEK-293 cells, and transiently in COS-7 cells and Xenopus oocytes Literature reference

Species h o m o l o g u e of SLC-1

Parental host cell

C h a m b e r s et al. (1999)

human

HEK-293

Saito et al. (1999)

human

CHO

L e m b o et al. (1999) S h i m o m u r a et al. (1999) B a c h n e r et al. (1999) M a c d o n a l d et al. (2000)

rat rat rat human

HEK-293 CHO oocyte COS-7

[Ca 2+]int

7.91 18.2 119 . -

.

Inositol phosphate

[cAMP]int

GTP-y-S binding

GIRK current

-

0.28

-

-

-

4.1

-

-

-

3.2 0.2

8.4 -

2.3

-

-

. 18.5

.

Values are given in nM. G I R K , G-protein gated i n w a r d l y rectifying p o t a s s i u m currents ( c o m p i l e d f r o m the literature).

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induced activation of both GIRK mediated currents and Ca2+-dependent chloride currents (Bachner et al., 1999).

5.4.2. MCH2 HEK293 cells transfected with MCH2 receptors responded to nanomolar concentrations of MCH with an increase in concentrations of intracellular Ca 2+ (An et al., 2001; Hill et al., 2001). Similar data were obtained in CHO cells transfected with MCH2 receptors (An et al., 2001; Mori et al., 2001; Sailer et al., 2001). Microphysiometry (Hill et al., 2001), binding studies (An et al., 2001; Hill et al., 2001; Sailer et al., 2001), inositol phosphate turnover assay (An et al., 2001; Sailer et al., 2001) and assay for inhibition of forskolin-induced intracellular accumulation of cAMP (Mori et al., 2001) were also used to show the specific response of MCH2 to MCH. Pretreatment with pertussis toxin had no effect on calcium mobilisation (An et al., 2001; Hill et al., 2001; Sailer et al., 2001) and inositol production (An et al., 2001) by MCH in these cells. MCH2 cannot reduce forskolin-stimulated cAMP production (Sailer et al., 2001). Those evidence suggest that the MCH2 receptor is coupled to G-proteins of the Gq/11 subfamily. 5.5. PHARMACOLOGY 5.5.1. SLC-1 SLC-1 was originally identified as a somatostatin receptor-like sequence, although somatostatin does not interact with SLC-1. Indeed, in the original identification of SLC-1 as an MCH receptor, over 500 known and putative mammalian neuropeptides were observed to lack detectable agonist activity (Chambers et al., 1999). SLC-1 is activated by [Phe13,Tyr19]MCH and salmon MCH with approximately three-fold less potency than mammalian MCH. A putative variant form of MCH (Breton et al., 1993a), differing from authentic MCH by four amino acids, and likely not naturally expressed (Miller et al., 1998; Viale et al., 2000), was 100-1000 times less active than MCH. This suggests that variant MCH is not a natural ligand for SLC- 1. Early work on the pharmacology of MCH has been limited to the fish heptadecapeptide homolog of MCH, using a melanophore pigment assay. These studies have demonstrated that reduction of the intramolecular disulphide bond of MCH to produce a linear molecule results in loss of activity (Kawazoe et al., 1987). Also, ring contraction analogues, demonstrated that the size of the cyclic peptide is important (Lebl et al., 1988), and the minimally active sequence of fish MCH, with activity equal to native peptide, is MCH(5-15) (Matsunaga et al., 1989). In addition, chemical modification of Tyr or Arg residues in fish MCH results in a significant loss of activity (Kawazoe et al., 1987). This led to the idea that radio-iodination of MCH on its natural tyrosine residue was resulting in a near complete loss of bioactivity in all possible situation, and that this ligand could not be used as a tracer to unravel the MCH receptor binding signature. It proved to be very wrong as [125I]MCH binds the SLC-1 protein quite as potently as [125I][Phe13,Tyr19]MCH (see Chambers et al., 1999). Interestingly, a number of pharmacological differences have been reported between the cloned MCH receptor and high affinity MCH binding sites on SVK14 keratinocytes (Burgaud et al., 1997) and mouse melanoma cells (Drozdz et al., 1995; Hintermann et al., 2001a). Binding on these sites was weakly displaced by a number of atrial, brain, and C-type natriuretic peptides (Ki values between 116 and 365 nM). However, such peptides had no 55

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detectable affinity for, or functional activity at SLC-1. In addition, the affinity of MCH for sites on keratinocytes and melanoma cells is over 275-fold lower than reported at SLC-1 (Ki values of 65-93 and 12-120 nM vs. 0.043 nM). These pharmacological differences may arise from the expression of SLC-1 in different hosts, or may be due to novel subtypes of MCH receptors in these cells. It will therefore be of great interest to investigate the expression of SLC-l-like sequences in these cells. Of interest, the recent characterisation of [lZSI]MCH binding in the human brain indicates a similar pharmacology to that reported for SLC-1, except that CNP-22 (but not ANP or BNP-32) was observed to partially inhibit binding at high concentrations (Sone et al., 2000). The low affinity of CNP-22 suggests it is unlikely to act as a natural agonist at MCH binding sites, but may prove a useful tool to discriminate between receptor subtypes.

5.5.2. MCH2 Rat atrial natriuretic peptide (ANP)(1-28), rat ANP(3-28), human C-type natriuretic peptide22, human brain natriuretic peptide-32, y-endorphin, ~-MSH, somatostatin-14, somatostatin28, cortistatin-14,; neuropeptide-EI (NEI), neuropeptide-GE (NGE), MCH-gene-overprinted peptide-14 (MGOP-14), and variant neuropeptide-EI (vNEI) were inactive as agonists or antagonists at concentrations up to 10 I~M when tested in an intracellular Ca 2+ assay. 5.6. LIGAND-RECEPTOR STRUCTURE-ACTIVITY RELATIONSHIPS The first mechanistic understanding of the interaction of mammalian MCH with SLC-1 was recently explored in a study by Schering-Plough which combined site-directed mutagenesis of SLC-1 and Ala-scanned mammalian MCH (Macdonald et al., 2000). This study confirmed some earlier observations: the entire MCH cyclic ring Cys7-Cys 16 is necessary for biological activity whereas the N- or C- linear portions are not critical. It also revealed that Arg 11 of MCH and Asp 123 in the third transmembrane domain of the SLC-1 protein are both required for the formation of peptide/receptor complex and that charge inversion does not reverse bioactivity (Asp 11 in MCH with D123R or D123K). Asp 123 is found in bioamine receptors as well as somatostatin and opioid receptors. The work also demonstrated that [Lys11]MCH is a partial agonist on SLC-l-transfected cells (67% of maximum response in calcium response as read out by FLIPR) whereas D-Arg 11 is a weak competitive functional antagonist (potency in the ~M range). All other substitutions at that location 11 in the MCH primary sequence abolished peptide bioactivity. Bednarek et al. (2001) reported that the sequence Arg-cyclo(S-S)(Cys-Met-Leu-Gly-Arg-Val-TyrArg-Pro-Cys) appears to constitute the 'active core' that is necessary for agonist potency at both human MCH receptor paralogues. Audinot et al. (2001a) who did the alanine scan of the dodecapeptide MCH(6-17) (MCH ring between Cys 7 and Cys 16, with a single extra amino acid at the N terminus (Arg 6) and at the C-terminus (Trp17), found it to be the minimal sequence required for a full and potent agonistic response on cAMP formation and [35S]GTP u binding. They showed that only 3 of 8 amino acids of the ring, namely Met 8, Arg 11, and Tyr 13, were essential to elicit full and potent responses in both tests. More recent peptide analog studies have shown that compounds with Ava in positions 9, 10 and/or 14, 15 revealed that the Leu9-Gly 1~ and Argl4-Prol5 segments of the disulphide ring are the principal structural elements determining hMCH-1R selectivity and ability to act as a hMCH-1R antagonist (Bednarek et al., 2002a). In addition, structural changes in positions 6 and 10 results in peptide conformations that allow for efficient interactions with hMCH-1R are 56

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unfavourable for molecular recognition at hMCH-2R (Bednarek et al., 2002b). The dipeptide H-NEI-MCH-OH is an agonist on MCH-R1 (Maulon-Feraille et al., 2002). Non-peptide antagonists at MCH-R1 such as the biphenyl carboxamide GSK compound (Witty et al., 2002), the Takeda compound T-226296 (Takekawa et al., 2002) or the synaptic compound SNAP-7941 (Borowsky et al., 2002) should allow to shed further light on the key interaction between the ligand and the receptor. 5.7. CENTRAL AND PERIPHERAL DISTRIBUTION OF THE MCH RECEPTOR SLC-1 IN THE MAMMALS

5.7.1. Overall distribution of SLC-1 mRNA and protein in the rodents The distribution of the SLC-1 mRNA has been reported by several teams (Kolakowski et al., 1996; Chambers et al., 1999; Lembo et al., 1999; Saito et al., 1999, 2001a; Hervieu et al., 2000; Tan et al., 2002), either by Northern blot and/or mRNA in situ hybridisation (ISH) as well as RT-PCR. In a study using in situ hybridisation with oligoprobes and immunohistochemistry, we reported the distribution of SLC-1 mRNA and its protein product in the rat brain and spinal cord (Hervieu et al., 2000). SLC-1 mRNA and protein were found to be widely and strongly expressed throughout the brain. Immunoreactivity was observed in areas that largely overlapped with regions mapping positive for mRNA. SLC-1 signals were observed in the cerebral cortex, caudate-putamen, hippocampal formation, amygdala, hypothalamus and thalamus, as well as in various nuclei of the mesencephalon and rhombencephalon. The distribution of the receptor mRNA and immunolabelling was in good general agreement with the previously reported distribution of MCH. In a study using riboprobe in situ hybridisation, Saito et al. reported similar results, except that SLC-1 gene expression could not be detected in the olfactory bulb, some diencephalic nuclei and the cerebellum (Saito et al., 2001a). Interestingly Saito et al. (1999) reported SLC-1 gene expression in the eye. Hintermann et al. (2001b) extended that observation and showed MCH-R expression was observed at both the mRNA and protein levels in primary porcine ciliary pigmented epithelial cells and on a human non-pigmented ciliary epithelial cell line. A crosslinking study resulted in a labelled 44-kDa protein, consistent with the molecular weight of the receptor. The retina is phylogenetically related to the hypothalamus and direct monosynaptic contact links both regions. Saito et al. (2001b) reported SLC-1 expression in the human melanoma cell line SK-MEL-37. Northern-blot experiments broadly confirm these data: the SLC-1 gene is strongly detected in the rat frontal cortex, striatum, thalamus and pons (but not in cerebellum) (Kolakowski et al., 1996) and in the whole brain as well as in the eye (Saito et al., 1999). These data are consistent with the known biological effects of MCH in the brain, such as modulation of the energy balance, stress response, sexual behaviour, anxiety, learning, seizure production, memory retention, grooming and sensory gating and with a role for SLC-1 in mediating these physiological actions.

5.7.2. Quantitative RT-PCR (Taqman analysis) of SLC-1 gene expression in rat CNS and PNS Our results show that SLC-1 is widely expressed in the rat nervous system, with mRNA detected in all tissues that were tested (Fig. 8A). However, some variation in expression is observed, with higher expression in amygdala, cerebral cortex (all divisions), hippocampus, 57

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hypothalamus and substantia nigra, and lower levels of expression in striatum, thalamus, cerebellum, rhombencephalon, spinal cord and dorsal root ganglia (DRG).

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5.7.3. lmmunochemical studies

In immunochemical experiments (Figs. 8-16), adult male Wistar rats (200-250 g, Charles River, UK) were used, kept in a fixed 12-h light-dark cycle with food and water provided ad libitum. All probe sequence (DNA and peptide) were checked for uniqueness to SLC-1 protein using BLAST. 5.7.3.1. mRNA in situ hybridisation (ISH)

The mRNA localisation experiments were performed with oligonucleotides designed from the rat SLC-1 orthologue sequence (Lakaye et al., 1998). A substantial amount of specific autoradiographic signal was obtained, with discrete anatomical localisation, using the radiolabelled antisense oligoprobes used in the study (Fig. 8B). In control experiments, sense oligoprobes produced no substantial tissue labelling (Fig. 8C). Furthermore, preincubation of radiolabelled antisense oligoprobes with an excess of cold antisense probes (Fig. 8D), and the use of RNase A pretreated sections (Fig. 8E) similarly resulted in a diminution of the signal intensity as compared to that obtained by using antisense oligoprobes (Fig. 8B). Autoradiographic signals were detected throughout the rat nervous system (brain and spinal cord). Thus specific signals corresponding to the SLC-1 mRNA were observed in the cortex (CTX), the olfactory regions (anterior olfactory nucleus and olfactory tubercle; respectively AON and OT), the basal ganglia (caudate-putamen and amygdala; respectively, CP and Amygd.), the hippocampal formation (hi), the diencephalon (thalamus, habenula and hypothalamus; respectively Th, H, Hyp) and various midbrain areas (superior and inferior colliculi, pons, reticular formation, nucleus of the solitary tract and cerebellum; respectively SC, IC, Pn, RF, NTS, Cb). The pattern of distribution was in agreement with the one reported by Taqman experiments. Also that mRNA distribution pattern was quite conserved in other small rodents such as the mouse (Fig. 8F,G) and the guinea pig (Fig. 8H), when compared to the rat.

+....._

Fig. 8. slc-1 gene expression in rodents. (A) SLC-1 mRNA expression in rat CNS and PNS by quantitative RT-PCR analysis. SLC-1 mRNA expression in rat CNS and PNS. SLC-1 expression has been normalised to the expression of the housekeeping gene GAPDH. Bars indicate the mean values derived from three independent RT-PCRs; error bars indicate standard error of the mean. SLC-1 mRNA is detectable in all areas tested with highest levels of expression observed in amygdala, cerebral cortex (all divisions), hippocampus, hypothalamus and substantia nigra. (B-E) mRNA in situ hybridisation in the rat brain for SLC-1. Consecutive rat brain sagittal sections were hybridised with a mix of SLC-1 radiolabelled antisense oligonucleotides (B), a mix of SLC-1 radiolabelled sense oligonucleotides (C), a mix of SLC-1 radiolabelled antisense oligonucleotides and a 100• excess of unlabelled radiolabelled antisense oligonucleotides (D). In E, rat brain sagittal sections were pretreated with the RNase A and were hybridised with a mix of SLC-1 radiolabelled antisense oligonucleotides. The use of sense oligoprobe competed radiolabelled antisense oligoprobes with an excess of cold antisense probes and the use of Rnase A pretreated sections resulted in an absence or diminution (D,E) of the signal intensity as compared to the one obtained by using antisense oligoprobes (B). Specific signals corresponding to the SLC-1 mRNA were observed in the cortex (CTX), the olfactory regions (anterior olfactory nucleus and olfactory tubercle; respectively, AON and OT), the basal ganglia (caudate-putamen and amygdala; respectively, CP and Amyg.), the hippocampal formation (hi), the diencephalon (thalamus and hypothalamus; respectively, Th, Hyp) and various midbrain areas (superior and inferior colliculi, pons, reticular formation, nucleus of the solitary tract and cerebellum; respectively, SC, IC, Pn, RF, NTS, Cbgr) (Hervieu et al., 2000). (F-H) mRNA in situ hybridisation in the mouse and guinea pig. Similar mRNA distribution patterns to the rat were observed in the mouse (F: antisense probe; G: antisense probe with an excess of cold probe) and guinea pig (H: rat antisense probe) brains. Abbreviations: same as in B-E; MRN: mesencephalic reticular nucleus. Scale bars: B-E, 0.35 cm; F-G, 0.3 cm; H, 0.55 cm.

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5. 7.3.2. Antiserum immunochemistry A rabbit polyclonal antiserum was raised against the extreme C-terminal hexadecapeptide H-SNAQTADEERTESKGT-OH (amino acids 338-353) derived from the human SLC-1 sequence (Kolakowski et al., 1996) with total sequence identity with the rat orthologue protein sequence (Lakaye et al., 1998). Immunohistochemistry and immunocytochemistry were carried out with an avidin-biotin complex system with either a peroxidase or a fluorescence reporter as previously described (Hervieu and Emson, 1998; Hervieu et al., 2000). Control experiments included the omission of the primary antiserum, the use of rabbit preimmune serum and preabsorbing the antiserum with the immunogenic peptide. Preabsorption controls were done with 10 gM of the immunogenic peptide (incubated overnight with the antiserum prior to the incubation on sections).

Specificity profile of the antiserum: Immunocytochemistry and Western blot using human SLC-1 transfected cells. Specificity of the antisera was investigated on SLC-1- HEK 293 transfected cells versus wild-type cells, with or without preabsorption, with or without primary antisera in a fluorescence procedure. For Western blot analysis, cell extracts were resolved by SDS-PAGE (4-20%), transferred onto nitrocellulose and revealed using an immunoglobulin fraction of the crude anti-SLC-1 antiserum at 1:2000 and a chemiluminescence detection. Specificity of the antibody was confirmed by preabsorption of antibody with the immunogenic peptide (2 gM) and by omitting the primary antibody. Specific immunocytochemical signals (Fig. 9A) largely confined to the plasma membrane, as opposed to the control condition using no primary serum (Fig. 9B) or the antiserum preabsorbed with the synthetic peptide (Fig. 9C), were generated with SLC-l-transfected HEK-293 cells incubated with the antiserum.

Fig. 9. Immunochemical specificity of the anti-SLC-1 antiserum. (A-C) Specificity of the SLC-1 antiserum: immunocytochemistry. HEK 293-SLC-l-transfected cells grown on chamber microscopic slides were incubated with affinity-purified MCH receptor antiserum (A), with no primary serum (B) and with affinity-purified MCH receptor antiserum preadsorbed with the synthetic peptide (C). Specific membrane-associated immunostaining can be in condition A (Hervieu et al., 2000). (D) Specificity of the SLC-1 antiserum: Western-blot. Immunoglobulin fraction of the crude MCH receptor antiserum detected several bands in SLC-1 transfected cells (lane 1) in comparison to untransfected (lane 3) HEK293 cells. Bands were identified at 60 kDa and may represent the glycosylated form of the receptor. High molecular weight forms may represent receptor aggregates. Pre-absorbed antibody failed to detect these bands in transfected (lane 2) or untransfected (lane 4) cells (Hervieu et al., 2000). (E,E') The MCH system in the hypothalamus. Strong MCH gene expression was detected in the lateral hypothalamic area and dopaminergic zone of the zona incerta (LHA and ZIda) by in situ hybridisation with rat MCH antisense oligoprobe (E). Strong gene expression was corroborated by strong immunohistochemical signals obtained with a rabbit polyclonal antiserum raised against the rat MCH. Immunostained cells (arrow) were intermingled within a rich network of labelled varicosities (arrowhead). (Bittencourt et al., 1992). (F-/) Specificity of the SLC-1 antiserum: Immunohistochemistry. The rat sagittal sections were incubated with the affinity-purified followed by immunohistochemistry reported with peroxidase (F,H,/). Immunosignals were observed in the cortex (CTX), the olfactory regions (AON, OB and OT), the basal ganglia (CP, ACB, HDB), the hippocampal formation (hi and SUB), the diencephalon (LHA, VP) and various midbrain and hindbrain areas (SC, IC, LL, NTS, PSV, SPV, Cb, MV). When the section was incubated with the antiserum preabsorbed with the synthetic peptide, no signals were observed (/). There was a good overlapping between the distribution of the SLC-1 protein-like immunoreactivity and the MCH peptide (G; adapted from Bittencourt et al., 1992; a schematic representation of the MCH projections through the rat brain). Calibration bars: A-C, 35 ~m; E, 0.3 cm; E', 70 txm; F, H, I, 0.30 cm. See Section 8 for abbreviations (Hervieu et al., 2000).

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Western blot analysis using the crude anti-SLC-1 antiserum revealed several specific bands in SLC-1 0verexpressing cells (Fig. 9D). Two prominent molecular forms were present at 60 and 120 kDa. Immunosignals were absent with wild-type HEK cells (lane 3) or when the antiserum had been preabsorbed with the synthetic peptide (2 ~M) and tested either with SLC-1 transfected (lane 2) or wild-type HEK 293 cell extracts (lane 4). Since the predicted molecular weight of SLC-1 is 38.9 kDa, it was likely that the 60-kDa band could represent a glycosylated form of the receptor. Indeed, amongst putative posttranslational modifications are three N-glycosylation sites in the human N-terminal presumed extracellular tail (namely NAS; NTS, NLT; respective position of the arginine residue: 13, 16 and 23). By treatment with N-glycosidase F, the apparent MW of the immunosignal was shifted from 60 kDa to 40 kDa (not shown). Also apparent mobility of the migrating protein could be affected by phosphorylation as several potential phosphorylation sites are present in the C-terminal presumed intracellular tail as follows: two protein kinase C phosphorylation sites: 317-319 TFR; 325-327 SVK and one casein kinase II phosphorylation site (342-345 TADE). It is likely that the 120-kDa band may represent receptor aggregates. Immunohistochemistry using rat brain tissue sections. A distinctive immunostaining pattern was obtained after incubation of brain sections in the affinity-purified anti-SLC-1 antiserum (Fig. 9F,H). SLC-1 immunosignals were observed in the cerebral cortex, caudate-putamen, hippocampal formation, amygdala, hypothalamus and thalamus, as well as in various nuclei of the mesencephalon and rhombencephalon. In control experiments using the anti-SLC-1 antiserum preabsorbed with the immunogenic peptide, a great reduction in immunosignal intensity could be observed (Fig. 9I). At the same anatomical level, the SLC-1 immunostained regions in the rat brain (Fig. 9F) were largely identical with MCH immunostained regions in the rat brain (Fig. 9I). Cellular morphological features of the SLC-1 immunostained rat brain cells SLC-l-like immunoreactivity was found in cells with the morphology of projection neurones (e.g. Fig. 12F) and interneurones (e.g. Fig. 12D). Glial cell immunostaining was observed, but labelling could not be fully competed with the preadsorbed antiserum. Furthermore, signals were mainly confined to plasma membranes of cells, as would be expected for a G-protein-coupled receptor. Confocal fluorescence analysis of tissue sections revealed mainly discrete punctate staining of plasma membranes (Fig. 12D) which was in contrast to the more continuous pattern of staining of entire cell membranes seen in SLC-1 transfected cells (Fig. 9A). This may well be explained by the different densities of receptors in these cells, with a much higher abundance of receptors in the transfected cells. The antiserum revealed some nerve fibre staining. Axons with collaterals and dendrites from the many interneurones were labelled in the cerebral cortex (e.g. Fig. 12A).

Fig. 10. The distribution of the SLC-1 mRNA and protein in the rat nervous system. Several plans of sections illustrating the rat distribution of the SLC-1 mRNA (A, C, E, G, L K) and protein (B, D, F, H, J, L) from the forebrain to the spinal cord. A good overlap between the mRNA and protein signals was observed. Key hypothalamic regions involved in feeding control such as the DMH and VMH are shown in G and H. Calibration bars: A, B, 0.2 cm; C, D, 0.33 cm; E, F, 0.2 cm; G, 0.3 cm; H, 280 Ixm; L J, 0.2 cm; K, L, 0.07 cm. See Section 8 for abbreviations (Hervieu et al., 2000).

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5. 7.3.3. A comparison between the mRNA and protein distribution pattern of SLC-1 products in the rat nervous system With the 'caveat' that neuronally expressed proteins are expected to be identified far away from the cell body they originate, receptor immunoreactivity was observed in areas that largely overlapped with regions mapping positive for mRNA. This is illustrated in Fig. 10 with a set of rat nervous system sections representative of the forebrain, brainstem and spinal cord, presented in parasagittal, sagittal and coronal views.

5.7.3.4. Neuroanatomical localisation studies: rat brain and spinal cord localisation of the MCH receptor SLC-1 Please also refer to Table 2 and Figs. 8B, 9F, H, 10-13, 15 and 16.

Cell groups Forebrain. A strong labelling was noticed at many subregional levels in the isocortex amongst them the primary and secondary motor area (MOp, MOs), primary and secondary somatosensory areas (SSp, SSs), gustatory area (GU), the infralimbic and prelimbic areas (ILA, PL), the orbital area (ORB), anterior cingulate area in both dorsal and ventral parts (ACAd, v), visceral area (VISC), agranular insular area in both dorsal and ventral parts (Ald and Alv), retrosplenial cortex (RSP) in both dorsal, ventral and lateral agranular parts (RSPd, v, agl), posterior-parietal region association area (PTLp), primary, ventral and dorsal auditory areas (AUDp, v, d), ventral temporal association area (TEv), the ectorhinal area (ECT), a number of visual areas (primary, anterolateral, rostrolateral and anteromedial visual areas (respectively, VISp, a, rl and am) and the claustrum (CLA). The olfactory cortex was one of the richest in terms of both SLC-1 mRNA and protein labelling. Heavy labelling of the olfactory region was seen in the main olfactory bulb (MOB), the anterior olfactory nucleus (AON) and olfactory tubercle (OT) where immunostaining of the layer 3 (polymorph layer) was more pronounced than in the molecular and pyramidal layers. Dense signals were observed in the taenia tecta (TT) both in the ventral part (TTv) and the dorsal part (TTd), mainly in layer 3, and in the major islands of Calleja (islm). Strong signals were observed in the piriform cortex (PIR) in the layer II (pyramidal) and the dorsal endopiriform nucleus (EPd) while much weaker immunosignals were seen in the post piriform transition area (TR).

Fig. 11. A rostrocaudal distribution of the SLC-1 protein in the rat brain. The SLC-1 mRNA was localised in some key-forebrain regions such as the isocortex, olfactory system/hippocampal formation, nucleus accumbens, caudateputamen and other basal ganglia (amygdala), medial septum, and nucleus of the diagonal band. A particularly dense staining was obtained in various hypothalamic (SO, AHN, PVH, MEPO, MPO, MPN, VMH, LHA, PM, PV, PH, LM, MM) and thalamic (RT, AV, AD, IAD, MH, ZI, CL, PVT, OP, STN, PF, VPL) nuclei. Note also the metathalamic signals obtained in the geniculate nuclei (lateral and medial) and in their functionally associated colliculus nuclei (SC, IC). Note also the signals obtained in the oculomotor nerve (III), the caudal part of the substantia nigra (SNpr), the specific staining of the caudal part of the interpeduncular nucleus (IPN) and the staining in other various midbrain nuclei (PAG, VTA, APN, RN, NB, DR, VTN, TRN, NTB, POR, PB-KF, DTN, VCO). Lastly, note the signals obtained in various nuclei of the reticular formation (PRN, MARN, GRN), in the cochlear nuclei (DCO, VCO), in the vestibular nuclei (MV), in the olivary nuclei (SOC1 and IO), in the trigeminal system (PSV, SPV), in the facial nerve (VII), in the locus coeruleus (LC) and the grey matter of the spinal cord. Calibration bars: A-U, 0.2 cm; V-W, 0.15 cm; X-Z', 0.1 cm. See Section 8 for abbreviations (Hervieu et al., 2000). 64

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Immunostaining was more concentrated in layers III (deep supragranular pyramidal) and V (infragranular pyramidal) with stained cells resembling multipolar interneurones and principal neurones (see Fig. 12). In the hippocampal formation, immunolabelled cells were observed in the dorsal parts (SUBd) and ventral parts (SUBv) of the subiculum (SUB). The post-subiculum (POST) and presubiculum (PRE) were also immunostained unlike the parasubiculum (PAR). The entorhinal area (ENT) was moderately immunostained in its lateral (ENT1) and medial (ENTm) parts. In the hippocampus (hi), immunosignals were mainly located in the stratum pyramidale (sp) in Ammon's horn and the granule cell layer of the dentate gyrus (DGsg). Also the crest of the dentate gyrus was immunostained both in the molecular (DGcr-mo) and granule cell layer (DGcr-gr) levels as seen at a more ventrocaudal level. Lastly, a pronounced immunostaining was present within the indusium griseum (IG). In the amygdala, immunostaining for the MCH receptor SLC-1 was observed in the nucleus of the olfactory tract (NLOT), basolateral, basomedial, intercalated nuclei (respectively BLA, BMA, IA) and anterior area (AAA). In the septal regions, the septum was particularly enriched in SLC-1 signals with labelling in the medial septum nucleus (MS), the nucleus of the diagonal band of Broca (NDB) and the lateral septum (LS). On a sagittal section, immunostaining could be seen in the horizontal limb of the diagonal band (HDB) and the vertical limb of the diagonal band (VDB). At the level of the corpus striatum, a very dense immunolabelling was seen in the striatum (caudate-putamen, fundus of the striatum and nucleus accumbens; CP, FS and NA) as well as in the pallidum where particularly strong signals were expressed within the substantia innominata (SI) including the magnocellular preoptic nucleus (MA) and the globus pallidus (GP) in contiguity with the labelling found in the SI. In the thalamus, at the epithalamic level, signals were more prominent in the medial habenula (MH) than in the lateral habenula (LH). The thalamus was reasonably enriched with SLC-1 signals. Progressing rostrally to caudally, in the dorsal thalamus, on a ventral-caudal axis, SLC-1 receptor signals were detected in the paraventricular nucleus thalamus (PVT) at low levels while being much more abundant in the anterodorsal (AD), anteroventral (AV) and lateroposterior (LP) thalamus nuclei. Immunosignals were also dense in the ventral thalamic complex including the ventral anterior-lateral complex thalamus (VAL), ventral posterior medial nucleus thalamus (VPM) and ventral posterolateral nucleus thalamus (VPL). At that level, immunosignals were also detected in the posterior complex thalamus (PO), and the parafascicular nucleus thalamus (PF). In the ventral part of the thalamus, the reticular nucleus thalamus (RT), the subthalamic nucleus (STN) and its overlying zona incerta (ZI) were also immunopositive for SLC-1 protein. More rostrally, the subdopaminergic cell group of the zona incerta (ZIda) also expressed the SLC-1 gene. In the metathalamus, the lateral geniculate complex (LG) including its dorsal, ventral, ventrolateral and ventromedial parts (LGd, v, vl, vm) as well as the intergeniculate leaflet (IGL) contained SLC-1 protein. Also, immunosignals were observed in the ventral part of the medial geniculate nuclei (MGv). Dense immunosignals were observed in the hypothalamus. In the periventricular zone, the median preoptic nucleus (MEPO) was the most rostral hypothalamic region to be labelled. Staining was also observed in the suprachiasmatic (SCN), supraoptic (SO), arcuate (AN), and paraventricular (PVH) as well as periventricular (PV) nuclei. In the medial hypothalamus, strong signals were obtained at the level of the anterior nucleus (AHN) in the anterior (AHNa) and central (AHNc) parts. In the tuberal area, staining was seen in the ventromedial (VMH) and the dorsomedial nuclei (DMH) while in the lateral zone, staining was noticed in the lateral area (LHA). Lastly, the posterior nucleus (PH) and 69

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TABLE 2. Distribution of slc-1 mRNA and immunoreactivity in the rat brain Region

mRNA

Immunoreactivity

Olfactory bulbs

+++

+++

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

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

+ ++ + ++

+ ++ + ++

++

++

+++ ++

++++ +++

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

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

+++ ++

+++ ++

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

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

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

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

Telencephalon Olfactory system Dorsal endopiriform nucleus Islands of Calleja Olfactory nuclei Piriform cortex Tenia tecta Neocortex Agranular insular cortex Frontal cortex Granular insular cortex Parietal cortex Metacortex Cingulate/retrosplenial cortex Basal Ganglia Caudate putamen Globus pallidus Hippocampal formation CA1 region CA2 region CA3 region Dentate gyrus Subiculum Amygdala Amygdaloid nuclei Substantia innominata Septal and basal magnocellular nuclei Accumbens nucleus Bed nucleus of the stria terminalis Lateral septal nucleus, dorsal part Lateral septal nucleus, ventral part Medial septal nucleus Nucleus of the horizontal limb of the diagonal band

Diencephalon Thalamus Anterodorsal thalamic nucleus Anteroventral thalamic nucleus Centrolateral thalamic nucleus Centromedial thalamic nucleus Geniculate nuclei Interanterodorsal thalamic nucleus Intermediodorsal thalamic nucleus Lateral habenular nucleus Medial habenular nucleus Parafascicular thalamic nucleus Paratenial thalamic nucleus Paraventricular thalamic nucleus Reticular thalamic nucleus Reuniens thalamic nucleus Submedius thalamic nucleus Ventral posterolateral thalamic nucleus Ventral posteromedial thalamic nucleus

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TABLE 2 (continued) Region Thalamus (continued) Ventrolateral thalamic nucleus Ventromedial thalamic nucleus Zona incerta Hypothalamus Anterior hypothalamic area Arcuate hypothalamic nucleus Dorsomedial hypothalamic nucleus Lateral hypothalamic area (LHA) Lateral mammillary nucleus Medial mammillary nucleus Medial preoptic area Medial preoptic nucleus Paraventricular hypothalamic nucleus Periventricular hypothalamic nucleus Posterior hypothalamic area Supraoptic nucleus Ventromedial hypothalamic nucleus

mRNA

Immunoreactivity

++ § §247247

++ §247 ++§

§247 +§ §247247 §247 §247247 +§247 §247 §247 §247247 §247 § §247247 §247247

§247 + §247247 §247 §247247 §247247 § + §247247 ++§ + §247247 §247247

§247 §247247 §247247 +§ +++ +§ + ++ ++ +§247 ++ ++

§247 §247247 §247247 +++ +++ +++ § +§ ++ §247 ++ §

++ n.d. ++ §247 + §247247 §247 n.d. n.d.

++ ++§ ++ ++§ + §247247 §247 §247247 §247247

+++ ++

+++ ++

Mesencephalon Anterior pretectal nucleus Dorsal tegmental nucleus Inferior colliculus Interpeduncular nuclei Oculomotor nucleus Periaqueductal grey Principal sensory trigeminal nucleus Raphe nuclei Red nucleus Substantia nigra Superior colliculus Ventral tegmental area

Rhombencephalon Cochlear nucleus complex Facial nucleus Parabrachial nuclei Locus coeruleus Nucleus of the solitary tract Olivary complex Pontine reticular nucleus Spinal trigeminal nucleus Vestibular nucleus

Cerebellum Cerebellar cortex Deep cerebellar nuclei

The relative density of labelling is classified as: absent ( - ) , sparse (+), moderate ( + + ) , extensive ( + + + ) , not determined (n.d.) (partially adapted from Hervieu et al., 2000).

mammillary body including the dorsal pre- (PM), medial (MM), lateral (LM), lateral supra(SUM1) and ventral tuberomammillary nucleus (TMv) displayed clear SLC-1 immunoreactive signals. 71

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Brainstem. In the sensory systems, many immunosignals were present in the visual areas

such as the superior colliculus (SC) (with a discernible enrichment of signals in the zonal, optic, intermediate (SCig-b/c) and deep (SCdg) grey layers). In the pretectal regions, immunostaining was present in the anterior pretectal nucleus (APN), the olivary pretectal nucleus (OP) and the nucleus of the optic tract (NOT). In the somatosensory areas, the principal sensory nucleus of the trigeminal (PSV) was stained as well as the spinal nucleus of the trigeminal (SPV), the cuneate (CU) and the external cuneate (ECU) nuclei. The oral (rostrodorsomedial and ventrolateral; SPVO rdm and vl), interpolar (SPVI) and caudal (SPVC) segments of the spinal nucleus were immunolabelled. In the auditory areas, both the dorsal (DCO) and ventral (VCO) cochlear nuclei were immunolabelled. Strong immunostaining was found in the nucleus of the trapezoid body (NTB) and the leminisci (LL) such as the nucleus of the lateral lemniscus (NLL). Also SLC-1 immunosignals were encountered in the superior olivary complex (SOC) including the complex SOC itself and the periolivary region (POR). The inferior colliculus (IC) in all its subdivisions (external, dorsal, central; e, d, c) was particularly rich in SLC-1 signals as was also the nucleus brachium (NB). The vestibular areas displayed high levels of SLC-l-like immunoreactivity. All vestibular nuclei including the medial (MV), superior (SUV), lateral (LV) and spinal vestibular nuclei (SPIV) were immunostained. Also, the nucleus prepositus (PRP) from the perihypoglossal nuclei was labelled. The gustatory areas exhibited immunolabelling in the medial zone of the nucleus of the solitary tract (NTSm). In the visceral areas, the nucleus of the solitary tract (NTS) contained protein in its intermediate (NTSi) and ventrolateral (NTSvl) parts. Similarly the lateral division of the parabrachial nucleus (PB1) contained immunostaining as well as its contiguous Kolliker-Fuse nucleus (KF). In the motor systems, the nucleus of oculomotor nerve III was immunostained as well as the facial nucleus (cranial nerve VII), the nucleus ambiguus (AMB), the superior salivatory nucleus (SSN) and the Edinger-Westphal nucleus (EW). A robust immunostaining was seen in the extrapyramidal system, particularly in the substantia nigra (SN) with signals in the pars reticulata and to a lesser extent in the pars compacta (SNpc). The ventral tegmental area (VTA) was immunostained. In the pre- and post-cerebellar nuclei, the red nucleus (RN) contained obvious immunolabelling as well as the pontine central grey (PG) and the tegmental reticular nucleus (TRN). More caudally, the inferior olivary complex (IO) was immunopositive in all its subdivisions. In the cerebellum, there was mRNA labelling in the granular cell layer of the cerebellar cortex (Cbgr) as well as immunolabelling. In addition, the interpositus cerebellar nucleus was immunostained in both its anterior and posterior subdivisions (IntA; IntP). In the reticular core, staining was strong in the central grey of the brain: in particular, SLC1 protein was detected in the many divisions of the peri-aqueductal grey (PAG); i.e. dorsal, ventrolateral and dorsolateral; d, vl and dl. The ventral (VTN) and dorsal (DTN) tegmental nuclei were also immunostained. The locus coeruleus (LC) was quite strongly positive for

Fig. 12. SLC-1 immunoreactivity in the neocortical areas of the rat brain. Numerous SLC-1 immunostained

cells were, respectively, detected throughout the isocortex such as in the ventral (A) and dorsal (B,F) parts of the retrosplenial cortex (respectively, RSPv and RSPd), the primary motor cortical area (MOp; C and D; D by confocal microscopy), the secondary motor cortical area, (MOs; B), the primary somatosensory cortex (SSs; E), the posterior-parietal region association area (PTLp; G) and the primary auditory cortex (AUDp; H). In all cases, cells were labelled on the membrane. Calibration bars (in Ixm):A, C, H, 70; B, 140; D, 20; E, F, G, 35 (Hervieu et al., 2OOO). 72

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SLC-1 protein staining. The LC staining was encompassed within the immunostained pontine central grey (PCG) In the raphe, immunostaining was observed in the dorsal nucleus raphe (DR), the central linear nucleus raphe (CL1), the nucleus raphe magnus (RM) and the medial and lateral part of the superior central nucleus raphe (CSm and CS1). More caudally, immunostaining was present in the nucleus raphe pallidus (RPA). There was a pronounced labelling of the interpeduncular nucleus (IPN), particularly in the central subnucleus (IPNc). In the reticular formation, staining was quite widespread. It was indeed observed in the mesencephalic reticular nucleus (MRN) including its retrotuberal area (RR), in the pedunculopontine nucleus (PPN), in the pontine reticular nucleus (PRN), in the gigantocellular reticular nucleus (GRN), the parvicellular reticular nucleus (PARN) the magnocellular reticular nucleus (MARN) and also found in the medullary reticular nucleus (MDRN). Spinal cord (lumbar segment). There was strong mRNA labelling and immunolabelling in the lumbar part of the spinal cord. All subdivisions of the grey matter (dorsal and ventral horns) were labelled (ventromedial, dorsomedial, intermediolateral, central, ventrolateral, dorsolateral and retrodorsolateral). The immunolabelling was particularly pronounced in the dorsal horn. Fibre tracts Most fibres were devoid of mRNA or immunolabelling such as the cranial nerve (olfactory limb of the anterior commissure, aco; spinal tract of the trigeminal nerve, sptV; the facial nerve, VIIn; the trapezoid body of the cochlear nerve, tb), the spinal nerves such as the medial lemniscus (ml) of the dorsal column, the cerebellum such as the middle cerebellar peduncle (mcp), the lateral forebrain bundle system such as the corpus callosum (cc) including the anterior forceps (fa), genu (ccg) and external capsule (ec), as well as the corticospinal tract (cst) including the pyramidal tract (py), the extrapyramidal fibre system such as the rubrospinal tract (rust), the medial forebrain bundle system (MFBS) such as the temporal limb of the anterior commissure (act), dorsal hippocampal commissure (dhc). However, the MFBS-habenula related fasciculus retroflexus (fr) was immunostained and the internal capsula (ic) was densely labelled. There was also dense immunoreactivity in some of the white matter tracts of the spinal cord such as the laterocorticospinal tract (lct), the laterospinocortical tract (lsc), the lateral spinothalamic tract (lst) and the anterior corticothalamic tract (act).

5.7.4. Peripheral and central distribution studies of SLC-1 regional gene expression sites in the human (Fig. 17) Northern blot experiments reported expression of the slc-1 gene in the frontal cortex, hypothalamus, basal forebrain, midbrain, amygdala, hippocampus, subthalamus, substantia

Fig. 13. SLC-1 immunoreactivityin the hippocampal formation. Hippocampal SLC-1 labelling was observed in the pyramidal layers of the Ammon's horns (CA1-3 spd) as well as in the dentate gyrus (DG). Immunostaining was seen in the CA1 near the fasciola cinerea (B) and in a more lateral position (C). A stronger immunostaining was seen in the CA2 (D, F) and CA3 (E). The immunolabelling was present on the membrane of the pyramidal cells (F). The dentate gyrus was stained in the granular (DGsg) and polymorph (DGpo) cell layers (G). Calibration bars (in Ixm):A, 600; B-E, G, 70; F, 35 (Hervieu et al., 2000).

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nigra, thalamus, corpus callosum, liver and heart while no signals were observed in the caudate-putamen, pancreas, kidney, muscle, lung and placenta (Kolakowski et al., 1996). The slc-1 gene is also expressed in adrenal glands as well as a variety of adrenal tumours, ganglioneuroblastomas and neuroblastoma (Takahashi et al., 2001). By Taqman, the SLC-1 receptor gene level expression was dramatically higher in the brain and the pituitary than in other tissues. The receptor was expressed at quite low levels in peripheral tissues regulating energy balance (stomach, intestine, adipose tissue, pancreas, skeletal muscle) and immune/haematopoietic system (spleen, lymphocytes, bone marrow, but not in macrophages) (Fig. 17A). In the human brain, the gene expression distribution pattern was similar to that found in the rat. Expression was high in the cerebral cortex (cingulate, medial frontal and superior frontal gyri), quite high in the hippocampus and hypothalamus, moderate to low in the basal ganglia (amygdala >> caudate nucleus, putamen, striatum, substantia nigra, globus pallidus) and low in the thalamus, spinal cord, cerebellum and medulla oblongata. (Fig. 17B). mRNA in situ experiments confirmed slc-1 gene expression in the neocortex (Fig. 17C), the hippocampus (Fig. 17D) and the cerebellum (Fig. 17F). Immunoreactive cells were also detected in the dentate gyms (Fig. 17E).

5.7.5. Autoradiographic ligand studies The synaptic compound SNAP-7941, an MCH-R1 non-peptidic antagonist, was used tritiated and applied to rat brain sections (Borowsky et al., 2002). Specific labelling was detected in the cerebral cortex, claustrum, several limbic structures (hippocampus, septum and nucleus of the diagonal band, bed nucleus of the stria terminalis, amygdala). Dense signals were recorded in dopaminergic regions such as the neostriatum and the nucleus accumbens. In the hypothalamus, discrete binding sites were revealed in the ventromedial and medial mammillary nuclei. The serotoninergic dorsal raphe and noradrenergic locus coerulus were also radiolabelled. This parallels the mRNA (Lembo et al., 1999; Saito et al., 1999, 2001a; Hervieu et al., 2000) and protein (Hervieu et al., 2000) distribution of the MCH-R1 gene product. 5.8. CENTRAL AND PERIPHERAL DISTRIBUTION OF THE MCH RECEPTOR MCH2 IN THE MAMMALS Several reports have provided data regarding the MCH-R2 gene expression sites in the human (An et al., 2001; Hill et al., 2001; Moil et al., 2001; Sailer et al., 2001) and the rhesus monkey (Sailer et al., 2001). Strikingly, several non-human species (rat, mouse, hamster, guinea, pig, and rabbit) do not have functional MCH-R2 receptors, or encode a non-functional MCH-R2 pseudogene while retaining MCH-R1 expression (Tan et al., 2002). MCH2 gene expression is

Fig. 14. SLC-1 immunoreactivity in the basal ganglia. A strong immunostaining was found in the basal ganglia as

illustrated in A with a sagittal section: dense immunosignals were seen in the substantia nigra, subthalamic nucleus (STN), lateral segment of the globus pallidus (GP1). Immunosignals were also observed in the caudate-putamen, shell of the nucleus accumbens (NacS) and ventral pallidum (VPal). Note also the labelling within the internal capsula (ic). Higher magnifications are presented for the medial and lateral segments of the globus pallidus (B), caudate-putamen (B,C), subthalamic nucleus (D) and substantia nigra pars reticulata (E). In the caudate-putamen, immunosignals are located on the cell membrane of aspiny neurones. Calibration bars (in Ixm): A, 800; B, 140; C, 35; D, E, 280 (Hervieu et al., 2000). 76

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much higher in the brain than in the periphery. The tissue distributions of MCH1 and MCH2 receptors using quantitative TaqMan RT-PCR (Hill et al., 2001; Moil et al., 2001) and Northern blot (An et al., 2001) were compared. The mRNA profiles of the two receptors were relatively similar, showing predominant expression in the brain. The distribution of the two receptors in the individual regions of the brain is similar but there are subtle differences. For example, the contribution of the hypothalamus, locus coeruleus, medulla oblongata and cerebellum to the MCH1 profile is greater than the contribution of these regions to the MCH2 profile (Hill et al., 2001). One of the more prominent differences was the increase in pituitary contribution to the MCH1 profile compared with the MCH2 profile (Hill et al., 2001). In the rhesus monkey, by in situ hybridisation, MCH2 gene expression was detected in the cerebral cortex, hippocampus, and hypothalamus, with lower levels in the caudate nucleus, putamen and thalamus. In the hypothalamus, MCH2 gene is strongly expressed in the anterior and lateral areas (unlike MCH1) but not in the dorsomedial area (unlike MCH1) (Sailer et al., 2001). 5.9. NEUROFUNCTIONAL ANALYSIS Functional implications based on SLC-1 localisation within the rat nervous system has been proposed in several reports (Hervieu et al., 2000; Kilduff and de Lecea, 2001; Saito et al., 2001a). The widespread and generally dense cortical distribution of the MCH receptor is consistent with the suggestion that the peptide is involved in generalised cortical arousal and sensorimotor integration (Bittencourt et al., 1992; Nahon, 1994). The substantia innominata and parabrachial nuclei receive dense MCH innervation and are both SLC-1 immunopositive. A particularity of these cell groups is that their cortical projections conform to the description of non-specific cortical afferents. Neurones in the lateral hypothalamus and zona incerta, where the MCH gene is strongly expressed, and where the mRNA and protein SLC-1 are strongly expressed, are also associated with diffused arousal functions and sensorimotor integration (see Bittencourt et al., 1992). Together with the presence of SLC-1 labelling in many parts of the limbic system and the medial septum, these findings may explain the role of MCH in sensory conditioning, since the peptide diminishes the ability of rats to appropriately filter sensory clues as found in a CNS auditory gating paradigm (Miller et al., 1993). The prediction is that MCH thus lessens the chances of a behavioural change in response to a new sound. Impairment in sensory processing (such as loss of activation in auditory association areas in response to external speech) is often encountered in people with schizophrenia who have a pronounced tendency to misinterpret significant sounds in a noisy environment. The subiculum, Ammon's horn (strata oriens and pyramidale) and dentate gyrus regions were strongly immunoreactive to the SLC-1 antisera in both the rat and human. Together with the immunostaining observed in other forebrain regions such as the amygdaloid regions and the

.......+

Fig. 15. SLC-1 immunoreactivity in the forebrain/diencephalon. A dense population of MCH receptor SLC-1

immunostained cells was detected in the supraoptic nucleus (SON; A), the suprachiasmatic nucleus (SCN; B), the posterior region of the anterior nucleus (AHNp; C). At the level of the paraventricular nucleus (PVH), there was a more pronounced staining of the lateral zone of the posterior magnocellular region (PVHpml) as opposed to a lighter immunostaining observed in the dorsal parvicellular, medio-parvicellular and the dorsal zone of the medial parvicellular region (resp. PVHdp, PVHmpv and PVHmpd; D). Immunostained cells were detected as well in the arcuate nucleus (AN; E), the posterior region of the dorsomedial nucleus (DMHp; F) and the lateral hypothalamic area (LHA; G). In the posterior hypothalamus, MCH receptor immunostained cells were seen in the tuberomammillary nucleus (TMv; H). Calibration bars: A-H, 140 txm (Hervieu et al., 2000). 78

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bed nucleus of the stria terminalis, these results may be functionally related to the action of MCH on passive avoidance, a behavioural paradigm associated with cognition and learning. In that work, MCH hastened the extinction of the passive avoidance response (commonly used to measure cognitive alterations). The ~-MSH in that paradigm showed the opposite action (McBride et al., 1994). MCH was also shown to affect memory retention when infused into the hippocampus and amygdala (Monzon et al., 1999). In keeping with this, our results appeared to indicate a particular representation of SLC-1 labelling in anatomical regions implicated in the control of vigilance. The SLC-1 protein is present in the locus coerulus, identified as a major nucleus to set levels of arousal and behavioural vigilance (Foote et al., 1983). MCH hypothalamic neurones project to the locus coerulus (Bittencourt et al., 1992). Moreover, immunostaining was also observed in many other regions that control the arousal state. It includes the metathalamic geniculate bodies, the thalamic paraventricular and reticular nuclei, the hypothalamic suprachiasmatic and tuberomammillary nuclei, the preoptic area, the mesencephalic reticular formation, and the pontine, raphe and full medullar nuclei. In all of these regions, SLC-1 gene was expressed as a protein with MCH-like associated immunoreactivity signatures (Bittencourt et al., 1992). Also the MCH receptor is present the diagonal band of Broca and medial septum in the forebrain, the laterotegmental nucleus and the pedunculopontine nucleus in the reticular formation in cholinergic regions. These nuclei are known to be involved in electroencephalogram (EEG) desynchronisation (as one of the indicators for wake/sleep cycles). Interestingly, a strong immunostaining was observed in the fasciculus retroflexus, a cholinergic fibre bundle originating in the habenula and projecting to the interpeduncular nucleus and various paramedian midbrain nuclei. MCH neurones have been shown to be responsive to acetylcholine stimulation, and carbachol has been shown to induce a rapid increase in hypothalamic MCH mRNA expression (Bayer et al., 1999a,b). MCH neurones are likely to be cholinoreceptive (see Section 3.4.2). There might be a more specific aspect of MCH control on sleep mechanisms as SLC-1 immunostaining was observed in the oculomotor nerve (III) and the red nucleus (RN). The third cranial nerve, passing through the red nucleus, controls the extrinsic ocular muscles as well as the pupillary sphincter and the ciliary muscles. Also immunostaining was found in the anterior pretectal area. This midbrain area receives afferents from the retina and the visual association cortex and controls pupillary reflexes. There may be grounds here for a role in sensorimotor integration played by SLC-1 at the level of the sensory systems. The dense labelling seen in the piriform cortex could be related to the anti-seizure activity of the MCH reported by Knigge and Wagner (1997). Indeed, i.c.v, injections of MCH given 15 min before pentylenetetrazole (PTZ) intraperitoneal injection prevents seizure activity triggered by PTZ in rat and guinea pig (Knigge and Wagner, 1997). SLC-1 labelling was also represented in the extrapyramidal motor system and throughout many areas of the mesencephalic, pontine and medullary reticular formations associated with locomotor activity. Much of this labelling may also be

._____.+

Fig. 16. SLC-1 immunoreactivity in the brainstem and lumbar spinal cord. Immunostained MCH receptor staining

was detected in the periaqueductal grey matter (PAG; A), the pontine grey (PG; B), the granular layer of the cerebellum (Gr; C) and in the locus coeruleus (LC; D). In the spinal cord, SLC-l-like immunoreactivity was present within the grey matter of the spinal cord with a more pronounced staining of the dorsal horn as opposed to the ventral one (E,F). In both horns, immunostained cells were detected (G,H). The spinal tracts such as the laterocorticospinal tract (lct), the laterospinocortical tract (lsc), the lateral spinothalamic tract (lst) and the anterior corticothalamic tract (act) were moderately labelled (E). Calibration bars (in Ixm): A, D, 70; B, C, 140; E, F, 280: G, H, 35 (Hervieu et al., 2000). 80

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concerned with a possible involvement of MCH in central pattern generator circuitry as well as brainstem-controlled motor behaviour. It should be noted, however, that targeted deletion of the MCH gene does not appear to affect locomotor behaviour in the mutant mice (Shimada et al., 1998) and MCH itself has no action on locomotor activity but antagonises MSH-induced hyperlocomotor behaviour (Sanchez et al., 1997). This sets a possible scene for MCH neurones being involved in the circuitry and activity of extrapyramidal motor pathways and it points out a possible role of MCH for the prevention of generalised seizure and basal ganglia disorders. Strong SLC-1 signals were reported in the basal ganglia for the messenger (Lembo et al., 1999; Saito et al., 1999; Hervieu et al., 2000; Saito et al., 2001a), the protein (Hervieu et al., 2000) and protein binding sites (Borowsky et al., 2002). Dense immunostaining was found in the anterior hypothalamic area and the zona incerta both involved in drinking behaviour. This could be linked with the observed effects of dehydration and salt-loading on MCH gene expression activity (see Section 3.2 and Section 5.3) and the effect of intestinal MCH on regulating the hydro-mineral balance in the gastrointestinal tract (Hervieu and Nahon, 1995). SLC-1 immunoreactivity also appears to be related to processing systems for visual and auditory stimuli. The visual pathway conveys regulatory information through the lateral geniculate nucleus, a thalamic relay receiving afferents from the retina, the parabrachial region, the hypothalamic tuberomammillary region and the superior colliculus, and sending projections to the visual cortex and the reticular thalamic nucleus. Information is then relayed back to the dorsal thalamus. All of these regions exhibit substantial SLC-1 immunoreactivity. Interestingly, Saito et al. (1999) have also reported slc-1 gene expression in the eye. SLC1 receptors are also probably involved in auditory processing since immunostaining was observed in the auditory cortex, medial geniculate nucleus, inferior colliculus, the lateral lemniscus, the ventral and dorsal cochlear nuclei and the olivary complex. SLC-1 is clearly involved in many diverse motor and sensory systems. As discussed earlier, staining observed in specific mid- and hindbrain nuclei (oculomotor nucleus, red nucleus and the anterior pretectal area) may further indicate that MCH, acting through SLC-1 receptors, plays a role in general sensorimotor integration in these systems. Attention has recently been very much focused on the involvement of MCH in feeding and energy balance. The presence of SLC-1 in the arcuate, ventromedial, dorsomedial and paraventricular nuclei of the hypothalamus, as well as many gustatory regions, indicates that the receptor could mediate the reported orexigenic effects of MCH (Qu et al., 1996; Rossi and Bloom, 1997; Ludwig et al., 1998) summarised earlier in Section 5.4. Interestingly,

Fig. 17. The SLC-1 gene expression pattern in the human. (A) By Taqman, the SLC-1 receptor gene level

expression was dramatically higher in the brain and the pituitary than in other tissues. The receptor was expressed at quite low levels in peripheral tissues regulating energy balance (stomach, intestine, adipose, pancreas, skeletal muscle) and immune/haematopoietic system (spleen, lymphocytes, bone marrow but not in macrophages). (B) In the human brain, the gene expression distribution pattern was similar to that found in the rat. Expression was high in the cerebral cortex (cingulate, medial frontal and superior frontal gyri), quite high in the hippocampus and hypothalamus, middle to low in the basal ganglia (amygdala >> caudate nucleus, putamen, striatum, substantia nigra, globus pallidus) and low in the thalamus, spinal cord, cerebellum and medulla oblongata. (C-F) mRNA in situ experiments confirmed human SLC-1 gene expression in the cortex (C), the hippocampus (D) and the cerebellum (F). Immunoreactive cells were also detected in the dentate gyms (E). Scale bars: C, F, 0.6 cm; D, 0.5 cm; E, 35 Ixm. Abbreviations: (C) Caud., caudate; Put, putamen; MOrG, medial orbital gyms; POrG, posterior orbital gyms; GR, gyms rectus; (D) ARG, Andreas Retzius gyms; sms, superficial medullary stratum; ICG, isthmus of the cingulate gyms; PHG, parahippocampal gyms; DG, dentate gyms. 82

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

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

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

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

7. ABBREVIATIONS

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

ac(o, t)

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

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

The melanin-concentrating hormone

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

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

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

Ch. H

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

Ch. II

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

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

The melanin-concentrating hormone

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

Ch. H

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

Ch. H

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

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

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

SEZ/RC SG SI sms

SN p(c) (r) so

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

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

The melanin-concentrating hormone

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

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

Ch. H

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

Miscellaneous

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

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

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SDS

(T)TBS v/v w/v

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

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

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

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Bayer L, Jacquemard C, Fellmann D, Griffond B (1999c): Survival of rat MCH neurons in hypothalamus slice culture: histological, pharmacological and molecular studies. Cell Tissue Res 297:23-33. Bednarek MA, Feighner SD, Hreniuk DL, Palyha OC, Morin NR, Sadowski SJ, MacNeil DJ, Howard AD, Van der Ploeg LHY (2001): Short segment of human melanin-concentrating hormone that is sufficient for full activation of human melanin-concentrating hormone receptors 1 and 2. Biochemistry 40 (31):9379-9386. Bednarek MA, Hreniuk DL, Tan C, Palyha OC, MacNeil DJ, Van der Ploeg LHY, Howard AD, Feighner SD (2002a): Synthesis and biological evaluation in vitro of selective, high affinity peptide antagonists of human melanin-concentrating hormone action at human melanin-concentrating hormone receptor 1. Biochemistry 41 (20):6383-6390. Bednarek MA, Tan C, Hreniuk DL, Palyha OC, MacNeill DJ, Van der Ploeg LHY, Howard AD, Feighner SD (2002b): Synthesis and biological evaluation in vitro of a selective, high potency peptide agonist of human melanin-concentrating hormone action at human melanin-concentrating hormone receptor 1. J Biol Chem 277 (16):13821-13826. Bernardis LL, Bellinger LL (1996): The lateral hypothalamic area revisited: ingesting behavior. Neurosci Behav Rev 20 (2): 189-287. Bittencourt JC, Elias CF (1998): MCH and NEI projections from the LHA and ZI to the medial septal nucleus and spinal cord: a study using multiple neuronal tracers. Brain Res 805:1-19. Bittencourt JC, Sawchenko PE (2000): Do centrally administered neuropeptides access cognate receptors?: an analysis in the central CRF system. J Neurosci 20 (3):1142-1156. Bittencourt JC, Presse F, Arias C, Peto C, Vaughan J, Nahon JL, Vale W, Sawchenko PE (1992): The melaninconcentrating hormone system of the rat brain: an immuno- and hybridization histochemical characterisation. J Comp Neurol 319:218-245. Bittencourt JC, Frigo R, Rissman RA, Casatti CA, Nahon JL, Bauer JA (1998): The distribution of MCH in the monkey brain. Brain Res 804:140-143. Bittner M, Meltzer P, Chen Y, Jiang MM, Seftor E, Hendrix M, Radmacher M, Trent J (2000): Molecular classification of cutaneous malignant melanoma by gene expression profiling. Nature 406:536-540. Blalock JE (1989): The bidirectional communication between the neural and immune systems. Physiol Rev 69 (1):1-32. Bluet-Pajot MT, Presse F, Voko Z, Hoeger C, Mounier J, Epelbaum J, Nahon JL (1995): Neuropeptide E-I antagonises the action of melanin-concentrating hormone on stress-induced release of adrenocorticotropin in the rat. J Neuroendocrinol 7:297-303. Borowsky B, Durkin M, Ogozaler K, Marzabadi MR, DeLeon J, Heurich R, Lightblau H, Shaposhnik Z, Daniewska I, Blackburn TP, Blanchek TA, Gerald C, Vaysse PJ, Forray C (2002): Antidepressant, anxiolytic and anorectic properties of MCH-R1 antagonist. Nat Med 8 (8):825-830. Borsu L, Presse F, Nahon JL (2000): The AROM gene, spliced mRNAs encoding new DNA/RNA-binding proteins are transcribed from the opposite strand of the MCH gene in mammals. J Biol Chem 275 (51):40576-40587. Boutin JA, Suply T, Audinot V, Rodriguez M, Beauverger P, Nicolas JP, Galizzi JP, Fauchfre JL (2002): Melaninconcentrating hormone and its receptors: state of the art. Can J Physiol Pharmacol 80 (5):388-395. Bray GA, Tartaglia LA (2000): Medicinal strategies in the treatment of obesity. Nature 404:672-677. Bradley RL, Kokkotou EG, Maratos-Flier E, Cheatham B (2000): MCH regulates leptin synthesis and secretion in rat adipocytes. Diabetes 49:1073-1077. Bresson JL, Clavequin MC, Fellman D, Bugnon C (1987): Donn6es ontog6n6tiques sur la population d'interneurones peptidergiques ~ immunor6activit6 de type GRF37 de l'hypothalamus postero-lat6ral humain. Etudes immunocytochimiques ~ l'aide d'un IS anti-GRF37 et d'un IS anti-MCH. CR Soc Biol 181:376-382. Bresson JL, Clavequin MC, Fellman D, Bugnon C (1989): Human hypothalamic neuronal system revealed with a salmon MCH antiserum. Neurosci Lett 102:39-43. Breton C, Fellman D, Bugnon C (1989): Clonage et sequencage d'ADNc du precurseur commun de trois neuropeptides hypothalamiques immunologiquement apparentes a la somatocrinine humaine 1-37, a l'alpha melanotropine e t a 1' hormone de melanoconcentration du saumon. CR Acad Sci Paris 309:749-754. Breton C, Schorpp M, Nahon JL (1993a): Isolation and characterization of the human melanin-concentrating hormone gene and a variant gene. Mol Brain Res 18:297-310. Breton C, Presse F, Hervieu G, Nahon JL (1993b): Structure and regulation of the mouse melanin-concentrating hormone mRNA and gene. Mol Cell Neurosci 4:271-284. Brischoux F, Fellmann D, Risold PY (2001): Ontogenetic development of the diencephalic MCH neurons: a hypothalamic 'MCH area' hypothesis. Eur J Neurosci 13:1733-1744. Broberger C (1999): Hypothalamic CART' neurons: histochemical relationship to TRH MCH, orexin/hypocretrin and NPY. Brain Res 848:101-113.

93

Ch. H

G.J. Hervieu et al.

Broberger C, de Lecea L, Sutcliffe JG, Hokfelt T (1998): Hypocretin/orexin- and melanin-concentrating hormoneexpressing cells form distinct populations in the rodent lateral hypothalamus: relationship to the neuropeptide Y and agouti gene-related protein systems. J Comp Neurol 402:460-474. Burgaud JL, Poosti R, Fehrentz JA, Martinez J, Nahon JL (1997): Melanin-concentrating hormone binding sites in human svkl4 keratinocytes. Biochem Biophys Res Commun 241:622-629. Campfield A (1998): Strategies and potential molecular targets for obesity treatment. Science 280:1383-1387. Chambers J, Ames RS, Bergsma D, Muir A, Fitzgerald LR, Hervieu G, Dytko GM, Foley JJ, Martin J, Liu WS, Park J, Ellis C, Ganguly S, Konchar S, Cluderay J, Leslie R, Wilson S, Sarau HM (1999): Melanin-concentrating hormone is the cognate ligand for the orphan G-protein-coupled receptor SLC-1. Nature 400:261-265. Chaffer CL, Morris MJ (2002): The feeding response to melanin-concentrating hormone is attenuated by antagonism of the NPYYl-receptor in the rat. Endocrinology 143 (1):191-197. Clapham JC, Arch JRS, Taddayyon M (2001): Anti-obesity drugs: a critical review of current therapies and future opportunities. Pharmacol Ther 89:81-121. Checler F, Dauch P, Barelli H, Nahon JL, Vincent JP (1992): Hydrolysis of rat melanin-concentrating hormone by endopeptidase 24.11 (neutral endopeptidase). J Biochem 286 (1):217-221. Chen YY, Hu CZ, Hsu CK, Zhang Q, Bi C, Asnicar M, Hsiung HM, Fox N, Slieker LJ, Yang DD, Heiman ML, Shi YG (2002): Targeted disruption of the melanin-concentrating hormone receptor-1 results in hyperphagia and resistance to diet-induced obesity. Endocrinology 143 (7):2469-2477. Chiocchio SR, Gallardo MGP, Louzan P, Gutnisky V, Tramezzani JH (2001): Melanin-concentrating hormone stimulates the release of luteinizing hormone-releasing hormone and gonadotropins in the female rat acting at both median eminence and pituitary levels. Biol Reprod 64 (5):1466-1472. Compagnone N, Fellman D, Bugnon C (1991): Expression of peptides derived from the MCH precursor in serum-free culture of rat fetal hypothalamic neurons: role of attachment factors. Dev Neurosci 13:417-423. Compagnone N, Fellman D, Bugnon C, Jacquemard C (1993): Diffusible factors from rat arcuate nucleus and Broca's diagonal band nucleus increase size and neurite outgrowth, respectively, of cultured MCH neurones. Brain Res 628:137-144. Courseaux A, Nahon JL (2001): Birth of two chimeric genes in the Hominidae lineage. Science 291 (5507):12931295. Crawley JN, McLean S (1996): Neuropeptides: basis and clinical advances. Ann New York Acad Sci 780:000-000. Dallvecchia-Adams S, Kuhar MJ, Smith Y (2002): CART peptide projections in the ventral midbrain: colocalisation with GABA, MCH, dynorphin, and synaptic interactions with dopamine neurons. J Comp Neurol 448:360-372. Darlinson MG, Richter D (1999): Multiple genes for neuropeptides and their receptors: co-evolution and physiology. Trends Neurosci 22 (2):81-88. Delecea L, Kilduff TS, Peyrin C, Foye PE, Danielson PE, Fukuhara C, Battenberg ELF, Gautvik VT, Bartlett FS, Frankel WN, van den Pol AN, Bloom FE, Gautvik KM, Sutcliffe JG (1998): The hypocretins: hypothalamusspecific peptides with neuroexcitatory activity. Proc Natl Acad Sci USA 95:322-327. Della-Zuana O, Presse F, Ortola C, Duhault J, Nahon JL, Levens N (2002): Acute and chronic administration od MCH enhances food intake and body weight in Wistar and Sprague-Dawley rats. Int J Obesity 26:1-7. Drozdz R, Eberle AN (1995a): Binding sites for melanin-concentrating hormone (MCH): In brain synaptosomes and membranes from peripheral tissues identified with highly tritiated MCH. J Receptor Signal Transduct Res 15:487-502. Drozdz R, Eberle AN (1995b): Synthesis and iodination of human (phenylalanine 13, tyrosine 19): melaninconcentrating hormone for radioreceptor assay. J Pept Sci 1:58-65. Drozdz R, Siegrist W, Baker BI, Chlubadetapia J, Eberle AN (1995): Melanin-concentrating hormone binding to mouse melanoma cells in vitro. FEBS Lett 359:199-202. Drozdz R, Hintermann E, Tanner H, Zumsteg U, Eberle AN (1999): (D-(p-benzoylphenylalanine):(13):, tyrosine(19):):-melanin-concentrating hormone, a potent analogue for MCH receptor crosslinking. J Pept Sci 5:234-242. Eberle AN (1988): The Melanotropins-Chemistry, Physiology and Mechanisms of Action. Karger, Basle. Edwards CMB, Abusnana S, Sunter D, Murphy KG, Ghatei MA, Bloom SR (1999): The effect of the orexins on food intake: comparison with NPY, MCH and galanin. J Endocrinol 160:R7-R12. Elias CF, Bittencourt JC (1997): Study of the origins of MCH and NEI immunoreactive projections to the periaqueductal gray matter. Brain Res 755:255-271. Elias CF, Saper CB, Eleftheria MF, Tritos NA, Lee C, Kelly J, Tatro JB, Hoffman GE, Ollmann MM, Barsh GS, Sakurai T, Yanagisawa M, Elmquist JK (1998): Chemically defined projections linking the mediobasal hypothalamus and the lateral hypothalamic area. J Comp Neurol 402:442-459.

94

The melanin-concentrating hormone

Ch. H

Elias CF, Lee C, Kelly JF, Ahima RS, Kuhar M, Saper CB, Elmquist JK (2001): Characterisaton of CART neurons in the rat and human hypothalamus. J Comp Neurol 432:1-19. Elmquist JK, Elias CF, Saper CB (1999): From lesions to leptin: Hypothalamic control of food intake and body weight. Neuron 22:221-223. Filglewicz DE Wood SC (2000): Adiposity signals and brain reward mechanisms. Trends Pharmacol Sci 21:235236. Flier JS, Maratos-Flier E (1998): Obesity and the hypothalamus: novel peptides for new pathways. Cell 92:437440. Foote SL, Bloom FE, Aston-Jones G (1983): Nucleus locus coeruleus: new evidence of anatomical and physiological specificity. Physiol Rev 63:844-914. Forsling ML, Zhou Y (1997): MCH and vasopressin release in the rat. J Physio1504:223. Fulton S (2000): Modulation of brain reward circuitry by leptin. Science 287:125-128. Gao B, Hagenbuch B, Kullak-Ublick GA, Benke D, Aguzzi A, Meier P (2000): Organic anion-transporting polypeptides mediate transport of opioid peptides across the blood-brain barrier. J Pharmacol Exp Ther 294 (1):73-79. Gao X-B, van den Pol AN (2001): MCH depresses synaptic activity of glutamate and GABA neurons from rat lateral hypothalamus. J Physiol 533 (1):237-252. Gautwik KM, de Lecea L, Gautwik VT, Danielson PE, Tranque P, Dopazo A, Bloom FE, Sutcliffe JG (1996): Overview of the most prevalent hypothalamus-specific mRNAs, as identified by directional tag PCR substraction. Proc Natl Acad Sci USA 88:10208-10212. Gonzalez MI, Vaziri S, Wilson CA (1996): Behavioral effects of alpha-MSH and MCH after central administration in the female rat. Peptides 17 (1):171-17'7. Gonzalez MI, Baker BI, Wilson CA (1997a): Stimulatory effect of MCH on LH release. Neuroendocrinology 66:254-262. Gonzalez MI, Kalia V, Hole DR, Wilson CA (1997b): Alpha-MSH and MCH modify monoaminergic levels in the preoptic area of the rat. Peptides 18 (3):387-392. Griffond B, Baker BI (2002): Cell and molecular cell biology of melanin-concentrating hormone. Int Rev Cytol 213:233-277. Griffond B, Deray A, Nguyen NU, Colard C, Fellmann D (1995): The synthesis of melanin-concentrating hormone is stimulated by ventromedial hypothalamic lesions in the rat lateral hypothalamus: a time-course study. Neuropeptides 28:267-275. Griffond B, Ciofi P, Bayer L, Jacquemard C, Fellman D (1997): Immunocytochemical detection of the neurokinin B receptor (NK3): on MCH neurons in rat brain. J Chem Neuranat 12:183-189. Griffond B, Grillon S, Herve C, Deray A, Fellman D (1998): Morphofunctional changes in MCH-producing neurones in relation to the control of food and water intake. Ann New York Acad Sci 839:219-222. Grillon S, Herve C, Griffond B, Fellmann D (1997): Exploring the expression of the melanin-concentrating hormone messenger RNA in the rat lateral hypothalamus after goldthioglucose injection. Neuropeptides 31:131136. Hakansson ML, Brown H, Ghilardi N, Skoda RC, Meister B (1998): Leptin receptor immunoreactivity in chemically defined target neurons of the hypothalamus. J Neurosci 18 (1):559-572. Hanada R, Nakazato M, Matsukara S, Murakami N, Yoshimatsu H, Sakata T (2000): Differential regulation of MCH and orexin genes in the agouti-related protein/MC-R4 system. Biochem Biophys Res Commun 268:88-91. Harris J, Bird DJ (2000): Supernatants from leucocytes treated with MCH and alpha-MSH have a stimulatory effect on rainbow trout phagocytes in vitro. Vet Immunol Immunopathol 76:117-124. Harrison DC, Medhurst AD, Bond BC, Campbell CA, Davis RE Philpott KL (2000): The use of quantitative RT-PCR to measure mRNA expression in a rat model of focal ischemia - - caspase-3 as a case study. Mol Brain Res 75:143-149. Hawes BE, Kil E, Green B, O'Neill K, Fried S, Graziano MP (2000): The melanin-concentrating hormone receptor couples to multiple G proteins to activate diverse intracellular signaling pathways. Endocrinology 141 (12):4524-4532. Haynes AC, Stevens AJ, Muir A, Avenell KY, Clapham JC, Tadayyon M, Darker JG, Wroblowski B, O'Toole CJ, Witty D, Arch JRS (2001): A MCH-R1 antagonist reduces food intake and body weight gain in rats and mice. North American Association for the Study of Obesity, Canada, Quebec. Published in Obesity Res 9 (Suppl. 9): O152. Henry BA, Tilbrook AJ, Dunshea FR, Rao A, Blache D, Martin GB, Clarke IJ (2000): Long-term alterations in adiposity affect the expression of melanin-concentrating hormone and enkephalin but not proopiomelanocortin in the hypothalamus of ovariectomized ewes. Endocrinology 141 (4): 1506-1514.

95

Ch. H

G.J. Hervieu et al.

Herv6 C, Fellmann D (1997): Changes in rat melanin-concentrating hormone and dynorphin messenger ribonucleic acids induced by food deprivation. Neuropeptides 31:237-242. Herv6 C, Colard C, Grillon S, Fellmann D, Griffond B (1998): Polyethylene glycol-induced hypovolemia affects the expression of MCH mRNA, but not dynorphin or secretogranin II mRNAs, in the rat lateral hypothalamus. Neurosci Lett 248:133-137. Hervieu G, Emson PC (1998): The localisation of somatostatin receptor 1 ssh immunoreactivity in the rat brain using an N-teminal specific antibody. Neuroscience 85 (4):1263-1284. Hervieu G, Nahon JL (1995): Pro-melanin-concentrating hormone messenger ribonucleic acid and peptides expression in peripheral tissues of the rat. Neuroendocrinology 61:348-364. Hervieu G, Volant K, Grishina O, Descroix-Vagne M, Nahon JL (1996a): Similarities in cellular expression and functions of melanin-concentrating hormone and atrial natriuretic factor in the rat digestive tract. Endocrinology 137 (2):561-571. Hervieu G, Segretain D, Nahon JL (1996b): Developmental and stage-dependent expression of melaninconcentrating hormone in the nuclei of rodent germ cell. Biol Reprod 54:1161-1171. Hervieu GJ, Cluderay JE, Harrison D, Meakin J, Maycox P, Nasir S, Leslie RA (2000): The distribution of the mRNA and protein products of the melanin-concentrating hormone (MCH): receptor gene, slc-1, in the central nervous system of the rat. Eur J Neurosci 12:1194-1216. Hetherington AW, Ranson SW (1940): Hypothalamic lesions and adiposity in the rat. Anat 78:149-172. Hill J, Duckworth M, Murdoch P, Rennie G, Sabido-David C, Ames RS, Szekeres P, Wilson S, Bergsma DJ, Gloger IS, Levy DS, Chambers JK, Muir AI (2001): Molecular cloning and functional characterisation of MCH2, a novel human MCH receptor. J Biol Chem 276 (23):20125-20129. Hintermann E, Drozdz R, Tanner H, Eberle AN (1999): Synthesis and characterization of new radioligands for the mammalian melanin-concentrating hormone (MCH): receptor. J Recept Signal Transd Res 19:411-422. Hintermann E, Tanner H, Talke-Messerer C, Schlumberger S, Zumsteg U, Eberle AN (2001a): Interaction of melanin-concentrating hormone (MCH):, neuropeptide E-I (NEI): 9 neuropeptide G-E (NGE), and alpha-MSH with melanocortin and MCH receptors on mouse B16 melanoma cells. J Recept Signal Transd Res 21 (1):93116. Hintermann E, Erb C, Talke-Messerer C, Liu R, Tanner H, Flammer J, Eberle AN (2001b): Expression of the melanin-concentrating hormone receptor in porcine and human ciliary epithelial cells. Invest Ophthal Vis Sci 42 (1):206-209. Hockfelt T (1991): Neuropeptides in perspective: the last ten years. Neuron 7:867-879. Hockfelt T, Broberger C, Xu DZ-Q, Sergeyev V, Ubink R, Diez M (2000): Neuropeptides, an overview. Neuropharmacology 39:1337-1356. Huang Q, Viale A, Picard F, Nahon JL, Richard D (1999): Effects of leptin on MCH expression in the brain of lean and obese mice. Neuroendocrinology 69:145-153. Iqbal J, Pompolo S, Murakami T, Grouzmann E, Sakurai T, Meister B, Clarke IJ (2001): Immunohistochemical characterization of localization of long-form leptin receptor (OB-Rb): in neurochemically defined cells in the ovine hypothalamus. Brain Res 920 (1-2):55-64. Jezova D, Bartanutsz V, Westergren I, Johansson B, Rivier J, Vale W, Rivier C (1992): Rat melanin-concentrating hormone stimulates adrenocorticotropin secretion: evidence for a site of action in brain regions protected by the blood brain barrier. Endocrinology 130:1021-1029. Kastin AJ, Pan WH, Maness LM, Banks WA (1999): Peptides crossing the blood-brain barrier: some unusual observations. Brain Res 848 (1-2):96-100. Kastin AJ, Akerstrom V, Hackler L, Zadina JE (2000): [Phe(13):,Tyr(19):]-melanin-concentrating hormone and the blood-brain barrier: role of protein binding. J Neurochem 74 (1):385-391. Kawauchi H, Kawazoe I, Tsubokawa M, Kishida M, Baker BI (1983): Characterisation of MCH in chum salmon pituitaries. Nature 305:321-323. Kawazoe I, Kawauchi H, Hirano T, Naito N (1987): Structure-activity relationships of melanin-concentrating hormone. Int J Pept Protein Res 29:714-721. Kemp EH, Waterman EA, Hawes BE, O'Neill K, Gottumukkala RVSRK, Gawkrodger DJ, Weetman AP, Watson PF (2002): The melanin-concentrating hormone receptor 1, a novel target of autoantibody responses in vitiligo. J Clin Invest 109 (7):923-930. Kennedy AR, Todd JF, Stanley SA, Abbott CR, Small CJ, Ghatei MA, Bloom SR (2001): Melanin-concentrating hormone (MCH): suppresses thyroid stimulating hormone (TSH): release, in vivo and in vitro, via the hypothalamus and the pituitary. Endocrinology 142 (7):3265-3268. Kilduff TS, de Lecea L (2001): Mapping of the mRNAs for the hypocretin/orexin and MCH receptors: network of overlapping peptide systems. J Comp Neurol 435:1-5.

96

The melanin-concentrating hormone

Ch. H

Kinoshita M, Morita T, Toyohara H, Hirata T, Sakaguchi M, Ono M, Inoue K, Wakamatsu Y, Ozato K (2001): Transgenic medaka overexpressing a melanin-concentrating hormone exhibit lightened body color but no remarkable abnormality. Mar Biotechnol 3 (6):536-543. Knigge KM, Wagner JE (1997): Melanin-concentrating hormone involvement in PTZ-induced seizure in rat and guinea pig. Peptides 18 (7):1095-1097. Knigge KM, Baxter-Grillo D, Speciale J, Wagner J (1996): Melanotropic peptides in the mammalian brain: the melanin-concentrating hormone. Peptides 17 (6): 1063-1073. Knollema S, Brown ER, Vale W, Sawchenko PE (1992): Novel hypothalamic and preoptic sites of pre-proMCH mRNA and peptide expression in lactating rats. J Neuroendocrinol 4:709-718. Kokkotou EG, Tritos NA, Mastaitis JW, Slieker L, Maratos-Flier E (2001): Melanin-concentrating hormone receptor is a target of leptin action in the mouse brain. Endocrinology 142 (2):680-686. Kolakowski LF, Jung BP, Nguyen T, Johnson MP, Lynch KR, Cheng R, Heng HHQ, George SR, O'Dowd BF (1996): Characterization of a human gene related to genes encoding somatostatin receptors. FEBS Lett 398:253258. Koob GF (1999): Corticotropin-releasing factor, norepinephrine, and stress. Biol Psychiatry 46:1167-1180. Kopelman PG (2000): Obesity as a medical problem. Nature 404:635-643. Lakaye B, Minet A, Zorzi W, Grisar T (1998): Cloning of the rat brain cdna encoding for the slc-1 g proteincoupled receptor reveals the presence of an intron in the gene. Biochim Biophys Acta Mol Cell Res 1401:216220. Lebl M, Hruby VJ, Castrucci AM, Visconti MA, Hadley ME (1988): Melanin concentrating hormone analogues: contraction of the cyclic structure. 1. Agonist activity. J Med Chem 31:949-954. Lembo PMC, Grazzini E, Cao J, Hubatsch DA, Pelletier M, Hoffert C, St Onge S, Pou C, Labrecque J, Groblewski T, O'Donnell D, Payza K, Ahmad S, Walker P (1999): The receptor for the orexigenic peptide melanin-concentrating hormone is a G-protein-coupled receptor. Nat Cell Biol 1:267-271. Lopez M, Seoane LM, Garcia MD, Dieguez C, Senaris R (2002): Neuropeptide Y, but not agouti-related peptide or melanin-concentrating hormone, is a target peptide for orexin-A feeding actions in the rat hypothalamus. Neuroendocrinology 75 (1):34-44. Ludwig DS, Mountjoy KG, Tato JB, Gillette JA, Frederich RC, Flier JS, Maratos-Flier E (1998): Melaninconcentrating hormone: a functional antagonist in the hypothalamus. Am J Physiol 274 [Endocrinol. Metab. 37]:E627-E633. Ludwig DS, Tritos NA, Mastaitis JW, Kulkarni R, Kokkotou E, Elmquist J, Lowell B, Flier JS, Maratos-Flier E (2001): MCH overexpression in transgenic mice leads to obesity and insulin resistance. J Clin Invest 107 (3):379-386. Macdonald D, Murgolo N, Zhang RM, Durkin JP, Yao XR, Strader CD, Graziano MP (2000): Molecular characterization of the melanin-concentrating hormone/receptor complex: identification of critical residues involved in binding and activation. Mol Pharmacol 58:217-225. Marsh DJ, Weingarth DT, Novi DE, Chen HY, Trumbauer ME, Chen Guan XM, Jiang MM, Feng Y, Camacho RE, Shen Z, Frazier EG, Yu H, Metzger JM, Kuca SJ, Shearman LP, Gopal-Truter S, MacNeil DJ, Strack AM, Maclntyre DE, Van der Ploeg LHT, Qian S (2002): Melanin-concentrating hormone 1 receptor-deficient mice are lean, hyperactive, and hyperphagic and have altered metabolism. Proc Natl Acad Sci USA 99 (5):3240-3245. Matsunaga TO, Castrucci AM, Hadley ME, Hruby VJ (1986): MCH: Structure-function aspects of its MSH-like activities. Peptides 10:773-778. Matsunaga TO, Castrucci AM, Hadley ME, Hruby VJ (1989): Melanin concentrating hormone (MCH): synthesis and bioactivity studies of MCH fragment analogues. Peptides 10:349-354. McBride RB, Becwith E, Swenson RR, Sawyer TK, Hadley ME, Matsungaga TO, Hruby VJ (1994): The actions of melanin-concentrating hormone on passive avoidance in rats: a preliminary study. Peptides 15 (4):757-759. Maulon-Feraille L, Della Zuana O, Suply T, Rovere-Jovene C, Audinot V, Leven N, Boutin JA, Duhault J, Nahon JL (2002): Appetite-boosting property of pro-melanin concentrating hormone(131-165): (neuropeptide-glutamic acid-isoleucine): is associated with proteolytic resistance. J Pharm Exp Ther 302 (2):766-773. Miller CL, Hruby VJ, Matsunaga TO, Bickford PC (1993): Alpha-MSH and MCH are functional antagonists in a CNS auditory gating paradigm. Peptides 14:431-440. Miller CL, Burmeister M, Thompson RC (1998): Antisense expression of the human preproMCH gene. Brain Res 803:86-94. Mizuno TM, Kleopoulos SP, Bergen HT, Roberts JL, Priest CA, Mobbs CV (1998): Hypothalamic POMC mRNA is reduced by fasting in ob/ob and db/db mice, but is stimulated by leptin. Diabetes 47:294-297. Mondal MS, Nakazato M, Matsukura S (2002): Characterization of orexins (hypocretins): and melaninconcentrating hormone in genetically obese mice. Reg Pept 104 (1-3 (Special Issue)):21-25.

97

Ch. H

G.J. Hervieu et al.

Monzon ME, de Souza MM, Izquierdo LA, Barros DM, de Bariglio SR (1999): MCH modifies memory retention in rats. Peptides 20:1517-1519. Monzon ME, Varas MM, De Barioglio SR (2001): Anxiogenesis induced by nitric oxide synthase inhibition and anxiolytic effect of melanin-concentrating hormone (MCH): in rat brain. Peptides 22 (7):1043-1047. Mori M, Harada M, Terao Y, Sugo T, Watanabe T, Shinomura Y, Abe M, Shintani Y, Onda H, Nishimura O, Fujino M (2001): Cloning of a novel GPCR SLT, a subtype of the MCH receptor. Biochem Biophys Res Commun 283:1013-1018. Moriguchi T, Sakurai T, Nambu T, Yanagisawa M, Goto K (1999): Neurons containing orexin in the lateral hypothalamic area of the adult rat brain are activated by insulin-induced acute hypoglycemia. Neurosci Lett 264:101-104. Morley JE (1987): Neuropeptide regulation of appetite and weight. Endocr Rev 8 (3):256-287. Murray JF, Mercer JG, Adan RAH, Datta J, Aldairy C, Moar KM, Baker BI, Stock MJ, Wilson CA (2000): The effect of leptin on LH release is exerted in the zona incerta and mediated by MCH. J Neuroendocrinol 12:1133-1139. Mystkowski P, Seeley RJ, Hahn TM, Baskin DG, Havel PJ, Matsumuto AM, Wilkinson CW, Peacock-Kinzig K, Blake KA, Schwatz M (2000): Hypothalamic MCH and estrogen-induced weight loss. J Neurosci 20 (22):86378642. Nahon JL (1994): The melanin-concentrating hormone: from the peptide to the gene. Crit Rev Neurobiol 8 (4):221262. Nahon JL, Presse F, Bittencourt JC, Sawchenko P, Vale W (1989): The rat melanin-concentrating hormone mRNA encodes multiple putative neuropeptides coexpressed in the dorsolateral hypothalamus. Endocrinology 125:20562065. Nahon JL, Presse F, Breton C, Hervieu G, Schorpp M (1993): Melanotropic peptides. In: Structure and regulation of the melanin-concentrating hormone gene. Ann New York Acad Sci 680:111-129. Navarra P, Tsagarakis S, Coy DH, Rees LH, Besser GP, Grossman AB (1990): Rat melanin concentrating hormone does not modify the release of CRH-41 from rat hypothalamus or ACTH from the anterior pituitary in vitro. J Endocrinol 127:R1-R4. Oldfield BJ, Giles ME, Watson A, Anderson C, Colvill LM, McKinley MJ (2002): The neurochemical characterisation of hypothalamic pathways projecting polysynaptically to brown adipose tissue in the rat. Neuroscience 110 (3):515-526. Oshima N, Wannitikul P (1996): Signal transduction of MCH in melanophores of the tilapia, Oreochromis Niloticus. Zool Sci 13:351-356. Parkes D, Vale W (1992a): Contrasting actions of MCH and NEI on posterior pituitary functions. In: Structure and regulation of the melanin-concentrating hormone gene. Ann New York Acad Sci 680:588-590. Parkes DG, Vale W (1992b): Secretion of melanin-concentrating hormone and neuropeptide-EI from cultured rat hypothalamic cells. Endocrinology 131:1826-1831. Parkes DG, Rivest S, Rivier C, Sawchenko PE, Vale W (1992): MCH and NEI activate hypothalamic CRF neurons in conscious rats. Soc Neurosci 18:120. Paxinos G, Watson C (1998): The Rat Brain in Stereotaxic Coordinates. 3rd edn. Academic Press, San Diego. Pedeutour F, Szpirer C, Nahon JL (1994): Assignment of the human proMCH gene (PMCH) to chromosome 12q23-q24 and two variant genes (PMCHL1 and PMCHL2) to chromosome 5p14 and 5q12-q13. Genomics 19(1):31-33. Peeke PM, Chrousos GP (1995): Hypercortisolism and obesity. Stress: basic mechanisms and clinical implications. Ann New York Acad Sci 711:665-676. Peyron C, Tighe DK, van den Pol AN, de Lecea L, Heller HC, Sutcliffe JG, Kilduff TS (1998): Neurons containing hypocretin (orexin): project to multiple neuronal systems. J Neurosci 18:9996-10015. Presse F, Nahon JL (1993): Differential regulation of Melanin-Concentrating Hormone gene expression in distinct hypothalamic areas under osmotic stimulation in rat. Neuroscience 55:709-720. Presse F, Hervieu G, Imaki T, Sawchenko PE, Vale W, Nahon JL (1992): Rat melanin-concentrating hormone messenger ribonucleic acid expression: marked changes during development and after stress and glucocorticoid stimuli. Endocrinology 131:1241-1250. Presse F, Sorokovsky I, Max JP, Nicolaidis S, Nahon JL (1996): MCH is a potent anorectic peptide regulated by food-deprivation and glucopenia in the rat. Neuroscience 71 (3):735-745. Presse F, Cardona B, Borsu L, Nahon JL (1997): Lithium increases melanin-concentrating hormone mRNA stability and inhibits tyrosine hydroxylase gene expression in PC12 cells. Mol Brain Res 52(2):270-283. Qu D, Ludwig DS, Gammeltoft S, Piper M, Pelleymounter MA, Cullen MJ, Mathes WF, Przypek J, Kanarek R,

98

The melanin-concentrating hormone

Ch. H

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

99

Ch. H

G.J. H e r v i e u et al.

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

The m e l a n i n - c o n c e n t r a t i n g h o r m o n e

Ch. H

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

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CHAPTER III

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

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

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

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

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

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

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

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

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

Neuroanatomical studies of the ORL1 receptor and OFQ

Ch. III

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

5.1. METHODS 5.1.1. Animals

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

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C.R. Neal Jr. et al.

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

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

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

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

Neuroanatomical studies of the ORL1 receptor and OFQ

Ch. III

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

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

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

5.2.1. Immunocytochemistry controls 5.2.1.1. Colchicine treatment

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

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C.R. Neal Jr. et al.

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

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

Neuroanatomical studies of the ORL1 receptor and OFQ

Ch. III

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

5.3.1. Cortex

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

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C.R. Neal Jr. et al.

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

Neuroanatomical studies of the ORL1 receptor and OFQ

Ch. III

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

CNS region

OFQ peptide

OFQ mRNA

Cells

ORL1 mRNA

OFQ binding

F/T

Neocortex Frontal Layer I

.

L a y e r II

+

. +

.

+++

. +++

+ +

L a y e r III

++

+

+++

++

++

Layer IV

-

-

-

++++

++++

Layer V

+

+

++

+

++

Layer VI

++

+

+++

+++

+++

Parietal Layer I

.

L a y e r II

+

. -

.

+++

. ++

+ +

L a y e r III

+

-

++

++

++

Layer IV

-

-

-

+++

+++

Layer V

+

+

+

++

++

Layer VI

++

+

+++

+++

++

Temporal Layer I

.

L a y e r II

-

. +

. +

.

. +++

++

L a y e r III

-

+

+

++

++

Layer IV

-

-

-

++++

+

Layer V

-

+

+

++

+++

Layer VI

+

+

++

+++

++

Occipital Layer I

.

L a y e r II

-

. -

.

++

. +++

++ +

L a y e r III

-

-

++

++

++

Layer IV

-

-

-

+++++

++

Layer V

+

+

+

++

+++++

Layer VI

++

+

++

+++

++++

+++

++++

+++ +

++++ ++

+++++ +++

+++

O~erco~icalregions AI

+

CC

.

Cg DP

++ -

+ -

-

-

Ent

.

+++ .

.

.

++++

+++

Epl

-

-

Ipl

-

-

++

+++

+++

+++

GI

+

-

G1

-

++++

GrO

-

-

IL

+

-

++

++

LO

+

-

+++

++

+++

++

++

Mi MO

-

-

++

+++

+++

Pir

+

++

++

++++

++++

RSA

++

+

+++

++++

+++++

RSG

++

+

++

++++

+++++

TT

++

-

++

+++

++

VO

++

-

+++

+++

+++

113

Ch. III

C.R. Neal Jr. et al.

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

O F Q peptide Cells

OFQ mRNA

ORL1 m R N A

O F Q binding

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

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

+++

+++

+

+++

+++ ++ +++ ++

++ ++ ++ ++

++++ +++ + ++

++ ++ +++

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

+ +++ + +++ ++

F/T

Ventral forebrain ac

.

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

+ + -

.

++++ ++ -

.

++ + -

.

++ + ++++ .

+ +++ ++ .

.

+ ++ + ++ ++++ .

. +++ + + +

.

.

.

df Ld LSD LSI LSV

. +++ ++ +

.

++ + ++ +++

m

Septum .

.

m

MS

+

-

+

PLd SFi

-

-

-

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

SHi

++

++

++

++++

SHy

+++

+++

++++

++

+++

++

++

+++

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

++ + ++ + ++ + + +

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

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

++

-

++

++++

++

+ +++

++++ +++

+ ++++

++ +++ + +++ +

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

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

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

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

++++ ++

+++

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

++ ++ +

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

Basal telencephalon AMPO AVPO BSTi BST1 BSTld BSTlj BSTlp BSTlv

114

m

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

Ch. III

(continued) OFQ mRNA

ORL1 m R N A

O F Q binding

++++

+++++

++

+

++

+++++

++++

+++

++++

++

+++++

++++

-

+++

++++

++++

++

+

++

+

Layer I

++

+++++

L a y e r II

++++

++

L a y e r III

++

++

CNS region

O F Q peptide Cells

F/T

BSTma

+++

BSTmpl

++++

BSTmpm BSTmv BSTv f

.

LOT

.

++

. +

.

.

++

LPO

++

+++

+++

++/+++

++

MCPO

+++

+

+++

++

++

++++

++++

MnPO MPA

++

++++

++++

+++

++

MPO

+++

++++

++++

++++

-

++

-

MPOC Pe

+

+++

+

+++

-

st

+

++

-

-

-

AHA

++

+

+++

++

++

Arc

+++

+++

+++

++++

++

DA

+

++

++

+

-

DM

++

+++

+++

+++

+

LA

++

+

+

++

+

LH

+++

+

++

+++

++

LM

++++

++

++

++

-

ME

-

++++

-

-

ML

.

Hypothalamus

.

.

.

+++

MM

-

-

-

+++

+++

MMn

++

+++

+

-

-

Mp

.

MTu

++

-

++

++

+++

Pa

++

++

++

+++

++

PaAP PaDC PaLM

++

++

++

+++ ++ ++++

+ ++ ++

.

.

.

.

+

+

+

PaPo PaV

+

+

+

++ ++++

++ +++

Pe PeF

+ +

+++ -

+ +

+++ +

+ +++

PH Pin

++ .

+++

++

.

Pit

.

.

PMD

++

+++

+++

+

++

PMV

++

+++

++

++++

+

RCh

+++

+

+

+

++

SCh

-

-

-

+

++++

SO

-

++

-

++++

+

+++

-

+++

-

++

+++

+++

+++

+

+++

SOR SuM

-

-

++

s u m x

.

TC

++

+++ . .

.

.

++

.

.

.

.

.

++

.

115

Ch. III T A B L E 1.

C.R. Neal Jr. et al.

(continued)

CNS region

O F Q peptide Cells

Te

++

TM

++

TMC

+

VMH

+

OFQ mRNA

ORL1 m R N A

OFQ binding

F/T

+ +++

++

+++

++

+++

++

+

+

-/+

+++++

++++

VMHC VMHDM

+++++

++++

+++++

++++

VMHVL

++++

++++

+ +

+

+

++

+ ++ +++

Amygdala AAA

+

ACo

+

AHi APir ++ +

BAOT BL BLA

+ ++

BLP +

BM

BMA

+++ ++ ++

++++ +++

++

+++

++

+

+++ +++ ++

BMP BSTIA

+

CeL

+

CeM CxA

+++

++ +++ ++++

I

+

++

La MeAD

++

++ ++

+ +

+

++++ ++

++

MeAV

+++

++

+ + ++

+

+

++++ ++

MePD

++++

++++

MePV

+

+++

+++++ +++

++

+

+ ++ +++ ++

++++

+++

++

++ ++

+++

opt

PLCo PMCo

Hippocampalformation CAlso CAlsp CAlsr CA2so CA2sp CA2sr

+ +++ ++ + ++ -

++ + ++ -

+ +++ + ++ +

-/+ ++ ++ +

+ ++ +

CA3sl

+++

++

+++

+

++

CA3so

-

+

-/+

+++

++++

CA3sp

++

+

++

++++

-

CA3sr DGgr

++

+ +

+ +++

+ ++++

-

DGhi

-

-

-

+

-

DGmo DGpo

+ +

++ +

+ +

++

+++ -

IG PaS PrS

+++ +

+ -

+++ ++

+ +

+ +++

+

+ +++

+ +++

+++ ++ ++

S

116

++++

Ch. III

Neuroanatomical studies of the ORL1 receptor and OFQ TABLE

1.

CNS region

(continued) OFQ peptide

OFQ mRNA

ORL1

mRNA

OFQ binding

Cells

F/T

SFO

+

++

++

+++

++

SHi

++

++

++

++++

++

AD

-

++

-

+

+++

AM

-

-

-

+

Ar

.

AV

-

Thalamus

.

.

.

m

+

++++

AVDM

+

++

AVVL

+ +

+++

+

++

+++

BSTS -

-

CM

.

F

-

-

-

+

++

-

+++

-

+

++

.

.

+

++

CL

++

.

G IAD

m

++

IAM IMD LD

+ -

-

+

LDDM

++

+

+++

+

+++

+

+++

LGN

-

-

+

++

+++

LHb

-

++

++

++

++

LP

-

-

-

+

++

MD

-

-

+

-

MDDC

.

.

.

MDDL

.

.

.

.

MDDM

.

.

.

.

LDVL

.

MGN

-

-

-

+

+

MHb

++

+++

+

+++

++++

Mt

.

pc

-

.

. + +

. -

PF

-/+

+++

+++

+++

PLi

+

-

++

Po

+

-

++

-

++

PR

-

-

+

+++

++

PrC

++

+++

++

+++

PT

-

+++

+++

++

+++

PVA

+

+++

++

++++

++++

PVP

-

+

++

+++

+++

Re

-

+

+

++

+++

Rh

-

+

-

++

++

RI

-

-

+

+++

++

Rt

++++

++

+++++

++

+++

SG

+++

-

++

+

+

SPFPC

++

+++

+++

++

SubG

-

-

++ +++

+

VL

-

-

-

+

++

VM

+

+++

-

-

VPL

++

+

-

-

VPM

++

-

-

-

ZI

++

++

+++

++

SubI

+

+++

117

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C.R. Neal Jr. et al.

TABLE 1. (continued) CNS region

OFQ peptide Cells

OFQ mRNA

+++ ++ +++ +

OFQ binding

F/T

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

ORL1 mRNA

++ ++ + +

++ ++ +++

++

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

++

++

+

m

D

+ +

++++ +

+++ +

++++ ++

+++ ++++

+++

+ ++

++

+ ++++

§

+

+ +++++

+ ++

+ +++++

+++ ++

+ ++ +++ ++

+ ++ + +

++ ++ ++++ +++

CSC

ctg DCIC Dk dlf DLL DpMe

DR (B7) DTg ECIC

EW

++ ++ +++++

+§

++ ++++

+

++ + ++ ++ +++

++

fr

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

Pa4

118

++

+

+++

+ ++

+ +++ + + ++ ++

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

+++ ++ +++

+

+++

++ ++

++

++ +

+

+

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

++

++++

+ ++

++ +++ + +

++

+§

Neuroanatomical studies of the ORL1 receptor and OFQ TABLE CNS

1.

Ch. III

(continued)

region

OFQ

peptide

OFQ

Cells

F/T +

PBG

+++

PBG

-

pc

.

.

PCom

+++

-

++++

+++

ORL1

-

mRNA

OFQ

+++

+

+++

++

++

+++

-

.

PF

mRNA

.

PL

+

-

-

PLi

+

-

++

.

PMR

++

-

+

+++

+++

Pn

-

+

-

++++

++++

PN

+++

+

++++

++++

+

PnO

+

++

++

+

++

PP

+++

++

++++

++

+++

PPT

++

-

++

+

++

PPTg

++

+

+++

++

+

PR

-

-

-

+++

++

Rbd

-

-

-

+++

-

RLi

++

+

++

+++

+++

RMC

+

+

++

+++++

+

RPC

-

+

+

++++

+

RR

++

-

+

++

-

RRF

++

+

++

++

-

rs

.

RtTg

-

-

-

++

+

SC(DpG)

-

-

+

+

++

.

.

.

.

SC(DpWh)

-

-

-

+

-

SC(InG)

+++

+

+++

++

+ -

SC(InWh)

-

-

-

++

SC(Op)

++

+

++

++

-

SC(SuG)

+

+

+

-

++++

-

++++

+

SC(Zo)

-

Scp

.

+

SG

++

-

+++

+

SPTg

-

-

-

++

-

Su3

++

+

++

+++

++++ -

.

.

.

binding

.

VLL

-

-

-

+

VLTg

-

-

-

+++

-

VTA

+++

++

+++

+++

+/++

VTg

+

+

++

+

-

Lobules

+

-

+

-

-

IntA

-

-

-

+++

-

IntDL

-

-

-

++

-

IntDM

-

-

-

++

-

IntP

-

-

-

++

-

Lat

-

-

-

++++

++

LatPC

-

-

-

+++

-

Med

+++

++

++

+++

+

MedDL

++

++

++

++

+

++++

++

+++++

+++

Cerebellum

Metencephalon 6

-

6n

.

7

-

.

.

-

. -

.

119

Ch. III

C.R. Neal Jr. et al.

TABLE 1. (continued) CNS region

OFQ peptide Cells

7

OFQ mRNA

120

OFQ binding

u

n

m

m

m

m

++

++

+++ ++

++

++ ++ ++

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

+++

+

++ +++ ++++

++

n

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

ORL 1 mRNA

F/T

++ ++ + + ++

+ + +

+ ++ +++

++ ++ ++ + ++ ++

++ ++ ++ +

++ ++ +++

++ + ++ + +

+ ++ +

++ + ++ ++ ++

+ ++ ++ ++

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

++ ++ +++ ++

+ + +

+ +++ ++ +++ +

++++ + ++ +

+ ++ ++

+

++ + +++ ++

+

++ +++

m

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

+++

++ ++++ +++

++++ +

++ +

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

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

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

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

+ +++

+++ ++++

m

Neuroanatomical studies of the ORL1 receptor and OFQ TABLE

1.

Ch. III

(continued)

CNS region

OFQ peptide

OFQ mRNA

Cells

ORL1 mRNA

OFQ binding

++++

+++

+++++

+

F/T

Myelenc~halon 9n

.

10

-

.

.

10n

.

12

-

12n

.

A1

++++

++

A2

+++

++

A7

-

-

-

++

++++

Amb

+++

++

++++

+++++

+++++

-

+++

-

.

.

.

.

.

.

-

.

. -

-

.

.

AP C1

-

-

-

++++

++

C2

-

-

-

+++

++

C3

.

CI

++

-

+++

-

-

Cu

-

-

-

+++

+++

DMSp5

+++

+++

+++++

+++

+++

DPGi

+

-

+

+

+

ECu

-

-

+

+

+++

Gi

+

-

++

++

++

GiV

+++

-

+++

++++

+++

Gr

-

-

-

+

+

.

.

.

.

In IOA

+

-

-

-

++

+

+++++

IOB

-

-

+

+

+++

IOD

+++

++

++

+++

+++

IODM

-

-

-

++

+++

IOM

-

-

-

++

+++++

IOPr

+++

+

+++

+++

++++

IRt

++

-

+++

++

-

LRt

+

-

++

++++

++

+

LVe

++

+

+++

+++

-

MdD

++

-

++

++

++++

MdV

++

++

++

mlf

.

-

++

.

.

.

.

MnA

-

++++

M~V

-

-

-

+++

++

M~

++

+

++

+++

+++

Pa5

+

-

++

+++

+++

PrH py

++++ .

+++

++++

++++

++++

RAmb

+++

-

+++++

++++

+++

RMg

+++

++

++++

+++++

+++

ROb (B2)

+

+

++

+++

++

R P a (B 1)

++

+

++

+++

++++

RVL

++++

++

SGe

++

-

++++

sol

.

Sol

++

++

++++

+++

++++

SolC

+++

++

++

++++

++++

SolL

+++

+++

+++

+++

++

SolM

++

+++

+++

+++

++

sp5

.

.

.

+++

.

+++

.

.

.

.

.

.

.

.

++++

121

Ch. III T A B L E 1.

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

CNS region

OFQ peptide

OFQ m R N A

ORL1 m R N A

OFQ binding

§247247247247 § §

§247247 §247247247 §247247

§247247247 §247247 §

Cells

F/T

§247247 §247

§247247247 §247247 §

I

-

§ 2 4 7

-

-

-

II

+§ ++ + § ++ §

+++§ +++ +++ + § + § +++

§247247 §247247 + § ++ + +§ §247247

+ ++ § +++ §247 ++ +++§ §247247 §247

++++ +++ ++ + +§247 ++ § §247 §247247

Sp5C Sp5I SpVe

Spinal cord Cervical

III IV V VI VII VIII IX X Thoracic I

-

+

-

-

-

II

§247 -

++§ +§ § §247 ++ §247 +++ +

§247 +++ + + ++ ++

+ + +++ § +++ §247247 §247247247 +++

§ § ++++ § +§ + ++++

++§247

-

+++§ §247247 +

§ §247247 -

§

-

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

§ ++ ++ .

.

.

.

.

.

.

+ . -

I M M

§

LatC lfu LSp vfu

+++ . ++ .

.

. -

+++ .

. +++

.

.

§247247 + . .

. .

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

5.3.1.2. OFQ immunohistochemistry

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

Neuroanatomical studies of the ORL1 receptor and OFQ

Ch. III

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

123

Ch. III

C.R. Neal Jr. et al.

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

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

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

Neuroanatomical studies of the ORL1 receptor and OFQ

Ch. III

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

Ch. III

C.R. Neal Jr. et al.

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

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

Neuroanatomical studies of the ORL1 receptor and OFQ

Ch. III

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

Ch. III

128

C.R. Neal Jr. et al.

Neuroanatomical studies of the ORL1 receptor and OFQ

Ch. III

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

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

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

.<._...._

Fig. 8. Darkfield autoradiograms comparing 125I-[14Tyr]OFQ binding (A,C,E) and orphanin receptor mRNA expression (B,D,F) in the pons and medulla. Scale bar: 1000 Ixm. 129

Ch. III

C.R. Neal Jr. et al.

lightly stained neurons, with diffuse fibers, intensifying caudally in its dorsal part. The accumbens shell contains scattered fibers and puncta, with occasional darkly stained cells. Immunoreactive fibers extend to the vertical limb of the diagonal band of Broca, and caudally as a continuum with the ventral division of the bed nucleus of the stria terminalis. In the ventral pallidum, lightly immunolabeled neurons are scattered within a prominent fiber plexus. A similar network of dense fibers and puncta are observed in the fundus striati. Neurons in layers II and III of the olfactory tubercle are moderately immunolabeled, with no labeling observed in the islands of Calleja. The vertical limb of the diagonal band of Broca contains occasional lightly labeled neurons. In the horizontal limb, OFQ-containing neurons are more numerous and darkly stained. Numerous large, darkly stained OFQ-containing neurons and fibers are observed in the substantia innominata, with scattered fibers extending dorsally into the basal nucleus of Meynert, where large, moderately stained neurons are also observed. 5.3.2.3. ORL1 in situ hybridization Orphanin receptor mRNA expression is generally low in the ventral forebrain. Rostrally, ORL1 mRNA expression is sparse to light in the anterior olfactory nucleus. There are no mRNA-containing neurons observed in the rostral pole of nucleus accumbens, with only sparse mRNA expression observed in the accumbens shell and core. The olfactory tubercle contains low mRNA expression scattered throughout layer III. At the level of the diagonal band, mRNA-expressing cells are lightly scattered in the ventral pallidum and ventral diagonal band, more moderate in the horizontal limb. The interstitial nucleus of the posterior limb of the anterior commissure and the substantia innominata have sparse expression and the islands of Calleja are devoid of mRNA expression.

5.3.3. Septum 5.3.3.1. PreproOFQ in situ hybridization Expression of OFQ mRNA in this region is strong. Light OFQ expression is observed in the rostral pole of the lateral septum. Messenger RNA expression is intense in the dorsal lateral septum. The intermediate lateral septum is also heavily labeled, but not as intensely as the dorsal part. Moderate mRNA expression is observed in the ventral lateral septum and septohippocampal nucleus. Light OFQ mRNA expression is observed in septohypothalamic nucleus and the medial septal nucleus. 5.3.3.2. OFQ immunohistochemistry In the lateral septum, a plexus of scattered fibers and terminals, and numerous darkly stained cells fills the dorsolateral portion. Immunolabeling is moderate in the intermediate lateral septum, slightly stronger in the ventral part. Fiber and terminal staining in the intermediate division is moderate to intense. The ventral lateral septum contains scattered, light neurons within a moderate fiber and terminal plexus. Lightly labeled neurons are scattered in the medial septum and fiber labeling is negligible. The septohippocampal region contains a moderate number of densely stained neurons and fibers, and the septohypothalamic nucleus contains moderately stained fibers, and several dark cells. No immunolabeling is noted in the lambdoid septal zone or paralambdoid septal nucleus.

130

Neuroanatomical studies of the ORL1 receptor and OFQ

Ch. III

5.3.3.3. ORL1 in situ hybridization Orphanin receptor mRNA expression in this region is moderate to dense. Dense expression is observed in the ventral lateral septum and the septohippocampal nucleus. Moderate expression is observed in the intermediate and dorsal lateral septal regions, and the medial septum. Expression is sparse in the lambdoid septal zone and paralambdoid septal nucleus. The septofimbrial nucleus and dorsal fornix are devoid of mRNA expression.

5.3.4. Basal ganglia 5.3.4.1. PreproOFQ in situ hybridization Dense mRNA expression is observed throughout the claustrum. Large neurons with dense expression are also observed throughout the globus pallidus and in the entopeduncular nucleus, with dense expression in pars reticulata, pars compacta and pars lateralis of the substantia nigra. Moderate mRNA expression is observed in the dorsal and ventral endopiriform nuclei and the ventromedial basal nucleus of Meynert. Expression is light in the subthalamic nucleus and very sparse in the striatum, observed primarily in the mediodorsal part.

5.3.4.2. OFQ immunohistochemistry Moderately labeled neurons and occasional scattered fibers persist throughout the claustrum. The dorsal endopiriform nucleus contains scattered, moderately labeled neurons and fibers, and the ventral endopiriform nucleus contains only scattered, lightly labeled neurons. The striatum contains sparse, darkly stained fibers throughout its rostral to caudal extent, most prominent ventromedially. Occasional lightly immunolabeled neurons are observed, and are very few in number. Large and intensely immunolabeled neurons are seen throughout the globus pallidus and the basal nucleus of Meynert. The entopeduncular nucleus contains scattered, darkly immunolabeled neurons, with sparse fibers and terminals. Sparse, lightly labeled fibers and neurons are observed in the subthalamic nucleus. Pars reticulata of substantia nigra has numerous, moderately labeled perikarya and fibers, with fewer immunolabeled perikarya observed in pars compacta. Large, darkly stained neurons are also observed in pars lateralis.

5.3.4.3. ORL1 in situ hybridization Orphanin receptor mRNA expression is strong in the ventral and dorsal endopiriform nuclei, and large neurons with abundant mRNA signal intensity are scattered throughout the globus pallidus. Dense expression is also observed in the substantia nigra pars compacta, where numerous cells with high mRNA expression are noted. Expression is moderate in the basal nucleus of Meynert, subthalamic nucleus and substantia nigra pars reticulata. Low mRNA expression is observed in the claustrum, entopeduncular nucleus and substantia nigra pars lateralis.

5.3.5. Basal telencephalon 5.3.5.1. PreproOFQ in situ hybridization Preproorphanin mRNA expression in the rat forebrain is highest in the basal telencephalon. Heavy mRNA levels are seen throughout the medial and lateral parts of the bed nucleus of 131

Ch. III

C.R. Neal Jr. et al.

the stria terminalis, strongest in the posterolateral part of the medial division. Heavy mRNA expression is also seen in the anteroventral preoptic nucleus, the medial preoptic area, the medial preoptic nucleus and the lateral preoptic area. Moderate mRNA expression is observed in the magnocellular preoptic nucleus and the nucleus of the lateral olfactory tract. Message expression is light in the juxtacapsular part of the bed nucleus and the anteromedial preoptic nucleus. No preproOFQ mRNA expression is observed in the fornix, the stria terminalis or the lateral olfactory tract. 5.3.5.2. OFQ immunohistochemistry

Paralleling mRNA expression, immunolabeling in this region is intense. Dense immunolabeled fibers, terminals and neurons fill the anterior and ventral parts of the medial division of the bed nucleus of the stria terminalis rostrally, and the posteromedial and posterolateral parts of the medial division caudally. In the posterolateral medial division, numerous darkly stained neurons extend medially to the lateral boundary of the paraventricular nucleus of the hypothalamus and lateral hypothalamic area. Fiber and terminal labeling in the lateral subdivision is intense rostrally. The juxtacapsular lateral subdivision contains occasional immunolabeled cell bodies, with light to moderate fiber and terminal staining. Fiber staining is prominent in the stria terminalis, with occasional immunostained neurons observed. The fornix is devoid of immunolabeling. The medial preoptic area contains scattered darkly stained neurons and moderate to heavy fibers throughout. Fewer immunolabeled neurons are seen in the medial preoptic nucleus and magnocellular preoptic nucleus. Scattered, lightly immunostained cells are observed in the anteromedial preoptic nucleus and darkly stained neurons in the anteroventral preoptic nucleus. The lateral preoptic area contains scattered, darkly stained neurons, with moderate fiber and terminal labeling. Immunostaining in the nucleus of the lateral olfactory tract is moderate. 5.3.5.3. ORL1 in situ hybridization

Orphanin receptor mRNA expression is strong in the posteromedial and posterolateral parts of the medial division of the bed nucleus of the stria terminalis, and the posterior part of the lateral division. High mRNA expression is also observed in the median preoptic nucleus, the anteromedial preoptic nucleus, the medial preoptic nucleus and layer II of the nucleus of the lateral olfactory tract. Moderate mRNA expression is observed in the dorsal part of the lateral bed nucleus, the periventricular hypothalamus, the anteroventral preoptic nucleus and the medial preoptic area. Lighter mRNA expression is observed in the anterior medial division of the bed nucleus, the lateral preoptic area and the magnocellular preoptic nucleus. Sparse expression is seen in the rostral pole and lateral and ventral divisions of the bed nucleus, and in layers I and III of the nucleus of the lateral olfactory tract. No expression is observed in the juxtacapsular part of the lateral bed nucleus.

5.3.6. Hypothalamus 5.3.6.1. PreproOFQ in situ hybridization

The majority of OFQ expression in the hypothalamic region lies primarily in the medial zone. Strong OFQ expression is only observed in the medial tuberal nucleus, the dorsomedial nucleus and the dorsal premammillary region. Expression is moderate in the arcuate nucleus, 132

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the anterior and lateral hypothalamic areas, the parvocellular part of the paraventricular nucleus, the tuberal magnocellular nucleus, the posterior hypothalamus, ventral premammillary nucleus, and the supramammillary and lateral mammillary nuclei. Low expression is observed in the tuber cinereum, retrochiasmatic area, magnocellular part of the paraventricular nucleus, dorsal hypothalamic area, perifornical nucleus and the median division of the medial mammillary nucleus. The median eminence, suprachiasmatic and supraoptic nuclei, the ventromedial nucleus, the lateral and medial divisions of the medial mammillary nucleus, the mammillary peduncle and the supramammillary decussation are devoid of mRNA expression. The pituitary and pineal glands are also devoid of OFQ mRNA expression. 5.3.6.2. OFQ immunohistochemistry Large, darkly stained neurons surround the fornix, lateral to the paraventricular nucleus. The parvocellular part of the paraventricular nucleus contains a modest number of lightly stained neurons and fibers, mostly in its medial aspect. Scattered, darkly stained fibers with occasional immunolabeled neurons are present within the magnocellular part. The lateroanterior hypothalamic nucleus, lateral hypothalamic area, anterior hypothalamic area and tuber cinereum contain a moderate number of labeled neurons. The supraoptic and retrochiasmatic supraoptic nuclei contain darkly immunolabeled fibers, but no labeled neurons. The retrochiasmatic area contains numerous darkly labeled neurons and fibers. No immunolabeled neurons or fibers are seen in the suprachiasmatic nucleus. The arcuate nucleus contains moderately stained cells and fibers, with numerous puncta throughout. The median eminence contains an intense immunolabeled fiber plexus filling the medial aspect of the median eminence in its rostral part and the lateral aspect caudally. The ventromedial hypothalamic nucleus contains moderate fiber and terminal labeling, with few labeled cells. Light immunolabeling is observed in the medial tuberal and tuberal magnocellular nuclei. Moderate labeling is noted in the dorsal hypothalamic area, dorsomedial hypothalamic nucleus, perifornical nucleus, posterior hypothalamus, terete hypothalamic nucleus, and tuberomammillary nucleus. Moderate to heavy fiber and terminal labeling is present in the ventral and dorsal premammillary nuclei, with moderate cell and fiber labeling in the lateral mammillary nucleus and supramammillary nucleus. The medial mammillary nucleus contains light to moderate cell and fiber labeling in its median division. The medial and lateral divisions are devoid of staining. 5.3.6.3. ORL1 in situ hybridization Strong mRNA expression is observed in the supraoptic nucleus and arcuate nucleus, and the ventromedial nucleus contains the densest mRNA expression in the hypothalamus. Strong mRNA expression is also observed in the ventral and lateral magnocellular parts of the paraventricular nucleus, and the ventral premammillary nucleus. Expression is moderate in the medial tuberal nucleus, the anterior parvicellular part of the paraventricular nucleus, the dorsomedial hypothalamic nucleus, the posterior hypothalamus, the medial part of the medial mammillary nucleus and rostral supramammillary nucleus. Lightly scattered mRNA expression is seen in the suprachiasmatic nucleus, retrochiasmatic region, tuber cinereum, medial tuberal nucleus, periventricular nucleus, the anterior and lateral hypothalamic areas, the posterior part and dorsal cap of the paraventricular nucleus, the dorsal hypothalamic area, perifornical nucleus and terete hypothalamic nucleus. Expression is sparse in the dorsal premammillary and tuberal magnocellular nuclei. No expression is observed in lateral part 133

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of the medial mammillary nucleus. The median eminence, anterior pituitary, intermediate pituitary and pineal gland are also devoid of mRNA expression.

5.3.7. Amygdala 5.3.7.1. PreproOFQ in situ hybridization Orphanin mRNA expression in the amygdaloid complex is relatively sparse in most nuclei, except for intense expression in the central and medial amygdaloid nuclei. Heavy mRNA expression is observed throughout the caudal medial division of the central nucleus and the posterodorsal division of the medial nucleus. Moderate mRNA expression is observed in the rostral medial division of the central nucleus, the intraamygdaloid division of the bed nucleus of the stria terminalis, the anterodorsal part of the medial nucleus, the posteroventral division of the medial nucleus, and the posteromedial cortical nucleus. Expression is light in the anterior amygdaloid area and the lateral division of the central nucleus, the basolateral and basomedial nuclei, the anteroventral part of the medial nucleus, the amygdalohippocampal area, the anterior cortical amygdaloid nucleus and posterior cortical nucleus. Negligible expression is observed in the intercalated nuclei, the lateral amygdaloid nucleus, the posterolateral cortical amygdaloid nucleus and amygdalopiriform transition area. 5.3. 7.2. OFQ immunohistochemistry In the rostral amygdaloid region, sparse immunolabeled fiber and puncta are observed in the anterior amygdaloid area and intercalated nuclei. Further caudal, scattered, moderately immunolabeled cells and fibers are observed in the intercalated nuclei, the bed nucleus of the lateral olfactory tract and the intraamygdaloid division of the bed nucleus of the stria terminalis. Immunolabeling in the basomedial and basolateral nuclei, posteromedial cortical nucleus and anteromedial cortical nucleus is sparse. The amygdalohippocampal and amygdalopiriform transition areas are devoid of immunolabeling. The central nucleus contains moderate to heavy fiber staining in its rostral pole and a dense plexus of fibers, terminals and neurons at mid to caudal levels. Strong cell and fiber immunolabeling predominates in the medial division, and fiber labeling predominates in the lateral division. Lightly labeled fibers are observed in the anteroventral division of the medial nucleus, with moderate to heavy fiber staining in the anterodorsal division. The posterodorsal division is filled with darkly stained fibers, terminals and neurons. The posteroventral division contains lightly labeled cells centrally, surrounded by a dense plexus of fibers and terminals. 5.3.7.3. ORL1 in situ hybridization Orphanin receptor mRNA expression in the amygdala is sparse in the anterior amygdaloid area and anterior cortical amygdala, anterior cortical nucleus, ventral and dorsal divisions of the lateral nucleus, intercalated nuclei, posteromedial and posterolateral cortical amygdala nuclei and the amygdalohippocampal area. Expression is moderate in the cortex-amygdala transition zone, bed nucleus of the accessory olfactory tract, basolateral nucleus, basomedial nucleus, posterolateral cortical nucleus and the amygdalopiriform transition area. The anterodorsal division of the medial nucleus contains low mRNA expression, with dense expression in the anteroventral division. In the most caudal part of the medial nucleus, the posterodorsal division contains low to moderate expression, whereas the posteroventral division is filled with 134

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numerous densely labeled mRNA-expressing cells. The central nucleus contains an occasional mRNA-containing neuron in its lateral part, but is essentially devoid of mRNA expression. The intraamygdaloid bed nucleus of the stria terminalis has no mRNA expression.

5.3.8. Hippocampal formation and related structures

5.3.8.1. PreproOFQ in situ hybridization Intense OFQ mRNA expression is observed in the indusium griseum, the subfornical organ and in the CA1 region of Ammon's horn, primarily within stratum pyramidale. In area CA3, mRNA expression is dense in stratum lucidum. Expression is moderate in the septohippocampal nucleus, the granule cell layer of the dentate gyrus, stratum pyramidale of area CA3 and the subiculum. Light orphanin mRNA expression is seen in the molecular and polymorph layers of the dentate gyrus, and in stratum radiatum and stratum oriens of area CA1. Sparse OFQ mRNA expression is observed in the pyramidal layer of area CA2, stratum radiatum of area CA3, the presubiculum and parasubiculum. The hilus of the dentate gyrus and fornix are devoid of preproOFQ mRNA expression.

5.3.8.2. OFQ immunohistochemistry The indusium griseum, septohippocampal nucleus, and tenia tecta contain densely immunolabeled fibers and cells. The subfornical organ contains moderate fiber labeling and scattered lightly labeled cells. In the dentate gyms, moderate-sized, darkly stained cells are scattered along the granule cell layer, localized primarily along the inner border of the polymorph layer with fibers extending perpendicularly into the molecular layer. In area CA3 of Ammon's horn, darkly immunolabeled cells are observed in stratum pyramidale. Moderately labeled fibers extend into stratum lucidum and oriens, and neuronal immunolabeling in stratum radiatum is very sparse. Near the transition to area CA2, darkly labeled neurons are observed in stratum lucidum. Minimal immunolabeling is observed in area CA2 throughout its extent. In area CA1, numerous, darkly labeled neurons are observed in stratum pyramidale, with moderate fibers radiating into both stratum oriens and stratum radiatum. In the dorsal subiculum, intensity of neuronal immunolabeling is strong. The ventral subiculum, presubiculum and parasubiculum contain scattered lightly labeled cells with no immunolabeled fibers.

5.3.8.3. ORL1 in situ hybridization Heavy ORL1 mRNA expression is observed in the septohippocampal nucleus, stratum pyramidale of area CA2, and stratum pyramidale and oriens of area CA3. Moderate to dense mRNA expression is observed in the subfornical organ, polymorph and granule cell layers of the dentate gyrus, stratum pyramidale of area CA2, and the dorsal and ventral subiculum. Messenger RNA expression is sparse the indusium griseum, hilus of the dentate gyrus, all of area CA1, stratum oriens and radiatum of area CA2, stratum lucidum, oriens and radiatum of area CA3, the presubiculum and the parasubiculum. The molecular layer of the dentate gyrus is devoid of mRNA expression.

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5.3.9. Thalamus

5.3.9.1. PreproOFQ in situ hybridization Rostrally, strong OFQ mRNA expression fills the paratenial nucleus and extends into the anterior paraventricular and reuniens nuclei where more moderate mRNA levels are observed. Strong mRNA expression is observed in the reticular nucleus, which contains the heaviest mRNA expression in the entire thalamic region. Dense expression is also present in the zona incerta. Moderate mRNA expression is observed in the anteroventral thalamic nucleus, the posterior division of the paraventricular nucleus and the lateral habenula. Messenger RNA levels are very light in the laterodorsal and mediodorsal thalamic nuclei, the medial habenula, the subincertal nucleus and the subgeniculate nucleus. No OFQ mRNA expression is observed in the fields of Forel, nor in the anterodorsal, anteromedial, interanterodorsal, paracentral, rhomboid, centromedial, centrolateral, lateroposterior, ventrolateral, ventromedial, ventral posterolateral and ventral posteromedial thalamic nuclei. In the midbrain thalamus, scattered mRNA expression in the rostral interstitial nucleus of the medial longitudinal fasciculus extends to the subparafascicular nucleus where mRNA levels are moderate. Scattered mRNA-expressing neurons are observed in the prerubral fields. There is no mRNA expression detected in the medial geniculate nucleus, and only occasional mRNA-expressing neurons in the lateral geniculate. The suprageniculate thalamic nucleus and the precommissural nucleus contain a moderate number of mRNA-expressing cells, and the posterior thalamic group contains scattered mRNA-containing neurons. 5.3.9.2. OFQ immunohistochemistry The anterior paraventricular nucleus contains a moderate accumulation of terminals, with moderate fibers and lightly labeled neurons. The paratenial, anterodorsal and interanterodorsal thalamic nuclei are filled with fine terminals and fibers, and no immunolabeled neurons. Lightly labeled fibers are scattered in the anteroventral nucleus, reuniens and rhomboid nuclei. The reticular nucleus contains numerous intensely stained neurons with lightly labeled, sparse fibers, extending caudally into the zona incerta. The subparafascicular thalamic nucleus contains prominent cell, fiber and terminal labeling. A dense immunoreactive fiber bundle and scattered darkly stained neurons fill the medial habenula. Lateral habenula and posterior paraventricular nuclei contain scattered, lightly labeled terminals and neurons. The precommissural nucleus contains moderate immunolabeled fibers and cells. There is light neuronal immunostaining in the posterior thalamic nuclear group, the lateral posterior, posterior limitans, suprageniculate, subgeniculate, and the lateral and medial geniculate nuclei. No immunolabeling is observed in the prerubral field or nuclear fields of Forel, the anteromedial, central medial, laterodorsal, mediodorsal or centrolateral nuclei, or the ventral nuclear group. 5.3.9.3. ORL1 in situ hybridization In the rostral thalamus, intense mRNA expression is observed in the anterior paraventricular thalamic nucleus. Moderate to high expression is observed in the subincertal nucleus. Message expression is moderate in the paratenial nucleus, medial habenula, reunions and rhomboid nuclei and the posterior paraventricular nucleus. Low to moderate expression is observed in the reticular nucleus, ventrolateral part of the anteroventral nucleus and zona 136

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incerta. The rhomboid, interanterodorsal and anterodorsal nuclei, and the interanteromedial, ventral posteromedial and ventral posterolateral nuclei contain low mRNA expression. Sparse expression is observed in the lateral habenula, ventrolateral and dorsomedial divisions of the anteroventral nucleus, the anterodorsal, centromedial, paracentral, ventromedial, ventrolateral and laterodorsal nuclei and the reticular nucleus. The rostral, anterodorsal and dorsomedial parts of the anteroventral nucleus, the reunions nucleus and the anteromedial and paracentral thalamic nuclei are devoid of mRNA expression. No mRNA expression is observed in the mediodorsal thalamic group, and the paracentral, centromedial, gelatinosus and posterior thalamic nuclei. In the mesencephalic thalamus, dense mRNA expression persists in the posterior paraventricular nucleus. Moderate to high expression is observed in the precommissural and perifornical nuclei. The rostral interstitial nucleus of the medial longitudinal fasciculus, the prerubral field, the lateral geniculate nucleus and the parvicellular part of the subparafascicular thalamic nucleus contain moderate preproOFQ mRNA expression. Sparse expression is observed in the lateroposterior thalamic nucleus, fields of Forel, medial geniculate nucleus and dorsal geniculate region. The posterior thalamic group and ethmoid nucleus, posterior limitans and suprageniculate thalamic nucleus also contain sparse mRNA expression. 5.4. DISTRIBUTION OF OFQ AND THE ORL1 RECEPTOR IN THE RAT BRAINSTEM AND SPINAL CORD

5.4.1. Mesencephalon 5.4.1.1. PreproOFQ in situ hybridization

OFQ mRNA expression is strong in the lateral and dorsal divisions of the interpeduncular nucleus, with intense expression in the ventral tegmental area, the paranigral nucleus and the nucleus of the posterior commissure. The nucleus of Darkschewitsch is completely filled with the most intense mRNA expression in the midbrain. Intense OFQ mRNA expression extends ventrolaterally to the interstitial nucleus of the medial longitudinal fasciculus. Expression is also strong in the ventral part of the central gray, the external cortex of the inferior colliculus, the peripeduncular nucleus and the nucleus of the trapezoid body. Moderate mRNA expression is observed in the rostrolateral division of the interpeduncular nucleus, the ventral anterior pretectal region, nucleus of the optic tract, posterior pretectal nucleus, and the optic and intermediate gray layers of the superior colliculus. Moderate expression is also observed in the retrorubral field, the dorsal and supraoculomotor divisions of the central gray, the oral and caudal pontine reticular nuclei, the pedunculopontine, dorsal, laterodorsal and ventral tegmental nuclei, the dorsal raphe and the rostral and caudal linear raphe nuclei. PreproOFQ mRNA expression is low in dorsal cortex of the inferior colliculus, the subbrachial nucleus, the median and paramedian raphe nuclei, the deep mesencephalic nucleus, and the magnocellular and parvocellular parts of the red nucleus. Sparse mRNA expression is observed in the superficial gray layer of the superior colliculus and no mRNA expression is found in the olivary pretectal nucleus, posterior commissure, central collicular nucleus, the intercollicular nucleus, prerubral field, parabrachial pigmented nucleus, mammillary peduncle, fasciculus retroflexus, rubrospinal tract, or the medial or lateral lemniscus. Negligible mRNA expression is observed in the brachium of the inferior colliculus, cuneiform nucleus, the nuclei of the lateral lemniscus, the mesencephalic trigeminal nucleus, paralemniscal nucleus and retrorubral nucleus. The Edinger-Westphal and medial accessory oculomotor nuclei, 137

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the paratrochlear nucleus, rhabdoid nucleus, microcellular, subpeduncular and ventrolateral tegmental nuclei, reticulotegmental nucleus, central trigeminal tract, dorsal longitudinal fasciculus, superior cerebellar peduncle and sensory root of the trigeminal nerve are devoid of mRNA expression. 5.4.1.2. OFQ immunohistochemistry

In the interpeduncular nucleus strongly immunolabeled neurons fill the rostral and dorsal divisions, with light immunolabeling in the caudal and lateral divisions. In the pedunculopontine tegmental nucleus darkly stained neurons are present. The ventral tegmental area and paranigral nucleus contain numerous darkly stained neurons. Numerous intensely stained fibers and neurons fill the peripeduncular nucleus. Moderate fibers are observed in the medial lemniscus and the deep mesencephalic nuclei. Moderately immunostained neurons are observed the nucleus of the posterior commissure, the posterior pretectal nucleus, olivary pretectal nucleus, nucleus of the optic tract, dorsal and ventral divisions of the anterior pretectal nucleus, the optic nerve and intermediate gray layers of the superior colliculus, and layers II and III of the external cortex of the inferior colliculus. Immunolabeling is light in the red nucleus, retrorubral field and the magnocellular nucleus of the posterior commissure. There are lightly labeled neurons in the superficial gray layer of the superior colliculus and lightly labeled fibers and cells are observed in the dorsal cortex, and the central nucleus of the inferior colliculus. No immunolabeling is observed in the prerubral field, the zonal, intermediate white, deep white or deep gray layers of the superior colliculus, the cerebral and mammillary peduncles, posterior commissure, commissure of the superior colliculus, central tegmental tract, intercollicular nucleus, brachium of the inferior colliculus or commissure of the inferior colliculus. At the midline, numerous, intensely stained neurons, fibers and terminals are observed in all divisions of the central gray. Numerous intensely immunolabeled cells, fibers and terminals fill the nucleus of Darkschewitsch and the interstitial nucleus of the medial longitudinal fasciculus. Numerous darkly labeled neurons and fibers are observed in the laterodorsal and dorsal tegmental nuclei, and the paramedian, rostral linear and caudal linear raphe. Immunolabeling is moderate in the dorsal and median raphe and the oral pontine reticular nucleus, paralemniscal nucleus, trapezoid body and rostral preolivary region contain scattered, moderately stained neurons. Lightly stained cells are scattered in the deep mesencephalic nuclei and the anterior and ventral tegmental nuclei. No immunolabeling is observed in the Edinger-Westphal nucleus, medial accessory oculomotor nucleus, supraoculomotor central gray, reticulotegmental, subpeduncular and ventrolateral tegmental nuclei, the ventral, dorsal or lateral lemniscal nuclei, cuneiform nucleus, mesencephalic trigeminal nucleus, paratrochlear nucleus, parabigeminal nucleus, longitudinal fasciculus, superior cerebellar peduncle, lateral lemniscus, rubrospinal tract and sensory root of the trigeminal nerve. 5.4.1.3. ORL1 in situ hybridization

In the mesencephalon, the dorsal division of the central gray contains high ORL1 mRNA expression and magnocellular division of the red nucleus contains the highest mRNA expression in the mesencephalon. High mRNA expression is also observed in the ventral tegmental area, paranigral nucleus and medial terminal nucleus of the accessory olfactory tract. Other areas of intense expression include the supraoculomotor central gray, EdingerWestphal nucleus, trochlear nucleus, dorsal raphe and caudal linear raphe. Moderate to high 138

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mRNA expression is observed in the oculomotor and medial accessory oculomotor nuclei, median raphe, rostral linear nucleus of the raphe, the parabrachial pigmented nucleus, caudal interpeduncular nucleus, magnocellular nucleus of the posterior commissure, paralemniscal nucleus, rhabdoid nucleus and laterodorsal tegmental nucleus. Moderate mRNA expression is observed in the dorsal cortex of the inferior colliculus, the anterior pretectal nucleus, the medial pretectal nucleus, nucleus of the posterior commissure, deep mesencephalic nucleus, reticulotegmental nucleus and the anterior, subpeduncular and ventrolateral tegmental nuclei. Light mRNA expression is observed in the intermediate gray, intermediate white and optic nerve layers of the superior colliculus, the pedunculopontine tegmental nucleus, the parvicellular division of the red nucleus, the nucleus of Darkschewitsch and interstitial nucleus of the medial longitudinal fasciculus. Low ORL1 mRNA expression is also observed in the paramedian raphe, rostral interpeduncular nucleus, retrorubral field, retrorubral nucleus and interfascicular nucleus. Sparse expression is observed in the deep gray and deep white layers of the superior colliculus, the external cortex of the inferior colliculus and the central and intercollicular nuclei of the inferior colliculus. Sparse mRNA expression is also observed in the nucleus of the optic tract, the posterior pretectal nucleus, olivary pretectal nucleus, oral part of the pontine reticular nucleus, lateral and medial geniculate nuclei, the peripeduncular nucleus, the dorsal, ventral and intermediate nuclei of the lateral lemniscus and the microcellular and ventral tegmental nuclei. The zonal and superficial granular layers of the superior colliculus, the dorsal tegmental nucleus, the cuneiform nucleus and the paratrochlear nucleus are devoid of mRNA expression. 5.4.2. Cerebellum

5.4.2.1. PreproOFQ in situ hybridization

Preproorphanin mRNA expression in the cerebellar hemispheres is confined to scattered neurons in the granular layer. No mRNA expression is detected within the molecular or Purkinje cell layers. A moderate number of neurons expressing OFQ mRNA are observed in the medial (fastigial) cerebellar nucleus and its dorsolateral protuberance. No mRNA expression was detected in the lateral (dentate) or the interposed cerebellar nuclei. 5.4.2.2. OFQ immunohistochemistry

No specific orphanin immunoreactivity is observed in the cerebellar hemispheres. A plexus of strongly immunolabeled cells and fibers is observed in the fastigial (medial) nucleus, with less labeling in its dorsolateral protuberance. No immunolabeling is observed in the dentate (lateral) or interposed cerebellar nuclei. 5.4.2.3. ORL1 in situ hybridization

Specific ORL1 mRNA expression is confined to the deep cerebellar nuclei. All mRNA expression detected in the cerebellar lobules is non-specific. The dentate nucleus is filled with numerous, large mRNA-expressing cells and the parvicellular part contains moderate mRNA expression. The anterior interposed nucleus contains moderate to high mRNA expression, with expression in the posterior, dorsolateral and dorsomedial divisions low to moderate. The fastigial cuneiform nucleus and paratrochlear nucleus and its dorsolateral protuberance contain high mRNA expression. 139

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5.4.3. Metencephalon 5.4.3.1. PreproOFQ in situ hybridization

In the rostral pons, strong OFQ mRNA expression is observed in the dorsomedial tegmental area, the laterodorsal and posterodorsal tegmental nuclei and the nucleus of the trapezoid body. Scattered neurons with intense mRNA levels reside in the region of the A5 cell group. Moderate mRNA expression is observed in the pontine central gray and dorsal raphe, locus coeruleus, lateral parabrachial nucleus and medioventral periolivary nucleus. Light mRNA expression is observed in the superior paraolivary nucleus, the lateroventral, dorsal and superior periolivary nuclei and lateral superior olive. Negligible expression is observed in the medial parabrachial nucleus, subcoeruleus nucleus, principal sensory trigeminal nucleus, Kolliker-Fuse nucleus, trigeminal motor nucleus, anterior ventral cochlear nucleus, caudal periolivary nucleus, medial superior olive, the A7, C1, C2 or C3 cell groups, medial longitudinal fasciculus, mesencephalic trigeminal nucleus, pyramidal tract and root of the abducens nerve. In the caudal pons, moderate to strong OFQ mRNA expression is observed in the lateral vestibular nucleus, with intense mRNA expression observed in the raphe magnus and prepositus hypoglossus. Moderate mRNA expression is observed in the paratrigeminal nucleus, the superior, medial and medioventral vestibular nuclei, supragenual nucleus, oral pontine reticular nucleus, gigantocellular nucleus, raphe pallidus and the ventral pontine reticular nucleus. Light mRNA expression is observed in the spinal vestibular nucleus, dorsal cochlear nucleus, the parvocellular, intermediate and dorsal paragigantocellular reticular nuclei, caudal pontine reticular nucleus, and the oral and interpolar parts of the spinal trigeminal nucleus. Negligible expression is observed in the ventral posterior cochlear nucleus, abducens and facial nuclei, principal sensory trigeminal nucleus, 6th, 7th or 8th nerves and the inferior cerebellar peduncle. 5.4.3.2. OFQ immunohistochemistry

In the pons, the nucleus of the trapezoid body is filled with darkly immunolabeled neurons and fibers, and strong immunolabeling is also observed in the pontine raphe nucleus, the raphe magnus, and the gigantocellular, lateral paragigantocellular and lateral reticular nuclei. The locus coeruleus, lateral parabrachial nucleus, dorsomedial tegmental nucleus, lateral rostral periolivary region, superior periolivary nucleus, lateroventral periolivary nucleus, medioventral periolivary nucleus, lateral superior olive, lateral, superior and spinal vestibular nuclei, and the dorsal gigantocellular, caudal pontine and lateral reticular nuclei contain moderate neuronal and fiber immunolabeling. Lightly labeled neurons are present in the pontine central gray, the laterodorsal and posterodorsal tegmental nuclei, lateral and dorsal superior olive, region of the A5 noradrenaline cell group, the medial, lateral and superior vestibular nuclei, dorsal cochlear nucleus, parvocellular reticular nucleus and the oral trigeminal nucleus. The mesencephalic trigeminal nucleus, subcoeruleus, medial parabrachial nucleus, A7 noradrenaline cell group, Kolliker-Fuse nucleus, medial superior olive, the C1, C2 and C3 adrenaline cell groups, anterior and posterior ventral cochlear nuclei, the rostral and superior paraolivary nuclei and cranial nerve nuclei and fibers are devoid of immunolabeling.

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5.4.3.3. ORL1 in situ hybridization Orphanin receptor mRNA expression is very high throughout the pons. The locus coeruleus is filled with numerous, densely labeled neurons and is one of the highest mRNA-expressing regions in the metencephalon. High mRNA expression is also observed in the metencephalic portion of the dorsal raphe, the lateral parabrachial nucleus, the motor trigeminal nucleus, principal sensory trigeminal nucleus and mesencephalic trigeminal nucleus. Strong mRNA expression is also observed in the abducens nucleus, medial vestibular nucleus, dorsomedial spinal trigeminal nucleus, pars oralis of the spinal trigeminal nucleus, lateral superior olive, nucleus of the facial nerve, the raphe magnus and raphe pallidus, and the caudal and ventral pontine reticular nuclei. Moderate mRNA expression is found in the A5 and A7 cell regions, the 0t division of the subcoeruleus nucleus, paraabducens nucleus, posterior ventral cochlear nucleus, the lateral and spinal vestibular nuclei, nucleus of the trapezoid body, the medioventral and lateroventral periolivary nuclei of the superior olive, superior and dorsal periolivary nuclei, pontine raphe nucleus, pontine reticular nucleus and the gigantocellular reticular nucleus. Messenger RNA expression is light in the pontine central gray, Kolliker-Fuse nucleus, posterodorsal tegmental nucleus, medial parabrachial nucleus, anterior ventral cochlear nucleus, dorsal cochlear nucleus, superior vestibular nucleus, the rostral and caudal periolivary regions, the parvicellular and intermediate reticular nuclei, and the lateral paragigantocellular reticular nucleus. No mRNA expression was observed in any of the major fiber bundles, the ventrolateral division of the principal sensory trigeminal nucleus, the dorsal and dorsomedial tegmental nuclei, Barrington's nucleus, supragenual nucleus or the medial superior olive.

5.4.4. Myelencephalon 5.4.4.1. PreproOFQ in situ hybridization In the medulla, OFQ mRNA expression is intense in the lateral and medial divisions of the solitary nucleus, prepositus hypoglossus, dorsal and ventral medullary reticular fields, lateral reticular and paragigantocellular nuclei, caudal interstitial nucleus of the medial longitudinal fasciculus, raphe obscurus, raphe magnus, spinal trigeminal nucleus, dorsomedial spinal trigeminal nucleus, nucleus ambiguous and the retroambiguous nucleus. Moderate mRNA expression is observed in the medial and lateral subdivisions of the rostral solitary nucleus, the commissural division of the solitary nucleus, lateral paragigantocellular nucleus and the raphe pallidus nucleus. Expression is light in the external cuneate nucleus, and the gigantocellular, gigantocellular alpha, intermediate and parvocellular reticular nuclei. Negligible preproOFQ mRNA expression is observed in the principal and dorsal nuclei of the inferior olive, subnucleus A and B of the medial subdivision of the inferior olive, cuneate nucleus, nucleus gracilus, dorsal motor nucleus of the 10th nerve, hypoglossal nucleus, medial and dorsomedial nuclei of the inferior olive and the paramedian reticular nucleus. 5.4.4.2. OFQ immunohistochemistry In the medulla, the nucleus ambiguous and retroambiguous and the prepositus hypoglossus contain numerous densely immunolabeled neurons, with moderate fiber staining, and strong neuronal labeling is observed in the supragenual nucleus and principal nucleus of the inferior olive. In the nucleus of the solitary tract, heavy terminal and fiber labeling is observed in 141

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the medial and lateral parts, and heavy cell staining in the medial and commissural parts. The caudal spinal trigeminal nucleus contains a heavily immunolabeled fiber and terminal field interspersed with numerous darkly labeled neurons. The caudal interstitial nucleus of the medial longitudinal fasciculus contains moderate neurons and fibers. Moderately labeled neurons are observed in the caudal gigantocellular reticular nucleus, the alpha and lateral paragigantocellular nucleus, the raphe magnus and pallidus, the midline raphe obscurus and the dorsomedial spinal trigeminal nucleus. There are lightly stained cells and scattered fibers within the anterior, medial and dorsal nuclei of the inferior olive. No immunolabeling is observed the in the cuneate and external cuneate nuclei, nucleus gracilus, the dorsomedial division of the inferior olive or the interpolar spinal trigeminal nucleus. 5.4.4.3. ORL1 in situ hybridization

In the medulla, high mRNA expression is seen in the region of the C1 cell group, the prepositus hypoglossus and the dorsal motor nucleus of the vagal nerve. The hypoglossal nucleus is filled with cells expressing high levels of mRNA, as is the nucleus ambiguous and retroambiguous nucleus. Intense expression is also observed in the central division of the nucleus of the solitary tract, the oral and interpolar divisions of the spinal trigeminal nucleus, the ventral division of the gigantocellular reticular nucleus, the rostroventriculolateral reticular nucleus and the lateral reticular nucleus. The raphe magnus is filled with dense mRNA expression as is the adjacent raphe pallidus nucleus. Moderate mRNA expression is observed in the region of the A2 and C2 cell groups, the medial and lateral vestibular nuclei, the lateral and medial divisions of the nucleus of the solitary tract, the cuneate nucleus, paratrigeminal nucleus, dorsomedial spinal trigeminal nucleus and pars caudalis of the spinal trigeminal nucleus. Moderate expression is also observed in the dorsal and ventral medullary reticular nuclei, the principal and dorsal nuclei of the inferior olivary complex and the raphe obscurus nucleus. Sparse mRNA expression is seen in the ventral medial and spinal vestibular nucleus, external cuneate nucleus, area postrema, median accessory nucleus of the medulla, gracile nucleus, parvicellular and intermediate reticular nuclei, the gigantocellular reticular nucleus, and subnuclei A and B of the inferior olivary complex. Low expression is observed in the medial and dorsomedial nuclei of the inferior olivary complex. No ORL mRNA expression is observed in any of the major fiber bundles in this region. Messenger RNA expression is also negligible in the region of the C3 cell group, the caudal interstitial nucleus of the medial longitudinal fasciculus, intercalated nucleus of the medulla, lateral paragigantocellular reticular nucleus and the intermediate reticular nucleus.

5.4.5. Spinal cord 5.4.5.1. PreproOFQ in situ hybridization

In the dorsal horn of the cervical cord, mRNA expression is intense in lamina II and very light in laminae III, V, VI and VIII. Intense mRNA expression is observed in lamina X, and extending ventrally, large, scattered neurons with intense mRNA expression reside in laminae VII and IX of the ventral horn, in what appear to be large motor neurons. The lateral cervical nucleus shows light mRNA expression. In the thoracic spinal cord, lamina I is devoid of mRNA expression the prepositus hypoglossus and lamina II has heavy expression. Other than sparse expression in laminae III and V, no other mRNA expression is observed in the dorsal horn. Moderate mRNA expression is observed in lamina X and the intermediomedial cell 142

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column. Large, intensely labeled cells reside in the ventral part of laminae IX of the ventral horn. The dorsal root ganglia contains scattered cells expressing OFQ mRNA, very few in number and confined centrally in the ganglia. No mRNA expression is observed in the lateral spinal nucleus, the intermediolateral cell column, dorsal corticospinal tract, gracile fasciculus, lateral funiculus or ventral funiculus in the thoracic cord region.

5.4.5.2. OFQ immunohistochemistry Intense immunostaining is observed in laminae II and III of the dorsal horn. Lamina I contains scattered terminals and fibers, but no cells. The lateral cervical nucleus is filled with a dense plexus of immunolabeled fibers with moderate cell staining. Moderate puncta and fibers are seen in lamina IV, and only lightly labeled, terminals and fibers seen in laminae V and VI. Fiber and terminal labeling increases in dorsal lamina VII, and is moderate in lamina X. Several moderately labeled neurons are also found in lamina X. Large immunolabeled neurons are scattered throughout ventral lamina VIII, ventrolateral lamina IX, and in ventral lamina VII. In the thoracic spinal cord, modest fiber and terminal staining is observed in laminae I and III, with moderately immunolabeled neurons in lamina II. The lateral spinal nucleus contains heavy fiber and moderate cell staining. Laminae IV, V, VII and VIII have moderate fiber and terminal labeling, but no immunolabeled neurons are evident. Terminal and fiber labeling in lamina X is strong, with scattered immunolabeled neurons also observed, as well as in the intermediomedial cell column. Moderately stained neurons are observed in the ventral horn, with large immunolabeled cells observed primarily in lamina IX, and fewer smaller neurons observed in lamina VIII. No immunolabeling is observed within the cuneate or gracile fasciculus, the dorsal corticospinal tract, the lateral funiculus or the ventral funiculus.

5.4.5.3. ORL1 in situ hybridization In the cervical spinal cord, ORL1 mRNA expression is highest in the ventral horn. No orphanin receptor mRNA expression is observed in lamina I, and only scattered mRNAcontaining cells are noted in lamina II. Expression in laminae III and IV is low to moderate, and in laminae V and VI, moderate to high. In lamina X, scattered neurons with low mRNA expression are observed. In laminae VIII and IX of the ventral horn, numerous large ventral horn cells containing abundant mRNA expression are widely distributed. In the thoracic spinal cord, ORL1 mRNA expression is very similar to that of the cervical cord. No mRNA expression is observed in lamina I, and laminae II and III contain only scattered mRNA-expressing cells. The ventral horn contains numerous large, densely labeled neurons. High mRNA expression is observed in the intermediomedial cell column and in the intermediolateral column. The central cervical nucleus contains low to moderate mRNA expression, and the lateral cervical and lateral spinal nuclei contain sparse mRNA expression. No mRNA expression is observed in the cuneate fasciculus, the dorsal corticospinal tract, lateral funiculus or ventral funiculus. The dorsal root ganglion is filled with numerous, large mRNA-expressing neurons. These neurons are located widely throughout the ganglion and contain high mRNA expression.

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6. ANATOMICAL STUDIES USING 12sI-[14Tyr]OFQ BINDING AND AGONIST STIMULATED [3sS]GTPyS RECEPTOR AUTORADIOGRAPHY 6.1. METHODS

6.1.1. Animals Adult male Sprague-Dawley rats (Charles River; 250-300 g) were used for all in vitro autoradiography and agonist-stimulated [35S]GTPyS binding studies. Handling and use of all animals strictly conformed to NIH guidelines.

6.1.2. Tissue preparation For receptor autoradiography, adult male Sprague-Dawley rats were killed by decapitation. Brains, pituitaries and spinal cords were removed and quick-frozen at -30~ Tissue was sectioned in the coronal plane at 15 ~m, thaw mounted on polylysine-treated microscope slides, then stored at -80~ until used. For agonist-stimulated [35S]GTPu receptor autoradiography, adult male Sprague-Dawley rats were killed by decapitation and their brains removed and quick-frozen at -30~ Brains were sectioned in the coronal plane at 20 Ixm and thaw-mounted onto polylysine-coated slides, then stored at -80~ until used.

6.1.3. Peptide synthesis and iodination The OFQ peptide analog used for iodination was synthesized by replacing the leucine in position 14 with a tyrosine residue (14Tyr-OFQ). The 125I-[14Tyr]OFQ peptide was labeled by the chloramine T method (Hunter and Greenwood, 1962), providing a monoiodinated species with an estimated specific activity of 2200 Ci/mmol after reverse phase HPLC purification. The 125I-[14Tyr]OFQ radioligand was stored in its elution buffer at -20~ until used. Binding analyses have demonstrated that 125I-[14Tyr]OFQ exhibits identical binding characteristics at the ORL1 receptor as [3H]OFQ, making this a suitable radioligand for pharmacological analysis of the ORL 1 receptor using in vitro assays (Ardati et al., 1997). The OFQl-17 peptide used for agonist-stimulated [35S]GTPyS receptor autoradiography was synthesized using an Applied Biosystems Model 431 Peptide Synthesizer. The synthesis procedure, fmoc-protected amino acids, reagents and solvents were used as supplied by Applied Biosystems. OFQ peptide was removed from the resin with simultaneous removal of protecting groups by treatment with trifluoroacetic acid. Crude peptide was precipitated with cold ether and purified by reverse phase chromatography. In all cases, peptide identity was confirmed by mass spectrometry.

6.1.4. Receptor autoradiography The in receptor autoradiography technique employed in the analysis of OFQ binding has been described in detail (Neal et al., 1999b). Tissue slides were removed from -80~ and, after reaching room temperature, transferred to incubation chambers to maintain ambient temperature and humidity (60-80%). Sections were then incubated in 125I-[14Tyr]OFQ for 60 min. The incubation buffer consisted of the 125I-[14Tyr]OFQ peptide, 50 mM Tris (pH 7.0), 1 mM EDTA, 0.1% bovine serum albumin, and a protease inhibitor. Incubation was terminated by four consecutive Tris washes, followed by a distilled water rinse. Scatchard 144

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analysis suggested a 0.1 nM Kd for the 125I-[14Tyr]OFQ peptide. Therefore, a concentration of 0.1-0.13 nM, which corresponds to a 50% receptor occupancy, was used as a labeling concentration for the competition and anatomical mapping studies. Upon completion of tissue treatment with the 125I-[14Tyr]OFQ ligand, brain and pituitary sections used for receptor autoradiography were apposed to Kodak XAR-5 X-ray film for 1-3 days. Sections were then exposed to paraformaldehyde vapors at 80~ for 2 h in an evacuated desiccator and lipids removed by a series of ethanol and xylene washes. The slides were then dipped in emulsion and developed 3 days later. Emulsion-dipped sections were Nissl counterstained with Cresyl violet and prepared for darkfield analysis.

6.1.5. Agonist-stimulated GTPyS receptor autoradiography The autoradiography technique used in these studies is a modification of that reported previously for the identification of OFQ-stimulated [35S]GTPyS binding in the rat and guinea pig brain (Sim et al., 1996; Sim and Childers, 1997). Tissue slides were removed from -80~ and, after reaching room temperature, transferred to humidifying chambers. At room temperature, assay buffer (50 mM Tris-HC1, 0.1% bovine serum albumin, 5 mM MgC12, 1 mM EGTA and 100 mM NaC1; pH 7.5) is directly applied to slide-mounted sections, which are incubated for 10 min. Assay buffer is removed and the sections are next incubated for 15 min in a solution containing 2 mM guanidine diphosphate (GDP; Sigma, St. Louis, MO) in the primary assay buffer. The GDP assay buffer is removed and the sections are then covered with incubation media (primary assay buffer containing 2 mM GDR 0.04 nM [35S]GTPyS [New England Nuclear, 1250 Ci/mM], 1 txM DTT, and a protease inhibitor) for 2 h. The incubation media is placed on tissue sections without agonist present, and adjacent sections are treated with incubation media plus added OFQ peptide at varying concentrations. After 2 h, incubation media is removed, the sections are rinsed in Tris buffer and water and air-dried. Slide mounted sections are opposed to Kodak XAR-5 X-ray film for 20-48 h. Emulsion films were digitized and analyzed using Scion Image (NIH image modified by Scion Corporation for the National Institutes of Health, Frederick, MD).

6.1.6. 125I-[14Tyr]OFQand agonist-stimulated [35S]GTPyS autoradiography controls For 125I-[14Tyr]OFQ autoradiography, competition studies were performed on slide-mounted sections. Each brain section was incubated with of 125I-[14Tyr]OFQ peptide in incubation buffer for 60 min, and a minimum of eight 8 competing ligand concentrations were examined (0.03 nM to 10 gM). Non-specific binding was evaluated by treating adjacent brain sections with the same 0.1 nM concentration of 125I-[14Tyr]OFQ peptide and a 1 txM final concentration of unlabeled native OFQ peptide. To pharmacologically characterize ORL1 receptor binding sites, a series of g (morphine, naloxone), ~ (DADL, DPDPE) and ~: (bremazocine, U69,593) agonists were evaluated. In addition, the affinity of ~-endorphin and OFQ peptide was examined. Receptor binding was quantified by liquid scintillation spectrophotometry. For agonist-stimulated GTPyS autoradiography, basal binding was measured on sections incubated with 2 mM GTP and 0.04 nM [35S]GTPyS in the absence of agonist in the incubation media. Non-specific binding was assessed when 10 ~tM unlabeled GTPyS was added to the incubation media containing 2 mM GTP and 0.04 nM [35S]GTPyS in the presence of agonist.

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6.2. CONTROL RESULTS 6.2.1. 12Sl-[14Tyr]OFQ autoradiography controls Comparison of total and non-specific binding demonstrated that binding is completely eliminated when a 1 ~M concentration of native OFQ peptide is added to the incubation media in the presence of a 0.1-0.13 nM concentration of 125I-[14Tyr]OFQ peptide (Fig. 10). 6.2.2. Agonist-stimulated [35S]GTPyS autoradiography controls Basal binding was minimal on sections incubated with 2 mM GTP and 0.04 nM [35S]GTPyS in the absence of agonist or antagonist in the incubation media. Additionally, specific binding

Fig. 10. Darkfield image of orphanin FQ receptor binding obtained in the rostral forebrain using 125I-[14Tyr]OFQ as the radioligand (A). Addition of a saturating concentration of unlabeled OFQ to the 125I-[lgTyr]OFQ receptor binding assay (B) generates negligible non-specific labeling. Scale bar: 900 I~m.

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Fig. 11. Color images of OFQ-stimulated GTPyS binding produced after exposing tissue sections to OFQ peptide at a 10 IxM concentration (A). Basal binding was minimal on sections incubated with [35S]GTPyS in the absence of agonist or antagonist in the incubation media (B). Additionally, specific binding was completely eliminated when 10 IxM unlabeled GTPyS was added to the incubation media in the presence of agonist (C). Scale bar: 2000 Ixm.

was completely eliminated when 10 IxM unlabeled GTPyS was added to the incubation media in the presence of agonist (Fig. 11). 6.3. P H A R M A C O L O G I C A L C H A R A C T E R I Z A T I O N OF R E C E P T O R B I N D I N G Under the above receptor autoradiographic conditions, only OFQI_17 peptide had high affinity (IC50 = 0.39 nM) for the orphanin receptor (Table 2). Specific ORL1 binding by 1251[14Tyr]OFQ represented greater than 85% of total binding. Prototypical Ix, 3 or • agonists fail to compete for orphanin receptor sites at all concentrations studied. Naloxone and ~-endorphin also fail to displace orphanin binding, suggesting that OFQ is not labeling a 'classical' opioid binding site. These findings are consistent with previous binding studies performed with 125I-[14Tyr]OFQ, suggesting that this ligand selectively binds ORL1 (Ardati et al., 1997). 6.4. DISTRIBUTION OF OFQ BINDING IN THE RAT F O R E B R A I N A detailed presentation of the distribution of 125I-[14Tyr]OFQ binding in the rat CNS is provided in Table 1. The table is organized regionally, providing a comparison of 1251TABLE 2. Results of competition studies (IC5o nM) comparing affinity of several opioid compounds to that of orphanin FQ peptide at the orphanin receptor Morphine Naloxone Bremazocine U69,593 DADLE DPDPE ~-Endorphinl_31 Orphanin FQ peptidel_17

> 10,000 > 10,000 > 10,000 > 10,000 > 10,000 > 10,000 > 10,000 0.39

None of the opioid agonists or antagonists tested were able to compete for the orphanin receptor binding site. Prototypical Ix (morphine), 3 (DPDPE, DADLE) or ~: (bremazocine, U69,593) agonists failed to compete for the orphanin receptor sites, even at a 10 IxM concentration. Similarly, the opiate antagonist naloxone and the endogenous peptide ~-endorphin failed to displace binding. In contrast, orphanin FQI-17 peptide had high affinity (IC50 = 0.39 nM) for the orphanin receptor binding site. 147

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[14Tyr]OFQ binding to that of the distribution of ORL1 mRNA, preproOFQ mRNA and OFQ immunolabeling. Examples of orphanin binding, compared with ORL1 mRNA distribution, are provided in Figs. 5-9. 6.4.1. Cortex Orphanin receptor binding is robust throughout the neocortex. Binding in frontal and parietal cortex is dense in layer IV, moderate in layer VI and low in the remaining layers. In temporal cortex, orphanin binding is not as pronounced as in other neocortical regions, with moderate binding in layers II, III and IV. Binding in the occipital cortex is moderate in layers I-IV, and dense in layers V and VI. In other cortical regions, binding is dense in the glomerular layer of the olfactory bulb and layer III of the piriform cortex. Binding in the cingulate cortex is the densest of all cortical regions, persisting caudally into the retrosplenial cortices, where dense binding is observed in the deeper layers of both the granular and agranular parts. Moderate binding is observed in the mitral cell layer of the olfactory bulb, medial, lateral and ventral orbital cortices, infralimbic and dorsal peduncular cortices, agranular and granular insular cortices and the entorhinal cortex. Orphanin binding is sparse in the ventral tenia tecta and no binding is observed in the corpus callosum. 6.4.2. Ventral forebrain Binding is dense in the external and ventral divisions of the anterior olfactory nucleus, moderate to dense in the olfactory tubercle, and dense in the caudal substantia innominata. Moderate binding is observed in the dorsal and lateral divisions of the anterior olfactory nucleus, the shell of nucleus accumbens and the horizontal limb of the diagonal band of Broca. Moderate binding also fills the interstitial nucleus of the posterior limb of the anterior commissure. Sparse to light OFQ binding is observed in the posterior division of the anterior olfactory nucleus, the core of nucleus accumbens and the vertical limb of the diagonal band of Broca. No binding is observed in the islands of Calleja. 6.4.3. Septum Similar to ORL1 mRNA expression, orphanin binding in the lateral septum is moderate. The dorsal tenia tecta contains moderate binding, as does the dorsal lateral septal nucleus and the septofimbrial nucleus. Low binding is observed in the ventrolateral and intermediate septal regions, the septohippocampal nucleus, medial septum and lambdoid nucleus. The intermediate lateral septum, paralambdoid nucleus and the septohypothalamic nucleus are devoid of binding. 6.4.4. Basal ganglia Dense orphanin binding fills the dorsal endopiriform nucleus, and is found throughout the claustrum. Moderate binding is observed in a diffuse, reticular pattern throughout the globus pallidus. Binding in the caudate-putamen is generally negligible, except for occasional sparse patches of binding located dorsomedially. In the substantia nigra, compacta contains moderate binding is observed throughout. In pars reticulata, moderate to heavy OFQ binding is observed in its rostral half, decreasing to become sparse caudally. In pars lateralis, orphanin binding is very low. The ventral endopiriform nucleus contains sparse to low levels of binding and 148

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low binding is also observed in the subthalamic nucleus. The basal nucleus of Meynert and entopeduncular nucleus are devoid of OFQ binding.

6.4.5. Basal telencephalon In comparison to ORL 1 mRNA expression, binding in the preoptic region and bed nucleus of the basal telencephalon is low. Orphanin binding is dense in the supracommissural and ventral parts of the median preoptic nucleus. Binding is moderate in the ventral medial preoptic region, the caudal anteromedial preoptic nucleus and the anteroventral preoptic nucleus and sparse to low in the rostral anteromedial preoptic nucleus, lateral preoptic region, medial preoptic nucleus and magnocellular preoptic nucleus. OFQ binding is negligible in the rostral preoptic region. Orphanin binding in the bed nucleus of the stria terminalis is generally moderate. Sparse OFQ binding is noted in the rostral bed nucleus, in the anterior part of the medial division, and in the ventral division. Moderate binding is observed in the dorsal and ventral parts of the lateral division. The posterolateral and posterointermediate parts of the medial division contain moderate to dense binding. The juxtacapsular part of the lateral division, the posteromedial part of the medial division and the posterior part of the lateral division of the bed nucleus are devoid of binding. In other areas, the nucleus of the lateral olfactory tract contains dense binding and the septohypothalamic nucleus contains low levels of OFQ binding.

6.4.6. Hypothalamus The distribution of orphanin binding in the hypothalamus is similar to that of ORL1 mRNA expression. Consistent with the paucity of ORL1 mRNA expression, no binding is seen in the median eminence, the anterior and intermediate pituitary, and the pineal gland. Dense binding is observed in the suprachiasmatic nucleus, and the ventromedial nucleus contains the densest binding in the hypothalamus. Dense binding is also seen in the terete hypothalamic nucleus and the supramammillary nucleus. Binding is moderate in the medial arcuate nucleus, the tuber cinereum, medial tuberal nucleus and the perifornical nucleus. Moderate binding is also observed in the paraventricular nucleus, the dorsal premammillary nucleus and the lateral part of the medial mammillary nucleus. Binding is low in the supraoptic nucleus, lateral arcuate nucleus, anterior and lateral hypothalamic areas, parvicellular paraventricular nucleus, the dorsomedial and posterior hypothalamic nuclei and the ventral premammillary nucleus. Negligible binding is observed in the retrochiasmatic supraoptic nucleus, retrochiasmatic area, the dorsal hypothalamus, tuberal magnocellular nucleus, the lateral mammillary nucleus, the median division of the medial mammillary nucleus and the mammillary peduncle.

6.4.7. Amygdala The central nucleus is devoid of binding at all levels. Intense binding is observed in the, bed nucleus of the accessory olfactory tract, the basolateral nucleus and the posteroventral division of the medial nucleus. Moderate orphanin binding is observed in the anterior cortical nucleus, anterodorsal division of the medial nucleus, lateral nucleus, the posteromedial cortical nucleus and the amygdalohippocampal area. Binding is low in the ventral and dorsal parts of the anterior amygdaloid area, the anterior medial nucleus, the region of the intercalated nuclei, posterodorsal division of the medial nucleus and the intraamygdaloid portion of the bed nucleus of the stria terminalis. Negligible binding is observed in the cortex-amygdala 149

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transition zone, posterolateral cortical nucleus, basomedial nucleus and the amygdalopiriform transition area. 6.4.8. Hippocampal formation and related structures

Orphanin binding in the hippocampal formation is generally unchanged from rostral to caudal levels. In the dentate gyms, no OFQ binding is observed in the granule cell layer, hilus or polymorph layer. The molecular layer is filled with moderate to dense binding. In Ammon's horn, area CA1 contains almost no orphanin binding, with only sparse signal observed in caudal stratum oriens. In area CA2, sparse receptor binding is observed in stratum oriens and stratum radiatum. No binding is observed in stratum pyramidale. In area CA3, very dense binding is observed in stratum oriens, with low to moderate binding observed in stratum lucidum, primarily at rostral levels. Stratum pyramidale and radiatum are devoid of OFQ receptor binding at all levels in this region. In other structures, orphanin binding is dense in the ventral and dorsal subiculum, equally intense in all layers. Binding is moderate to dense in the indusium griseum, and low in the subfornical organ, septohippocampal nucleus, presubiculum and parasubiculum. The fornix is devoid of orphanin binding at all levels. 6.4.9. Thalamus

In the rostral thalamus, dense binding filling the anterior part of the paraventricular nucleus and the anteroventral nucleus. Binding is moderate to dense in the paratenial nucleus, the ventrolateral part of the anteroventral thalamic nucleus, the reticular nucleus, paracentral nucleus, medial habenula and the anterodorsal nucleus. Orphanin binding is low to moderate in the dorsomedial part of the anteroventral thalamic nucleus, the interanterodorsal and reunions nuclei, zona incerta, laterodorsal nucleus and the ventrolateral and ventromedial nuclei. Binding is sparse in the anteromedial, rhomboid, centromedial and interanteromedial nuclei. The rostral anterodorsal nucleus, mediodorsal nuclear group, anteromedial nucleus and gelatinosus nucleus are devoid of binding. In the caudal thalamus, binding is most intense in the medial habenula, paraventricular nucleus and the lateral geniculate nucleus. Moderate binding is observed in the laterodorsal and reunions nuclei, the centrolateral nucleus, the lateroposterior thalamic nuclei, posterior and perifornical nuclei, prerubral field and rostral interstitial nucleus of the medial longitudinal fasciculus. Light to sparse orphanin binding is seen in the ventromedial, ventrolateral, ventral posterolateral, ventral posteromedial and subincertal nuclei, the fields of Forel, medial geniculate body and suprageniculate nucleus. The mediodorsal thalamus, ventral posteromedial nucleus, parvicellular subparafascicular thalamic nucleus, the precommissural nucleus and the posterior limitans nucleus are devoid of binding. 6.5. DISTRIBUTION OF OFQ BINDING IN THE RAT BRAINSTEM AND SPINAL CORD 6.5.1. Mesencephalon

Orphanin binding in the mesencephalon is diffuse. In the rostral midbrain, orphanin binding is dense in the olivary pretectal nucleus, the zonal and superficial gray layers of the superior colliculus, the peripeduncular nucleus and the laterodorsal tegmental nucleus. Moderate levels of OFQ binding are observed in the perifornical nucleus, the deep gray layer of the superior 150

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colliculus, the oral part of the pontine reticular formation, lateral geniculate nucleus and anterior tegmental nucleus. Low levels of binding are observed in the nucleus of the posterior commissure, the posterior and medial pretectal nuclei, the central and intercollicular nuclei of the inferior colliculus, as well as its dorsal and external cortices. Binding is sparse in the dorsal and ventral parts of the anterior pretectal nucleus, intermediate gray layer of the superior colliculus, the nucleus of the brachium of the inferior colliculus, the deep mesencephalic nucleus, the medial geniculate nucleus, dorsal nucleus of the lateral lemniscus, the pedunculopontine tegmental nucleus, and the reticulotegmental nucleus. No binding is noted in the magnocellular nucleus of the posterior commissure, nucleus of the optic tract or the optic nerve, intermediate and deep white layers of the superior colliculus. Negligible binding is observed in the recess of the inferior colliculus, paralemniscal nucleus, lateral and ventral nuclei of the lateral lemniscus, microcellular tegmental nucleus, subpeduncular tegmental nucleus, the ventrolateral, dorsal and ventral tegmental nuclei, and the rhabdoid nucleus. The brachium and commissure of the inferior and superior colliculi, the cerebral peduncle, central tegmental tract, dorsal longitudinal fasciculus, fasciculus retroflexus, longitudinal fasciculus, lateral or medial lemniscus, mammillary peduncle, posterior commissure, rubrospinal tract, superior cerebellar peduncle and spinal trigeminal tract are devoid of orphanin binding. In the mid to caudal mesencephalon, the central gray contains dense binding ventrally, most dense in its dorsal division. Dense binding is also observed in the supraoculomotor central gray, Edinger-Westphal nucleus, dorsal raphe, rostral and caudal linear nuclei of the raphe, the caudal and lateral divisions of the interpeduncular nucleus and the medial terminal nucleus of the accessory olfactory tract. Moderate binding is observed in the paramedian and median raphe, the medial accessory oculomotor nucleus and the interfascicular nucleus. Binding is light in the nucleus of Darkschewitsch, the red nucleus and ventral tegmental area, with sparse binding in the paranigral and parabrachial pigmented nucleus. Binding is negligible in the cuneiform nucleus, interstitial nucleus of the medial longitudinal fasciculus, the paratrochlear, oculomotor, and trochlear nuclei, the dorsal and rostral divisions of the interpeduncular nucleus, the retrorubral field and the retrorubral nucleus.

6.5.2. Cerebellum No specific orphanin binding is observed in any of the cell layers of the cerebellar lobules. In the deep cerebellar nuclei, the interposed nucleus is devoid of OFQ binding in its anterior, dorsomedial, dorsolateral and posterior parts. The lateral (dentate) nucleus contains low to moderate binding, and sparse, patchy binding is observed in the medial (fastigial) nucleus. The dorsolateral protuberance of the fastigial nucleus, has no detectable orphanin binding.

6.5.3. Metencephalon In the metencephalon, no OFQ binding was observed in major fiber bundles, including the superior cerebellar peduncle, inferior cerebellar peduncle, pyramidal tract, abducens nerve, facial nerve, vestibulocochlear nerve, medial longitudinal fasciculus, sensory root of the trigeminal nerve and trapezoid body. Rostrally, binding is high in the pontine central gray, the caudal dorsal raphe and the dorsomedial division of the principal sensory trigeminal nucleus. The locus coeruleus is diffusely filled with intense OFQ binding, and dense binding is also observed in the lateral parabrachial nucleus. Binding is moderate in the motor trigeminal nucleus, ventrolateral division of the principal sensory trigeminal nucleus, A7 cell region and Kolliker-Fuse nucleus, and the granular layer of the anterior ventral cochlear nucleus. 151

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Sparse to low binding is observed in the medial parabrachial nucleus, the ~ division of the subcoeruleus nucleus and Barrington's nucleus. No detectable orphanin binding is observed in the dorsal, dorsomedial or posterodorsal tegmental nuclei, or the mesencephalic trigeminal nucleus. Caudally, binding is high in the posterior ventral cochlear nucleus and dorsal cochlear nucleus, and the supragenual nucleus. Binding is moderate in the vestibular nuclear complex in the superior and medial vestibular nuclei, and in the dorsomedial nucleus and pars oralis of the trigeminal nuclear complex. Moderate binding is also observed in the nucleus of the trapezoid body, the rostral periolivary area, caudal periolivary nucleus, region of the A5 cell group, facial nucleus, the ventral pontine reticular nucleus, the raphe magnus and raphe pallidus. Low binding is observed in the ventral vestibular nucleus, the superior olivary nuclear complex, parvicellular reticular nucleus and gigantocellular reticular nucleus. Binding is sparse in the abducens nucleus, the spinal vestibular nucleus, the superior and dorsal paraolivary nuclei, the caudal pontine reticular nucleus and intermediate reticular nucleus. No detectable OFQ binding is observed in the paraabducens nucleus, lateral vestibular nucleus, the medioventral or lateroventral periolivary nuclei, the lateral or medial superior olive, lateral paragigantocellular reticular nucleus or the ~ division of the gigantocellular reticular nucleus.

6.5.4. Myelencephalon In the myelencephalon, orphanin binding is widespread. Dense orphanin binding is observed in the area postrema, prepositus hypoglossus, external cuneate nucleus, the central division of the nucleus of the solitary tract, median accessory nucleus of the medulla, the dorsal medullary reticular nucleus, nucleus ambiguous, the raphe pallidus nucleus and the interpolar and dorsomedial parts of the medullary spinal trigeminal nucleus. Intense binding fills pars caudalis of the spinal trigeminal nucleus, extending from the spinal trigeminal nucleus into the caudal spinal trigeminal tract, making this the only major fiber bundle in the CNS with orphanin receptor mRNA expression or OFQ binding. The medial and principal nuclei of the inferior olive, and subnucleus A contain dense OFQ binding, persisting to the caudal pole of the inferior olive where the medial nucleus is filled with dense OFQ binding. Binding is more moderate in the dorsal motor vagal nucleus, the medial and ventral medial vestibular nuclei, the lateral and medial divisions of the nucleus of the solitary tract, the cuneate and paratrigeminal nuclei and pars oralis of the spinal trigeminal nucleus. Moderate binding is also observed in the gigantocellular reticular nucleus, ventral medullary reticular nucleus, retroambiguous nucleus, dorsal nucleus of the inferior olive, subnucleus B of the olivary complex and raphe magnus. Low orphanin binding is observed in the regions of the A1, A2, C1 and C2 cell groups, the parvicellular reticular nucleus, rostroventriculolateral reticular nucleus, lateral reticular nucleus and raphe obscurus. Binding is sparse in the hypoglossal nucleus, spinal vestibular nucleus, gracile nucleus, intermediate reticular nucleus and dorsomedial paragigantocellular reticular nucleus. Orphanin binding is negligible in the region of the C3 cell group, the intercalated nucleus of the medulla, caudal interstitial nucleus of the medial longitudinal fasciculus, lateral vestibular nucleus and lateral paragigantocellular reticular nucleus.

6.5.5. Spinal cord In contrast to ORL1 mRNA distribution, orphanin binding is higher in the dorsal horn than in the ventral horn. In the cervical spinal cord, lamina I contains no OFQ binding. Binding is 152

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dense in lamina II and moderate to dense in lamina III. Binding intensity is low in lamina IV and V, moderate in lamina VI. In the ventral horn density of binding is sparse in laminae VII, VIII and IX. Lamina X contains moderate binding. In the thoracic spinal cord, no binding is noted in lamina I and low binding is noted in lamina II. The density of binding is moderate in lamina III, with high levels noted in lamina IV. In lamina X very dense orphanin binding is noted. In the ventral horn, low levels of binding are noted in laminae VII and VIII. In marked contrast to the intense ORL1 mRNA expression observed in the dorsal root ganglia, no orphanin binding is noted in this structure. In remaining spinal cord structures, low to moderate binding is observed in the intermediomedial cell column, and moderate to dense binding is observed in the intermediolateral cell column. Other than sparse labeling noted in the central cervical nucleus, the remainder of the spinal cord has no detectable orphanin binding. 6.6. DISTRIBUTION OF OFQ-STIMULATED GTPyS BINDING IN THE RAT CNS Orphanin-stimulated GTPyS binding closely matches the pattern of 125I-[14Tyr]OFQ binding observed throughout the rat brain. With the exception of regional variations in intensity, the distribution of OFQ-stimulated GTPyS binding is essentially the same as that of binding observed using 125I-[14Tyr]OFQ (Fig. 12). Like ORL1 binding, OFQ-stimulated GTPyS binding is robust throughout the neocortex. In other cortical regions, binding is intense in the prelimbic and orbital cortices, infralimbic and dorsal peduncular cortices, the insular, piriform, cingulate and entorhinal cortices. In the ventral forebrain and basal telencephalon, binding is dense in the anterior olfactory nucleus, moderate in the accumbens shell, the diagonal band of Broca, the lateral preoptic area, medial preoptic nucleus and bed nucleus of the stria terminalis. Binding is strong in the nucleus of the lateral olfactory tract. Strong GTPyS binding is observed in the tenia tecta, indusium griseum and septohippocampal nucleus. The dorsal endopiriform nucleus and claustrum contain dense GTPyS binding. The caudate-putamen is devoid of GTPyS binding and binding in the entopeduncular nucleus is negligible. In the hypothalamus dense binding is observed in the suprachiasmatic nucleus, ventromedial nucleus, terete nucleus, arcuate nucleus, medial mammillary nucleus and lateral mammillary nucleus. In the supraoptic and paraventricular nuclei GTPyS binding is light. In the amygdala, the central nucleus is devoid of GTPyS binding. Strong binding is observed in the basomedial and anterior cortical nuclei and the medial nucleus. Moderate to dense GTPyS binding is observed in the posteromedial and posterolateral cortical nuclei, the lateral amygdaloid nucleus and the amygdalohippocampal area. Orphanin-stimulated GTPyS binding is pronounced in the hippocampal formation and related structures. Stimulated GTPyS binding is light in the dentate gyms, moderate in areas CA1 and CA2 of Ammon's horn. Moderate binding is observed in the subiculum. In the thalamus, binding is dense in the anteroventral nucleus, the paraventricular nucleus and the paratenial nucleus. Moderate GTPyS binding is also observed in the anterodorsal, central medial and rhomboid nuclei. Medial habenula binding is moderate to strong and lateral habenula binding is light. The lateral geniculate nucleus contains moderate binding and the medial geniculate body is devoid of GTPyS binding. Binding in the remainder of the caudal thalamus is negligible. In the mesencephalon, binding is strong in the subthalamic nucleus and in the substantia nigra pars reticulata and compacta. Moderate binding is observed in the olivary and anterior pretectal nuclei, the precommissural nucleus, the parabrachial pigmented nucleus, the superior colliculus and the dorsal medial geniculate body. Strong GTPyS binding is observed in 153

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Fig. 12. Color images comparing basal binding (left) to OFQ-stimulated GTPyS binding (right). Signal was generated after exposing tissue sections to OFQ peptide at a 10 IxM concentration. Coronal images presented are at representative levels of the forebrain and brainstem (A-F). Scale bar: 2000 Ixm.

the paranigral nucleus, the lateral interpeduncular nucleus and the peripeduncular nucleus. In more caudal midbrain regions binding is strong in the lateral interpeduncular nucleus and caudal superior colliculus. Strong GTPyS binding is also observed in the dorsolateral periaqueductal gray, the nucleus of the brachium of the inferior colliculus, the caudal linear raphe nucleus and the oculomotor nucleus. Binding in the red nucleus is minimal. In the metencephalon, intense GTPyS binding is observed rostral in the pontine nuclei, the caudal part of the dorsal central gray and the dorsal cortex of the inferior colliculus. Moderate to strong GTPyS binding is observed in the dorsal raphe and the dorsal and lateral nuclei of the lemniscus. The locus coeruleus is filled with moderate GTPyS binding, and dense binding 154

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is observed in the lateral parabrachial nucleus. Strong binding is observed in the dorsal and lateral dorsal tegmental nuclei. Minimal binding is observed in the pontine reticular nucleus and superior olivary nuclei. No specific GTPyS binding is observed in any of the cell layers of the cerebellar lobules. In the deep cerebellar nuclei, the interposed nucleus the lateral (dentate) nucleus and the medial (fastigial) nucleus contain no detectable GTPu binding. In the myelencephalon, moderate binding is observed in the inferior olive, area postrema, prepositus hypoglossus and external cuneate nucleus. Strong GTPu binding is observed in the spinal trigeminal nucleus. Binding is light in the dorsal motor vagal nucleus, the vestibular nuclei, nucleus of the solitary tract, reticular formation and raphe nuclei. No GTPu binding is observed in the regions of the A1, A2, C1 and C2 cell groups. 7. ONTOGENY STUDIES 7.1. METHODS

7.1.1. Animals Time-pregnant adult female Sprague-Dawley rats were obtained from Charles River at various gestational ages. Prior to sacrifice, handling and use of all animals strictly conformed to NIH guidelines.

7.1.2. Rat developmental tissue preparation Age of gestation of pregnant females was provided by Charles River, and fetal age was assigned as embryonic (E) days 12-22 prior to sacrifice. Postnatal (P) pups were assigned ages based on day of birth being day P1. Pregnant rats and postnatal pups were deeply anesthetized with sodium pentobarbital and transcardially perfused with 0.9% NaC1 containing 2% sodium nitrite, followed with Zamboni's fixative. After perfusion was completed, whole embryos (El 1-El4) or fetal brains (E16-E22) were dissected from pregnant females. Whole brains were removed from postnatal pups after perfusion. All embryos, embryonic brains and postnatal brains were post-fixed in Zamboni fixative after perfusion. After post-fixation, all tissue was soaked in a buffered 10% sucrose solution, quick-frozen at -30~ Tissue was sectioned at 15 txm and thaw-mounted on polylysine-treated microscope slides, then stored at -80~ until used.

7.1.3. Human developmental brain tissue procurement Consent for use of donated materials was obtained from the University of Michigan Institutional Review Board for human subject use. Whole human brains (range 16-36 weeks' gestation) were collected from teratology donations to the University of Michigan Pathology Department. Tissues most commonly are donated to teratology after fetal demise, spontaneous loss, or neonatal death due to non-viable gestational age. These are primarily specimens that did not survive in the neonatal intensive care setting and, therefore, have not been exposed to prolonged therapy or stresses. During autopsy, whole brain specimens were removed, weighed, and immersed in Zamboni's fixative. In all specimens used, external examination of the brain was unremarkable. All human brain specimens remain immersed in Zamboni's fixative for a minimum of 80 days 155

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before use. Prior to processing, whole brains are placed in 10% sucrose for 72 h, with fresh sucrose change every 24 h. They are then photographed, blocked into 3-5-cm-thick sections and quick-frozen at -50~ in isopentane. Selected blocks of tissue were sectioned in the coronal plane at 15 txm and thaw-mounted on polylysine-treated slides then stored at -80~ until used. 7.1.4. Preproorphanin and ORL1 cRNA probes Hybridization of rat developmental brain tissue was performed using the same 35S-labeled riboprobes used in adult studies; the OFQ cRNA riboprobe was generated against a 580bp cDNA fragment containing the entire open reading frame of the preproOFQ precursor molecule, and the ORL1 cRNA riboprobe was generated from a 700 base cDNA extending from the 5' UT region to 611 bases within the coding region of the ORL1 receptor. In situ hybridization on human developmental tissue was performed using 35S-UTP and 35SCTP-labeled riboprobes generated against a 1200-base cDNA fragment encompassing the entire reading frame of the human ORL1 receptor, and a 900-base cDNA encoding human preproorphanin. 7.1.5. In situ hybridization The in situ hybridization technique employed for both developmental rat and human tissue, is identical to that described above for the detection of preproorphanin and ORL1 receptor mRNA in the adult rat brain. Briefly, slide-mounted 15-1xm sections were hybridized overnight using 35S-labeled probes, followed on day 2 with treatment in RNase A and decreasing salt concentration washes, then dehydrated. After hybridization, sections were opposed to Kodak XAR-5 X-ray film for 5 days (rat), or 21-30 days (human), then dipped in NTB2 film emulsion. Slides were developed following a 30-day exposure to NTB2 for rat tissue, and 60-90 days for human tissue. Sections were then Nissl counterstained with Cresyl violet and prepared for darkfield analysis and photography. 7.1.6. In situ hybridization controls The specificity of the human and rat preproOFQ and ORL1 riboprobes was determined using standard control measures. As described above, tissue pretreatment with RNase A, or use of 35S-labeled mRNA (sense) in the hybridization reaction were used as internal controls (see Fig. 15). 7.2. EXPRESSION OF OFQ IN THE DEVELOPING RAT BRAIN (Fig. 13) 7.2.1. E12-E22 PreproOFQ mRNA expression is initially observed on day 12 of embryonic development in early hypothalamic neuroepithelium medullary and pontine neuroepithelium and cervical spinal neuroepithelium. On embryonic day 14, expression is observed in the olfactory bulb, rhinencephalic neuroepithelia, cingulate cortex neuroepithelium and hippocampal neuroepithelium. Expression is also observed in the pallidal and striatal neuroepithelia, hypothalamic neuroepithelium and in the amygdala. In the El4 diencephalon expression is strong in the hypothalamus, zona incerta, medial and lateral geniculate bodies and pretectum. Strong 156

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Fig. 13. Distribution of preproOFQ mRNA expression in the developing rat brain at representativegestational and postnatal ages. Scale bar: 900 Ixm.

mRNA expression is also observed in many brainstem structures at this developmental stage, including the superior colliculus neuroepithelium, tegmentum, isthmus region, vestibular area neuroepithelium, spinal trigeminal nucleus, the hypoglossal nucleus, medulla and intermediate reticular zone. PreproOFQ is also expressed strongly in the spinal cord. By embryonic day 16, preproorphanin mRNA expression fills the septal region and diagonal band, olfactory tubercle and piriform cortex. Expression is observed in the neocortex, cingulate neuroepithelium and insular cortical region. The claustrum, bed nucleus of the stria terminalis, pallidum and hippocampal formation also have strong expression at this gestational age. The striatum is devoid of signal. Expression is strong in the hypothalamic, preoptic and thalamic neuroepithelia, the reticular thalamic nucleus, the arcuate nucleus and ventral region of the hypothalamus. In the amygdaloid region, OFQ expression is observed in the central and medial nuclei. Strong OFQ mRNA expression is also observed in the differentiating tegmental field, the medial geniculate body, the premammillary and early differentiating mammillary regions, the subthalamic nucleus and substantia nigra and pretectum and central gray. In the day 19 embryo, mRNA expression remains diffusely localized with intense expression in the septum, piriform and cingulate cortices, the olfactory tubercle, insular cortex, the frontal and parietal neocortex and the bed nucleus of the stria terminalis and medial 157

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preoptic area. Orphanin mRNA expression is also strong at this age in the central and medial nuclei of the amygdala, the anterior and posterolateral cortical amygdaloid nuclei, and in the thalamus, with intense label noted in the paraventricular and reticular nuclei. In the hypothalamus, expression is strong in the anterior hypothalamus, the arcuate and ventromedial nucleus, the dorsomedial and posterior regions and the paraventricular nucleus. Hippocampal expression remains strong. In the neocortex, mRNA expression is strong in the temporal and occipital cortical plate and subventricular cortical layer. Strong expression is also noted in the retrosplenial cortex, subiculum and entorhinal cortex. Strong expression is also noted in the medial geniculate nucleus, zona incerta, supramammillary and medial mammillary nuclei, the interpeduncular nucleus, the central gray, substantia nigra, nucleus of Darkschewitsch and interstitial nucleus of the medial longitudinal fasciculus. At parturition (E22/P1), orphanin expression is well defined and significantly more intense than is observed in the adult brain. Preproorphanin mRNA is expressed in similar structures as is observed in the El9 brain. Expression is still observed rostrally in the olfactory tubercle, tenia tecta, septal differentiating field and the cortical plate and subventricular zone of the neocortex. The cingulate, orbital, insular and piriform cortices contain strong expression, as does the septal region, indusium griseum, bed nucleus of the stria terminalis, pallidum, preoptic region, thalamic reticular nucleus, and the anterodorsal, centrolateral, anteromedial and periventricular thalamic nuclei. Strong expression is still found in the hippocampal formation, the dentate gyrus and areas CA1-CA3. Expression in the amygdala and hypothalamus is similar to that observed in the El9 embryo. Expression is also strong in the temporal and occipital neocortex, with strong expression in the retrosplenial cortex, subiculum and entorhinal cortex. In the thalamus, expression is strong in the posterior paraventricular and medial geniculate nuclei, the reticular nucleus and zona incerta. In the brainstem, OFQ is expressed strongly at this age in the pretectum, tegmental field, superior colliculus, mammillary region, ventral tegmental area, paranigral nucleus, substantia nigra, interpeduncular nucleus, nucleus of Darkschewitsch and interstitial nucleus of the medial longitudinal fasciculus. Diffuse and strong expression is observed throughout the midbrain reticular formation. 7.2.2. P7-adult

After 2 weeks of life, preproorphanin expression is in a distribution similar to that observed in the adult brain, but with more intensity in many structures. Expression is strong in the olfactory tubercle, cingulate and orbital cortex, lateral septum, tenia tecta, indusium griseum, diagonal band, bed nucleus of the stria terminalis, medial preoptic region, globus pallidus, granule cell layer of the dentate gyrus, areas CA1-CA3 of Ammon's horn, subiculum, thalamic reticular nucleus, the anterodorsal and anteroventral nuclei, the paratenial nucleus, the paraventricular nucleus and nucleus reunions. In the hypothalamus, expression is strong in the anterior, ventromedial and dorsomedial areas, and intense in the mammillary region. Robust expression is also noted in the medial, central, anterior and posterior cortical, and lateral amygdaloid nuclei. In the P7 midbrain, orphanin distribution is more localized. In the pretectum, tectum and geniculate bodies expression is moderate, and in the interpeduncular nucleus, substantia nigra, paranigral nucleus, nucleus of Darkschewitsch and interstitial nucleus of the medial longitudinal fasciculus, mRNA expression is intense. Strong expression is also observed in the ventral tegmental area, reticular formation, the central gray and dorsal raphe. In the P21 animal, orphanin distribution is essentially the same as in the fully developed 158

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animal. However, intensity of expression is still markedly increased in some areas of the P21 brain in comparison to that of the adult, including the cingulate cortex, the claustrum, the paraventricular, paratenial and anteroventral thalamic nuclei, subiculum and hippocampus, interpeduncular nucleus, substantia nigra, paranigral nucleus, ventral tegmental area, nucleus of Darkschewitsch and interstitial nucleus of the medial longitudinal fasciculus. By P21, mRNA expression in the medial septal nucleus and the anterodorsal and reunions thalamic nuclei become very light and remains so into the adult. PreproOFQ mRNA expression in the neocortex is also stronger in the P21 brain compared to the adult. This intense expression is likely due to strong labeling throughout the cortical subplate. In the cortical plate, distribution is similarly light to moderate as observed in the fully layered cortex. 7.3. EXPRESSION OF ORL1 IN THE DEVELOPING RAT BRAIN (Fig. 14) 7.3.1. E12-E22

Similar to preproOFQ expression, ORL1 mRNA is initially observed on day 12 of embryonic development. At this age, mRNA expression is observed in the cortical, hypothalamic,

Fig. 14. Distribution of ORL1 mRNA expression in the developing rat brain at representative gestational and postnatal ages. Scale bar: 900 lxm. 159

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thalamic and pontine neuroepithelia, the pretectum and isthmus, the medulla and spinal cord. Orphanin receptor mRNA expression becomes less diffuse by embryonic day 14. Expression is observed in the olfactory bulb, cingulate region, neocortical neuroepithelium, hippocampus, thalamus, hypothalamus and amygdala. In brainstem structures, expression is observed in the superior colliculus neuroepithelium, the isthmus region, the inferior colliculus, the tegmentum, central gray, the differentiating medulla, the spinal trigeminal nucleus and the ventral and dorsal horns of the differentiating spinal cord. On embryonic day 16, ORL1 mRNA is observed in the neocortex, developing septum, piriform and cingulate cortices, orbital and insular areas, the pallidum and the subicular area and developing Ammon's horn. The striatum is devoid of mRNA expression. Intense mRNA expression is observed in the preoptic region, the anterior hypothalamus, the habenula, the epithalamus, reticular nucleus and developing thalamic neuroepithelium, the medial, basomedial and basolateral amygdaloid nuclei, the hippocampus and the subiculum. Caudally, orphanin expression is intense in the arcuate and ventromedial nuclei of the hypothalamus, the intermediate thalamus, reticular nucleus and the medial and lateral geniculate nuclei. In the day 19 embryo, ORL1 mRNA expression is strong in the cortical plate of the cingulate, insular, piriform and neocortices. Expression is also strong in the anterior olfactory nucleus, the septal region, tenia tecta, the diagonal band, preoptic region, pallidum, the bed nucleus of the stria terminalis medial and lateral parts, the hippocampal formation and the subiculum. The hypothalamus and developing neuroepithelium contain strong mRNA expression, particularly in the ventromedial and arcuate nuclei. Expression at this stage is also observed in the paraventricular nucleus, anterior and lateral hypothalamus, the retrochiasmatic supraoptic nucleus the habenula, the paraventricular, mediodorsal and centromedial thalamic nuclei, the medial amygdaloid nucleus, the bed nucleus of the lateral olfactory tract, Ammon's horn and dentate gyrus. Caudally, cortical expression is strong in the cortical plate in the temporal and occipital cortices, with lighter signal in the subplate region. Expression in the retrosplenial cortex and subiculum is strong. Signal is also observed in the subthalamic nucleus, lateral geniculate nucleus, midbrain pretectum, interpeduncular nucleus, ventral tegmentum, substantia nigra, anterior pons and medulla, particularly in the raphe and inferior olive. At parturition (E22/P1), ORL1 expression is well defined in most brain regions, with a distribution similar to that of the adult. Expression is observed in the anterior olfactory nucleus the orbital and insular cortices, the insular and piriform cortices, the cingulate cortex, tenia tecta and cortical plate of the neocortex. Expression is also strong in the claustrum, lateral septum and septal neuroepithelium, endopiriform cortex, the bed nucleus of the stria terminalis, the preoptic region, the hippocampal formation in CA1-CA3 of Ammon's horn and the dentate gyrus, and the paraventricular, anteromedial and anteroventral nuclei of the thalamus. The rostral accumbens and differentiating striatum contain moderate mRNA expression at this age, although this signal becomes very light at more caudal levels. Expression is moderate in the globus pallidus, the entopeduncular nucleus, the nucleus of the lateral olfactory tract the medial nucleus, the basomedial nucleus, the anterior cortical nucleus, the posterior cortical nucleus of the amygdala and the amygdalohippocampal area. In the hypothalamus, mRNA expression is observed in the anterior hypothalamic area, the paraventricular nucleus, ventromedial nucleus, arcuate nucleus and the mammillary nuclei. In the cortex, expression is strong in the cortical plate of the temporal and occipital neocortices. The entorhinal cortex contains intense mRNA expression. Signal is also observed in the subthalamic nucleus, the lateral geniculate nucleus, the parafascicular nucleus, medial geniculate nucleus and central gray, the substantia nigra and ventral tegmental area, the 160

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superior colliculus, the interpeduncular nucleus, red nucleus, medial geniculate nucleus, the dorsal raphe and midbrain reticular formation.

7.3.2. P7-adult By 2 weeks of life, ORL 1 mRNA expression is quite similar to that observed in the adult brain with the exception of some increased intensity in some structures, particularly the neocortex, hippocampus and thalamus. In the P14 brain, strong mRNA expression is observed in the anterior olfactory nucleus, the orbital cortex, cingulate, piriform and neocortical regions, the lateral septum, the medial and lateral divisions of the bed nucleus of the stria terminalis, the medial preoptic region, the medial preoptic nucleus, areas CA1-CA3 of Ammon's horn, the dentate gyrus, subiculum, medial, basomedial and anterior cortical amygdaloid nuclei, anterior hypothalamus, paraventricular, ventromedial and arcuate nuclei and the mammillary nuclei. In the thalamus, robust expression is observed in the paraventricular nucleus and the anteromedial and anteroventral nuclei rostrally, and in the medial habenula, posterior paraventricular nucleus, laterodorsal, ventrolateral and reunions nuclei and medial geniculate nucleus caudally. In the caudal cortical regions, expression remains strong in the retrosplenial, entorhinal and temporal cortices. In the brainstem, mRNA expression is robust in the midbrain in the parafascicular nucleus, medial geniculate nucleus, rostral central gray, superior colliculus, substantia nigra, ventral tegmental area, interpeduncular nucleus and red nucleus. By postnatal day P21, ORL1 mRNA distribution is essentially identical to that of the fully developed animal. Expression remains strong in the anterior olfactory nucleus and orbital cortex. In the cingulate, retrosplenial, insular and piriform cortices and the neocortex signal is generally stronger in intensity than that observed in the adult. Expression is also strong in the lateral septum, horizontal limb of the diagonal band of Broca, claustrum, endopiriform nucleus, the bed nucleus of the stria terminalis and the medial preoptic area. In the thalamus, strong signal is observed in the paratenial and paraventricular thalamic nuclei, the anteromedial and anteroventral nuclei, the medial habenula, the posterior paraventricular nucleus, the laterodorsal, ventrolateral and reunions nuclei and the medial geniculate nucleus. The hippocampal formation, subiculum, medial, basomedial and anterior cortical amygdaloid nuclei, suprachiasmatic and supraoptic nuclei, the paraventricular nucleus, ventromedial nucleus, arcuate nucleus and mammillary region contain strong mRNA expression. Caudally, ORL 1 mRNA expression remains strong in the retrosplenial, entorhinal and temporal cortices. Expression is also strong in the parafascicular nucleus, medial geniculate nucleus, central gray, superior colliculus, substantia nigra, ventral tegmental area, red nucleus and interpeduncular nucleus, in a pattern intensity identical to that observed in the adult. 7.4. OFQ AND ORL1 mRNA EXPRESSION OF IN THE DEVELOPING HUMAN BRAIN

7.4.1. OFQ (Figs. 15 and 16) Preproorphanin mRNA expression is robust in the human brain by 16 weeks' gestation. Clear, strong expression is observed in the reticular thalamic nucleus and zona incerta, the ventral and medial developing thalamic neuroepithelium, the internal pallidum, differentiating medial and central nuclei of the amygdala and the parahippocampal amygdaloid transition area. Strong expression is also seen in the entorhinal cortex and the hypothalamus in the region of the developing paraventricular nucleus and dorsal hypothalamic area. By 19 weeks' 161

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Fig. 15. Distribution of preproOFQ mRNA expression in the developing human brain at gestational ages of 16 and 21 weeks (top panels). Corresponding sense and RNase controls are shown in the bottom panels. Scale bar: 5250 I~m.

162

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Fig. 16. Distribution of preproOFQ mRNAexpression in the developing human brain at gestational ages of 19 and 30 weeks. Scalebar: 5250 Ixm.

gestation specific expression is noted in the cortical plate, with minimal subplate expression. Strong expression is also observed in the globus pallidus, the reticular nucleus and zona incerta, the ventral thalamic neuroepithelium, and the amygdala and hypothalamus. By 2122 weeks' gestation, expression increases dramatically in the developing cortical plate and subplate region. No expression is noted in the caudate, putamen, claustrum or accumbens region. Strong mRNA expression is noted in the septal region, reticular nucleus, zona incerta, globus pallidus, mammillary bodies, medial and central amygdaloid nuclei, hippocampus, subthalamic nucleus, substantia nigra and medial geniculate body. There is little remarkable change in these expression patterns up to 36 weeks' gestation in the forebrain. At 25 weeks' gestation cortical preproOFQ mRNA expression remains strong and is more organized in a laminar distribution in the cortical plate. Strongest cortical expression is noted in the cingulate region. Expression remains strong in the reticular nucleus and zona incerta, and abundant preproOFQ is also observed in the anterior thalamic nucleus. At 30 weeks' gestation, expression is noted in the cortical plate, in the reticular thalamic nucleus, the centromedian nucleus, the globus pallidus, the claustrum and the amygdala. At 35 weeks' gestation, expression remains laminar in distribution and strong in the cortical plate. Specific OFQ mRNA expression is also observed early in the developing brainstem. At 16 weeks' gestation, mRNA expression is noted in the pons near the vestibular nuclei, the pontine neuroepithelium near the developing locus coeruleus and midline raphe, the developing medullary reticular formation, nucleus ambiguous and nucleus of the solitary tract. In the 21-week brainstem, mRNA expression is observed in the lateral and ventral midbrain tegmental region, the pontine neuroepithelium near the ventral aqueduct, the lateral developing reticular fields, the inferior olive, midline reticular region, and adjacent to the 163

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Fig. 17. Distribution of ORL1 mRNA expression in the developing human brain at gestational ages of 16, 25 and 30 weeks. Scale bar: 5250 I~m.

fourth ventricle, near the developing vestibular nuclei and locus coeruleus. In the 30-week brainstem, orphanin expression is observed in the basal neuroepithelium and peri-aqueductal region of the rostral pons, the medullary spinal trigeminal nucleus and the caudal dorsal medulla. 7.4.2. ORL1 (Fig. 17) Similar to preproorphanin, ORL1 mRNA expression is robust in the human brain by 16 weeks' gestation. At this stage of development, no mRNA expression is found in the cortical plate, subplate region or germinal matrix. Signal is detected in the head of the caudate, the 164

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putamen, the claustrum, the region of the developing olfactory tubercle, the region of the anterior and ventral anterior thalamic nuclei, the reticular thalamus and the developing rostral hypothalamus. At 19 weeks' gestational age, strong signal is detected in the cortical plate, with light subplate expression. Expression is still observed in the head of the caudate, the putamen, the anterior and ventral anterior thalamus and rostral hypothalamus. In the 21-22-week brain ORL1 expression remains strong in the cortical plate and is stronger and more laminar than that observed at 16 weeks. Expression remains strong in the olfactory region, the head of the caudate and the putamen. The principal anterior, ventral anterior, anteromedial, paratenial and paraventricular thalamic nuclei contain mRNA expression. The dorsal, dorsomedial, ventromedial and paraventricular hypothalamic nuclei, the subthalamic nucleus, the mediodorsal, centromedial, laterodorsal and parafascicular thalamic nuclei, and the red nucleus are also moderately labeled. Intense label is observed by this gestational age in the dentate gyms of the hippocampus, Ammon's horn, and in caudal cortical regions, including entorhinal, temporal and occipital lobes. In the 25-week brain, ORL1 mRNA expression is observed in the caudate and putamen, the claustrum, and in the cortical plate of the neocortex, cingulate cortex and insular region. In the 29-week brain, strong expression is still observed in the developing hippocampus and subiculum. Cortical and thalamic mRNA expression remain strong. In the 35-week brain, cortical ORL1 expression remains strong, particularly in the deeper layers. Specific signal is still observed in the caudate and putamen, but decreased from that observed in earlier gestation. Expression is still observed in the hippocampus, subiculum and entorhinal cortex. Orphanin receptor mRNA expression is observed in the developing brainstem, though less clearly than that observed with preproOFQ. In the 16-week brainstem, ORL1 expression is observed in the lateral midbrain reticular formation, ventral tegmentum, the ventral pontine midline, the region of the nucleus ambiguous, the hypoglossal nucleus, the caudal trigeminal nucleus and the developing solitary nucleus and midline reticular formation. In the 22-week brainstem, expression is observed in the dorsal peri-aqueductal region, the geniculate bodies, the peripeduncular and ventral tegmental region, the reticular formation, in the inferior olive, the vestibular and cochlear nuclei, the parabrachial nucleus, the pontine reticular formation and the midline raphe. In the 35-week brainstem, light expression is observed in the region of the dorsal raphe, in the ventral tegmentum, the ventral pontine peri-aqueductal region and reticular formation, the region of the vestibular nuclei and in trigeminal nucleus. 8. PHYSIOLOGICAL IMPLICATIONS OF OFQ AND THE ORL1 RECEPTOR

8.1. COMPARISONS WITH ENDOGENOUS OPIOID SYSTEMS The orphanin receptor has been shown to differ markedly from the known opioid receptors in its neuroanatomical distribution (Chen et al., 1994; Fukuda et al., 1994; Lachowicz et al., 1994; Anton et al., 1996; Neal et al., 1999b). Likewise, the distribution of OFQ is distinct from that of the endogenous opioid peptides (Neal et al., 1999a). The distribution of the endogenous opioid peptide precursors, proopiomelanocortin, proenkephalin and prodynorphin (Kachaturian et al., 1985) and that of the corresponding ~, K and ~ receptors (Mansour et al., 1987, 1994, 1995b) has been studied in detail. Analysis and comparison of the distributions of these peptide-receptor systems does point to several brain regions where the opioid and orphanin systems may interact. However, it should be noted that the possible modulation 165

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of opioid and orphanin systems by one another will not be direct, as opioid peptides have essentially no affinity for ORL1 receptor (Bunzow et al., 1994; Chen et al., 1994; Fukuda et al., 1994; Lachowicz et al., 1994; Wick et al., 1994; Ma et al., 1997; Nicholson et al., 1998), and OFQ has no affinity for the endogenous opioid receptors (Reinscheid et al., 1995). There may be some interactions between these systems, however, due to their localization in, or interactions with similar neuronal systems. 8.1.1. Proopiomelanocortin and the IX receptor Proopiomelanocortin gives rise to ~-endorphin, which has a limited cell body distribution, but has a wide CNS distribution within fibers and terminals (Kachaturian et al., 1985). In contrast, the IX receptor is widely distributed within both fibers and cell bodies (Mansour et al., 1987, 1994, 1995a,b). The arcuate nucleus of the hypothalamus and nucleus of the solitary tract are the only structures where cell bodies may express both OFQ and [3-endorphin. Additionally, these structures have been shown to contain ORL1 receptor mRNA expression and exhibit orphanin binding. Orphanin may play a modulatory role on [3-endorphin-containing neurons in these brain regions. Within the bed nucleus of the stria terminalis, central and medial nuclei of the amygdala, dorsomedial nucleus of the hypothalamus, paraventricular nucleus of the thalamus, central gray, dorsal raphe, lateral parabrachial nucleus and medullary raphe system, abundant ~-endorphin-containing fibers are present. Orphanin immunolabeling and ORL1 receptor activity are present in these areas as well, providing further regions where OFQ may play a modulatory role on the [~-endorphin system. Similar to possible modulatory effects on the ~-endorphin system, orphanin may modulate IX receptor effects as well. Both IX and ORL1 receptor binding is present in the neocortex, medial amygdala, dentate gyms, thalamus, substantia nigra, pars compacta, superior colliculus, parabrachial nucleus, nucleus ambiguous, solitary nucleus, spinal trigeminal nucleus and lamina II of the spinal dorsal horn. In the dentate gyms, the IX receptor localizes to interneurons within the granule cell layer in a pattern very similar to OFQ immunoreactivity. tx Receptor expression has been reported in cell bodies within the central gray, raphe nuclei, lateral parabrachial nucleus, nucleus ambiguous, solitary nucleus and lamina II of the dorsal horn, in a distribution very similar to what has been observed for orphanin. Orphanin is also localized in neurons in pars compacta of the substantia nigra, another area where the Ix receptor is localized within cell bodies and binding is dense. Other areas where OFQ is strongly expressed and Ix receptor expression is robust include the medial amygdaloid nucleus and paraventricular nucleus of the thalamus. 8.1.2. Prodynorphin and the K receptor Prodynorphin-containing cell bodies and fibers, and ~: receptor localization and binding are widely distributed throughout the rat CNS (Kachaturian et al., 1985; Mansour et al., 1987, 1994, 1995b). There are several brain regions that may be sites of dynorphin and orphanin system interactions. Regions where orphanin-containing cell bodies could co-localize with dynorphin-containing neurons, include the dentate gyms, central nucleus of the amygdala, paraventricular and arcuate nuclei of the hypothalamus, lateral hypothalamic area, pars reticulata of substantia nigra, the central gray, interpeduncular nucleus, raphe magnus, lateral reticular nucleus, solitary nucleus, spinal trigeminal nucleus and dorsal horn of the spinal cord. Within area CA3 of Ammon's horn, the central gray, parabrachial nucleus, caudal spinal trigeminal nucleus, nucleus of the solitary tract and dorsal horn of the spinal cord, regions 166

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with significant dynorphin fiber labeling and K receptor localization, orphanin is strongly expressed in cells and fibers. Orphanin binding is also strong in these regions. There are also several regions where OFQ binding could co-exist with dynorphincontaining cell bodies, including the dentate gyrus, the arcuate and ventromedial hypothalamic nuclei, substantia nigra, central gray, parabrachial nucleus, nucleus of the solitary tract, caudal spinal trigeminal nucleus and dorsal horn of the spinal cord. In such regions, ORL1 receptor activation may directly influence the dynorphin system. Within the medial preoptic area, bed nucleus of the stria terminalis, paraventricular thalamic nucleus, medial nucleus of the amygdala, ventromedial hypothalamus, superficial gray layer of the superior colliculus, central gray, dorsal raphe, parabrachial nucleus, nucleus of the solitary tract and spinal trigeminal nucleus, robust ORL1 and K receptor localization and binding is observed. These areas provide sites for possible cross modulation of ORL1 and K activation. The core of nucleus accumbens, the medial preoptic area, bed nucleus of the stria terminalis, paraventricular and ventromedial nuclei of the hypothalamus, median eminence and medial nucleus of the amygdala contain abundant OFQ fiber and cell staining and robust K receptor localization and binding. These distribution patterns provide evidence for possible modulation of orphanin FQ within these structures.

8.1.3. Proenkephalin and the 8 receptor Proenkephalin peptides are the most widely distributed endogenous opioids (Kachaturian et al., 1985). Distribution of the 3 receptor is more modest (Mansour et al., 1987, 1994, 1995b). Enkephalin-containing neurons may be co-localized with OFQ in several structures where orphanin-containing neurons are also present, including the cingulate and piriform cortices, diagonal band, bed nucleus of the stria terminalis, central nucleus of the amygdala, hippocampal formation, paraventricular and arcuate nuclei of the hypothalamus, central gray, interpeduncular nucleus, paramedian raphe and raphe magnus, lateral reticular nucleus, solitary nucleus, spinal trigeminal nucleus and dorsal horn of the spinal cord. Many of these structures also demonstrate ORL1 expression and binding. The function of enkephalincontaining neurons could be influenced by activation of the ORL1 receptor where both are expressed, including the cingulate and piriform cortices, paraventricular and arcuate nuclei of the hypothalamus, hippocampal formation, central gray, paramedian raphe and raphe magnus, solitary nucleus, spinal trigeminal nucleus and dorsal horn of the spinal cord. Enkephalincontaining fibers in the ventromedial nucleus of the hypothalamus, hippocampal formation, central gray, interpeduncular nucleus, solitary nucleus and dorsal horn of the spinal cord are also within regions where OFQ binding is present, providing another milieu where ORL1 activation may influence enkephalin activity. In myenteric plexus preparations, OFQ has been shown to modulate enkephalin release (Gintzler et al., 1997), providing evidence that such an interaction may also occur in the CNS. Dense ORL1 and 3 receptor binding is observed in the ventromedial nucleus of the hypothalamus, medial nucleus of the amygdala, interpeduncular nucleus, spinal trigeminal nucleus and substantia gelatinosa of the dorsal horn. Orphanin cell and/or fiber immunolabeling is observed in these regions and also in the cingulate cortex, the neocortex and nucleus accumbens. This overlapping distribution of the 3 receptor with orphanin receptor and peptide provides sites for possible cross modulation of 3 and ORL 1 receptor activation, or enkephalin or other opioid modulation of the orphanin peptide.

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8.2. FUNCTIONAL CONSIDERATIONS OF ORPHANIN FQ AND ORL1 CIRCUITRY

8.2.1. The limbic hypothalamic-pituitary-adrenal (L-HPA) axis The L-HPA axis is a steroid-sensitive system with many nuclei sensitive to circulating glucocorticoid levels. The function of the stress axis at the behavioral, anatomical and cellular level has been extensively studied (Herman et al., 1996; Herman and Cullinan, 1997). In the initiation of the stress response, the two types of stressors involved include an immediate physiologic threat (systemic stressors), and those requiting interpretation by higher brain systems (processive stressors). Whereas processive stressors are channeled through limbic forebrain circuits then to the hypothalamic paraventricular nucleus, systemic stressors are likely relayed directly to the paraventricular nucleus via brainstem cholinergic systems. Although the paraventricular nucleus contains only light orphanin-like immunoreactivity, it demonstrates strong ORL1 mRNA expression and binding and receives afferent input from several stress-related structures with orphanin-like immunoreactivity. Considerable evidence supports a role for brainstem catecholaminergic neurons in the regulation of the stress axis (Plotsky et al., 1989). The stimulatory action of these neurons is likely by way of ~l-adrenergic receptors. Direct noradrenergic input to the parvocellular paraventricular nucleus is via the ventral noradrenergic bundle, supplied primarily by the A2, C1, C2 and C3 brainstem cell groups. Orphanin peptide is not expressed in these regions, making it unlikely that OFQ plays a modulatory role in these direct inputs. However, the locus coeruleus is one of the most stress-responsive nuclei in the brain (Herman et al., 1996) and contains moderate OFQ and intense ORL1 expression and binding. Evidence supports the notion that this nucleus likely modulates L-HPA activation indirectly via multisynaptic connections through forebrain regions such as the prefrontal cortex, hippocampus and amygdala. Orphanin and ORL1 are strongly expressed in these regions, providing areas where OFQ may modulate noradrenergic input into the stress axis. Orphanin and ORL1 are also localized within many elements within the raphe system, providing possible involvement of OFQ in the modulation of serotonergic activation of the L-HPA axis. Orphanin immunolabeling is prominent in many forebrain regions with projections to the paraventricular nucleus, including the subfornical organ, median preoptic nucleus, medial preoptic area, bed nucleus of the stria terminalis, periventricular nucleus, arcuate nucleus, dorsomedial nucleus lateral hypothalamus, medial nucleus of the amygdala and central nucleus of the amygdala. Of these forebrain nuclei, mineralocorticoid receptors are found in the medial, central and posterior cortical nuclei of the amygdala, and the lateral bed nucleus of the stria terminalis. Glucocorticoid receptors are found in the medial preoptic area, arcuate nucleus, medial bed nucleus of the stria terminalis, ventromedial nucleus of the hypothalamus and central nucleus of the amygdala. These paraventricular inputs are extensive and the orphanin system may play a modulatory role in the stress response via these connections, given the robust ORL1 binding observed in the glucocorticoid responsive paraventricular nucleus. Other forebrain regions with input in the L-HPA circuitry and prominent OFQ immunolabeling and ORL1 expression are the ventral subiculum, prefrontal cortex and hippocampal formation. All of these regions also contain glucocorticoid receptors. Recent evidence does support a possible role for orphanin in the stress response (Devine et al., 2001). In the rat, orphanin administration modulates anxiety states generated by acute stress (Griebel et al., 1999; Jenck et al., 1997, 2000; Redrobe et al., 2001; Gavioli et al., 2002; Kapusta et al., 2002) and prevents stress-induced ethanol-seeking behavior (Martin-Fardon et al., 2000). Additionally, targeted disruption of the OFQ gene in mice appears to increase 168

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stress susceptibility and impair stress adaptation (Koster et al., 1999). Given the abundance of possible OFQ-containing inputs into the stress axis and the possible anxiolytic role of orphanin in behavioral studies, orphanin's modulatory role on the stress response may be via an inhibitory influence on L-HPA activation.

8.2.2. Learning and memory The hippocampal formation is not only intimately involved in the function of the L-HPA axis, it is also a critical structure in learning and memory. Orphanin peptide, orphanin receptor mRNA expression and ORL 1 binding are found throughout the hippocampal formation. They are also found in the entorhinal cortex, which has direct projections to the entire hippocampal formation. Orphanin has been reported to reversibly inhibit voltage-gated calcium channels in pyramidal neurons from area CA1 and CA3 (Knoflach et al., 1996), augment potassium currents in hippocampal CA1 neurons (Madamba et al., 1999; Amano et al., 2000), and postsynaptically inhibit synaptic transmission and long-term potentiation in area CA1 and the dentate gyrus (Yu et al., 1997; Yu and Xie, 1998). Scopolamine-induced impairment of learning has been shown to be improved by OFQ administration (Hiramatsu and Inoue, 1999). Additionally, microinjection of OFQ into the CA3 region of adult rats has been shown to impair spatial learning (Sandin et al., 1997), and knockout mice lacking the orphanin receptor demonstrate enhancement of spatial attention in the water-finding test (Mamiya et al., 1998) and facilitation of long-term potentiation and memory (Manabe et al., 1998). Behavioral studies appear to support a role for this peptide system in learning and memory and its distribution throughout the hippocampal formation and entorhinal cortex supports this concept as well (Higgins et al., 2002).

8.2.3. Motor systems Orphanin-containing neurons and fibers are located in the globus pallidus, entopeduncular nucleus and substantia nigra. In addition, the lateral bed nucleus of the stria terminalis, a component of the extended central amygdaloid complex (Alheid et al., 1995), has abundant OFQ peptide content and ORL1 binding. This structure projects to the retrorubral field, ventral tegmental area and substantia nigra pars compacta, structures which possess abundant ORL1 receptor binding and give rise to major dopaminergic projections. Interestingly, in pars compacta, very few dopamine-containing neurons co-localize with OFQ, while virtually every dopamine-containing neuron also contains ORL1 mRNA expression (Norton et al., 2002; Maidment et al., 2002). The presence of orphanin in motor circuitry and co-existence of ORL 1 activity with dopamine in substantia nigra provides anatomical evidence for an orphanin modulatory role in motor systems. Several behaviors observed after central administration of this peptide may reflect a role of OFQ in the modulation of gross locomotive behavior (Devine et al., 1996a, Rizzi et al., 2001 a). Orphanin peptide and receptor binding is also localized within brainstem structures that direct several aspects of fine motor control and balance, including vestibular nuclei, deep cerebellar nuclei and the inferior olive. Proprioceptive input from the dorsal columns of the spinal cord is integrated in the dorsal olivary nucleus, which relays information to the cerebellar vermis, which in turn has very strong inputs into the medial (fastigial) cerebellar nucleus. The fastigial nucleus projects to nuclei involved with visual integration, and the spinal vestibular nucleus. Orphanin-containing cell bodies are consistently expressed throughout this circuitry and orphanin binding is prominent in many of these structures. Input into the 169

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principal olivary nucleus primarily originates in visual integration areas and association cortices. Orphanin FQ content is very heavy in the visual nuclei which project to the principal olivary nucleus (nucleus of Darkschewitsch, interstitial nucleus of the medial longitudinal fasciculus and the medial accessory oculomotor nucleus), which has moderate ORL1 mRNA expression, but demonstrates intense OFQ binding. This nucleus in turn projects to the lateral cerebellar hemispheres which have heavy input to vestibular nuclei. The vestibular nuclei, via immense efferent projections to the thalamus, inferior olive, cerebellum, reticular formation and spinal cord, provide substantial integration of proprioceptive information. Orphanin's effects on gross motor function are largely unknown at this point. Orphanin has been implicated in producing hypolocomotion in rats (Devine et al., 1996a) and mice (Rizzi et al., 2001a) and in stimulating locomotion and exploratory behavior in mice (Florin et al., 1996). Orphanin peptide and ORL1 receptor binding is observed within many brain structures intimately involved in locomotion, balance and proprioception. It remains to be seen exactly what role orphanin plays in motor function, but the presence of OFQ and ORL 1 binding in the basal ganglia, and the vestibular and visual motor systems supports a modulatory role for this peptide in these functions and the effects of OFQ on motor function should be considered in analysis of other behavioral effects. 8.2.4. Reinforcement and reward

The nucleus accumbens has been implicated in a number of functions, including drug reinforcement and locomotor behavior (Koob et al., 1991). Little is known regarding possible motivational effects of OFQ in relationship to this region. Rats have been shown to develop tolerance to locomotor effects induced by orphanin (Devine et al., 1996a), but they fail to demonstrate conditioned place preference when given intraventricular injections of this peptide (Devine et al., 1996b). Orphanin is strongly expressed in several structures involved in this circuitry, including the substantia nigra and ventral tegmental area. These regions contain direct projections to the nucleus accumbens and ventral pallidum. Although some OFQ fiber labeling in the accumbens nucleus is observed, expression within cell bodies in this structure is negligible. Accumbens has also been reported to contain ORL 1 immunoreactivity (Anton et al., 1996), but OFQ binding is minimal. However, it has been reported that injections of OFQ into the lateral ventricle of the rat suppresses dopamine release in the nucleus accumbens (Murphy et al., 1996). As mentioned above, although few dopamine-containing neurons in the substantia nigra pars compacta possess OFQ mRNA expression, virtually every dopaminecontaining neuron co-localizes with ORL 1 (Maidment et al., 2002; Norton et al., 2002; Zheng et al., 2002). Therefore, orphanin effects on this circuitry may lie upstream in the dopaminecontaining neurons of pars compacta of the substantia nigra or the ventral tegmental area, rather than directly in the accumbens nucleus itself. Little is known concerning behavioral effects of OFQ in relationship to these structures, but the abundance of orphanin expression and ORL1 expression and binding within this mesoaccumbens circuitry may indicate a modulatory role for this peptide system in motivational and motor-related behaviors. One example of a rewarding activity that appears to be influenced by the orphanin system is feeding behavior. Feeding as a behavioral act is complicated, requiting the integration of olfactory, visual and auditory information, and many brainstem centers involved in the acts of mastication and digestion. Pomonis et al. (1996) demonstrated that injections of OFQ into the lateral ventricle of the rat stimulates feeding to a degree similar to that seen with injection of other opiates. This response, however, was blocked by naloxone, making it more likely that it may have been an opiate-driven event. More recently, Stratford et al. (1997) demonstrated 170

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that injections of orphanin into the ventromedial hypothalamic nucleus or nucleus accumbens increases food intake in rats. The role of OFQ in stimulation of feeding behavior gains further support by studies demonstrating a reduction of orphanin-induced hyperphagia (Ciccocioppo et al., 2001; Olszewski et al., 2002; Pietras and Rowland, 2002) by central administration of an ORL1 antisense probe (Leventhal et al., 1998) or nocistatin, a preproorphanin peptide known to have antagonistic effects to OFQ. In addition, following intraventricular administration of OFQ in rats, c-fos expression is observed in several feeding related brain areas including the nucleus of the solitary tract, paraventricular and supraoptic nuclei of the hypothalamus, central nucleus of the amygdala, lateral septum and lateral habenula (Olszewski et al., 2000). Other than the central amygdaloid nucleus, where ORL 1 expression and binding is negligible, orphanin binding to the ORL1 receptor is strong in these nuclei. The lateral septum, which contains both high OFQ content and ORL1 mRNA expression and binding, has direct projections to the medial hypothalamus, and the lateral hypothalamic area, structures that may be vital in the initiation and control of food intake (King and Nance, 1986). Much remains to be answered concerning the possible role of OFQ in mechanisms of motivation and reinforcement-driven behaviors, such as feeding. Behavioral findings, coupled with the presence of OFQ and ORL1 receptor binding in structures fundamental to these behaviors, warrants further investigation into the role of the orphanin system in the integration of this circuitry. 8.2.5. Sexual behavior

The anatomic distribution of the orphanin system in sexually dimorphic pathways supports a possible functional role of OFQ in sexual behavior. Evidence for sexual dimorphism was initially reported in the medial preoptic area (Gorski et al., 1980), emerging early in development with differences in neuronal production and regulated by circulating gonadal steroids (Davis et al., 1996; Jacobson and Gorski, 1981). A steroid-sensitive, enkephalinergic sexual dimorphism has also been described in the medial preoptic region (Watson et al., 1986; Simerly et al., 1988). Several sexually dimorphic nuclei have major connections to the medial preoptic area, including the medial amygdala (Nishizuka and Arai, 1983), medial and lateral parts of the bed nucleus of the stria terminalis (Guillamon et al., 1988), and the arcuate and ventromedial hypothalamic nuclei (Borisova et al., 1996). The circuitry primarily involving the medial preoptic area, bed nucleus of the stria terminalis and medial nucleus of the amygdala is not only sexually dimorphic, but it has also been shown to be critical for normal sexual behavior in the male hamster (Neal and Newman, 1989). The lateral septum, also a sexually dimorphic and sex hormone-sensitive nucleus (Jakab and Leranth, 1995), provides significant hypothalamic projections and receives concentrated input from the hippocampal formation, another sexually dimorphic region (Madeira et al., 1991). The lateral septum, medial preoptic area, medial amygdaloid nucleus, bed nucleus of the stria terminalis and arcuate nucleus contain intense orphanin and ORL1 mRNA expression, and strong orphanin binding. The ventromedial hypothalamus contains the most intense ORL1 mRNA expression and OFQ binding in the hypothalamus. Although little behavioral evidence to date has demonstrated a strong connection between sexual activity and the orphanin system, microinjections of OFQ into the ventromedial nucleus of the hypothalamus has been shown to facilitate lordosis in the female rats (Sinchak et al., 1997). What role orphanin plays in sexual behavior is not known. However, its expression throughout this sexually dimorphic circuitry makes it likely that orphanin will play a major role in the development or maintenance of this activity. 171

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8.2.6. Pain perception The nature of orphanin's effects on the pain response remain controversial (Henderson and McKnight, 1997; Mogil and Pasternak, 2001), with studies demonstrating orphanin-induced analgesia in some models (King et al., 1997; Rossi et al., 1997; Tian et al., 1997a; Yamamoto et al., 1997; Rizzi et al., 2001b), and orphanin-induced nociception in others, tempting some to depict its actions as anti-opioid (Grisel et al., 1996; Mogil et al., 1996a,b; Xu et al., 1996; Dawson-Basoa and Gintzler, 1997; Heinricher et al., 1997; Morgan et al., 1997; Jhamandas et al., 1998; Yu et al., 2002). Orphanin has also been implicated as an antagonist of morphine analgesia (Hao et al., 1997; Tian et al., 1997b; Zhu et al., 1997). In spite of conflicting studies, the anatomical localization of OFQ and ORL1 within vital proximity to the ascending and descending opioid pain pathways, and mounting physiologic and behavioral evidence appear to support a major role for the orphanin system in pain modulation. Much is known concerning the ascending and descending pain pathways involving the endogenous opioid systems (Mansour et al., 1995b). Orphanin peptide, ORL1 mRNA expression and orphanin binding are robustly present along all parts of this pain circuitry, with OFQ-containing neurons and fibers and ORL1 activity found in the central gray, the raphe nuclei, lateral parabrachial nucleus, reticular formation nuclei, spinal trigeminal nucleus and the superficial laminae of the spinal cord. Interestingly, although co-localization has not been demonstrated, OFQ and its ORL1 receptor have been shown to have an overlapping distribution with opioid peptides in several of these regions involved in opioid pain modulation, including the dorsal horn of the spinal cord, spinal trigeminal nucleus, raphe nuclei, locus coeruleus and central gray (Schulz et al., 1996). In addition, there is an accumulating body of evidence supporting modulatory influences of OFQ on painful inputs into the central nervous system, as both an agonist and antagonist to analgesia. Microinjections of morphine and OFQ into the central gray demonstrate OFQ attenuation of morphine-induced analgesia in awake rats (Morgan et al., 1997), and microinjections of OFQ alone demonstrates inhibitory actions on central gray neurons (Vaughan et al., 1997). Opioid- and orphanin-containing cell bodies are also numerous in the rostral ventromedial medulla, a region where ORL1 receptor mRNA expression and OFQ binding has been identified. This region includes reticular formation and raphe neurons that are known to have an antinociceptive influence to noxious stimuli. Electrophysiological studies in this region have demonstrated an inhibitory role of OFQ on the firing of these neurons in the presence of morphine activation, leading investigators to hypothesize that OFQ exerts an 'antiopioid' effect by suppressing firing of these cells (Heinricher et al., 1997). Orphanin immunoreactivity has also been quantitatively demonstrated in lamina II of the dorsal horn, and OFQ-containing fibers paralleling endogenous opioids have been identified (Riedl et al., 1996; Schuligoi et al., 1997; Ahmadi et al., 2001a,b; Luo et al., 2001). Cell recordings from these substantia gelatinosa neurons have shown that exogenous OFQ depresses excitatory postsynaptic potentials evoked by stimulation of dorsal root ganglia (Lai et al., 1997; Liebel et al., 1997; Jennings, 2001). Additionally, intrathecally administered OFQ has been shown to inhibit C-fiber evoked discharge of dorsal horn neurons (Stanfa et al., 1996). These studies to date clearly support a role for the orphanin system within the pain pathways of the rat CNS. Given the overlapping distribution of orphanin with opioid peptides in several of these regions (Schulz et al., 1996) and the observation that ORL 1 and tx antisera label different fibers in pain processing regions of the rat brain (Monteillet et al., 1998), it is most likely that orphanin and endogenous opioid influences on pain perception occur via predominantly different fiber systems. 172

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8.2.7. Autonomic and physiologic functions Several studies have suggested a role for the orphanin system in basic autonomic and physiologic mechanisms. Orphanin activation of the ORL1 receptor has been shown to induce vasorelaxation (Gumusel et al., 1997), hypotension (Champion and Kadowitz, 1997a,b) and decreased cardiac output (Champion et al., 1997) in rats. Intraventricular administration of OFQ induces diuresis and antinatriuresis in rats (Kapusta et al., 1997), while peripheral infusion of orphanin produces diuresis and inhibits salt wasting. Orphanin receptor mRNA is highly expressed in the supraoptic and paraventricular nucleus of the hypothalamus, as is binding. The diuretic and antinatriuretic effects of OFQ may be vasopressin mediated as OFQ has been shown to inhibit firing of vasopressin-containing neurons in the supraoptic nucleus of the hypothalamus (Doi et al., 1998a,b). Due to the differential effects of central versus peripheral orphanin administration, peripheral diuretic effects via OFQ modulation of renal sympathetic nerves should also be considered. Orphanin and ORL 1 content in the noradrenergic A5 cell group and nucleus of the solitary tract of the caudal mesencephalon and metencephalon is abundant. The A5 cell group is known to project to autonomic nuclei of the brainstem and spinal cord (Byrum and Guyenet, 1987), with direct spinal projections to the intermediate gray and intermediolateral cell column in the spinal cord and the nucleus of the solitary tract. Chemical stimulation of neurons in the A5 region has been reported to induce a fall in blood pressure (Neil and Loewy, 1982). Autonomic efferents from the solitary nucleus project to the intermediolateral cell column of the spinal cord, nucleus ambiguous, dorsal motor nucleus of the vagal nerve, ventrolateral medulla, A5 cell group, parabrachial nucleus and numerous forebrain regions (Saper, 1995). Solitary nucleus projections to the preganglionic parasympathetic neurons in the medulla and sympathetic neurons in the spinal cord influence numerous autonomic responses. Additionally, efferents to the medullary reticular formation influences gastrointestinal, cardiovascular and respiratory reflexes. Although initial studies have demonstrated an OFQ influence on primarily cardiovascular function and water balance, it is evident from the diffuse localization of OFQ and ORL1 throughout these nuclei that the orphanin system may be involved in several autonomic and physiologic functions.

8.2.8. Special sensory systems Orphanin and ORL 1 activity are highly expressed in nuclei which integrate visual information, including the nucleus of Darkschewitsch, pretectum and superior colliculus. The lateral mammillary nucleus contains little ORL1 activity, but has a very dense plexus of OFQcontaining neurons and fibers. This nucleus received heavy input from both visual and auditory centers and contains direct cortical connections (Simerly, 1995). Although the majority of brainstem auditory regions have little OFQ and ORL1 content (Neal et al., 1999a,b; Kakimoto et al., 2001), the trapezoid body, which is involved with the integration of high frequency sound in the rat (Aitkin et al., 1984), contains abundant OFQ and ORL1 expression. The trapezoid body projects directly to the lateral superior olive, which in turn projects to the inferior colliculus. These structures contain moderate OFQ and ORL1 activity. Given the presence of OFQ and the ORL1 receptor within these auditory nuclei, as well as the inferior olive and vestibular system, the orphanin system may be involved in the integration of auditory and vestibular information, and possibly play a major role in the animal's ability to adapt to visual, proprioceptive and auditory cues in its environment. Little is known concerning a orphanin's role in visual or auditory processing. However, ORL1173

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deficient mice do demonstrate an insufficient recovery of hearing ability after adaptation to sound exposure (Nishi et al., 1997). Cranial nerves which carry gustatory and somatosensory axons from the oral cavity are distributed throughout the nucleus of the solitary tract (Altschuler et al., 1989). Gustatory information from the solitary nucleus is relayed to the parabrachial nucleus which projects via the ventral posteromedial thalamus to the dorsal insular cortex and central nucleus of the amygdala, all areas with dense OFQ immunoreactivity and heavy ORL1 expression. Neurons in the olfactory bulb and the piriform cortex relay substantial olfactory information to the entorhinal cortex, which in turn projects via the perforant pathway to the hippocampal formation and other higher centers (Schwerdtfeger et al., 1990). Similar to gustatory centers, these olfactory structures contain dense OFQ and ORL1 expression. To date, there is no data supporting a role for orphanin in the processing of olfactory or gustatory information. However, though injections of OFQ into the lateral ventricle of the rat appear to directly stimulate feeding behavior (Pomonis et al., 1996), given the vital role that signals from gustatory and olfactory centers have in the complex series of behaviors involved with feeding, the stimulatory effects of intraventricular orphanin on this activity may be secondary to its influence within major nuclei in the gustatory and/or olfactory systems.

9. CONCLUDING REMARKS Orphanin FQ and its endogenous ORL 1 receptor are widely distributed throughout the central nervous system of the adult male rat. Orphanin FQ and the ORL1 receptor are expressed in critical components of numerous neural circuits, supporting its involvement in various CNS systems, including nociception, modulation of the L-HPA stress axis, motivation and reward, learning and memory, gross motor control, balance and proprioception, sexual behaviors, control of autonomic and physiologic functions and integration of special sensory input. Existing information on the distribution of ORL1 and OFQ provide an anatomical basis for study of specific neural systems in deciphering the many possible behavioral and physiologic roles of this neuropeptide. Orphanin FQ is often referred to as nociceptin due to its possible antagonism of analgesic modalities. However, the orphanin system has a diffuse distribution in numerous forebrain and brainstem circuits and systems, making it likely that OFQ and its receptor most likely play a major role in many behaviors and physiologic functions. With behavioral evidence mounting in support of a diverse role for the orphanin system, nociceptin has become too limiting a nomenclature for such an important neuropeptide. We support the continued use of 'orphanin FQ' when referring to this neuropeptide as we continue to unravel its role in the central nervous system.

10. ABBREVIATIONS 3 4 4V 6 6n 7 7n 174

oculomotor nucleus trochlear nucleus fourth ventricle abducens nucleus root of the abducens nerve facial nucleus facial nerve

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9n 10 10n 12 12n A1 A2 A5 A7 AAA ac

Acb AcbC AcbSh ACo AD AHA AHi AI A1 alv AM Amb AMPO AOD AOE AOL AOM AOP AOV AP APir APT APTD APTV Aq ar

Arc ATg AV AVDM AVVL AVPO B

BAOT Bar bas

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vestibulocochlear nerve glossopharyngeal nerve dorsal motor nucleus of the vagal nerve vagus nerve or its root hypoglossal nucleus root of the hypoglossal nerve A1 noradrenaline cells A2 noradrenaline cells A5 noradrenaline cells A7 noradrenaline cells anterior amygdaloid area anterior commissure accumbens nucleus accumbens nucleus, core accumbens nucleus, shell anterior cortical amygdaloid nucleus anterodorsal thalamic nucleus anterior hypothalamic area amygdalohippocampal area agranular insular anterior lobe, pituitary gland alveus anteromedial thalamic nucleus nucleus ambiguous anterior medial preoptic nucleus anterior olfactory nucleus, dorsal anterior olfactory nucleus, external anterior olfactory nucleus, lateral anterior olfactory nucleus, medial anterior olfactory nucleus, posterior anterior olfactory nucleus, ventral area postrema amygdalopiriform transition area anterior pretectal nucleus anterior pretectal nucleus, dorsal part anterior pretectal nucleus, ventral part aqueduct acoustic radiation arcuate nucleus anterior tegmental nucleus anteroventral thalamic nucleus anteroventral thalamic nucleus, dorsomedial part anteroventral thalamic nucleus, ventrolateral part anteroventral preoptic nucleus basal nucleus of Meynert bed nucleus of the accessory olfactory tract Barrington's nucleus basilar artery 175

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bic BIC BL BLA BLP BM BMA BMP bsc BST BSTi BSTIA BST1 BSTld BSTlj BSTlp BSTlv BSTma BSTmpl BSTmpm BSTmv BSTS BSTv C C1 C2 C3 CA1-CA3 CA1-3so CA1-3sp CA1-3sr CA3sl cc

CC CeCv CeL CeM Cer Cg CG CGD CGPn CI cic CIC C1 CL CLi (B 8) 176

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brachium of the inferior colliculus nucleus of the brachium of the inferior colliculus basolateral amygdaloid nucleus basolateral amygdaloid nucleus, anterior part basolateral amygdaloid nucleus, posterior part basomedial amygdaloid nucleus basomedial amygdaloid nucleus, anterior part basomedial amygdaloid nucleus, posterior part brachium of the superior colliculus bed nucleus of the stria terminalis bed nucleus of the stria terminalis, intermediate division bed nucleus of the stria terminalis, intraamygdaloid division bed nucleus of the stria terminalis, lateral division bed nucleus of the stria terminalis, lateral division, dorsal part bed nucleus of the stria terminalis, lateral division, juxtacapsular part bed nucleus of the stria terminalis, lateral division, posterior part bed nucleus of the stria terminalis, lateral division, ventral part bed nucleus of the stria terminalis, medial division, anterior part bed nucleus of the stria terminalis, medial division, posterolateral part bed nucleus of the stria terminalis, medial division, posteromedial part bed nucleus of the stria terminalis, medial division, ventral part bed nucleus of the stria terminalis, supracapsular part bed nucleus of the stria terminalis, ventral division caudate nucleus C 1 adrenaline cells C2 adrenaline cells C3 adrenaline cells fields CA1-CA3 of Ammon's horn fields CA1-CA3 of Ammon's horn, stratum oriens fields CA1-CA3 of Ammon's horn, stratum pyramidale fields CA1-CA3 of Ammon's horn, stratum radiatum field CA3 of Ammon's horn, stratum lucidum corpus callosum central canal central cervical nucleus ~:. central amygdaloid nucleus, lateral central amygdaloid nucleus, medial cerebellum cingulate gyms central gray central gray, dorsal pontine central gray caudal interstitial nucleus of the medial longitudinal fasciculus commissure of the inferior colliculus central nucleus of the inferior colliculus claustrum centrolateral thalamic nucleus caudal linear nucleus of the raphe

Neuroanatomical studies of the ORL1 receptor and OFQ CM CnF cp CPO CPu csc

ctg cu

Cu Cx CxA CxP CxS DA DC DCIC des DEn df DG DGgr DGhi DGmo DGpo DH Dk dlf DLL DM DMSp5 DMTg DP DPGi DpMe DPO DR (B6, B7) DRG Dsc

DTg ECIC ECu Ent EP EP1 EW f F fr

Ch. III

central medial thalamic nucleus cuneiform nucleus cerebral peduncle caudal periolivary nucleus caudate putamen (striatum) commissure of the superior colliculus central tegmental tract cuneate fasciculus cuneate nucleus cortical layer cortex-amygdala transition zone cortical plate cortical subplate dorsal hypothalamic area dorsal cochlear nucleus dorsal cortex of the inferior colliculus dorsal corticospinal tract dorsal endopiriform nucleus dorsal fornix dentate gyrus dentate gyms, granule cell layer dentate gyms, hilum dentate gyms, molecular layer dentate gyms, polymorph layer dorsal horn of the spinal cord nucleus of Darkschewitsch dorsal longitudinal fasciculus dorsal nucleus of the lateral lemniscus dorsomedial hypothalamic nucleus dorsomedial spinal trigeminal nucleus dorsomedial tegmental area dorsal peduncular cortex dorsal paragigantocellular nucleus deep mesencephalic nucleus dorsal periolivary nucleus dorsal raphe dorsal root ganglion lamina dissecans of the entorhinal cortex dorsal tegmental nucleus external cortex of the inferior colliculus external cuneate nucleus entorhinal cortex entopeduncular nucleus external plexiform layer, olfactory bulb Edinger-Westphal nucleus fomix nucleus of the fields of Forel fasciculus retroflexus 177

Ch. III Fr

FStr g7 G Gi GI GiA GiV G1 GP gr Gr GrO Hb HDB Hipp Hyp I

IAD IAM IMD ic ICj icp IF IG I1 IL ILL IML IMLF IMM In InCo InfS IntA IntDL IntDM IntP Ins IO IOA IOB IOD IODM IOM IOPr IPAC 178

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frontal cortex fundus striati genu of the facial nerve gelatinosous thalamic nucleus gigantocellular reticular nucleus granular insular cortex gigantocellular reticular nucleus, alpha gigantocellular reticular nucleus, ventral glomerular layer, olfactory bulb globus pallidus gracile fasciculus gracile nucleus granular cell layer, olfactory bulb habenula nucleus of the horizontal limb of the diagonal band of Broca hippocampus hypothalamus intercalated nuclei of the amygdala interanterodorsal thalamic nucleus interanteromedial thalamic nucleus intermediodorsal thalamic nucleus internal capsule islands of Calleja inferior cerebellar peduncle interfascicular nucleus indusium griseum intermediate lobe, pituitary gland infralimbic cortex intermediate nucleus of the lateral lemniscus intermediolateral cell column interstitial nucleus of the medial longitudinal fasciculus intermediomedial cell column intercalated nucleus of the medulla intercollicular nucleus infundibular stem interposed cerebellar nucleus, anterior part interposed cerebellar nucleus, dorsolateral part interposed cerebellar nucleus, dorsomedial part interposed cerebellar nucleus, posterior part insular cortex inferior olive inferior olive, medial subnucleus A inferior olive, medial subnucleus B inferior olive, dorsal inferior olive, dorsomedial inferior olive, medial inferior olive, principal interstitial nucleus of the posterior limb of the anterior commissure

Neuroanatomical studies of the ORL1 receptor and OFQ IPC IPD IPL IP1 IPN IPR IRt KF La LA Lat LatC LatPC LC Ld LD LDDM LDVL LDTg lfp lfu LGN LH LHb 11 LM LO lo LOT LP LPB LPGi LPO LRt LS LSD LSI LSO LSp LSV LVe LVPO MA3 MCPC MCPO MD MDC MDL

Ch. III

interpeduncular nucleus, caudal interpeduncular nucleus, dorsal interpeduncular nucleus, lateral internal plexiform layer, olfactory bulb interpeduncular nucleus interpeduncular nucleus, rostral intermediate reticular nucleus Kolliker-Fuse nucleus lateral amygdaloid nucleus lateroanterior hypothalamic nucleus lateral (dentate) cerebellar nucleus lateral cervical nucleus lateral cerebellar nucleus, parvicellular part locus coeruleus lambdoid septal zone laterodorsal thalamic nucleus laterodorsal thalamic nucleus, dorsomedial part laterodorsal thalamic nucleus, ventrolateral part laterodorsal tegmental nucleus longitudinal fasciculus of the pons lateral funiculus of the spinal cord lateral geniculate nucleus lateral hypothalamic area lateral habenula lateral lemniscus lateral mammillary nucleus lateral orbital cortex lateral olfactory tract nucleus of the lateral olfactory tract lateral posterior thalamic nucleus lateral parabrachial nucleus lateral paragigantocellular nucleus lateral preoptic area lateral reticular nucleus lateral septal nucleus lateral septal nucleus, dorsal lateral septal nucleus, intermediate lateral superior olive lateral spinal nucleus lateral septal nucleus, ventral lateral vestibular nucleus lateroventral periolivary nucleus medial accessory oculomotor nucleus magnocellular nucleus of the posterior commissure magnocellular preoptic nucleus mediodorsal thalamic nucleus mediodorsal thalamic nucleus, central part mediodorsal thalamic nucleus, lateral part 179

Ch. III

MDM MdD MdV Me ME Me5 MeAD MeAV Med MedDL MePD MePV MGN MHb Mi MiTg ml ML mlf MM MMn MnA MnPO MnR (B5) MO Mo5 mp MPA MPB MPO MPOC MPT MRe MS MSO mt MT MTu Mtx MVe MVeV MVPO Oc opt OPT OT ox

Pa 180

C.R. Neal Jr. et al.

mediodorsal thalamic nucleus, medial part medullary reticular nucleus, dorsal medullary reticular nucleus, ventral medial amygdaloid nucleus medial eminence mesencephalic trigeminal nucleus medial amygdaloid nucleus, anterodorsal medial amygdaloid nucleus, anteroventral medial (fastigial) cerebellar nucleus medial cerebellar nucleus, dorsolateral protuberance medial amygdaloid nucleus, posterodorsal medial amygdaloid nucleus, posteroventral medial geniculate nucleus medial habenula mitral cell layer, olfactory bulb microcellular tegmental nucleus medial lemniscus medial mammillary nucleus, lateral part medial longitudinal fasciculus medial mammillary nucleus, medial part medial mammillary nucleus, median part median accessory nucleus of the medulla median preoptic nucleus median raphe nucleus medial orbital cortex motor trigeminal nucleus mammillary peduncle medial preoptic area medial parabrachial nucleus medial preoptic nucleus medial preoptic nucleus, central part medial pretectal nucleus mammillary recess, third ventricle medial septal nucleus medial superior olive mammillothalamic tract medial terminal nucleus of the accessory optic tract medial tuberal nucleus germinal matrix medial vestibular nucleus medial vestibular nucleus, ventral medioventral periolivary nucleus occipital cortex optic tract olivary pretectal nucleus nucleus of the optic tract optic chiasm paraventricular hypothalamic nucleus

Neuroanatomical studies of the ORL1 receptor and OFQ

Pa4 Pa5 Pa6 Pa PaAM PaAP PaDC PaLM PaMP PaPo Par PaS PaV PBG PBP pc PC PCom PCRt PDTg Pe PeF PF PH Pin Pir Pit PL PLCo PLd PLi PMCo PMD PMR PMV Pn PN PnC PnO PnR PnV Po PoT PP PPT PPTg PR Pr5

Ch. III

paratrochlear nucleus paratrigeminal nucleus paraabducens nucleus paraventricular hypothalamic nucleus paraventricular hypothalamic nucleus, anterior magnocellular part paraventricular hypothalamic nucleus, anterior parvicellular part paraventricular hypothalamic nucleus, dorsal cap paraventricular hypothalamic nucleus, lateral magnocellular part paraventricular hypothalamic nucleus, medial parvicellular part paraventricular hypothalamic nucleus, posterior part parietal cortex parasubiculum paraventricular hypothalamic nucleus, ventral part parabigeminal nucleus parabrachial pigmented nucleus posterior commissure paracentral thalamic nucleus nucleus of the posterior commissure parvicellular reticular nucleus posterodorsal tegmental nucleus periventricular hypothalamic nucleus perifornical nucleus parafascicular thalamic nucleus posterior hypothalamus pineal gland piriform cortex pituitary gland paralemniscal nucleus posterolateral cortical amygdaloid nucleus paralambdoid septal nucleus posterior limitans thalamic nucleus posteromedial cortical amygdaloid nucleus premammillary nucleus, dorsal paramedian raphe nucleus premammillary nucleus, ventral pontine nuclei paranigral nucleus pontine reticular nucleus, caudal pontine reticular nucleus, oral pontine raphe nucleus pontine reticular nucleus, ventral posterior thalamic nucleus group posterior thalamic nuclear group, triangular part peripeduncular nucleus posterior pretectal nucleus pedunculopontine tegmental nucleus prerubral field principal sensory trigeminal nucleus 181

Ch. III

PrC PrH PrS PT Pu PV PVA PVP PY R

RAmb Rbd RCh Re ReIC Rh RI RLi RMC RMg (B3) ROb (B2) RPa (B 1) RPC RPO RR RRF rs

RSA RSG Rt RtTg RVL S s5 SC SC(DpG) SC(DpWh) SC(InG) SC(InWh) SC(Op) SC(SuG) SC(Zo) SCh scp SFi SFO SG SGe 182

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precommissural nucleus prepositus hypoglossal nucleus presubiculum paratenial nucleus putamen paraventricular thalamic nucleus paraventricular thalamic nucleus, anterior paraventricular thalamic nucleus, posterior pyramidal tract red nucleus retroambiguous nucleus rhabdoid nucleus retrochiasmatic area reuniens thalamic nucleus recess of the inferior colliculus rhomboid thalamic nucleus rostral interstitial nucleus of the medial longitudinal fasciculus rostral linear nucleus of the raphe red nucleus, magnocellular raphe magnus nucleus raphe obscurus nucleus raphe pallidus nucleus red nucleus, parvicellular rostral periolivary region retrorubral nucleus retrorubral field rubrospinal tract retrosplenial agranular cortex retrosplenial granular cortex reticular thalamic nucleus reticulotegmental nucleus of the pons rostroventriculolateral reticular nucleus subiculum sensory root of the trigeminal nerve superior colliculus superior colliculus, deep gray layer superior colliculus, deep white layer superior colliculus, intermediate gray layer superior colliculus, intermediate white layer superior colliculus, optic nerve layer superior colliculus, superficial gray layer superior colliculus, zonal layer suprachiasmatic area superior cerebellar peduncle septofimbrial nucleus subfornical organ suprageniculate nucleus supragenual nucleus

Neuroanatomical studies of the ORL1 receptor and OFQ

SHi SHy SI sm

SN SNC SNL SNR SO sol Sol SolC SoIL SolM SOR sp5 Sp5C Sp5I Sp50 SPFPC SPO SPTg SpVe st STh Su3 SubC SubG SubI SuM sumx

SuVe T TC Te Thai TM TMC TT Tu tz Tz VCA VCP VDB VEn vfu VH

Ch. III

septohippocampal nucleus septohypothalamic nucleus substantia innominata stria medullaris of the thalamus substantia nigra substantia nigra, pars compacta substantia nigra, pars lateralis substantia nigra, pars reticulata supraoptic nucleus solitary tract nucleus of the solitary tract nucleus of the solitary tract, commissural nucleus of the solitary tract, lateral nucleus of the solitary tract, medial supraoptic nucleus, retrochiasmatic (diffuse) spinal trigeminal tract spinal trigeminal nucleus, caudal spinal trigeminal nucleus, interpolar spinal trigeminal nucleus, oral subparafascicular thalamic nucleus, parvicellular superior paraolivary nucleus subpeduncular tegmental nucleus spinal vestibular nucleus stria terminalis subthalamic nucleus supraoculomotor central gray subcoeruleus nucleus subgeniculate nucleus subincertal nucleus supramammillary nucleus supramammillary decussation superior vestibular nucleus temporal cortex tuber cinereum terete hypothalamic nucleus thalamus tuberomammillary nucleus tuberal magnocellular nucleus tenia tecta olfactory tubercle trapezoid body nucleus of the trapezoid body ventral cochlear nucleus, anterior ventral cochlear nucleus, posterior nucleus of the vertical limb of the diagonal band of Broca ventral endopiriform nucleus ventral funiculus of the spinal cord ventral horn of the spinal cord 183

Ch. III

VL VLL VLTg VM VMH VMHC VMHDM VMHVL VO VP VPL VPM VTA VTg ZI

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ventrolateral thalamic nucleus ventral nucleus of the lateral lemniscus ventrolateral tegmental nucleus ventromedial thalamic nucleus ventromedial hypothalamic nucleus ventromedial hypothalamic nucleus, central part ventromedial hypothalamic nucleus, dorsomedial part ventromedial hypothalamic nucleus, ventrolateral part ventral orbital cortex ventral pallidum ventral posterolateral thalamic nucleus ventral posteromedial thalamic nucleus ventral tegmental area ventral tegmental nucleus zona incerta

11. ACKNOWLEDGEMENTS We wish to thank Sharon Burke, Lisa Bain and James Stewart for their superb technical assistance. This work was supported by a Robert Wood Johnson Foundation Fellowship to C R N J (RWJ 030811), a National Institute of Child Health and D e v e l o p m e n t Junior Investigator Award to C R N J (P30-HD28820) and a National Institute of Drug Abuse grant to H.A. and S.J.W. (NIDA RO1 DA08920).

12. REFERENCES Abdulla FA, Smith PA (1997): Nociceptin inhibits T-type Ca2+ channel current in rat sensory neurons by G-protein-independent mechanisms. J Neurosci 17:8721-8728. Ahmadi S, Kotalla C, Guhring H, Takeshima H, Pahl A, Zeilhofer HU (2001a): Modulation of synaptic transmission by nociceptin/orphanin FQ and nocistatin in the spinal cord dorsal horn of mutant mice lacking the nociceptin/orphanin FQ receptor. Mol Pharmacol 59:612-618. Ahmadi S, Liebel JT, Zeilhofer HU (2001b): The role of the ORL1 receptor in the modulation of spinal neurotransmission by nociceptin/orphanin FQ and nocistatin. Eur J Pharmaco1412:39--44. Aitkin LM, Irving DRF, Webster WR (1984): Central neural mechanisms of hearing. In: Smith ID (Ed), Handbook of Physiology. New York, New York: American Physiological Society, pp. 675-737. Alheid GF, de Olmos JS, Beltramino CA (1995): The amygdala and extended amygdala. In: Paxinos G (Ed), The Rat Nervous System. Sydney: Academic Press, pp. 495-578. Allen CN, Jiang ZG, Teshima K, Darland T, Ikeda M, Nelson CS, Quigley DI, Yoshioka T, Allen RG, Rea MA, Grandy DK (2000): Orphanin-FQ/nociceptin (OFQ/N) modulates the activity of suprachiasmatic nucleus neurons. J Neurosci 19:2152-2160. Altschuler SM, Bao X, Bieger D, Hopkins DA, Miselis RR (1989): Viscerotopic representation of the upper alimentary tract in the rat: sensory ganglia and nuclei of the solitary tract and spinal trigeminal tracts. J Comp Neurol 283:248-268.

Amano T, Matsubayashi H, Tamura Y, Takahashi T (2000): Orphanin FQ-induced outward current in rat hippocampus. Brain Res 853:269-274. Anton B, Fein J, To T, Li X, Silberstein L, Evans CJ (1996): Immunohistochemical localization of ORL-1 in the central nervous system of the rat. J Comp Neurol 368:229-251. Ardati A, Henningsen RA, Higelin J, Reinscheid RK, Civelli O, Monsma FJ (1997): Interaction of [3H]orphanin FQ and 125I-Tyr14-orphanin FQ with the orphanin FQ receptor: kinetics and modulation by cations and guanine nucleotides. Mol Pharmaco151:816-824. 184

Neuroanatomical studies of the ORL1 receptor and OFQ

Ch. III

Armstead WM (1999): Nociceptin/orphanin FQ dilates pial arteries by K(ATP) and K(ca) channel activation. Brain Res 835:315-323. Armstead WM (2000a): Relationship between orphanin FQ and cerebral hemodynamics after hypoxia-ischemia in piglets. Am J Physiol Heart Circ Physio1278:H477-H483. Armstead WM (2000b): Altered release of prostaglandins contributes to hypoxic/ischemic impairment of NOC/OFQ cerebrovasodilation. Brain Res 859:104-112. Armstead WM (2000c): NOC/OFQ contributes to hypoxic-ischemic impairment of N-methyl-D-aspartate-induced cerebral vasodilation. Brain Res 868:48-55. Armstead WM (2000d): Role of NOC/OFQ in impaired opioid-induced pial artery dilation following brain injury. Brain Res 869:231-235. Armstead WM (2001): Age dependent endothelin contribution to NOC/OFQ induced impairment of NMDA cerebrovasodilation after brain injury. Peptides 22:39-46. Armstead WM (2002): NOC/OFQ and NMDA contribute to piglet hypoxic ischemic hypotensive cerebrovasodilation impairment. Pediatr Res 51:586-591. Arndt ML, Wu D, Soong Y, Szeto HH (1999): Nociceptin/orphanin FQ increases blood pressure and heart rate via sympathetic activation in sheep. Peptides 20:465-470. Borgland SL, Connor M, Christie, MJ (2001): Nociceptin inhibits calcium channel currents in a subpopulation of small nociceptive trigeminal ganglion neurons in mouse. J Physiol 536:35-47. Borisova NA, Proshlyakova EV, Sapronova AY, Ugrumov MV (1996): Androgen-dependent sex differences in the hypothalamic serotoninergic system. Eur J Endocrinol 134:232-235. Bunzow JR, Saez C, Mortrud M, Bouvier C, Williams JT, Low M, Grandy DK (1994): Molecular cloning and tissue distribution of a putative member of the rat opioid receptor gene family that is not a mu, delta or kappa opioid receptor type. FEBS Lett 347:284-288. Bucher B (1998): ORLl-mediated inhibition by nociceptin of noradrenaline release from perivascular sympathetic nerve endings of the rat tail artery. Arch Pharmacol 358:682-685. Butour JL, Moisand C, Mazarguil H, Mollereau C, Meunier JC (1997): Recognition and activation of the opioid receptor-like ORL 1 receptor by nociceptin, nociceptin analogs and opioids. Eur J Pharmacol 321:97-103. Byrum CE, Guyenet PG (1987): Afferent and efferent connections of the A5 noradrenergic cell group in the rat. J Comp Neurol 261:529-542. Ceccatelli S, Cortes R, Hrkfelt T (1991): Effect of reserpine and colchicine on mRNA levels in the rat hypothalamic paraventricular nucleus. Brain Res Mol Brain Res 9:57-69. Champion HC, Kadowitz PJ (1997a): Nociceptin, an endogenous ligand for the ORL1 receptor, has novel hypotensive activity in the rat. Life Sci 60:PL241-245. Champion HC, Kadowitz PJ (1997b): [Tyrl]-Nociceptin, a novel nociceptin analog, decreases systemic arterial pressure by a naloxone-insensitive mechanism in the rat. Biochem Biophys Res Commun 234:309-312. Champion HC, Czapla MA, Kadowitz PJ (1997): Nociceptin, an endogenous ligand for the ORL1 receptor, decreases cardiac output and total peripheral resistance in the rat. Peptides 18:729-732. Champion HC, Pierce RL, Kadowitz PJ (1998): Nociceptin, a novel endogenous ligand for the ORL1 receptor, dilates isolated resistance arteries from the rat. Regul Pept 30:69-74. Chen Y, Fan Y, Liu J, Mestek A, Tian M, Kozak CA, Yu L (1994): Molecular cloning, tissue distribution and chromosomal localization of a novel member of the 0pioid receptor gene family. FEBS Lett 347:279-283. Chen X, McClatchy DB, Geller EB, Liu-Chen L, Tallarida RJ, Adler MW (2001): Possible mechanism of hypothermia induced by intracerebroventricular injection of orphanin FQ/nociceptin. Brain Res 904:252-258. Chu XNX, Li P, Mao L, Wang JQ (1999): Inhibition of cardiovascular activity following microinjection of novel opioid-like neuropeptide nociceptin (orphanin FQ) into the rat rostral ventrolateral medulla. Brain Res 829:134142. Ciccocioppo R, Angeletti S, Sanna PP, Weiss F, Massi M (2000): Effect of nociceptin/orphanin FQ on the rewarding properties of morphine. Eur J Pharmacol 404:153-159. Ciccocioppo R, Martin-Fardon PP, Weiss F, Massi M (2001): Nociceptin/orphanin FQ inhibits stress- and CRFinduced anorexia in rats. NeuroReport 12:1145-1149. Ciccocioppo R, Polidori CL, Salvadori S, Guerrini R, Massi M (2002): Pharmacological characterization of the nociceptin receptor which mediates reduction of alcohol drinking in rats. Peptides 23:117-125. Civelli O, Nothacker H-P, Bourson A, Ardati A, Monsma F, Reinscheid R (1997): Orphan receptors and their natural ligands. J Recept Signal Transduct Res 17:545-550. Connor M, Christie MJ (1998): Modulation of Ca2+ channel currents of acutely dissociated rat periaqueductal grey neurons. J Physiol 508:47-58.

185

Ch. I I I

C.R. N e a l Jr. et al.

Connor M, Vaughan CW, Chieng B, Christie MJ (1996): Nociceptin receptor coupling to potassium conductance in locus coeruleus neurons in vitro. Br J Pharmacol 119:1614-1618. Connor M, Vaughan CW, Jennings EA, Allen RG, Christie MJ (1999): Nociceptin, Phe(1)psi-nociceptin(1-13), nocistatin and prepronociceptin(154-181) effects on calcium channel currents and a potassium current in rat locus coeruleus in vitro. Br J Pharmacol 128:1779-1787. Cortes R, Ceccatelli S, Schalling M, H6kfelt T (1990): Differential effects of intracerebroventricular colchicine administration on the expression of mRNAs for neuropeptides and neurotransmitter enzymes, with special emphasis on galanin: an in situ hybridization study. Synapse 6:369-391. Curro D, Yoo JH, Anderson M, Song I, Del Valle J, Owyang C (2001): Molecular cloning of the orphanin FQ receptor gene and differential tissue expression of splice variants in rat. Gene 266:139-145. Danielson PB, Dores RM (1999): Molecular evolution of the opioid/orphanin gene family. Gen Comp Endocrinol 113:169-186.

Danielson PB, Hoversten MT, Fitzpatrick M, Schreck C, Akil H, Dores RM (2001): Sturgeon orphanin, a molecular "fossil" that bridges the gap between the opioids and orphanin FQ/nociceptin. J Biol Chem 276:22114-22119. Davis EC, Popper P, Gorski RA (1996): The role of apoptosis in sexual differentiation of the sexually dimorphic nucleus of the preoptic area. Brain Res 734:10-18. Dawson-Basoa M, Gintzler A (1997): Nociceptin (Orphanin FQ) abolishes gestational and ovarian sex steroidinduced antinociception and induces hyperalgesia. Brain Res 750:48-52. Devine DP, Taylor L, Reinscheid RK, Monsma FJ, Civelli O, Akil H (1996a): Rats rapidly develop tolerance to the locomotor-inhibiting effects of the novel neuropeptide orphanin FQ. Neurochem Res 21:1387-1396. Devine DP, Reinscheid RK, Monsma FJ, Civelli O, Akil H (1996b): The novel neuropeptide orphanin FQ fails to produce conditioned place preference or aversion. Brain Res 727:225-229. Devine DP, Watson SJ, Akil H (2001): Nociceptin/orphanin FQ regulates neuroendocrine function of the limbichypothalamic-pituitary-adrenal axis. Neuroscience 102:541-553. Dhandapani KM and Brann DW (2002): Orphanin FQ inhibits GnRH secretion from rat hypothalamic fragments but not GT1-7 neurons. NeuroReport 13:1247-1249. Di Giannuario A, Pieretti S, Catalani A, Loizzo A (1999): Orphanin FQ reduces morphine-induced dopamine release in the nucleus accumbens: a microdialysis study in rats. Gen Comp Neurosci Lett 272:183-186. Doi N, Dutia MB, Brown CH, Leng G, Russell JA (1998a): Inhibitory actions of nociceptin (orphanin FQ) on rat supraoptic nucleus oxytocin and vasopressin neurons in vitro. Adv Exp Med Bio1449:147-151. Doi N, Dutia MB, Russell JA (1998b): Inhibition of rat oxytocin and vasopressin supraoptic nucleus neurons by nociceptin in vitro. Neuroscience 84:913-921. Dooley CT, Houghten RA (1996): Orphanin FQ: receptor binding and analog structure activity relationships in rat brain. Life Sci 59:PL23-PL29. Dooley CT, Spaeth CG, Berzetei-Gurske IP, Craymer K, Adapa ID, Brandt SR, Houghten RA, Toll L (1997): Binding and in vitro activities of peptides with high affinity for the nociceptin/orphanin FQ receptor, ORL1. J Pharm Exp Ther 283:735-741.

Evans CJ (1999): Corrigendum. J Comp Neurol 412:708. Faber ES, Chambers JP, Evans RH, Henderson G (1996): Depression of glutamatergic transmission by nociceptin in the neonatal rat hemisected spinal cord preparation in vitro. Br J Pharmacol 119:189-190. Florin S, Suaudeau C, Meunier JC, Costentin J (1996): Nociceptin stimulates locomotion and exploratory behaviour in mice. Eur J Pharmaco1317:9-13. Florin S, Suaudeau C, Meunier JC, Costentin J (1997a): Orphan neuropeptide NoclI, a putative pronociceptin maturation product, stimulates locomotion in mice. NeuroReport 8:705-707. Florin S, Leroux-Nicollet I, Meunier JC, Costentin J (1997b): Autoradiographic localization of [3H]nociceptin binding sites from telencephalon to mesencephalic regions in the mouse brain. Neurosci Lett 230:33-36. Fukuda K, Kato S, Mori K, Nishi M, Takeshima H, Iwabe N, Miyata T, Houtani T, Sugimoto T (1994): cDNA cloning and regional distribution of a novel member of the opioid receptor family. FEBS Lett 343:42-46. Gavioli EC, Rae GA, Calo G, Guerrini R, De Lima TC (2002): Central injections of nocistatin or its Cterminal hexapeptide exert anxiogenic-like effect on behaviour of mice in the plus-maze test. Br J Pharmacol 136:764-772.

Gintzler AR, Adapa ID, Toll L, Medina VM, Wang L (1997): Modulation of enkephalin release by nociceptin. Eur J Pharmacol 325:29-34.

Giuliani S, Maggi CA (1996): Inhibition of tachykinin release from peripheral endings of sensory nerves by nociceptin, a novel opioid peptide. Br J Pharmacol 118:1567-1569. Gorski RA, Harlan RE, Jacobson CC, Shryne JE, Southam AM (1980): Evidence for the existence of a sexually dimorphic nucleus in the preoptic area of the rat. J Comp Neurol 193:529-539. 186

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Griebel G, Perrault G, Sanger DJ (1999): Orphanin FQ, a novel neuropeptide with anti-stress-like activity. Brain Res 836:221-224. Grisel JE, Mogil JS, Belknap JK, Grandy DK (1996): Orphanin FQ acts as a supraspinal, but not a spinal, anti-opioid peptide. NeuroReport 7:2125-2129. Guerrini R, Calo G, Rizzi A, Bianchi C, Lazarus LH, Salvadori S, Temussi PA, Regoli D (1997): Address and message sequences for the nociceptin receptor: a structure-activity study of nociceptin-(1-13)-peptide amide. J Med Chem 40:1789-1793. Guillamon A, Segovia S, del Abril A (1988): Early effects of gonadal steroids on the neuron number in the medial posterior region of the lateral division of the bed nucleus of the stria terminalis in the rat. Brain Res Dev Brain Res 44:281-290. Gumusel B, Hao Q, Hyman A, Chang JK, Kapusta DR, Lippton H (1997): Nociceptin: an endogenous agonist for central opioid like l (ORL1) receptors possesses systemic vasorelaxant properties. Life Sci 60:PL141-PL145. Gupta DS, Kelson AB, Polgar WE, Toll L, Szucs M, Gintzler AR (2001): Ovarian sex steroid-dependent plasticity of nociceptin/orphanin FQ and opioid modulation of spinal dynorphin release. J Pharmacol Exp Ther 298:12131220. Halford WP, Gebhardt BM, Carr DJ (1995): Functional role and sequence analysis of a lymphocyte orphan opioid receptor. J Neuroimmunol 59:91-101. Hao JX, Wiesenfeld-Hallin Z, Xu XJ (1997): Lack of cross-tolerance between the antinociceptive effect of intrathecal orphanin FQ and morphine in the rat. Neurosci Lett 223:49-52. Hara N, Minami T, Okuda-Ashitaka E, Sugimoto T, Sakai M, Onaka M, Mori H, Imanishi T, Shingu K, Ito S (1997): Characterization of nociceptin hyperalgesia and allodynia in conscious mice. Br J Pharmacol 121:401408. Heinricher MM, McGaraughty S, Grandy DK (1997): Circuitry underlying antiopioid actions of orphanin FQ in the rostral ventromedial medulla. J Neurophysiol 78:3351-3358. Henderson G, McKnight AT (1997): The orphan opioid receptor and its endogenous ligand nociceptin/orphanin FQ. Trends Pharmacol Sci 18:293-300. Herman JP, Cullinan WE (1997): Neurocircuitry of stress: central control of the hypothalamo-pituitaryadrenocortical axis. Trends Neurosci 20:78-84. Herman JP, Chantal MF, Cullinan WE (1996): Neuronal circuit regulation of the hypothalamo-pituitaryadrenocortical stress axis. Crit Rev Neurobiol 10:371-394. Higgins GA, Kew JN, Richards JG, Takeshima H, Jenck F, Wichmann J, Kemp JA, Grottick AJ (2002): A combined pharmacological and genetic approach to investigate the role of orphanin FQ in learning and memory. Eur J Neurosci 15:911-922. Hiramatsu M, Inoue K (1999): Effects of nocistatin on nociceptin-induced impairment of learning and memory in mice. Eur J Pharmacol 367:151-155. Houtani T, Nishi M, Takeshima H, Nukada T, Sugimoto T (1996): Structure and regional distribution of nociceptin/orphanin FQ precursor. Biochem Biophys Res Commun 219:714-719. Houtani T, Nishi M, Takeshima H, Sato K, Sakuma S, Kakimoto S, Ueyama T, Noda T, Sugimoto T (2000): Distribution of nociceptin/orphanin FQ precursor protein and receptor in brain and spinal cord: a study using in situ hybridization and X-gal histochemistry in receptor-deficient mice. J Comp Neurol 424:489-508. Hunter WM, Greenwood FC (1962): Preparation of iodine-131 labeled human growth hormone of high specific activity. Nature 194:495-496. Ikeda K, Kobayashi K, Kobayashi T, Ichikawa T, Kumanishi T, Kishida H, Yano R, Manabe T (1997): Functional coupling of the nociceptin/orphanin FQ receptor with the G-protein-activated K+ (GIRK) channel. Brain Res Mol Brain Res 45:117-126. Ikeda K, Watanabe M, Ichikawa T, Kobayashi T, Yano R, Kumanishi T (1998): Distribution of prepronociceptin/orphanin FQ mRNA and its receptor mRNA in developing and adult mouse central nervous system. J Comp Neurol 399:139-151. Inoue MI, Shimohira A, Yoshida A, Zimmer H, Takeshima T, Sakurada T, Ueda H (1999): Dose-related opposite modulation by nociceptin/orphanin FQ of substance P nociception in the nociceptors and spinal cord. J Pharmacol Exp Ther 291:308-313. Jacobson CD, Gorski RA (1981): Neurogenesis of the sexually dimorphic nucleus of the preoptic area in the rat. J Comp Neurol 196:519-529. Jagolino A and Armstead WM (2001): Nociceptin/orphanin FQ contributes to hypoxic/ischemic impairment of hypercapnic cerebrovasodilation. Brain Res Bull 55:465-468. Jakab RL, Leranth C (1995): The septum. In: Paxinos G (Ed), The Rat Nervous System. Sydney: Academic Press, pp. 405-442. 187

Ch. III

C.R. Neal Jr. et al.

Jenck E Moreau JL, Martin JR, Kilpatrick GJ, Reinscheid RK, Monsma FJ, Nothacker H-E Civelli O (1997): Orphanin FQ acts as an anxiolytic to attenuate behavioral responses to stress. Proc Natl Acad Sci USA 94:1485414858. Jenck F, Wichmann J, Dautzenberg F, Moreau JL, Ouagazzal A, Martin JR, Lundstrom K, Cesura A, Poli S, Roever S, Kolczewski S, Adam G, Kilpatrick G (2000): A synthetic agonist at the orphanin FQ/nociceptin receptor ORLI: anxiolytic profile in the rat. Proc Natl Acad Sci USA 97:4938-4943. Jennings EA (2001): Postsynaptic K+ current induced by nociceptin in medullary dorsal horn neurons. NeuroReport 12:645-648. Jhamandas KH, Sutak M, Henderson G (1998): Antinociceptive and morphine modulatory actions of spinal orphanin FQ. Can J Physiol Pharmacol 76:314-324. Kachaturian H, Lewis ME, Schafer MK-H, Watson SJ (1985): Anatomy of the CNS opioid systems. Trends Neurosci 8:111-119.

Kakimoto S, Houtani T, Sato K, Ueyama T, Sakuma S, Munemoto Y, Ohishi H, Kase M, Yamashita T, Takeshima H, Sugimoto T (2001): Brainstem auditory regions in mice: expression of nociceptin/orphanin FQ precursor mRNA in select neurons. Neurosci Lett 314:37-40. Kapusta DR, Kenigs VA (1999): Cardiovascular and renal responses produced by central orphanin FQ/nociceptin occur independent of renal nerves. Am J Physio1277:R987-R995. Kapusta DR, Sezen SF, Chang JK, Lippton H, Kenigs VA (1997): Diuretic and antinatriuretic responses produced by the endogenous opioid-like peptide, nociceptin (orphanin FQ). Life Sci 60:PL15-PL21. Kapusta DR, Dayan LA, Kenigs VA (2002): Nociceptin/Orphanin FQ modulates the cardiovascular, but not renal, responses to stress in spontaneously hypertensive rats. Clin Exp Pharmacol Physio129:254-259. Kest B, Hopkins E, Palmese CA, Chen ZE Mogil JS, Pintar JE (2001): Morphine tolerance and dependence in nociceptin/orphanin FQ transgenic knock-out mice. Neurosci 104:217-222. King TR, Nance DM (1986): Neuroestrogenic control of feeding behavior and body weight in rats with kainic acid lesions of the lateral septal area. Physiol Behav 37:475-481. King MA, Rossi GC, Chang AH, Williams L, Pasternak GW (1997): Spinal analgesic activity of orphanin FQ/nociceptin and its fragments. Neurosci Lett 223:113-116. Kiyama H, Emson PC (1991): Colchicine-induced expression of proneurotensin mRNA in rat striatum and hypothalamus. Brain Res Mol Brain Res 9:353-358. Knoflach F, Reinscheid RK, Civelli O, Kemp JA (1996): Modulation of voltage-gated calcium channels by orphanin FQ in freshly dissociated hippocampal neurons. J Neurosci 16:6657-6664. Kolesnikov YA, Pasternak GW (1997): Peripheral orphanin FQ/nociceptin analgesia in the mouse. Life Sci 64:2021-2028. Koob GF, Swerdlow NR, Vaccarino F, Hubner C, Pulvirenti L, Weiss F (1991): Functional output of the basal forebrain. In: Napier TC, Kalivas PW, Hanin I (Eds), The Basal Forebrain: Anatomy to Function. New York: Plenum Press, pp. 291-305. Koster A, Montkowski A, Schulz S, Stube E, Knaudt K, Jenck F, Moreau JL, Nothacker H-P, Civelli O, Reinscheid RK (1999): Targeted disruption of the orphanin FQ/nociceptin gene increases stress susceptibility and impairs stress adaptation in mice. Proc Natl Acad Sci USA 96:10444-10449. Kotlinska J, Suder E Legowska A, Rolka K, Silberring J (2000): Orphanin FQ/nociceptin inhibits morphine withdrawal. Life Sci 66:PL 119-PL 123. Kotlinska J, Wichmann J, Legowska A, Rolka K, Silberring J (2002): Orphanin FQ/nociceptin but not Ro 65-6570 inhibits the expression of cocaine-induced conditioned place preference. Behav Pharmacol 13:229-235. Kusaka T, Yamada S, Kimura R (2001): Characterization of specific [3H]nociceptin binding in rat brain and spinal cord. Biol Pharm Bull 24:902-905. Lachowicz JE, Shen Y, Monsma FJ, Sibley DR (1994): Molecular cloning of a novel G protein-coupled receptor related to the opiate receptor family. J Neurochem 64:34-40. Lai CC, Wu SY, Dun SL, Dun NJ (1997): Nociceptin-like immunoreactivity in the dorsal horn and inhibition of substantia gelatinosa neurons. Neuroscience 81:887-891. Laudenbach V, Calo G, Guerrini R, Lamboley G, Benoist JF, Evrard P, Gressens P (2001): Nociceptin/orphanin FQ exacerbates excitotoxic white-matter lesions in the murine neonatal brain. J Clin Invest 107:457-466. Letchworth SR, Mathis JP, Rossi GC, Bodnar RJ, Pasternak GW (2000): Autoradiographic localization of 125I[Tyr14]orphanin FQ and 125I[Tyr10]orphanin FQ(1-11) binding sites in rat brain. J Comp Neurol 423:319329. Leventhal L, Mathis JP, Rossi GC, Pasternak GW, Bodnar RJ (1998): Orphanin opioid receptor antisense probes block orphanin FQ-induced hyperphagia. Eur J Pharmaco1349:R1-R3.

188

N e u r o a n a t o m i c a l s t u d i e s o f the O R L 1 r e c e p t o r a n d O F Q

Ch. I I I

Liebel JT, Swandulla D, Zeilhofer HU (1997): Modulation of excitatory synaptic transmission by nociceptin in superficial dorsal horn neurons of the neonatal rat spinal cord. Br J Pharmacol 121:425-432. Lou LG, Ma L, Pei G (1997): Nociceptin/orphanin FQ activates protein kinase C, and this effect is mediated through phospholipase C/Ca 2+ pathway. Biochem Biophys Res Commun 240:304-308. Lutfy K, Hossain SM, Khaliq I, Maidment NT (2001a): Orphanin FQ/nociceptin attenuates development of morphine tolerance in rats. Br J Pharmacol 134:529-534. Lutfy K, Do T, Maidment NT (2001b): Orphanin FQ/nociceptin attenuates motor stimulation and changes in nucleus accumbens extracellular dopamine induced by cocaine in rats. Psychopharmacology (Berl) 154:1-7. Luo C, Kumamoto E, Furue H, Yoshimura M (2001): Nociceptin-induced outward current in substantia gelatinosa neurons of the adult rat spinal cord. Neurosci 108:323-330. Ma L, Cheng ZL, Fan GH, Cai YC, Jiang LZ, Pei G (1997): Functional expression, activation and desensitization of opioid receptor-like receptor ORL1 in neuroblastoma • glioma NG 108-15 hybrid cells. FEBS Lett 403:91-94. Madamba SG, Schweitzer P, Siggins GR (1999): Nociceptin augments K(+) currents in hippocampal CA1 neurons by both ORL-1 and opiate receptor mechanisms. J Neurophysiol 82:1776-1785. Madeira MD, Sousa N, Paula-Barbosa MM (1991): Sexual dimorphism in the mossy fiber synapses of the rat hippocampus. Exp Brain Res 87:537-545. Maidment NT, Chen Y, Tan AM, Murphy NP, Leslie FM (2002): Rat ventral midbrain dopamine neurons express the orphanin FQ/nociceptin receptor ORL-1. NeuroReport 13:1137-1140. Makman MH, Lyman WD, Dvorkin B (1997): Presence and characterization of nociceptin (orphanin FQ) receptor binding in adult rat and human fetal hypothalamus. Brain Res 762:247-250. Mamiya T, Noda M, Takeshima H, Nabeshima T (1998): Enhancement of spatial attention in nociceptin/orphanin FQ receptor-knockout mice. Brain Res 783:236-240. Mamiya T, Noda Y, Ren X, Nagai T, Takeshima H, Ukai M, Nabeshima T (2001): Morphine tolerance and dependence in the nociceptin receptor knockout mice. J Neural Transm 108:1349-1361. Manabe T, Noda Y, Mamiya T, Katagirl H, Houtani T, Nishi M, Noda T, Takahashi T, Sugimoto T, Nabeshima T, Takeshima H (1998): Facilitation of long-term potentiation and memory in mice lacking nociceptin receptors. Nature 394:577-581. Mansour A, Kachaturian H, Lewis ME, Akil H, Watson SJ (1987): Autoradiographic differentiation of mu, delta, and kappa opioid receptors in the rat forebrain and midbrain. J Neurosci 7:2445-2464. Mansour A, Thompson RC, Akil H, Watson SJ (1993): Delta opioid receptor mRNA distribution in the brain: comparison to delta receptor binding and proenkephalin mRNA. J Chem Neuroanat 6:351-362. Mansour A, Fox CA, Burke S, Meng F, Thompson RC, Akil H, Watson SJ (1994): Mu, delta and kappa opioid receptor mRNA expression in the rat CNS: an in situ hybridization study. J Comp Neurol 350:412-438. Mansour A, Fox CA, Burke S, Akil H, Watson SJ (1995a): Immunohistochemical localization of the cloned tx opioid receptor in the rat CNS. J Chem Neuroanat 8:283-305. Mansour A, Fox CA, Akil H, Watson SJ (1995b): Opioid receptor mRNA expression in the rat CNS: anatomical and functional implications. Trends Neurosci 18:22-29. Mao L, Wang JQ (2000): Microinjection of orphanin FQ into nucleus tractus solitarii elevates blood pressure and heart rate in both anesthetized and conscious rats. J Pharmacol Exp Ther 294:255-262. Marchese A, Docherty JM, Nguyen T, Heiber M, Cheng R, Heng HH, Tsui LC, Shi X, George SR, O'Dowd BF (1994): Cloning of human genes encoding novel G protein-coupled receptors. Genomics 23:609-618. Martin-Fardon R, Ciccocioppo R, Massi M, Weiss F (2000): Nociceptin prevents stress-induced ethanol- but not cocaine-seeking behavior in rats. NeuroReport 11:1939-1943. Maslov LN, Krylatov AV, Lishmanov I, Albrecht E (1999): The anti-arrhythmia properties of the functional opioid agonist orphanin FQ (nociceptin). Eksp Klin Farmakol 62:21-24 (in Russian). Mathis JP, Ryan-Moro J, Chang A, Hom JS, Scheinberg DA, Pasternak GW (1997): Biochemical evidence for orphanin FQ/nociceptin receptor heterogeneity in mouse brain. Biochem Biophys Res Commun 230:462-465. Mathis JP, Goldberg IE, Letchworth SR, Ryan-Moro J, Pasternak GW (1999): Identification of a high-affinity orphanin FQ/nociceptin(1-11) binding site in mouse brain. Synapse 34:181-186. Mathis JP, Rossi GC, Pellegrino MJ, Jimenez C, Pasternak GW, Allen RG (2001): Carboxyl terminal peptides derived from prepro-orphanin FQ/nociceptin (ppOFQ/N) are produced in the hypothalamus and possess analgesic bioactivities. Brain Res 895:89-94. Meis S and Pape HC (2001): Control of glutamate and GABA release by nociceptin/orphanin FQ in the rat lateral amygdala. J Physiol 532:701-712. Meng F, Taylor LP, Hoversten MT, Ueda Y, Ardati A, Reinscheid RK, Monsma FJ, Watson SJ, Civelli O, Akil H (1996): Moving from the orphanin FQ receptor to an opioid receptor using four point mutations. J Biol Chem 271:32016-32020. 189

Ch. III

C.R. Neal Jr. et al.

Menzies JR, Corbett AD (2000): Nociceptin inhibits tonic nitric oxide release in the mouse isolated proximal colon. Eur J Pharmacol 388:183-186.

Meunier JC, Mollereau C, Toll L, Suaudeau C, Moisand C, Alvinerie P, Butour JL, Guillemot JC, Ferrara P, Monsarrat B, Mazarguil H, Vassart G, Parmentier M, Costentin J (1995): Isolation and structure of the endogenous agonist of opioid receptor-like ORL1 receptor. Nature 377:532-535. Minami T, Okuda-Ashitaka E, Nishizawa M, Mori H, Ito S (1997): Inhibition of nociceptin-induced allodynia in conscious mice by prostaglandin D2. Br J Pharmacol 122:605-610. Mogil JS and Pasternak GW (2001): The molecular and behavioral pharmacology of the orphanin FQ/nociceptin peptide and receptor family. Pharmacol Rev 53:381-415. Mogil JS, Grisel JE, Zhangs G, Belknap JK, Grandy DK (1996a): Functional antagonism of mu-, delta- and kappa-opioid antinociception by orphanin FQ. Neurosci Lett 214:131-134. Mogil JS, Grisel JE, Reinscheid RK, Civelli O, Belknap JK, Grandy DK (1996b): Orphanin FQ is a functional anti-opioid peptide. Neuroscience 75:333-337. Mogil JS, Nessim LA, Wilson SG (1999): Strain-dependent effects of supraspinal orphanin FQ on thermal nociceptive sensitivity in mice. Neurosci Lett 261:147-150. Mollereau C, Parmentier M, Mailleux P, Butour JL, Moisand C, Chalon P, Caput D, Vassart G, Meunier JC (1994): ORL 1, a novel member of the opioid receptor family. Cloning, functional expression and localization. FEBS Lett 341:33-38.

Mollereau C, Moisand C, Butour JL, Parmentier M, Meunier JC (1996a): Replacement of Gln28~ by His in TM6 of the human ORL1 receptor increases affinity but reduces intrinsic activity of opioids. FEBS Lett 395:17-21. Mollereau C, Simons MJ, Soularue P, Liners F, Vassart G, Meunier JC, Parmentier M (1996b): Structure, tissue distribution, and chromosomal localization of the prepronociceptin gene. Proc Natl Acad Sci USA 93:8666-8670. Monteillet G, Fein J, Anton B, Evans C (1998): ORL-1 and mu opioid receptor antisera label different fibers in areas involved in pain processing. J Comp Neurol 399:373-383. Morgan MM, Grisel JE, Robbins CS, Grandy DK (1997): Antinociception mediated by the periaqueductal gray is attenuated by orphanin FQ. NeuroReport 8:3431-3434. Murphy NP, Maidment NT (1999): Orphanin FQ/nociceptin modulation of mesolimbic dopamine transmission determined by microdialysis. J Neurochem 73:179-186. Murphy NP, Ly HT, Maidment NT (1996): Intracerebroventricular orphanin FQ/nociceptin suppresses dopamine release in the nucleus accumbens of anaesthetized rats. Neuroscience 75:1-4. Murphy NP, Lee Y, Maidment NT (1999): Orphanin FQ/nociceptin blocks acquisition of morphine place preference. Brain Res 832:168-170. Nakano H, Minami T, Abe K, Arai T, Tokumura M, Ibii N, Okuda-Ashitaka E, Mori H, Ito S (2000): Effect of intrathecal nocistatin on the formalin-induced pain in mice versus that of nociceptin/orphanin FQ. J Pharmacol Exp Ther 292:331-336.

Narayanan S, Maidment NT (1999): Orphanin FQ and behavioral sensitization to cocaine. Pharmacol Biochem Behav 63:271-277. Narayanan S, Lutfy K, Maidment NT (2002): Sensitization to cocaine after a single intra-cerebral injection of orphanin FQ/nociceptin. Behav Brain Res 131:97-103. Neal CR, Newman SW (1989): Prodynorphin peptide distribution in the forebrain of the Syrian hamster and rat: a comparative study with antisera against dynorphin A, dynorphin B and the C-terminus of the prodynorphin precursor molecule. J Comp Neurol 288:353-386. Neal CR, Mansour A, Reinscheid R, Nothacker H-P, Civelli O, Watson SJ (1999a): Localization of orphanin FQ (nociceptin) peptide and messenger RNA in the central nervous system of the rat. J Comp Neurol 406:503-547. Neal CR, Mansour A, Reinscheid R, Nothacker HP, Civelli O, Akil H, Watson SJ (1999b): Opioid receptor-like (ORL1) receptor mRNA expression in the rat CNS: comparison of 125I-[laTyr]-orphanin FQ binding with the ORL1 receptor. J Comp Neurol 412:563-605. Neil JJ, Loewy AD (1982): Decreases in blood pressure in response to L-glutamate microinjections into the A5 catecholamine cell group. Brain Res 241:271-278. Nicol B, Lambert DG, Rowbotham DJ, Smart D, McKnight AT (1996): Nociceptin induced inhibition of K+ evoked glutamate release from rat cortical slices. Br J Pharmacol 119:1081-1083. Nicholson JR, Peterson SJ, Menzies JRW, Corbett AD, McKnight AT (1998): Pharmacological studies on the 'orphan' opioid receptor in central and peripheral sites. Can J Physiol Pharmacol 76:304-313. Nishi M, Houtani T, Noda Y, Mamiya T, Sato K, Doi T, Kuno J, Takeshima H, Nukada T, Nabeshima T, Yamashita T, Noda T, Sugimoto T (1997): Unrestrained nociceptive response and dysregulation of heating ability in mice lacking the nociceptin/orphanin FQ receptor. EMBO J 16:1858-1864.

190

Neuroanatomical studies of the ORL1 receptor and OFQ

Ch. III

Nishizuka M, Arai Y (1983): Male-female differences in the intra-amygdaloid input to the medial amygdala. Exp Brain Res 52:328-332. Norton CS, Neal CR, Kumar S, Akil H, Watson SJ (2002): Nociceptin/orphanin FQ and opioid receptor-like receptor mRNA expression in dopamine systems. J Comp Neurol 444(4):358-368. Nothacker H-P, Reinscheid RK, Mansour A, Henningsen RA, Ardati A, Monsma FJ, Watson SJ, Civelli O (1996): Primary structure and tissue distribution of the orphanin FQ precursor. Proc Natl Acad Sci USA 93:8677-8682. Okuda-Ashitaka E, Ito S (2000): Nocistatin: a novel neuropeptide encoded by the gene for the nociceptin/orphanin FQ precursor. Peptides 21:1101-1109. Okuda-Ashitaka E, Minami T, Tachibana S, Yoshihar Y, Nishiuchi Y, Kimura T, Ito S (1998): Nocistatin, a peptide that blocks nociceptin action in pain transmission. Nature 392:286-289. Olszewski PK, Billington CJ, Levine AS (2000): Fos expression in feeding-related brain areas following intracerebroventricular administration of orphanin FQ in rats. Brain Res 855:171-175. Olszewski PK, Grace MK, Sanders JB, Billington CJ, Levine AS (2002): Effect of nociceptin/orphanin FQ on food intake in rats that differ in diet preference. Pharmacol Biochem Behav 73:529-535. Osinski MA, Pampusch MS, Murtaugh MP, Brown DR (1999): Cloning, expression and functional role of a nociceptin/orphanin FQ receptor in the porcine gastrointestinal tract. Eur J Pharmacol 365:281-289. Pan YX, Xu J, Pasternak GW (1996): Cloning and expression of a cDNA encoding a mouse brain orphanin FQ/nociceptin precursor. Biochem J 315:11-13. Pan Z, Hirakawa N, Fields HL (2000): A cellular mechanism for the bi-directional pain-modulating actions of orphanin FQ/nociceptin. Neuron 26:515-522. Pietras TA and Rowland NE (2002) Effect of opioid and cannabinoid receptor antagonism on OFQ-induced hyperphagia in rats. Eur J Pharmacol 442:237-239. Plotsky PM, Cunningham ET, Widmaier EP (1989): Catecholaminergic modulation of corticotrophin-releasing factor and adrenocorticotropin. Endocr Rev 10:437-451. Pomonis JD, Billington CJ, Levine AS (1996): Orphanin FQ, agonist of orphan opioid receptor ORL1, stimulates feeding in rats. NeuroReport 8:369-371. Przewlocki R and Przewlocka B (2001): Opioids in chronic pain. Eur J Pharmacol 429:79-91. Pu L, Bao GB, Ma L, Pei G (1999): Acute desensitization of nociceptin/orphanin FQ inhibition of voltage-gated calcium channels in freshly dissociated hippocampal neurons. Eur J Neurosci 11:3610-3616. Quigley DI, McDougall J, Darland T, Zhang G, Ronnekleiv O, Grandy DK, Allen RG (1998): Orphanin FQ is the major OFQ 1-17-containing peptide produced in the rodent and monkey hypothalamus. Peptides 19:133-139. Redrobe JP, Calo G, Regoli D, Quirion R (2002): Nociceptin receptor antagonists display antidepressant-like properties in the mouse forced swimming test. Naunyn Schmiedebergs Arch Pharmacol 365:164-167. Reinscheid RK, Nothacker H-P, Bourson A, Ardati A, Henningsen RA, Bunzow JR, Grandy DK, Langen H, Monsma FJ, Civelli O (1995): Orphanin FQ: a neuropeptide that activates an opioid-like G protein-coupled receptor. Science 270:792-794. Reinscheid RK, Ardati A, Monsma FJ, Civelli O (1996): Structure-activity relationship studies on the novel neuropeptide orphanin FQ. J Biol Chem 271:14163-14168. Reinscheid RK, Higelin J, Henningsen RA, Monsma FJ, Civelli O (1998): Structures that delineate orphanin FQ and dynorphin A pharmacologic selectivities. J Biol Chem 273:1490-1495. Riedl M, Shuster S, Vulchanova L, Wang J, Loh HH, Elde R (1996): Orphanin FQ/nociceptin-immunoreactive nerve fibers parallel those containing endogenous opioids in rat spinal cord. NeuroReport 7:1369-1372. Rizzi A, Bigoni R, Marzola G, Guerrini R, Salvadori S, Regoli D, Calo G (2001a): Characterization of the locomotor activity-inhibiting effect of nociceptin/orphanin FQ in mice. Naunyn Schmiedebergs Arch Pharmacol 363:161-165. Rizzi A, Marzola G, Bigoni R, Guerrini R, Salvadori S, Mogil JS, Regoli D, Calo G (2001b): Endogenous nociceptin signaling and stress-induced analgesia. NeuroReport 12:3009-3013. Rossi GC, Leventhal L, Pasternak GW (1996): Naloxone sensitive orphanin FQ-induced analgesia in mice. Eur J Pharmaco1311:R7-R8. Rossi GC, Leventhal L, Bolan E, Pasternak GW (1997): Pharmacological characterization of orphanin FQ/nociceptin and its fragments. J Pharmacol Exp Ther 282:858-865. Rossi GC, Mathis JP, Pasternak GW (1998): Analgesic activity of orphanin FQ2, murine preproorphanin FQ141-157, in mice. NeuroReport 9:1165-1168. Saito Y, Maruyama K, Saido TC, Kawashima S (1995): N23K, a gene transiently up-regulated during neural differentiation, encodes a precursor protein for a newly identified neuropeptide nociceptin. Biochem Biophys Res Commun 217:539-545. Saito Y, Maruyama K, Kawano H, Hagino-Yamagishi K, Kawamura K, Saido TC, Kawashima S (1996): Molecular

191

Ch. III

C.R. Neal Jr. et al.

cloning and characterization of a novel form of neuropeptide gene as a developmentally regulated molecule. J Biol Chem 271:15615-15622. Saito Y, Maruyama K, Saido TC, Kawashima S (1997): Over expression of a neuropeptide nociceptin/orphanin FQ precursor gene, N23K/N27K, induces neurite outgrowth in mouse NS20Y cells. J Neurosci Res 48:397-406. Sakurada T, Sakurada S, Katsuyama S, Hayashi T, Sakurada C, Tan-No K, Johansson H, Sandin J, Terenius L (2000): Evidence that N-terminal fragments of nociceptin modulate nociceptin-induced scratching, biting and licking in mice. Neurosci Lett 279:61-64. Sandin J, Georgieva J, Schott PA, Ogren SO, Terenius L (1997): Nociceptin/orphanin microinjected into hippocampus impairs spatial learning in rats. Eur J Neurosci 9:194-197. Saper CB (1995): The central autonomic system. In: Paxinos G (Ed), The Rat Nervous System. Sydney: Academic Press, pp. 107-135. Schuligoi R, Amann R, Angelberger P, Peskar BA (1997): Determination of nociceptin-like immunoreactivity in the rat dorsal spinal cord. Neurosci Lett 224:136-138. Schulz S, Schreff M, Nuss D, Gramsch C, Hollt V (1996): Nociceptin/orphanin FQ and opioid peptides show overlapping distribution but not co-localization in pain-modulatory brain regions. NeuroReport 7:3021-3025. Schwerdtfeger WK, Buhl EH, Germroth P (1990): Disynaptic olfactory input to the hippocampus mediated by stellate cells in entorhinal cortex. J Comp Neurol 292:163-177. Shimohigashi Y, Hatano R, Fujita T, Nakashima R, Nose T, Sujaku T, Saigo A, Shinjo A, Nagahisa A (1996): Sensitivity of opioid receptor-like receptor ORL1 for chemical modification on nociceptin, a naturally occurring nociceptive peptide. J Biol Chem 271:23642-23645. Shu YS, Zhao ZQ, Li MY, Zhou GM (1998): Orphanin FQ/nociceptin modulates glutamate- and kainic acidinduced currents in acutely isolated rat spinal dorsal horn neurons. Neuropeptides 32:567-571. Sim LJ, Childers SR (1997): Anatomical distribution of mu, delta, and kappa opioid- and nociceptin/orphanin FQ-stimulated [35S]Guanylyl-5'-O-(y-thio)-triphosphate binding in guinea pig brain. J Comp Neurol 386:562572. Sim LJ, Xiao R, Childers SR (1996): Identification of opioid receptor-like (ORL1) peptide-stimulated [35S]GTP gamma S binding in rat brain. NeuroReport 7:729-733. Simerly RB (1995): Anatomical substrates of hypothalamic integration. In: Paxinos G (Ed), The Rat Nervous System. Sydney: Academic Press, pp. 353-376. Simerly RB, McCall LD, Watson SJ (1988): Distribution of opioid peptides in the preoptic region: immunohistochemical evidence for a steroid-sensitive enkephalin sexual dimorphism. J Comp Neuro1276:442-459. Sinchak K, Hendricks DG, Baroudi R, Micevych P (1997): Orphanin FQ/nociceptin in the ventromedial nucleus facilitates lordosis in female rats. NeuroReport 8:3857-3860. Slugg RM, Ronnekleiv OK, Grandy DK, Kelly MJ (1999): Activation of an inwardly rectifying K+ conductance by orphanin-FQ/nociceptin in vasopressin-containing neurons. Neuroendocrinology 69:385-396. Standifer KM, Cheng J, Brooks AI, Honrado CP, Su W, Visconti LM, Biedler JL, Pasternak GW (1994): Biochemical and pharmacological characterization of mu, delta and kappa 3 opioid receptors expressed in BE(2)-C neuroblastoma cells. J Pharmacol Exp Ther 270:1246-1255. Stanfa LC, Chapman V, Kerr N, Dickenson AH (1996): Inhibitory action of nociceptin on spinal dorsal horn neurons of the rat, in vivo. Br J Pharmacol 118:1875-1877. Stratford TR, Holahan MR, Kelly AE (1997): Injections of nociceptin into nucleus accumbens shell or ventromedial hypothalamic nucleus increase food intake. NeuroReport 8:423-426. Sulaiman MR, Niklasson M, Tham R, Dutia MB (1999): Modulation of vestibular function by nociceptin/orphanin FQ: an in vivo and in vitro study. Brain Res 828:74-82. Sun RQ, Zhao CS, Wang HJ, Yang K, Chang JK, Han JS (2001): Nocistatin, a peptide reversing acute and chronic morphine tolerance. NeuroReport 12:1789-1792. Takahashi T, Mizuta Y, Owyang C (2000): Orphanin FQ but not dynorphin A, accelerates colonic transit in rats. Gastroenterology 119:71-79. Tian JH, Xu W, Fang Y, Mogil JS, Grisel JE, Grandy DK, Han JS (1997a): Bi-directional modulatory effect of orphanin FQ on morphine-induced analgesia: antagonism in brain and potentiation in spinal cord of the rat. Br J Pharmacol 120:676-680. Tian JH, Xu W, Zhang W, Fang Y, Grisel JE, Mogil JS, Grandy DK, Han JS (1997b): Involvement of endogenous orphanin FQ in electroacupuncture-induced analgesia. NeuroReport 8:497-500. Vanderah TW, Raffa RB, Lashbrook J, Burritt A, Hruby V, Porreca F (1998): Orphanin-FQ/nociceptin: lack of antinociceptive, hyperalgesic or allodynic effects in acute thermal or mechanical tests following intracerebroventricular or intrathecal administration to mice or rats. Eur J Pain 2:267-280.

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Vaughan CW, Christie MJ (1996): Increase by the ORL1 receptor (opioid receptor-likel) ligand, nociceptin, of inwardly rectifying K conductance in dorsal raphe nucleus neurons. Br J Pharmacol 117:1609-1611. Vaughan CW, Ingram SL, Christie MJ (1997): Actions of the ORL1 receptor ligand nociceptin on membrane properties of rat periaqueductal gray neurons in vitro. J Neurosci 17:996-1003. Vaughan CW, Connor M, Jennings EA, Marinelli S, Allen RG, Christie MJ (2001): Actions of nociceptin/orphanin FQ and other prepronociceptin products on rat rostral ventromedial medulla neurons in vitro. J Physiol 534:849859. Wagner EJ, Ronnekleiv OK, Grandy DK, Kelly MJ (1998): The peptide orphanin FQ inhibits beta-endorphin neurons and neurosecretory cells in the hypothalamic arcuate nucleus by activating an inwardly-rectifying K+ conductance. Neuroendocrinology 67:73-82. Walker JR, Terenius L, Koob GF (2002): Conditioned opioid withdrawal decreases nociceptin/orphanin FQ levels in the frontal cortex and olfactory tubercle. Neuropsychopharmacol 27:203-211. Wang JB, Johnson PS, Imai Y, Persico AM, Ozenberger BA, Eppler CM, Uhl GR (1994): cDNA cloning of an orphan opiate receptor gene family member and its splice variant. FEBS Lett 348:75-79. Wang XM, Zhang KM, Mokha SS (1996): Nociceptin (orphanin FQ), an endogenous ligand for the QRL1 (opioid-receptor-likel) receptor; modulates responses of trigeminal neurons evoked by excitatory amino acids and somatosensory stimuli. J Neurophysiol 76:3568-3572. Wang YQ, Zhu CB, Wu GC, Cao XD, Wang Y, Cui DF (1999): Effects of orphanin FQ on endomorphin-1 induced analgesia. Brain Res 835:241-246. Watson RE, Hoffmann GE, Wiegand SJ (1986): Sexually dimorphic opioid distribution in the preoptic area: manipulation by gonadal steroids. Brain Res 398:157-163. Wei WZ, Xie CW (1999): Orphanin FQ suppresses NMDA receptor-dependent long-term depression and depotentiation in hippocampal dentate gyms. Learn Mem 6:467-477. Wick MJ, Minnerath SR, Liana X, Elde R, Law PY, Loh HH (1994): Isolation of a novel cDNA encoding a putative membrane receptor with high homology to the cloned mu, delta, and kappa receptors opioid. Brain Res Mol Brain Res 27:37-44. Wu Y, Pu L, Ling K, Zhao J, Cheng Z, Ma L, Pei G (1997): Molecular characterization and functional expression of opioid receptor-like 1 receptor. Cell Res 7:69-77. Xu XJ, Hao JX, Wiesenfeld-Hallin Z (1996): Nociceptin or antinociceptin: potent spinal antinociceptive effect of orphanin FQ/nociceptin in the rat. NeuroReport 7:2092-2094. Xu IS, Hashemi M, Calo G, Regoli D, Wiesenfeld-Hallin Z, Xu XJ (1999): Effects of intrathecal nocistatin on the flexor reflex and its interaction with orphanin FQ nociceptin. NeuroReport 10:3681-3684. Yakimova KS, Pierau FK (1999): Nociceptin/orphanin FQ: effects on thermoregulation in rats. Methods Find Exp Clin Pharmacol 21:345-352. Yamamoto T, Nozaki-Taguchi N, Kimura S (1997): Analgesic effect of intrathecally administered nociceptin (orphanin FQ), an opioid receptor-like 1 receptor agonist, in the rat formalin test. Neuroscience 81:249-254. Yamamoto T, Nozaki-Taguchi N, Sakashita Y, Kimura S (1999): Nociceptin/orphanin FQ: role in nociceptive information processing. Prog Neurobiol 57:527-535. Yu TP, Xie CW (1998): Orphanin FQ/nociceptin inhibits synaptic transmission and long-term potentiation in rat dentate gyrus through postsynaptic mechanisms. J Neurophysiol 80:1277-1284. Yu TP, Fein J, Phan T, Evans CJ, Xie CW (1997): Orphanin FQ inhibits synaptic transmission and long-term potentiation in rat hippocampus. Hippocampus 7:88-94. Yu LC, Lu JT, Huang YH, Meuser T, Pietruck C, Gabriel A, Grond S, Pierce-Palmer P (2002): Involvement of endogenous opioid systems in nociceptin-induced spinal antinociception in rats. Brain Res 945:88-96. Zamboni L, DeMartino C (1967): Buffered picric acid formaldehyde, a rapid new fixative for electron microscopy. J Cell Bio135:148A. Zaveri N, Polgar WE, Olsen CM, Kelson AB, Grundt P, Lewis JW, Toll L (2001): Characterization of opiates, neuroleptics, and synthetic analogs at ORL 1 and opioid receptors. Eur J Pharmacol 428:29-36. Zeilhofer HU, Selbach UM, Guhring H, Erb K, Ahmadi S (2000): Selective suppression of inhibitory synaptic transmission by nocistatin in the rat spinal cord dorsal horn. J Neurosci 20:4922-4929. Zhao CS, Li BS, Zhao GY, Liu HX, Luo F, Wang Y, Tian JH, Chang JK, Han JS (1999): Nocistatin reverses the effect of orphanin FQ/nociceptin in antagonizing morphine analgesia. NeuroReport 10:297-299. Zheng F, Grandy DK, Johnson SW (2002): Actions of orphanin FQ/nociceptin on rat ventral tegmental area neurons in vitro. Br J Pharmacol 136:1065-1071. Zhu CB, Cao XD, Xu SF, Wu GC (1997): Orphanin FQ potentiates formalin-induced pain behavior and antagonizes morphine analgesia in rats. Neurosci Lett 235:37-40.

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CHAPTER IV

Localization of galanin receptor subtypes in the rat CNS D. O'DONNELL, E MENNICKEN, C. HOFFERT, D. HUBATSCH, M. PELLETIER, P. WALKER AND S. AHMAD

1. INTRODUCTION Galanin is a 29 amino acid peptide (30 aa in human) that is widely distributed in central and peripheral tissues. Galanin is proposed to be involved in a broad spectrum of biological effects including neuroendocrine control, food intake, sensory transmission, memory and learning, central cardiovascular regulation and has been implicated in a variety of different diseases. Due to its wide range of actions and its potential for various therapeutic interventions, galanin research has attracted much interest since its discovery in the early 1980s. To date, three novel and distinct galanin receptor (GALR) subtypes have been cloned, each encoded by separate genes and located on different human chromosomes. Knowledge of their sequence has enabled the generation of receptor-specific probes for mRNA localization studies. The tissue distribution patterns of GALR1 and GALR2 are well characterized; however, localization of GALR3 is somewhat more controversial. This paper reviews our current knowledge of the distribution of GALRs with a particular focus on the rat CNS. For comparative purposes, we have performed receptor autoradiography binding with 125I-galanin (which displays similar affinity for all three receptor subtypes) and in situ hybridization analyses with receptor subtype selective probes to GALR1, GALR2 and GALR3 on series of consecutive sections spanning the rostrocaudal extent of the rat brain. Although there are some areas of overlap, each of the three galanin receptors exhibits its own characteristic pattern of expression, suggesting individual receptor subtypes are likely to mediate different effects of galanin within the CNS. 2. GALANIN 2.1. HISTORICAL PERSPECTIVE Galanin was discovered in 1983 by Victor Mutt and his colleagues at the Karolinska Institute in Sweden (Tatemoto et al., 1983). The name galanin is derived from the first (glycine) and last (alanine) amino acids of the pig galanin sequence. Though initially isolated from pig intestine (Tatemoto et al., 1983), galanin was subsequently found to be present within peripheral and central nervous systems where it acts as a neurotransmitter and/or neuromodulator (see sections below). As is the case with many peptides, galanin is synthesized as part of a

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

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precursor protein, preprogalanin, which is cleaved to yield galanin and galanin messageassociated peptide (GMAP) in a 1:1 ratio (Kaplan et al., 1988; Sillard et al., 1992). Although galanin is unique in that it shares little homology with any known peptide, it is nonetheless highly conserved across several species. To date, galanin has been identified in 15 vertebrate species. Its N-terminus is particularly well conserved and appears to be critical for biological activity (for review see Langel and Bartfai, 1998; Wynick et al., 1998b). Like most neuropeptides, galanin is amidated at the carboxy terminus (Tatemoto et al., 1983). The human sequence is an exception however since it differs from sequences in other species in that it contains 30 amino acids instead of 29 and is not amidated at the N-terminus (Evans and Shine, 1991). The significance of this evolutionary transformation is unclear since galanin from rat, pig and human bind to and activate with similar affinity the different cloned rat and human GALRs (see Branchek et al., 1998). Since its discovery in the early 1980s, more than 2000 reports have appeared implicating galanin in a wide spectrum of biological processes, underscoring the importance of this peptide. A landmark finding was the demonstration of a marked up-regulation of galanin in dorsal root ganglia (DRG) and spinal cord neurons following sciatic nerve injury (Hokfelt et al., 1987), which led to much speculation as to the role of galanin in nociceptive sensory function in both normal and neuropathic states. In the early 1990s, Bartfai and colleagues developed a series of high affinity chimeric peptides (Bartfai et al., 1991, 1992, 1993b) providing valuable research tools, which have significantly contributed to elucidating galanin's roles in feeding, memory and other physiological processes. The mid-to-late 1990s were marked by the cloning of three distinct galanin receptor subtypes (see Section 3), which led to their pharmacological characterization, elucidation of signaling pathways, and anatomical mapping. 2.2. DISTRIBUTION Early studies using immunohistochemistry and radioimmunoassay demonstrated that galaninlike immunoreactivity was widely distributed in central and peripheral tissues of several species including human (Rokaeus et al., 1984; Ch'ng et al., 1985; Melander et al., 1985, 1986a,b; Skofitsch and Jacobowitz, 1985, 1986; Melander and Staines, 1986; Gentleman et al., 1989; Michener et al., 1990; Elmquist et al., 1992; Kordower et al., 1992). For a comprehensive description of the anatomy and physiology of central galanin-containing pathways in various species and of the co-localization of galanin with other neurotransmitters see review by Merchenthaler et al. (1993). In the rat, galanin-immunoreactive cell bodies and/or fibers are present, at least to some extent, in virtually all brain regions. Highest levels of galanin-immunoreactive profiles were detected in the telencephalon, hypothalamus, thalamus, pons and medulla. Within the telencephalon, moderate numbers of galanin-immunoreactive cell bodies were detected in the cingulate and medial prefrontal cortex (Skofitsch and Jacobowitz, 1985). The medial septum and diagonal band also contain moderate numbers of galanin-immunoreactive neurons (Skofitsch and Jacobowitz, 1985); however, double-labeling techniques have demonstrated that only a subpopulation of these galanin-containing neurons are co-localized with choline acetyltransferase (CHAT) and that these neurons in fact project to the hippocampal formation (Crawley and Wenk, 1989; Miller et al., 1998). Galanin immunoreactivity in the hippocampus is relatively low by comparison to other brain regions and is restricted largely to the ventral hippocampus (Skofitsch and Jacobowitz, 1985). Galanin-immunoreactive fibers within the hippocampal formation originate from septal and diagonal band cholinergic neurons as 196

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mentioned above, but also from locus coeruleus noradrenergic neurons (Melander et al., 1985, 1986c). In the diencephalon, a high abundance of galanin-immunoreactive cell bodies and/or fibers was reported within the medial preoptic area, periventricular, suprachiasmatic, paraventricular, and arcuate nuclei as well as the lateral hypothalamic area and at the base of the mammillary body (Skofitsch and Jacobowitz, 1985). In contrast, only galanin-immunoreactive fibers were detected within the thalamus, and were predominantly located within the medial thalamic area including the parafascicular, reuniens, rhomboid and medial nuclei (Skofitsch and Jacobowitz, 1985). In the mesencephalon, a sparse network of galanin fibers was observed in the periaqueductal gray, ventral tegmental area, and midbrain reticular formation, whereas the dorsal raphe and the lateral part of the substantia nigra contained moderate numbers of immunoreactive cells and fibers (Skofitsch and Jacobowitz, 1985). The cerebellum was devoid of galanin immunoreactivity (Merchenthaler et al., 1993). In the pons, a dense accumulation of galanin-containing cell bodies and fibers was observed in the locus coeruleus with fewer immunoreactive profiles seen within the parabrachial nucleus and the inferior colliculus (Skofitsch and Jacobowitz, 1985). In the medulla, immunoreactive profiles were detected in the ventral raphe nuclei, the nucleus of the solitary tract and the lateral reticular nucleus (Skofitsch and Jacobowitz, 1985). Overall, the spinal cord contained few galanin-immunoreactive fibers with the exception of higher densities observed in the superficial laminae I and II of the dorsal horn and in the area surrounding the central canal (Ch'ng et al., 1985; Skofitsch and Jacobowitz, 1985). It is noteworthy that a recent study has now shown that the distribution of galaninergic immunoreactivity in the mouse brain is quite similar to that in the rat (P6rez et al., 2001). This finding not only corroborates earlier immunohistochemical findings in the rat but also is of significant importance for the interpretation of recent studies using galanin knock-out and transgenic mice. 2.3. BIOLOGICAL ROLES Consistent with its widespread distribution, galanin exerts a multitude of biological effects. Historically, the first two reported physiological responses to galanin were its ability to contract rat isolated smooth muscle preparations and to induce mild hyperglycemia in dogs following an i.v. injection (Tatemoto et al., 1983). Galanin has since been implicated in feeding, cognitive function, somatosensory processing/nociception, hormone release and neuroendocrine secretion (Melander et al., 1987; Ottlecz et al., 1988; Merchenthaler et al., 1990; Bartfai et al., 1993a,b; Crawley, 1993; Bedecs et al., 1995; Cheung et al., 1996), as well as central cardiovascular regulation (Narvaez et al., 1994, 2000; Chen et al., 1996) and body fluid homeostasis (Koenig et al., 1989; Balment and al Barazanji, 1992). The roles of galanin in feeding, cognition and nociception have likely received the most attention and are discussed in more detail below.

2.3.1. Feeding Several reports indicate that central injections of galanin have a potent stimulatory effect on feeding behavior in rats and that there is macronutrient specificity in this response, which is mediated at the level of the paraventricular nucleus of the hypothalamus (PVN; Tempel et al., 1988; Kyrkouli et al., 1990; Tempel and Leibowitz, 1990). Results from behavioral studies looking at macronutrient choice preference following galanin injection into the PVN (Tempel 197

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et al., 1988; Smith et al., 1997) and expression studies examining hypothalamic mRNA levels in response to different diets (Brady et al., 1990) suggest that galanin is linked specifically to the ingestion of fat, whereas neuropeptide Y and norepinephrine, also potent stimulators of food intake, are associated preferentially with the ingestion of carbohydrates (Tempel et al., 1988; Tempel and Leibowitz, 1990). Galanin's stimulatory effects on fat ingestion are independent of circulating levels of glucocorticoids (Tempel and Leibowitz, 1993) but appear to be significantly modulated by endogenous opioid levels (Dube et al., 1994; Horvath et al., 1995), presumably acting via the g-opioid receptor (Barton et al., 1996). Several studies have also demonstrated that galanin receptor antagonists, when administered alone or in combination with galanin, reduce spontaneous ingestion of fat (Leibowitz and Kim, 1992) and block galanin-induced feeding when injected into the hypothalamus or amygdala of rats (Corwin et al., 1993; Crawley et al., 1993), suggesting that a galanin receptor antagonist may control obesity. Anatomically, galanin was most effective at increasing food intake in the satiated rat when injected directly into the PVN and adjacent periventricular region (Kyrkouli et al., 1990; Tempel and Leibowitz, 1990). Similar intrahypothalamic galanin injections into the lateral hypothalamus and ventromedial hypothalamus also increase food consumption (Schick et al., 1993), suggesting that galanin's orexigenic effects may be mediated in part by inhibiting satiety signals at the level of the lateral hypothalamus and ventromedial hypothalamic nucleus. This hypothesis is consistent with the observations that galanin's appetite-stimulating effects are observed in satiated but not fasted animals (Schick et al., 1993). Moreover, injection of galanin into the PVN stimulates dopamine release in the nucleus accumbens, suggesting that galanin initiates feeding in part by activating the mesolimbic dopamine reward system (Rada et al., 1998). Several extrahypothalamic sites known to be involved in feeding, such as the amygdala, the nucleus of the solitary tract (NTS), the lateral parabrachial nucleus and the area postrema, possess high levels of galanin peptide and galanin-binding sites and have also been demonstrated to mediate galanin-induced effects on feeding behavior (Kyrkouli et al., 1990; Corwin et al., 1993; Koegler and Ritter, 1996, 1998). Collectively, these neuroanatomical and behavioral findings suggest a pervasive mode of action and prominent role for galanin in feeding. 2.3.2. Cognition and memory

Several lines of evidence support a role for galanin in memory. As mentioned above, galaninlike immunoreactivity is widely distributed within the basal forebrain of rats, monkeys and humans (Melander and Staines, 1986; Melander et al., 1986a; Kordower et al., 1992) and coexists with acetylcholine in medial septal neurons projecting to the hippocampal formation (Melander et al., 1986c), a key pathway involved in learning and memory. Galanin has potent inhibitory effects on the evoked release of acetylcholine both in vitro and in vivo (Fisone et al., 1987). Central administration of galanin impairs acquisition but not retrieval of spatial memory in the Morris swim maze (Sundstrom et al., 1988) and hinders working memory in a choice accuracy T-maze task when administered directly into the medial septal area (Givens et al., 1992). Likewise, co-administration of galanin attenuated the ability of exogenous acetylcholine to improve spatial memory deficits induced by neurochemical lesions (Mastropaolo et al., 1988). Finally, administration of galanin antagonists has been shown to improve acquisition in the Morris swim maze test (Ogren et al., 1992) and to completely block choice-accuracy deficits induced by galanin administration in a rodent working-memory paradigm (McDonald and Crawley, 1996). Taken together, these data infer that galanin has 198

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inhibitory actions on cholinergic function and that high levels of galanin can be detrimental to cognitive processes. Consistent with this notion, several studies demonstrated a galanin hyperinnervation in the brain of Alzheimer's patients, suggesting galanin may play an underlying role in the pathophysiology of Alzheimer's disease (Crawley, 1996; Mufson et al., 1998). The recent demonstrations that galanin over-expressing transgenic mice have fewer cholinergic basal forebrain neurons and display selective performance deficits in the Morris spatial swim test (Steiner et al., 2001), provide convincing arguments that sustained high levels of galanin may indeed play a part in the neurochemical and cognitive impairments characteristic of Alzheimer's disease. Therefore, it is reasonable to surmise that galanin antagonists may provide a therapeutic approach to enhance memory processes in patients with Alzheimer's disease.

2.3.3. Sensory transmission/nociception The role of galanin in sensory transmission has also received much attention. Anatomical mapping studies have demonstrated the presence of galanin-like immunoreactivity and galanin-binding sites in the spinal cord of several mammals (see Sections 2.2 and 4.1). Galanin has been shown to depress spinal nociceptive reflexes in rats (Wiesenfeld-Hallin et al., 1989a); this latter effect is enhanced after nerve section (Xu et al., 1990; Wiesenfeld-Hallin et al., 1989b, 1992b). The galanin peptide is also present in a small number of DRG neurons (Ch'ng et al., 1985; Skofitsch and Jacobowitz, 1985), and sciatic nerve section increases galanin-like immunoreactivity and galanin mRNA in these neurons (Hokfelt et al., 1987, 1994; Villar et al., 1989). Recent studies have demonstrated that the pattern and extent of injury-induced galanin up-regulation in primary sensory neurons is differentially regulated following partial and complete sciatic nerve injuries (Ma and Bisby, 1997). For instance, partial sciatic nerve injuries induced greater galanin up-regulation in medium- and large-size DRG neurons than complete sciatic nerve injury (Ma and Bisby, 1997), suggesting there may be different mechanisms involved. Chronic administration of the putative galanin receptor antagonist M35 (Verge et al., 1993) and of antisense oligonucleotides directed to galanin (Ji et al., 1994) markedly increased the severity of the autotomy (self mutilation) behavior in rats following nerve injury, suggesting that endogenous levels of galanin suppress axotomyinduced autotomy in rats and may play a role in controlling the development of neuropathic pain. Intrathecal administration of galanin has been reported to induce antinociception (Post et al,, 1988; Wiesenfeld-Hallin et al., 1989b, 1993; Liu and Hokfelt, 2000) and to potentiate the analgesic effect of morphine (Wiesenfeld-Hallin et al., 1990). Likewise, lumbar transplant of: neurons genetically modified to secrete galanin reversed nerve injury-induced pain-like behaviors (Eaton et al,, 1999). Collectively, these data suggest that galanin is involved in pain and that a non-peptide galanin agonist may be a potent analgesic for treating neuropathic pain. , ,

2,4. THERAPEUTIC IMPLICATIONS Due to galanin's involvement in many diverse physiological processes described above and its link with various diseases, galanin receptors are perceived as potential drug targets for a variety: of different therapeutic applications. These include obesity, psychiatric disorders, Alzheimer's disease;-analgesia, diabetes, cardiovascular diseases, sexual dysfunction and growth disorders (for review see Wang et al., 2000; as well as Volume 863 of the Annals of the New York Academy of Sciences). Galanin receptor agonists are thought to have therapeutic application in treatment of chronic pain and prevention of ischemic damage; ~199

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galanin receptor antagonists have therapeutic potential in the treatment of Alzheimer's disease, depression, and feeding disorders (see Bartfai et al., 1993a; Wang et al., 2000). Although several pharmaceutical companies have embarked on high throughput screening campaigns to identify non-peptide galanin receptor agonists and/or antagonists, only one such compound has been reported thus far in the literature. Sch 202596 was isolated from a fungal fermentation culture Aspergillus sp. by the Schering-Plough research group and represents the first non-peptide antagonist putatively selective for GALR1 (Chu et al., 1997). However, it has reportedly low affinity, with an IC50 of 1.7 ~M in an in vitro GALR1 assay (Chu et al., 1997). Moreover, with the exception of one other study, which describes an efficient procedure for its chemical synthesis (Katoh and Ohmori, 2000), there have been no reports describing the pharmacological properties of this compound on the other cloned galanin receptors or its ability to antagonize galanin effects in vivo. 2.5. GALANIN ANTAGONISTS The knowledge that the N-terminus of galanin, galanin(1-15), is highly conserved among several species and that it is sufficient for high affinity binding led to the design and synthesis of several high affinity chimeric peptides in the early 1990s (see review by Bedecs et al., 1995). These included M15 (galanin(1-13)-substance P(5-11) amide), M32 (galanin(113)-neuropeptide Y(25-36) amide), M35 (galanin(1-13)-bradykinin(2-9) amide), M40 (galanin(1-13)-(Ala-Leu)2-Ala amide) and C7 (galanin(1-13)-spantide), each containing the N-terminal portion of galanin, galanin(1-13), and another sequence covalently attached to the carboxy terminus, usually the C-terminus portion of other endogenous neuropeptides (Bartfai et al., 1991; Langel et al., 1992, 1996). The affinity (Kd) of these chimeric peptides for hypothalamic galanin receptors ranges from 0.1 to 10 nM (Langel and Bartfai, 1998). These chimeric peptides have also been demonstrated to act as putative, high affinity antagonists (IC50 = 0.1-10 nM) of exogenous galanin in various in vivo studies (Langel and Bartfai, 1998). For instance, M40 has been reported to inhibit the effects of galanin in rat in vivo models of feeding behavior (Leibowitz and Kim, 1992; Crawley et al., 1993; Corwin et al., 1993, 1995) and memory tasks (McDonald and Crawley, 1996; McDonald et al., 1997, 1998). Paradoxically however, most of these putative galanin antagonists have also been shown by several groups to possess agonist properties on all of the cloned receptors in various in vitro functional assays (Smith et al., 1997; Ahmad et al., 1998; Floren et al., 2000). Moreover, due to the chimeric nature of these peptides, some display bi-receptor recognition (see Bedecs et al., 1995) thus making the interpretation of in vivo results uncertain. Since the pharmacological profile, selectivity and mechanisms of action of current antagonists are questionable, subtype-selective preferably non-peptide antagonists are needed. The development of receptor subtype-specific antagonists and/or agonists is critical not only for clarifying the physiological roles of these receptors, but also may be of therapeutic usefulness. However, the only reported putative non-peptide antagonist is the aforementioned Sch 202595 compound, and it has reportedly low affinity for galanin receptors (Chu et al., 1997). In addition to the pharmacological approaches described above, receptor-specific antisense oligonucleotides have also been used to selectively target the GALR1 receptor subtype. In the rat, intrathecal administration of cell-penetrating peptide nucleic acid (PNA) constructs to GALR1 suppressed galanin receptor levels in spinal cord and altered pain transmission (Pooga et al., 1998), suggesting GALR1 plays a key role mediating spinal sensory transmission.

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2.6. GENETIC MANIPULATIONS OF GALANIN EXPRESSION In a further attempt to elucidate the role of galanin, several groups have genetically manipulated expression of galanin in mice. Galanin knockout mice have, at least to some extent, confirmed a neuroendocrine role for galanin as well as provided supporting evidence of a role for galanin in nociception and possibly in neuronal growth and survival. Wynick and colleagues observed that mice lacking a functional galanin gene exhibited a marked reduction in both pituitary content and circulating plasma levels of prolactin which resulted in the loss of lactation in female mice (Wynick et al., 1998a,b). They also reported a second phenotype whereby the response to nerve injury in the galanin knockout mice was altered (Wynick et al., 1998b). The mutant mouse displayed a marked reduction in both peripheral nerve regeneration and the development of chronic neuropathic pain following peripheral nerve damage as compared to wild-type mice (Wynick et al., 1998b; Holmes et al., 2000; Kerr et al., 2000a). Moreover, galanin knockout mice exhibited a reduction in the number of neurons in the DRG, suggesting galanin may also be critical for neuronal development and survival (Holmes et al., 2000). Conversely, mice over-expressing galanin appear normal in all aspects however exhibit significant elevation of nociceptive threshold to heat (hypoalgesia) which is reversed by administration of the galanin receptor antagonist M-35 (Blakeman et al., 2001). Unexpectedly, this alteration appeared to be specific to heat nociceptive threshold since no change in response to mechanical or cold stimulation was observed (Blakeman et al., 2001). These findings are somewhat consistent with those of Kerr et al. (2000a) who reported that galanin knockout mice demonstrated reduced latency to noxious heat (hyperalgesia) however they also reported reduced latency to mechanical withdrawal thresholds compared to wild-type. Crawley and colleagues also developed transgenic mice that overexpressed galanin (Steiner et al., 2001) in an attempt to mimic the increased galanin innervation observed in the brain of Alzheimer's patients (Crawley, 1996; Mufson et al., 1998), using the same dopamine ~-hydroxylase promoter as Blakeman et al. (2001). Over-expression of galanin was associated with a decrease in the number of cholinergic basal forebrain neurons and selective performance deficits in the Morris spatial swim test, suggesting sustained high levels of galanin may play a part in the neurochemical and cognitive impairments characteristic of Alzheimer's disease (Steiner et al., 2001). Similar genetic manipulations aiming at the selective disruption of the different galanin receptor subtypes are in all likelihood ongoing but have not yet been reported. 2.7. GALANIN-RELATED PEPTIDES

2.7.1. Galanin message-associated peptide (GMAP) Galanin message-associated peptide (GMAP) is a biologically active peptide, which is cleaved from preprogalanin along with galanin (Rokaeus and Brownstein, 1986). Hokfelt and colleagues have demonstrated, using GMAP-specific antibodies, the presence of GMAPimmunoreactive cell bodies and/or nerve fibers within the CNS of the rat namely in the hypothalamus, septum, pons and spinal cord (Hokfelt et al., 1992; Xu et al., 1994) as well as in a limited number of DRG neurons (Xu et al., 1995). The intimate overlap between GMAP- and galanin-immunoreactive profiles suggested that these two peptides are likely expressed in a 1 : 1 ratio (Hokfelt et al., 1992). GMAP-immunoreactive nerve fibers have also been observed throughout the gastrointestinal tract (Xu et al., 1994), and a strong GMAP201

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immunoreactive signal was detected in prolactin cells of the anterior pituitary gland as well as in the insulin-secreting cells located in the islets of Langerhans of the pancreas (Hokfelt et al., 1992). In contrast to the wealth of knowledge on galanin, very little is known with respect to the role of GMAP in the CNS. It appears however that GMAP may have several features in common with galanin. For instance, GMAP, like galanin, is significantly up-regulated in DRG neurons following sciatic nerve injury (Xu et al., 1995) and intrathecal administration of GMAP (Xu et al., 1995) as well some, but not all, GMAP fragments (Xu et al., 1996) produced a moderate facilitation of the flexor reflex. GMAP, like galanin, relieves mechanical allodynia, however, is less potent than galanin (Hao et al., 1999). Moreover, as was the case with galanin fragments, several biologically active fragments of GMAP have been identified and shown to exhibit distinct pharmacological profiles in vivo, thus suggesting the possibility of different GMAP receptor subtypes, analogous to the multi-receptor system for galanin (Xu et al., 1996).

2.7.2. Galanin-like peptide (GALP) Undoubtedly a significant finding was the recent discovery of GALP, a novel galanin-like peptide, isolated from porcine hypothalamus extracts using a functional [35S]GTPyS binding assay (Ohtaki et al., 1999). GALP is a 60 amino acid peptide and unlike most neuropeptides (but akin to human galanin) has a non-amidated C-terminus. Cloning of the pig, rat and human sequences confirmed the molecular identity of GALP and revealed that it is reasonably well conserved across these species (Ohtaki et al., 1999). This discovery is even more interesting in that a portion of GALP, the amino acid sequence of GALP(9-21), is in fact identical to that of GAL(1-13) and more importantly, unlike GMAP, GALP(1-60) binds to the cloned galanin receptors, activating preferentially the GALR2 relative to GALR1 (Ohtaki et al., 1999). Unfortunately, due to technical reasons the binding affinity of GALP for GALR3 was not determined in the study by Ohtaki et al. (1999) nor to our knowledge has it been reported elsewhere. It would be interesting to determine if GALP is indeed the endogenous ligand for GALR3 since galanin has been shown to have lower affinity for the cloned human GALR3 (Smith et al., 1998). In situ hybridization studies indicate that GALP mRNA is expressed rather exclusively in the arcuate nucleus and median ~eminence of the hypothalamus and in the posterior lobe of the pituitary (Larm and Gundlach, 2000; Kerr et al., 2000b). Consistent with these findings, immunohistochemical studies reveal that GALP-immunoreactive neurons are only detected in the arcuate nucleus, median eminence and infundibular stalk (Takatsu et al., 2001). Further characterization, using double label immunohistochemistry have demonstrated that the majority (>85%) of GALP neurons within the arcuate nucleus coqocalized:with the leptin receptor, but not with c~-melanotropin-stimulating hormone, somatostatin, neuropeptide Y, agouti,related protein, or galanin ~(Takatsu et al., 2001). In~ addition, dense staining GALPcontaining fibers are present in the p~avemricular hypothalamic nucleus, lateral septum as well as in the bed nucleus of the stria terminalis and medial preoptic area where they are in close contact to gonadotropin=releasing hormone-immunoreactive fibers (Takatsu et al,, 2001). Interestingly, the overall distribution of GALP-i~unoreactive profiles coincides with the expresfion of GALR3 mRNA that we reported (Mennicken et al., 2002; herein). Although thephysiological roles of GALP remainto be elucidated, these neuroanatomical data suggest that GALP, like galanin, may play a role in regulation of feeding behavior and reproduction. Furthermore, the &scovery of a novel galanin-like peptide reinforces the notion that other 202

Localization of galanin receptor subtypes in the rat CNS

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

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

, ,

,

203

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

I

i

..........[

,,

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

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

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

Localization of galanin receptor subtypes in the rat CNS

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

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

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

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

Localization of galanin receptor subtypes in the rat CNS

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

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

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

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

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

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

rat CNS

Ch. IV

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

Region

125I-Galanin

GALR 1

GALR2

+

GALR3

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

++++

+++

External plexiform layer

+++

-

-

Internal plexiform layer

++

++

+

Granular layer

++++

++

+

++

++

+

Islands of Calleja

+++

(+)

(+)

M o l e c u l a r layer

+

-

-

++++

++++

-

(+)

Anterior olfactory nucleus Olfactory tubercle

Nucleus of the accessory olfactory tract

Cerebral cortex Neocortex

(+)

-

Entorhinal cortex

+++

++

-

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

+++

+++

-

Insular cortex

++

++

-

Piriform cortex

+++

++

+

Retrosplenial cortex

+

-

++

Limbic and basal forebrain Lateral septum

+++

++

-

Medial septum

+

+

-

Bed nucleus of the stria terminalis

+++

++

+

Stria terminalis

++++

-

-

Nucleus vertical limb of the diagonal band

+++

++

+

Nucleus horizontal limb of the diagonal band

+++

++

+

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

+++

++

-

Medial nucleus

+++

++

+

Central nucleus

+++

+++

+

Cortical nucleus

+++

++

-

Shell

+++

+

-

Core

++

-

-

+ +

+

-

Dorsal C A cell fields

-

-

-

Dorsal dentate g y m s Dorsal subiculum Ventral CA1

++ +++

+++

+++ +

Ventral CA2, CA3 Ventral dentate g y m s

+ +

+ -

+ ++

Ventral subicu lum

+++

-

-

Centrolateral nucleus

++++

+++

-

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

++++

+++

-

Intermediodorsal nucleus

++

++

-

Laterodorsal nucleus

++

+

+

Paracentral nucleus

++

+++

+

Paraventricular nucleus

++

++++

-

Reuniens nucleus

++

++

+

(+)

A c c u m b e n s nucleus

Striatum Globus pallidus

Hippocampus

Thalamus

213

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TABLE 1 (continued) Region

125i_Galanin

GALR1

GALR2

GALR3

Habenula

+++

Zona incerta

+++

+++

+

(+)

++

+

-

Arcuate nucleus Paraventricular hypothalamic nucleus

+

+

++

(+)

+

+++

++

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

(+)

+

+

+

++

Hypothalamus

Supraoptic nucleus

+++

+++

-

-

Ventromedial hypothalamic nucleus

+++

++

++

++

P r e o p t i c area

+

++

+

++

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

++

++

+

(+)

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

+++

+++

++

+

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

++

++

++

++

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

++

++

++

+

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

++

-

+++

+

Medial mammillary nucleus

+

-

+++

-

Supramammillary nucleus

++

++

+

(+)

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

+++

-

++

-

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

-

-

+

-

Ventral t e g m e n t a l area

+++

-

++

-

R a p h e linearis

++

-

+

-

Midbrain

Central gray

+++

++

+

+

Dorsal raphe

+++

+

+

-

Mesencephalic V nucleus

+

+

+

-

Superficial layers

++

+

(+)

-

Optic l a y e r

+++

++

(+)

-

-

-

Superior colliculus

C o r t e x of the inferior colliculus

++

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

.

+ .

.

.

Pons and medulla Parabrachial nucleus

+++

+++

++

+

Locus coeruleus

+

++

+

(+)

Sensory trigeminal nucleus

++

-

++

-

Motor trigeminal nucleus

++

++

+

-

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

++ + ++

++ ++

(+) (+) +

(+) + -

Raphe obscursus nucleus

++

++

(+)

-

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

++

-

(+)

-

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

++

-

++

-

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

+

+

++

Hypoglossal nucleus

+

-

++

-

Vestibular c o m p l e x

+

(+)

+

-

Facial n u c l e u s

-

-

+

-

Ambiguus nucleus

+

-

++

-

Lateral reticular nucleus

++

+

+

-

External cuneate nucleus

-

-

++

-

N u c l e u s of the solitary tract

+++

++

-

-

Cerebellum Molecular layer

.

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

-

-

+++

-

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

-

-

(+)

-

214

.

.

.

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Ch. IV

TABLE 1 (continued) Region

125i_Galanin

GALR1

GALR2

GALR3

++++ +++ ++

+++ + ++

+++ ++ ++-t++

(+) + +

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

+ ++ . . .

+at++ -

++ -

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

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

. . .

. . .

Dorsal root ganglia Small cells Medium cells Large cells

* * *

++ ++ ++++

++++ at-++ +

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

~m

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

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v

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

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

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

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

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

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

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

~m

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

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

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

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

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

<_._..._

Fig. 7. Brightfield photomicrographs showing the cellular distribution of GALR1 (A), GALR2 (B) and GALR3 (C) mRNA in the locus coeruleus. Adjacent coronal rat brain sections were hybridized with 35S-labeled riboprobes directed to GALR1, GALR2 or GALR3 and counterstained with hematoxylinand eosin. Neuroanatomical structures were identified according to the rat brain atlas of Paxinos and Watson (1998) as indicated on the schematic drawing. Moderate expression of GALR1 mRNA is observed in the majority of neurons (A), whereas GALR2 mRNA is expressed at lower levels (B). In contrast, only a few LC neurons express GALR3 mRNA albeit at very low levels. See Section 9 for abbreviations. Scale bar: 50 Ixm. 227

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to large reticular neurons at the level of the dorsal paragigantocellular reticular field (DPGi) (Fig. 6H). Interestingly, reticular neurons give rise to branching and highly collateralized axons with long descending and ascending projections, which are believed to exert widespread influence on the spinal cord and brain, especially at the level of the sensory-motor integration of the posture and movement control (for review see Jones, 1995). In the same way, the parabrachial nucleus plays an important role in the central autonomic system as an interface between medullary reflex control and forebrain behavioral and integrative regulation of the autonomic system (Saper, 1995). Moreover the GALR3-expressing cells were observed in the most lateral parts of the parabrachial nucleus (Fig. 6G), from which the descending projections originate and which play a role in various cardiovascular responses (Saper, 1995) suggesting a potential role for GALR3 in cardiovascular control (see also Section 6). Although not readily discernible on film autoradiograms, a few faintly labeled GALR3-expressing cells were detected by emulsion autoradiography in the locus coeruleus (Fig. 7C) and the caudal part of pontine reticular formation. All other regions in the pons and medulla were noticeably free of GALR3-expressing cells. And, akin to GALR1, no specific expression of GALR3 was observed within the cerebellar cortex.

4.4.5. Spinal cord GALR3 mRNA was also expressed in spinal cord but at very low levels, as evidenced by a weak hybridization signal in the superficial dorsal horn on film autoradiograms (Table 1). Microscopic examination of emulsion-processed sections confirmed a specific but very low accumulation of silver grains over cells within laminae I-II. Additionally, a few moderately labeled cells were seen scattered throughout the dorsal horn particularly in laminae IV-VI as well as around the central canal corresponding to laminae X (Mennicken et al., 2002; Table 1). Based on morphological appearance alone, these labeled cells exhibited all the characteristic features of neurons. However, double labeling studies are required to confirm the precise cell phenotype of GALR3 mRNA-expressing cells. No GALR3 labeled cells were detected in the ventral horn. Thus the overall pattern of GALR3 expression, though much weaker, is reminiscent of that of GALR1, and suggests that GALR3 may also play a role in mediating galanin's effects on pain transmission.

5. EXPRESSION OF GALRs BY G L I A L CELLS Although the expression of GALRs by neurons is well established, less is known with respect to their presence on glial cells. Interestingly, a recent study has demonstrated the presence of 125I-galanin-binding sites on astrocytes (and neurons) in explant cultures of rat neocortex, cerebellum, locus coeruleus and spinal cord (Hosli et al., 1997), suggesting that galanin may also mediate physiological responses in glial cells. This notion is corroborated by another study which demonstrated the presence of functional GALRs in cultures of rat astrocytes, using the expression of immediate early genes as a model for receptor-mediated transcriptional activation (Priller et al., 1998). Whether galanin receptors are present on glial cells in the intact animal, however, remains to be demonstrated. In our studies, all three GALR probes showed clearer accumulations of silver grains over neurons with very few grains distributed over white matter (Figs. 3 and 7). Although the resolution is not sufficient to unequivocally exclude glial cell labeling, no obvious labeled glial cells were detected for any of the three GALR subtypes, suggesting that under physiological conditions, transcripts may be expressed 228

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exclusively in neuronal cell populations within the CNS. It is, however, possible that, under resting conditions, mRNA levels for the different GALRs are present in glial cells but in very low abundance and therefore may not have been detected in our in situ hybridization analysis. It is also possible that glial expression of GALRs may be up-regulated under certain pathophysiological conditions or when placed in culture, similar to what has been observed with other neuropeptide receptors (Mantyh et al., 1989b; Nouel et al., 1999).

6. L O C A L I Z A T I O N OF GALANIN R E C E P T O R S IN T H E C I R C U M V E N T R I C U L A R ORGANS OF T H E RAT Circumventricular organs are located within the brain; however, they are not considered part of the CNS since the capillaries of these structures are fenestrated and by definition lie outside the blood-brain barrier (Oldfield and Mckinley, 1995). There are seven such organs in the rat. They are all located in the walls of the lateral, third and fourth ventricles and play important roles in homeostasis and various neuroendocrine functions (Oldfield and Mckinley, 1995). Several immunohistochemical studies have indicated the presence of galanin-containing fibers and the occasional galanin-immunoreactive neuron at least to some extent within most circumventricular organs in the rat (Skofitsch and Jacobowitz, 1985; Melander et al., 1986c; Merchenthaler et al., 1993) as well as in other mammals (Elmquist et al., 1992; Kordower et al., 1992; P6rez et al., 2001), suggesting that galanin may mediate water balance, food intake, blood pressure and reproduction. There is, however, little or no data in the literature on the localization of GALRs in these structures. Our findings revealed that several of the circumventricular organs in the rat do in fact exhibit galanin-binding sites and/or mRNA for one or more of the GALR subtypes (Fig. 8 and Table 1). Of particular interest is the subfornical organ (SFO) since it exhibited high levels of 12SI-galanin-binding sites as well as moderate to low levels of all three galanin receptor subtype mRNAs (GalR2 ~ GalR3 > GalR1, Figs. 2G and 8). Even more striking is the observation that among the few structures within the CNS that express GALR3 mRNA, the SFO contains amongst the highest levels of GALR3 mRNA observed (see Figs. 2G and 8C, and Table 1). The overall preponderance of GALR3 mRNA in the SFO suggests that GALR3 may play a prominent role in body fluid homeostasis and cardiovascular regulation. The median eminence also displayed high levels of galanin-binding sites along with moderate levels of GALR1 and GALR2 mRNA, but was devoid of GALR3 mRNA expression (Fig. 2I). Moderate to low 125I-galanin-binding site densities were observed over the subcommissural organ and area postrema; however, expression of the different GALR subtypes mRNAs was not detected in these structures. No significant amounts of galanin-binding or GALR subtype hybridization signal were detected in the three remaining circumventricular organs, namely the vascular organ of the lamina terminalis (VOLT), the pineal gland and the choroid plexus.

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

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

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

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

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

8. CONCLUDING REMARKS

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

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

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

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

E/or

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

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

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

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

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

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

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

10. ACKNOWLEDGEMENTS

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

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11. REFERENCES Ahmad S, Shen SH, Walker R Wahlestedt C (1996): Molecular cloning of a novel widely distributed galanin receptor subtype (GALR2). 8th World Congress on Pain, IASP Press: Seattle, 81,134 (abstract). Ahmad S, O'Donnell D, Payza K, Ducharme J, Menard D, Brown W, Schmidt R, Wahlestedt C, Shen SH, Walker P (1998): Cloning and evaluation of the role of rat GALR-2, a novel subtype of galanin receptor, in the control of pain perception. Ann NY Acad Sci 863:108-119. Amiranoff B, Servin AL, Rouyer-Fessard C, Couvineau A, Tatemoto K, Laburthe M (1987): Galanin receptors in a hamster pancreatic beta-cell tumor: identification and molecular characterization. Endocrinology 121:284-289. Amiranoff B, Lorinet AM, Laburthe M (1989): Galanin receptor in the rat pancreatic beta cell line Rin m 5E Molecular characterization by chemical cross-linking. J Biol Chem 264:20714-20717. An SZ, Cutler G, Zhao JJ, Huang SG, Tian H, Li WB, Liang LM, Rich M, Bakleh A, Du J, Chen JL, Dai K (2001): Identification and characterization of a melanin-concentrating hormone receptor. Proc Natl Acad Sci USA 98:7576-7581. Annals of the New York Academy of Sciences (1998): Galanin: Basic Research Discoveries and Therapeutic Implications. Conference proceedings. Stockholm, Sweden, May 3-5, 1998. Annals of the New York Academy of Sciences, Vol. 863, pp. 1-469. Arvidsson U, Ulfhake B, Cullheim S, Bergstrand A, Theodorson E, Hokfelt T (1991): Distribution of 125I-galanin binding sites, immunoreactive galanin, and its coexistence with 5-hydroxytryptamine in the cat spinal cord: biochemical, histochemical, and experimental studies at the light and electron microscopic level. J Comp Neurol 308:115-138. Azumaya Y, Tsutsui K (1996): Localization of galanin and its binding sites in the quail brain. Brain Res 727:187195. Balment RJ, al Barazanji K (1992): Renal, cardiovascular and endocrine effects of centrally administered galanin in the anaesthetised rat. Regul Pept 38:71-77. Bartfai T, Bedecs K, Land T, Langel U, Bertorelli R, Girotti P, Consolo S, Xu XJ, Wiesenfeld-Hallin Z, Nilsson S (1991): M-15: high-affinity chimeric peptide that blocks the neuronal actions of galanin in the hippocampus, locus coeruleus, and spinal cord. Proc Natl Acad Sci USA 88:10961-10965. Bartfai T, Fisone G, Langel U (1992): Galanin and galanin antagonists: molecular and biochemical perspectives. Trends Pharmacol Sci 13:312-317. Bartfai T, Hokfelt T, Langel U (1993a): Galanin: a neuroendocrine peptide. Crit Rev Neurobiol 7:229-274. Bartfai T, Langel U, Bedecs K, Andell S, Land T, Gregersen S, Ahren B, Girotti P, Consolo S, Corwin R (1993b): Galanin-receptor ligand M40 peptide distinguishes between putative galanin-receptor subtypes. Proc Natl Acad Sci USA 90:11287-11291. Barton C, York DA, Bray GA (1996): Opioid receptor subtype control of galanin-induced feeding. Peptides 17:237-240. Bedecs K, Berthold M, Bartfai T (1995): Galanin: 10 years with a neuroendocrine peptide. Int J Biochem Cell Biol 27:337-349. Birgtil N, Weise C, Kreienkamp HJ, Richter D (1999): Reverse physiology in Drosophila: identification of a novel allatostatin-like neuropeptide and its cognate receptor structurally related to the mammalian somatostatin/galanin/opioid receptor family. EMBO J 18:5892-5900. Blakeman KH, Holmberg K, Hao JX, Xu XJ, Kahl U, Lendahl U, Bartfai T, Wiesenfeld-Hallin Z, Hokfelt T (2001): Mice over-expressing galanin have elevated heat nociceptive threshold. NeuroReport 12:423-425. Bloomquist BT, Beauchamp MR, Zhelnin L, Brown SE, Gore-Willse AR, Gregor P, Cornfield LJ (1998): Cloning and expression of the human galanin receptor GalR2. Biochem Biophys Res Commun 243:474-479. Borowsky B, Walker MW, Huang LY, Jones KA, Smith KE, Bard J, Branchek TA, Gerald C (1998): Cloning and characterization of the human galanin GALR2 receptor. Peptides 19:1771-1781. Brady LS, Smith MA, Gold PW, Herkenham M (1990): Altered expression of hypothalamic neuropeptide mRNAs in food-restricted and food-deprived rats. Neuroendocrinology 52:441-447. Branchek T, Smith KE, Walker MW (1998): Molecular biology and pharmacology of galanin receptors. Ann NY Acad Sci 863:94-107. Branchek TA, Smith KE, Gerald C, Walker MW (2000): Galanin receptor subtypes. Trends Pharmacol Sci 21:109117. Burazin TC, Larm JA, Ryan MC, Gundlach AL (2000): Galanin-R1 and -R2 receptor mRNA expression during the development of rat brain suggests differential subtype involvement in synaptic transmission and plasticity. Eur J Neurosci 12:2901-2917. 236

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Burazin TC, Larm JA, Gundlach AL (2001): Regulation by osmotic stimuli of galanin-R1 receptor expression in magnocellular neurones of the paraventricular and supraoptic nuclei of the rat. J Neuroendocrinol 13:358-370. Burgevin MC, Loquet I, Quarteronet D, Habert-Ortoli E (1995): Cloning, pharmacological characterization, and anatomical distribution of a rat cDNA encoding for a galanin receptor. J Mol Neurosci 6:33-41. Ch'ng JL, Christofides ND, Anand P, Gibson SJ, Allen YS, Su HC, Tatemoto K, Morrison JF, Polak JM, Bloom SR (1985): Distribution of galanin immunoreactivity in the central nervous system and the responses of galanin-containing neuronal pathways to injury. Neuroscience 16:343-354. Chambers J, Ames RS, Bergsma D, Muir A, Fitzgerald LR, Hervieu G, Dytko GM, Foley JJ, Martin J, Liu WS, Park J, Ellis C, Ganguly S, Konchar S, Cluderay J, Leslie R, Wilson S, Sarau HM (1999): Melanin-concentrating hormone is the cognate ligand for the orphan G-protein-coupled receptor SLC-1. Nature 400:261-265. Chen Y, Fournier A, Couvineau A, Laburthe M, Amiranoff B (1993): Purification of a galanin receptor from pig brain. Proc Natl Acad Sci USA 90:3845-3849. Chen YL, Chan SH, Chan JY (1996): Participation of galanin in baroreflex inhibition of heart rate by hypothalamic PVN in rat. Am J Physiol 271:8. Cheung CC, Clifton DK, Steiner RA (1996): Galanin: an unassuming neuropeptide moves to center stage in reproduction. Trends Endocrinol Metab 7:301-306. Chu M, Mierzwa R, Truumees I, King A, Sapidou E, Barrabee E, Terracciano J, Patel MG, Gullo VP, Burrier R, Das PR, Mittelman S, Puar MS (1997): A new fungal metabolite, sch 202596, with inhibitory activity in the galanin receptor galrl assay. Tetrahedron Lett 38:6111-6114. Corwin RL, Robinson JK, Crawley JN (1993): Galanin antagonists block galanin-induced feeding in the hypothalamus and amygdala of the rat. Eur J Neurosci 5:1528-1533. Corwin RL, Rowe PM, Crawley JN (1995): Galanin and the galanin antagonist M40 do not change fat intake in a fat-chow choice paradigm in rats. Am J Physiol 269:8. Crawley JN (1993): Functional interactions of galanin and acetylcholine: relevance to memory and Alzheimer's disease. Behav Brain Res 57:133-141. Crawley JN (1996): Minireview. Galanin-acetylcholine interactions: relevance to memory and Alzheimer's disease. Life Sci 58:2185-2199. Crawley JN, Wenk GL (1989): Co-existence of galanin and acetylcholine: is galanin involved in memory processes and dementia?. Trends Neurosci 12:278-282. Crawley JN, Robinson JK, Langel U, Bartfai T (1993): Galanin receptor antagonists M40 and C7 block galanininduced feeding. Brain Res 600:268-272. Deecher DC, Mash DC, Staley JK, Mufson EJ (1998): Characterization and localization of galanin receptors in human entorhinal cortex. Regul Pept 73:149-159. Depczynski B, Nichol K, Fathi Z, Iismaa T, Shine J, Cunningham A (1998): Distribution and characterization of the cell types expressing GALR2 mRNA in brain and pituitary gland. Ann NY Acad Sci 863:120-128. Diaz-Cabiale Z, Narvaez JA, Finnman UB, Bellido I, Ogren SO, Fuxe K (2000): Galanin-(1-16) modulates 5-HT1A receptors in the ventral limbic cortex of the rat. NeuroReport 11:515-519. Diaz Z, Narvaez JA, Hedlund PB, Aguirre JA, Gonzalez-Baron S, Fuxe K (1996): Centrally infused galanin-(1-15) but not galanin-(1-29) reduces the baroreceptor reflex sensitivity in the rat. Brain Res 741:32-37. Dube MG, Horvath TL, Leranth C, Kalra PS, Kalra SP (1994): Naloxone reduces the feeding evoked by intracerebroventricular galanin injection. Physiol Behav 56:811-813. Dutriez I, Lagny-Pourmir I, Epelbaum J, Beauvillain JC (1996): Autoradiographic quantitation and anatomical mapping of GTP sensitive-galanin receptors in the guinea pig central nervous system. J Chem Neuroanat 12:85-104. Dutriez I, Lagny-Pourmir I, Epelbaum J, Beauvillain JC (1997): Ultrastructural localization of galanin and galanin receptors in the guinea pig median eminence. Brain Res 753:36-46. Eaton MJ, Karmally S, Martinez MA, Plunkett JA, Lopez T, Cejas PJ (1999): Lumbar transplant of neurons genetically modified to secrete galanin reverse pain-like behaviors after partial sciatic nerve injury. J Periph Nerv Syst 4:245-257. Elmquist JK, Fox CA, Ross LR, Jacobson CD (1992): Galanin-like immunoreactivity in the adult and developing Brazilian opossum brain. Dev Brain Res 67:161-179. Evans HF, Shine J (1991): Human galanin: molecular cloning reveals a unique structure. Endocrinology 129:16821684. Fathi Z, Cunningham AM, Iben LG, Battaglino PB, Ward SA, Nichol KA, Pine KA, Wang J, Goldstein ME, Iismaa TP, Zimanyi IA (1997): Cloning, pharmacological characterization and distribution of a novel galanin receptor. [Erratum appears in Brain Res Mol Brain Res 1998 Jan;53(1-2)348]. Mol Brain Res 51:49-59. Fathi Z, Battaglino PM, Iben LG, Li H, Baker E, Zhang D, McGovern R, Mahle CD, Sutherland GR, Iismaa 237

Ch. I V

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

TP, Dickinson KE, Zimanyi IA (1998): Molecular characterization, pharmacological properties and chromosomal localization of the human GALR2 galanin receptor. Mol Brain Res 58:156-169. Fisone G, Wu CF, Consolo S, Nordstrom O, Brynne N, Bartfai T, Melander T, Hokfelt T (1987): Galanin inhibits acetylcholine release in the ventral hippocampus of the rat: histochemical, autoradiographic, in vivo, and in vitro studies. Proc Natl Acad Sci USA 84:7339-7343. Fisone G, Berthold M, Bedecs K, Unden A, Bartfai T, Bertorelli R, Consolo S, Crawley J, Martin B, Nilsson S (1989): N-terminal galanin-(1-16) fragment is an agonist at the hippocampal galanin receptor. Proc Natl Acad Sci USA 86:9588-9591. Floren A, Land T, Langel U (2000): Galanin receptor subtypes and ligand binding. Neuropeptides 34:331-337. Gaymann W, Falke N (1990): Galanin lacks binding sites in the porcine pituitary and has no detectable effect on oxytocin and vasopressin release from rat neurosecretory endings. Neurosci Lett 112:114-119. Gentleman SM, Falkai P, Bogerts B, Herrero MT, Polak JM, Roberts GW (1989): Distribution of galanin-like immunoreactivity in the human brain. Brain Res 505:311-315. Givens BS, Olton DS, Crawley JN (1992): Galanin in the medial septal area impairs working memory. Brain Res 582:71-77.

Gundlach AL, Burazin TC (1998): Galanin-galanin receptor systems in the hypothalamic paraventricular and supraoptic nuclei. Some recent findings and future challenges. Ann NY Acad Sci 863:241-251. Gustafson EL, Smith KE, Durkin MM, Gerald C, Branchek TA (1996): Distribution of a rat galanin receptor mRNA in rat brain. NeuroReport 7:953-957. Habert-Ortoli E, Amiranoff B, Loquet I, Laburthe M, Mayaux JF (1994): Molecular cloning of a functional human galanin receptor. Proc Natl Acad Sci USA 91:9780-9783. Hao JX, Shi TJ, Xu IS, Kaupilla T, Xu XJ, Hokfelt T, Bartfai T, Wiesenfeld-Hallin Z (1999): Intrathecal galanin alleviates allodynia-like behaviour in rats after partial peripheral nerve injury. Eur J Neurosci 11:427-432. Hedlund PB, Yanaihara N, Fuxe K (1992): Evidence for specific N-terminal galanin fragment binding sites in the rat brain. Eur J Pharmacol 224:203-205. Hedlund PB, Finnman UB, Yanaihara N, Fuxe K (1994): Galanin-(1-15), but not galanin-(1-29), modulates 5-HT1A receptors in the dorsal hippocampus of the rat brain: possible existence of galanin receptor subtypes. Brain Res 634:163-167.

Heuillet E, Bouaiche Z, Menager J, Dugay P, Munoz N, Dubois H, Amiranoff B, Crespo A, Lavayre J, Blanchard JC (1994): The human galanin receptor: ligand-binding and functional characteristics in the Bowes melanoma cell line. Eur J Pharmaco1269:139-147. Hill J, Duckworth M, Murdock P, Rennie G, Sabido-David C, Ames RS, Szekeres P, Wilson S, Bergsma DJ, Gloger IS, Levy DS, Chambers JK, Muir AI (2001): Molecular cloning and functional characterization of MCH2, a novel human MCH receptor. J Biol Chem 276:20125-20129. Hokfelt T, Wiesenfeld-Hallin Z, Villar M, Melander T (1987): Increase of galanin-like immunoreactivity in rat dorsal root ganglion cells after peripheral axotomy. Neurosci Lett 83:217-220. Hokfelt T, Aman K, Arvidsson U, Bedecs K, Ceccatelli S, Hulting AL, Langel U, Meister B, Pieribone V, Bartfai T (1992): Galanin message-associated peptide (GMAP)- and galanin-like immunoreactivities: overlapping and differential distributions in the rat. Neurosci Lett 142:139-142. Hokfelt T, Zhang X, Wiesenfeld-Hallin Z (1994): Messenger plasticity in primary sensory neurons following axotomy and its functional implications. [See comments]. Trends Neurosci 17:22-30. Holmes FE, Mahoney S, King VR, Bacon A, Kerr NC, Pachnis V, Curtis R, Priestley JV, Wynick D (2000): Targeted disruption of the galanin gene reduces the number of sensory neurons and their regenerative capacity. Proc Natl Acad Sci USA 97:11563-11568.

Holmqvist BI, Carlberg M (1992): Galanin receptors in the brain of a teleost: autoradiographic distribution of binding sites in the Atlantic salmon. J Comp Neurol 326:44-60. Horvath TL, Kalra SP, Naftolin F, Leranth C (1995): Morphological evidence for a galanin-opiate interaction in the rat mediobasal hypothalamus. J Neuroendocrinol 7:579-588. Hosli E, Ledergerber M, Kofler A, Hosli L (1997): Evidence for the existence of galanin receptors on cultured astrocytes of rat CNS: colocalization with cholinergic receptors. J Chem Neuroanat 13:95-103. Howard AD, Tan C, Shiao LL, Palyha OC, McKee KK, Weinberg DH, Feighner SD, Cascieri MA, Smith RG, Van Der Ploeg LH, Sullivan KA (1997): Molecular cloning and characterization of a new receptor for galanin. FEBS Lett 405:285-290.

Hulting AL, Meister B, Carlsson L, Hilding A, Isaksson O (1991): On the role of the peptide galanin in regulation of growth hormone secretion. Acta Endocrinol 125:518-525. Iismaa TP, Shine J (1999): Galanin and galanin receptors. Results Probl Cell Different 26:257-291. 238

Localization of galanin receptor subtypes in the rat CNS

Ch. IV

Iismaa TP, Fathi Z, Hort YJ, Iben LG, Dutton JL, Baker E, Sutherland GR, Shine J (1998): Structural organization and chromosomal localization of three human galanin receptor genes. Ann NY Acad Sci 863:56-63. Ikeda M, Dewar D, McCulloch J (1991): Preservation of [125I]galanin binding sites despite loss of cholinergic neurones to the hippocampus in Alzheimer's disease. Brain Res 568:303-306. Ikeda M, Dewar D, McCulloch J (1995): Galanin receptor binding sites in the temporal and occipital cortex are minimally affected in Alzheimer's disease. Neurosci Lett 192:37-40. Ji RR, Zhang Q, Bedecs K, Arvidsson J, Zhang X, Xu XJ, Wiesenfeld-Hallin Z, Bartfai T, Hokfelt T (1994): Galanin antisense oligonucleotides reduce galanin levels in dorsal root ganglia and induce autotomy in rats after axotomy. Proc Natl Acad Sci USA 91:12540-12543. Johard HA, Lundquist CT, Rokaeus A, Nassel DR (1992): Autoradiographic localization of 125I-galanin binding sites in the blowfly brain. Regul Pept 42:123-134. Jones BE (1995): Reticular formation: cytoarchitecture, transmitters, and projections. In: Paxinos G (Ed), The Rat Nervous System, 2nd ed. San Diego: Academic Press, pp. 155-171. Kaplan LM, Spindel ER, Isselbacher KJ, Chin WW (1988): Tissue-specific expression of the rat galanin gene. Proc Natl Acad Sci USA 85:1065-1069. Kar S, Quirion R (1994): Galanin receptor binding sites in adult rat spinal cord respond differentially to neonatal capsaicin, dorsal rhizotomy and peripheral axotomy. Eur J Neurosci 6:1917-1921. Kar S, Quirion R (1995): Neuropeptide receptors in developing and adult rat spinal cord: an in vitro quantitative autoradiography study of calcitonin gene-related peptide, neurokinins, mu-opioid, galanin, somatostatin, neurotensin and vasoactive intestinal polypeptide receptors. J Comp Neurol 354:253-281. Katoh T, Ohmori O (2000): Studies toward the total synthesis of Sch 202596, an antagonist of the galanin receptor subtype GalRI: synthesis of geodin, the spirocoumaranone subunit of Sch 202596. Tetrahedron Lett 41:465-469. Kerr BJ, Cafferty WB, Gupta YK, Bacon A, Wynick D, McMahon SB, Thompson SW (2000a): Galanin knockout mice reveal nociceptive deficits following peripheral nerve injury. Eur J Neurosci 12:793-802. Kerr NC, Holmes FE, Wynick D (2000b): Galanin-like peptide (GALP) is expressed in rat hypothalamus and pituitary, but not in DRG. NeuroReport 11:3909-3913. King SC, Slater P, Turnberg LA (1989): Autoradiographic localization of binding sites for galanin and VIP in small intestine. Peptides 10:313-317. Koegler FH, Ritter S (1996): Feeding induced by pharmacological blockade of fatty acid metabolism is selectively attenuated by hindbrain injections of the galanin receptor antagonist, M40. Obesity Res 4:329-336. Koegler FH, Ritter S (1998): Galanin injection into the nucleus of the solitary tract stimulates feeding in rats with lesions of the paraventricular nucleus of the hypothalamus. Physiol Behav 63:521-527. Koenig JI, Hooi S, Gabriel SM, Martin JB (1989): Potential involvement of galanin in the regulation of fluid homeostasis in the rat. Regul Pept 24:81-86. Kohler C, Chan-Palay V (1990): Galanin receptors in the post-mortem human brain. Regional distribution of 125I-galanin binding sites using the method of in vitro receptor autoradiography. Neurosci Lett 120:179-182. Kohler C, Hallman H, Melander T, Hokfelt T, Norheim E (1989a): Autoradiographic mapping of galanin receptors in the monkey brain. J Chem Neuroanat 2:269-284. Kohler C, Persson A, Melander T, Theodorsson E, Sedvall G, Hokfelt T (1989b): Distribution of galanin-binding sites in the monkey and human telencephalon: preliminary observations. Exp Brain Res 75:375-380. Kolakowski Jr. LF, O'Neill GP, Howard AD, Broussard SR, Sullivan KA, Feighner SD, Sawzdargo M, Nguyen T, Kargman S, Shiao LL, Hreniuk DL, Tan CP, Evans J, Abramovitz M, Chateauneuf A, Coulombe N, Ng G, Johnson ME Tharian A, Khoshbouei H, George SR, Smith RG, O'Dowd BF (1998): Molecular characterization and expression of cloned human galanin receptors GALR2 and GALR3. J Neurochem 71:2239-2251. Kordower JH, Le HK, Mufson EJ (1992): Galanin immunoreactivity in the primate central nervous system. J Comp Neurol 319:479-500. Kyrkouli SE, Stanley BG, Seirafi RD, Leibowitz SF (1990): Stimulation of feeding by galanin: anatomical localization and behavioral specificity of this peptide's effects in the brain. Peptides 11:995-1001. Lakaye B, Minet A, Zorzi W, Grisar T (1998): Cloning of the rat brain cDNA encoding for the SLC-1 G protein-coupled receptor reveals the presence of an intron in the gene. Biochim Biophys Acta Mol Cell Res 1401:216-220. Landry M, Aman K, Hokfelt T (1998): Galanin-R1 receptor in anterior and mid-hypothalamus: distribution and regulation. J Comp Neurol 399:321-340. Langel U, Bartfai T (1998): Chemistry and molecular biology of galanin receptor ligands. Ann NY Acad Sci 863:86-93. Langel U, Land T, Bartfai T (1992): Design of chimeric peptide ligands to galanin receptors and substance P receptors. Int J Pept Protein Res 39:516-522.

239

Ch. I V

D. O ' D o n n e l l

et al.

Langel U, Pooga M, Kairane C, Zilmer M, Bartfai T (1996): A galanin-mastoparan chimeric peptide activates the Na+,K(+)-ATPase and reverses its inhibition by ouabain. Regul Pept 62:47-52. Larm JA, Gundlach AL (2000): Galanin-like peptide (GALP) mRNA expression is restricted to arcuate nucleus of hypothalamus in adult male rat brain. Neuroendocrinology 72:67-71. Lee DK, Nguyen T, O'Neill GP, Cheng R, Liu Y, Howard AD, Coulombe N, Tan CP, Tang-Nguyen AT, George SR, O'Dowd BF (1999): Discovery of a receptor related to the galanin receptors. FEBS Lett 446:103-107. Leibowitz SF, Kim T (1992): Impact of a galanin antagonist on exogenous galanin and natural patterns of fat ingestion. Brain Res 599:148-152. Lembo PM, Grazzini E, Cao J, Hubatsch DA, Pelletier M, Hoffert C, St Onge S, Pou C, Labrecque J, Groblewski T, O'Donnell D, Payza K, Ahmad S, Walker P (1999): The receptor for the orexigenic peptide melaninconcentrating hormone is a G-protein-coupled receptor. Nature Cell Biol 1:267-271. Lenz C, Sondergaard L, Grimmelikhuijzen CJ (2000a): Molecular cloning and genomic organization of a novel receptor from Drosophila melanogaster structurally related to mammalian galanin receptors. Biochem Biophys Res Commun 269:91-96. Lenz C, Williamson M, Grimmelikhuijzen CJ (2000b): Molecular cloning and genomic organization of a second probable allatostatin receptor from Drosophila melanogaster. Biochem Biophys Res Commun 273:571-577. Liu H, Hokfelt T (2000): Effect of intrathecal galanin and its putative antagonist M35 on pain behavior in a neuropathic pain model. Brain Res 886:67-72. Liu Q, Pong SS, Zeng Z, Zhang Q, Howard AD, Williams Jr. DL, Davidoff M, Wang R, Austin CP, McDonald TE Bai C, George SR, Evans JF, Caskey CT (1999): Identification of urotensin II as the endogenous ligand for the orphan G-protein-coupled receptor GPR14. Biochem Biophys Res Commun 266:174-178. Lundquist CT, Johard HA, Rokaeus A, Nassel DR (1993): Galanin immunoreactivity and 125I-galanin binding sites in the blowfly brain. Acta Biol Hung 44:51-54. Ma W, Bisby MA (1997): Differential expression of galanin immunoreactivities in the primary sensory neurons following partial and complete sciatic nerve injuries. Neuroscience 79:1183-1195. Mantyh PW, Catton MD, Boehmer CG, Welton ML, Passaro EP, Maggio JE, Vigna SR (1989a): Receptors for sensory neuropeptides in human inflammatory diseases: implications for the effector role of sensory neurons. Peptides 10:627-645. Mantyh PW, Johnson DJ, Boehmer CG, Catton MD, Vinters HV, Maggio JE, Too HE Vigna SR (1989b): Substance P receptor binding sites are expressed by glia in vivo after neuronal injury. Proc Natl Acad Sci USA 86:51935197. Mantyh PW, Catton MD, Allen CJ, Labenski ME, Maggio JE, Vigna SR (1992): Receptor binding sites for cholecystokinin, galanin, somatostatin, substance P and vasoactive intestinal polypeptide in sympathetic ganglia. Neuroscience 46:739-754. Mastropaolo J, Nadi NS, Ostrowski NL, Crawley JN (1988): Galanin antagonizes acetylcholine on a memory task in basal forebrain-lesioned rats. Proc Natl Acad Sci USA 85:9841-9845. McDonald ME Crawley JN (1996): Galanin receptor antagonist M40 blocks galanin-induced choice accuracy deficits on a delayed-nonmatching-to-position task. Behav Neurosci 110:1025-1032. McDonald ME Wenk GL, Crawley JN (1997): Analysis of galanin and the galanin antagonist M40 on delayed non-matching-to-position performance in rats lesioned with the cholinergic immunotoxin 192 IgG-saporin. Behav Neurosci 111:552-563. McDonald ME Gleason TC, Robinson JK, Crawley JN (1998): Galanin inhibits performance on rodent memory tasks. Ann NY Acad Sci 863:305-322. Melander T, Staines WA (1986): A galanin-like peptide coexists in putative cholinergic somata of the septum-basal forebrain complex and in acetylcholinesterase-containing fibers and varicosities within the hippocampus in the owl monkey (Aotus trivirgatus). Neurosci Lett 68:17-22. Melander T, Staines WA, Hokfelt T, Rokaeus A, Eckenstein F, Salvaterra PM, Wainer BH (1985): Galanin-like immunoreactivity in cholinergic neurons of the septum-basal forebrain complex projecting to the hippocampus of the rat. Brain Res 360:130-138. Melander T, Hokfelt T, Rokaeus A (1986a): Distribution of galaninlike immunoreactivity in the rat central nervous system. J Comp Neurol 248:475-517. Melander T, Hokfelt T, Rokaeus A, Cuello AC, Oertel WH, Verhofstad A, Goldstein M (1986b): Coexistence of galanin-like immunoreactivity with catecholamines, 5-hydroxytryptamine, GABA and neuropeptides in the rat CNS. J Neurosci 6:3640-3654. Melander T, Staines WA, Rokaeus A (1986c): Galanin-like immunoreactivity in hippocampal afferents in the rat, with special reference to cholinergic and noradrenergic inputs. Neuroscience 19:223-240. Melander T, Fuxe K, Harfstrand A, Eneroth P, Hokfelt T (1987): Effects of intraventricular injections of galanin on 240

Localization of galanin receptor subtypes in the rat CNS

Ch. IV

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

241

Ch. I V

D. O'Donnell et al.

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

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

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

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Orexin receptors TAKESHI SAKURAI, GUILLAUME HERVIEU AND MASASHI YANAGISAWA

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

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

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

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

human

MNLPSTKVSW

AAVTLLLLLL

LLPPALLSSG

AAA Q P L P D C C

RQKTCSCRLY

ELLHGAGNHA

60

pig

MNPPFAKVSW

ATVTLLLLLL

LLPPAVLSPG

AAA Q P L P D C C

RQKTCSCRLY

ELLHGAGNHA

60

dog

MNPPSTKVPW

AAVTLLLLLL

L-PPALLSPG

AAA ~ P L P D C C

RQKTCSCRLY

ELLHGAGNHA

59

rat

FINLPSTKVPW A A V T L L L L L L

L-PPALLSLG

VDA ~ P L P D C C

RQKTCSCRLY

ELLHGAGNHA

59

mouse

MNFPSTKVPW

AAVTLLLLLL

L-PPALLSLG

VDA ~ P L P D C C

RQKTCSCRLY

ELLHGAGNHA

59

.

*

* * * * * * * * * *

* * * * * * * * * *

**

.

********

***

**

.

********

s i g n a l peptide human

AGILTL3KR

SGPPGLQGRL

QRLLQASGNH

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

LGRRCSAPAA

120

pig

AGILTLZKR

PGPPGLQGRL

QRLLQASGNH

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

PGRRCLAAAA

120

dog

AGILT/3KR

PGPPGLQGRL

QRLLQASGNH

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

PGRRCPVVAV

119

rat

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

QRLLQANGNH

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

PGRRCPTATA

119

mouse

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

QRLLQANGNH

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

SGRGCPTVTT

119

* * * * * * * * * *

******

* * * * * * * * * *

* * * * * * * * *

human

ASVAPGGQSG

I

131

pig

SSVAPGGRSG

I

131

dog

PSAAPGGRSG V

130

rat

TALAPRGGSR V

130

mouse

TALAPRGGSG

130

V

***

****

*

*

****

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

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

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

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

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

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

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

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

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

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

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

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

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

Orexin receptors

Ch. V

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

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

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

251

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3. OREXIN RECEPTORS

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

Orexin receptors

120

.~.

Ch. V

i

OX1R

OX2R 120

10X-A

I

OX-B

OX-A

~

100.

OX-B

100

< .=-

x

80

? u')

c,,I

60

60

"o t.:3 o 133

40

40

! 20

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

10

9

8

20

7

Concentration (-log[M])

6

5

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

10

9

8

7

6

5

Concentration (-log[M])

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

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

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

2

P

Peptide

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

Sequence

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

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

ECSO(nM)

PECSO(peptide) - PEGO(OXW

PEG0

OXlR

OX2R

OXlR

OX2R

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

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

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

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

-

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

OXlR

0x2r

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

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

q

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

of

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

Sequence

RSGPPGLQGRLQRLLQASGNHAAGILTM-amide

ASGPPGLQGRLQRLLQ ASGNHAAGILTM-amide

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

RSGPPGLQGRAQRLLQASGNHAAGILTM-amide RSGPPGLQGRLARLLQASGNHAAGILTM-amide RSGPPGLQGRLQ ALLQASGNHAAGILTM-amide

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

EC50 (nM)

0

PEGO

OXlR

OX2R

OXlR

OX2R

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

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

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

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

~~

~~

__

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

~

pEC5~(peptide) - pEC5o (OXB)

$.

OXlR

OX2R

2 CI

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

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

20 rd

2

Ch. V

T. Sakurai et al.

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

OX1R-ir

OX1R mRNA

OX2R m R N A

+++ +++ ++

++ +++

++ +

+ + + +

+ + + +

++ ++ -

++

++

-

+ 4-4++ ++ -

+ + -

+ + ++ +

+ ++ + +++

+ ++ ++

+++ +

++ ++

++ +

++ +

+++ ++ ++ 4-44-

+++ ++ 4-

4-4+ +++ ++44-4-

+4-+ 4-4-444-4+ ++44-4-44-+

++ 44+++ 4-44-4-

+ 4-44-4+ ++ 4-4-

++

+

+ + 4-44+ -

4-44-+ 4-44+ +

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

256

:_..

++

44-44+ -

-~

Orexin receptors

Ch. V

TABLE 3 (continued) Region

OX 1R-ir

OX 1R mRNA

OX2R mRNA

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

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

++

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

-

+ + + -

++ ++ + -

_

+ + +

+ +

-

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

++ + + ++ ++ ++ +

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

+ + +

-

+ +

+ + + +

+ + + +

-

+ +

-

+ +

+ +

-

+ +

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

Rhombencephalon Facial nucleus Locus coeruleus Pontine reticular nucleus Spinal trigeminal nucleus

Cerebellum Cerebellar cortex Deep cerebellar nuclei

D

++

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

+++ +++

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

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

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

Ch. V

T. Sakurai et al.

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

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

4.2.1. Telencephalon 4.2.1.1. Isocortex

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

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

Orexin receptors

Ch. V

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

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

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

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

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

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4.2.2. Diencephalon 4.2.2.1. Thalamus

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

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

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

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

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

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

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

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

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

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OX2R mRNA was also prominent in the arcuate nucleus, PVN, LHA, and tuberomammillary nucleus. In these regions there was little or no OX1R signal. On the other hand, OX1R mRNA was highly expressed in the anterior hypothalamic area and VMH, where OX2R mRNA was not so evident. Many nuclei in the brainstem also showed a differential pattern of expression. The densest signal for OX1R was found in the locus coeruleus, while OX2R mRNA was not detected in this region. On the other hand, cholinergic groups, including the laterodorsal and pedunculopontine tegmental nuclei, preferentially expressed OX2R mRNA. However, several regions contain mRNA for both receptors. For example, in the thalamus, the two receptors showed a similar distribution. Both receptors were located in the paraventricular thalamic nuclei. The ventral tegmental area, a region containing dopaminergic neurons, contained both OX1R and OX2R expression. The dorsal raphe nucleus also contained both OX1R and OX2R mRNA.

6. COMPARISON OF OX1R mRNA AND PROTEIN DISTRIBUTION

There was generally good agreement between the results of in situ hybridization to examine OX1R mRNA expression profiles and immunohistochemical analysis to examine OX1R protein distribution within the rat brain. A few areas of discrepancy between mRNA distribution and protein distribution, for example in the olfactory tubercle, piriform cortex, tuberomammillary nucleus and paraventricular hypothalamic nucleus, were observed. In these regions, OX1R mRNA was not observed, although OX1R-like immunoreactivity was found. The reason for these discrepancies is unclear, but may be due to several factors. First, in situ hybridization may not demonstrate the complete pattern of expression because of low sensitivity. In addition, the locations with dense OX1R-immunoreactivity but little receptor mRNA signal may reflect presynaptic expression of OX1R protein on terminals, whose cell bodies are located at some distance.

7. COMPARISON BETWEEN LOCALIZATION OF OREXIN RECEPTOR AND SITES OF c-Fos ACTIVATION UPON CENTRAL ADMINISTRATION OF OREXINS IN RAT

Central injection of orexins produced a characteristic anatomical pattern of Fos, an immediate early gene product that is correlated with the functional activation of neurons (Date et al., 1999). Fos distribution was similar in orexin-A- and -B-injected rats as well as in 3- and 30-~tg orexin-injected rats. Fos-immunoreactive neurons were observed in the PVT, LC, central gray and dorsal raphe nucleus, nucleus tractus solitarius (NTS), and dorsal motor nucleus of the vagus. In the hypothalamus, Fos-like signals were observed in the arcuate nucleus, ventral and dorsolateral parts of the SCN, SON, and PVN except the lateral magnocellular division. Most of these regions express OX1R and/or OX2R, suggesting that observed Fos-like immunoreactivity was due to direct activation of orexin receptors by injected orexins. 8. HOW MANY OREXIN RECEPTORS?

Two genes for orexin receptors have been identified in mammalian species thus far. The phenotypes of OX1R and OX2R double-deficient mice were analyzed and shown to have 312

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sleep state abnormality, which was indistinguishable from that of prepro-orexin gene-deficient mice (Yanagisawa, unpublished results). This observation suggests that only two receptors for orexins might exist in mammals, at least in vigilance state control. However, we cannot exclude the possibility that there are other subtypes of receptors produced from OX1R or OX2R genes by alternative splicing.

9. PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL IMPLICATIONS OF OREXIN RECEPTORS 9.1. FEEDING BEHAVIOR Orexin receptors are observed in various nuclei of the hypothalamus. This is consistent with the observations that orexins have roles in regulating feeding behavior and energy homeostasis (Sakurai et al., 1998, 1999). For instance, OX1R mRNA is observed in the VMH and DMH in the hypothalamus. OX1R-like immunoreactivity is present in the PVN, arcuate nucleus, VMH and DMH. On the other hand, OX2R mRNA is observed in the PVN, VMH, DMH, arcuate nucleus and LHA. All these nuclei have been implicated in feeding behavior. Thus, the overall feeding effect of the peptides could be mediated through both receptors. In fact we observed that an OX2R-selective agonist (Alal 1-orexin-B) increased food intake when administered intracerebroventricularly, although it was less potent as compared with orexin-A (unpublished result). 9.2. REGULATION OF WATER BALANCE OX1R was detected in the zona incerta, a region thought to regulate drinking behavior, which might be linked to the effect of orexins on water intake as we reported (Kunii et al., 1999). The dense signal for OX1R seen in the supraoptic nucleus might indicate that orexin regulates the antidiuretic vasopressin system. 9.3. NEUROENDOCRINE REGULATION Studies have shown that i.c.v, injection of orexin-A causes a dose-dependent decrease in plasma growth hormone, thyroid-stimulating hormone and prolactin levels while triggering a dose-dependent increase in corticosterone (Hagan et al., 1999; Ida et al., 2000; Mitsuma et al., 1999). Orexins have also been found to modulate secretion of luteinizing hormone-releasing hormone (Pu et al., 1998). The presence of OXRs in the arcuate nucleus might mediate neuroendocrine responses evoked upon injection of orexins. The presence of OX2R mRNA in the medial parvocellular paraventricular hypothalamic nucleus, where thyrotropin-releasing hormone- and corticotropin-releasing hormone-producing neurons are found, might also have roles in regulating the release of these hormones. 9.4. REGULATION OF AUTONOMIC NERVOUS SYSTEM It is reported that orexins increase heart rate and blood pressure in conscious rats (Samson et al., 1999). The presence of OX1R and OX2R in the PVN, which has been implicated in the central regulation of sympathetic outflow, especially in the parvocellular region, is consistent 313

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with these results. Orexin receptors in the nucleus of the solitary tract might also have roles in the regulation of blood pressure. The presence of OX1R in the spinal cord might also have a role in regulating the parasympathetic and sympathetic activities of the autonomic nervous system. 9.5. VIGILANCE STATE CONTROL OX1R in the PVT, LHA, tuberomammillary hypothalamic nucleus as well as in the preoptic area, pontine reticular formation, raphe nuclei, dorsal tegmental nuclei and locus coeruleus, and OX2R in the PVT, LHA, tuberomammillary hypothalamic nucleus and median raphe nucleus might have roles in regulating arousal and vigilance, as recently reported (Chemelli et al., 1999; Hagan et al., 1999; Lin et al., 1999; Nishino et al., 2000; Peyron et al., 2000). Thus, both receptors might be involved in the control of arousal and vigilance state. The functional significance of OXRs in the cerebral cortex is unknown, but they may also be related to orexin-mediated control of arousal state. The importance of OX2R in sleep/wakefulness regulation was demonstrated by the finding that narcolepic dogs carry mutations in the OX2R gene (Lin et al., 1999). The finding that OX2R mRNA is distributed throughout several sites implicated in sleep regulation is consistent with these observations. For example, OX2R mRNA was observed in the tuberomammillary nucleus, VTA, raphe nuclei, pedunculopontine nucleus (PPN), and laterodorsal tegmental nucleus (LDT). The tuberomammillary nucleus is the source of neuronal histamine, while raphe nuclei are the source of serotonin, and the VTA is the source of dopamine. All these monoaminergic neurons have been implicated in the regulation of sleep and vigilance. The PPN and LDT contain cholinergic cells, which are also implicated in the maintenance of arousal and sleep state regulation. Especially, the dopaminergic neurons in the VTA might be implicated in the cataplexy in narcolepsy. For example, pharmacological studies of drug injections into the VTA highlight the role of the D2/D3 dopamine autoreceptor in the regulation of cataplexy in the narcoleptic dog (Reid et al., 1996). The role of the LC in mediating the effects of orexin on sleep/wake states is unclear, because, little OX2R mRNA signal was found in this region in rats. However, a dense OX1R signal was observed in the LC. Since the LC is thought to be implicated in the regulation of arousal, the presence of OX1R in this region may also have a role in the regulation of wakefulness. In fact, administration of orexin-A directly onto the LC increases wakefulness and reduces REM sleep in rats (Bourgin et al., 2000). Thus, together with the observation that orexinA application strongly stimulates firing rate in the LC suggest a role of the LC in the wake-promoting effects of rexins (Hagan et al., 1999). The fact that prepro-orexin ligand deficient mice are more severely affected with narcolepsy than OX2R gene deficient mice agree with the suggestion of an enhancing role of OX1R in the sleep abnormality. 9.6. OTHER FUNCTIONS OX1R was expressed in regions regulating pain, including the cingulate cortex, anterior thalamus and posterior hypothalamus, peri-aqueductal gray matter, trigeminal nucleus, and dorsal root ganglia, suggesting that orexins have roles in nociception. 314

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Both receptors were found within the amygdaloid nuclei and other forebrain limbic regions such as the septum and entorhinal cortex, traditionally referred to as control centers for emotion. This suggests that orexins regulate emotion, as already proposed (Piper et al., 2000). The presence of orexin receptors in monoaminergic neurons in the LC, ventral tegmental area, and raphe nuclei suggest that orexins may contribute to the development of psychotic disorders including bipolar disorder and schizophrenic disorder.

10. CONCLUSION In accordance with the diffuse projection of orexin neurons, orexin receptors have a widespread distribution in the brain. This suggests diverse and complex physiological roles of the orexins in brain function. The expression patterns of the two orexin receptors are quite different and usually complementary, suggesting differential roles for each subtype.

11. ABBREVIATIONS

1-10 3-V 4-V 10 I-VI aca Acb AcbC AcbSh aci Aco Acp AD af AHC AhiAL AhiPM AIP alv AM Amy AOB AOD AOL AOM AOV AP Apir Aq Arc

laminae 1-10 of the spinal cord 3rd ventricle 4th ventricle dorsal motor nucleus of vagus cortical layer 1-6 anterior commissure, anterior part accumbens nucleus accumbens nucleus, core accumbens nucleus, shell anterior commissure, intrabulbar part anterior cortical amygdaloid nucleus anterior commissure, anterior part anterodorsal thalamic nucleus amygdaloid fissure anterior hypothalamic area, central part amygdalohippocampal area, anterolateral part amygdalohippocampal area, posteromedial part agranular insular cortex, posterior part alveus of the hippocampus anteromedial thalamic nucleus amygdaloid complex accessory olfactory bulb accessory olfactory nucleus, dorsal part accessory olfactory nucleus, lateral part accessory olfactory nucleus, medial part accessory olfactory nucleus, ventral part area postrema amygdalopiriform transition area aqueduct (Sylvius) arcuate nucleus 315

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Aul AuD AuV AV AVVL Bas BL BLA BMA BMP BST BSTMPI BSTMPL BSTMPM BSTMV BSTS Ca CA1 CA2 CA3 cc

CC Ce Cer cg Cg Cgl Cg2 CG CL C1 CM Cpu Cx CxA D3V Den df DG DI DLG DLO DMH dr DR ec

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primary auditory cortex secondary auditory cortex, dorsal area secondary auditory cortex, ventral area anteroventral thalamic nucleus anteroventral thalamic nucleus, ventrolateral part basilar artery 43-64 basolateral amygdaloid nucleus basolateral amygdaloid nucleus, anterior part basomedial amygdaloid nucleus, anterior part basomedial amygdaloid nucleus, posterior part bed nucleus of the stria terminalis bed nucleus of the stria terminalis, medial division, posterointermediate bed nucleus of the stria terminalis, medial division, posterolateral part bed nucleus of the stria terminalis, medial division, posteromedial part bed nucleus of the stria terminalis, medial division, ventral part bed nucleus of the stria terminalis, supracapsular part caudate field CA1 of hippocampus field CA2 of hippocampus field CA3 of hippocampus corpus callosum central canal central amygdaloid nuclei cerebellum cingulum cingulate cortex cingulate cortex, area 1 cingulate cortex, area 2 central gray centrolateral thalamic nucleus claustrum central medial thalamic nucleus caudate putamen (striatum) cortex cortex-amygdala transition zone dorsal 3rd ventricle dorsal endopiriform nucleus dorsal fornix dentate gyrus dysgranular insular cortex dorsal lateral geniculate nucleus dorsolateral orbital cortex dorsomedial hypothalamic nucleus dorsal root spinal nerve dorsal raphe nucleus exterminal capsule ectorhinal cortex entorhinal cortex

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external plexiform layer of the olfactory bulb ependyma and subependymal layer of the olfactory ventricle fornix fimbria of the hippocampus forceps minor of the corpus callosum forceps major of the corpus callosum frontal cortex granular insular cortex glomerular layer of the olfactory bulb 1-4 gracile fasciculus granular cell layer of the accessory olfactory bulb granular layer of the dentate gyrus 33-45 habenular nucleus nucleus of the horizontal limb of the diagonal band hippocampus hilus of the dentate gyrus hypothalamus interanteromedial thalamic nucleus internal capsule inferior colliculus islands of Calleja lateral funiculus spinal cord indusium grisemia intermediomedial cell column inferior olive interpeduncular nucleus locus coeruleus laterodorsal thalamic nucleus lateral entorhinal cortex lateral geniculate nucleus lateral globus pallidus lateral hypothalamic area lacunosum moleculare layer of the hippocampus lateral orbital cortex lateral olfactory tract lateral posterior thalamic nuclei lateral posterior thalamic nucleus, mediocaudal part lateral posterior thalamic nucleus, mediorostral part lateral preoptic area lateral recess of the 4th ventricle lateral septal nucleus lateral septal nucleus, dorsal part lateral septal nucleus, intermediate part lateral spinal nucleus lateral ventricle primary motor cortex secondary motor cortex magnocellular preoptic nucleus 317

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MD ME Me mfb MG MM MnPO MO Mol MPA MPn MPO NS ON opt Or ox

Par PDTg PH Pir plf PLCo PMCo PMV PnC PnO Po Post PRh PrL PT PtA Put PVA PVP PVN Py PY Rad Re ff Rh RMg RSA RSGa RSGb Rt 318

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mediodorsal thalamic nucleus median eminence medial amygdaloid nuclei medial forebrain bundle medial geniculate nucleus medial mammillary nucleus, medial part median preoptic nucleus medial orbital cortex molecular layer of the dentate gyrus medial preoptic area medial pontine nucleus medial preoptic nucleus non-specific binding olfactory nerve layer optic tract oriens layer of the hippocampus optic chiasm parietal cortex posterodorsal tegmental nucleus posterior hypothalamic area piriform cortex posterolateral fissure posterolateral cortical amygdaloid nucleus (C2) posteromedial cortical amygdaloid nucleus (C3) premammillary nucleus, ventral part pontine reticular nucleus, caudal part pontine reticular nucleus, oral part posterior thalamic nuclear group postsubiculum 40-51 perirhinal cortex prelimbic cortex paratenial thalamic nucleus parietal association cortex putamen paraventricular thalamic nucleus, anterior part paraventricular thalamic nucleus, posterior part paraventricular hypothalamic nucleus pyramidal cell layer of the hippocampus pyramidal tract stratum radiatum of the hippocampus reuniens thalamic nucleus rhinal fissure rhomboid thalamic nucleus raphe magnus nucleus retrosplenial agranular cortex retrosplenial granular a cortex retrosplenial granular b cortex reticular thalamic nucleus

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reticulotegmental nucleus of the pons primary somatosensory cortex, barrel field primary somatosensory cortex, dysgranular region primary somatosensory cortex, forelimb region primary somatosensory cortex, hindlinb region secondary somatosensory cortex splenium of the corpus callosum superior colliculus suprachiasmatic nucleus septofimbrial nucleus 18-22 subfornical organ septohippocampal nucleus substantia innominata stria medullaris of the thalamus substantia nigra substantia nigra, compact part substantia nigra, lateral part substantia nigra, reticular part supraoptic nucleus nucleus of the solitary tract supraoptic nucleus stria terminalis superficial gray layer of the superior colliculus thalamus temporal association cortex triangular septal nucleus tenia tecta olfactory tubercle nucleus of the trapezoid body trapezoid body primary visual cortex, binocular area primary visual cortex, monocular area secondary visual cortex, lateral area secondary visual cortex, mediolateral area secondary visual cortex, mediomedial area ventral anterior thalamic nucleus vestibular nucleus ventral endopiriform nucleus ventral funiculus spinal cord ventral hippocampal commissure ventrolateral hypothalamic nucleus ventromedial hypothalamic nucleus ventral median fissure spinal cord ventral orbital cortex ventral posteromedial thalamic nucleus ventral posterior thalamic nucleus ventral root spinal nerve ventral tegmental area 319

Ch.

VTT ZI

V

T. S a k u r a i et al.

ventral tenia tecta z o n a incerta

12. ACKNOWLEDGEMENTS T h e authors w o u l d like to t h a n k Dr. Joel E l m q u i s t , Ms. J u n k o H a r a and Dr. A k i h i r o Y a m a n a k a for v a l u a b l e discussions.

13. REFERENCES Anand BK, Brobeck JR (1951): Hypothalamic control of food intake in rats and cats. Yale J Biol Med 24:123-140. Asahi S, Egashira S, Matsuda M, Iwaasa H, Kanatani A, Ohkubo M, Ihara M, Sakurai T, Morishima H (1999): Structure-activity regulation studies on the novel neuropeptide orexin. Peptide Sci:37-40. Bittencourt JC, Presse F, Arias C, Peto C, Vaughan J, Nahon JL, Vale W, Sawchenko PE (1992): The melaninconcentrating hormone system of the rat brain: an immuno- and hybridization histochemical characterization. J Comp Neurol 319:218-245.

Bourgin P, Huitron-Resendez S, Spier AD, Morte BM, Criado JR, Henriksen KM, de Lecea L (2000): Hypocretin-1 modulates REM sleep through activation of locus coeruleus. J Neurosci 20:7760-7765. Broberger C, De Lecea L, Sutcliffe JG, Hokfelt T (1998): Hypocretin/orexin- and melanin-concentrating hormoneexpressing cells form distinct populations in the rodent lateral hypothalamus: relationship to the neuropeptide Y and agouti gene-related protein system. J Comp Neurol 402:460-474. Chemelli RM, Willie JT, Sinton CM, Elmquist JK, Scammell T, Lee T, Richardson JA, Williams SC, Xiong Y, Kisanuki Y, Fitch TE, Nakazato M, Hammer RE, Saper CB, Yanagisawa M (1999): Narcolespsy in orexin knockout mice: molecular genetics of sleep regulation. Cell 98:437-451. Chou TC, Lee CE, Lu J, Elmquist JK, Hara J, Willie JT, Beuckmann CT, Chemelli RM, Sakurai T, Yanagisawa M, Saper CB, Scammell TE (2001): Orexin (hypocretin) neurons contain dynorphin. J Neurosci 21:RC168. Date Y, Ueta Y, Yamashita H, Yamaguchi H, Matsukura S, Kangawa K, Sakurai T, Yanagisawa M, Nakazato M (1999): Orexins, orexigenic hypothalamic peptides interact with autonomic neuroendocrine and neuroregulatory systems. Proc Natl Acad Sci USA 96:748-753. de Lecea L, Kilduff TS, Peyron C, Gao X, Foye PE, Danielson PE, Fukuhara C, Battenberg EL, Gautvik VT, Bartlett FS, Frankel WN, van den Pol AN, Bloom FE, Gautvik KM, Sutcliffe JG (1998): The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity. Proc Natl Acad Sci USA 95:322-327. Elias CF, Saper CB, Maratos-Flier E, Tritos NA, Lee C, Kelly J, Tatro JB, Hoffman GE, Ollmann MM, Barsh GS, Sakurai T, Yanagisawa M, Elmquist JK (1998): Chemically defined projections linking the mediobasal hypothalamus and the lateral hypothalamic area. J Comp Neurol 402:442-459. Griffond B, Deray A, Fellmann D, Ciofi D, Croix D, Bugnon C (1993): Colocalization of prolactin-and dynorphinlike substances in a neuronal population of the rat lateral hypothalamus. Neurosci Lett 156:91-95. Griffond B, Deray A, Jacquemard C, Fellmann D, Bugnon C (1994): Prolactin immunoreactive neurons of the rat lateral hypothalamus immunocytochemical and ultrastructural studies. Brain Res 635:179-186. Griffond B, Risold PY, Jacquemard C, Colard C, Fellmann D (1999): Insulin-induced hypoglycemia increases preprohypocretin (orexin) mRNA in the rat lateral hypothalamic area. Neurosci Len 262:77-80. Hagan JJ, Leslie RA, Patel S, Evans ML, Wattam TA, Holmes S, Benham CD, Taylor SG, Routledge C, Hemmati P, Munton RP, Ashmeade TE, Shah AS, Hatcher PD, Jones ML, Smith MI, Piper AJ, Hunter AJ, Porter RA, Upton N (1999): Orexin A activates locus coeruleus cell firing and increases arousalin the rat. Proc Natl Acad Sci USA 96:10911-10916. Hakansson ML, Brown H, Ghilardi N, Skoda RC, Meister B (1998): Leptin receptor immunoreactivity in chemically defined target neurons of the hypothalamus. J Neurosci 18:559-572. Hara J, Beuckmann CT, Nambu T, Willie JT, Chemelli RM, Sinton CM, Sugiyama F, Yagami K, Goto K, Yanagisawa M, Sakurai T (2001): Genetic ablation of orexin neurons in mice results in narcolepsy, hypophagia, and obesity. Neuron 30:345-354. Horvath TL, Diano S, Van den Pol AN (1999): Synaptic interaction between hypocretin (orexin) and neuropeptide Y cells in the rodent and primate hypothalamus: a novel circuit implicated in metabolic and endocrine regulations. J Neurosci 19:1072-1087. 320

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Hosoya Y, Matsushita M (1981): Brainstem projections from the lateral hypothalamic area in the rat, as studied with autoradiography. Neurosci Lett 24:111-116. Ida T, Nakahara K, Katayama T, Murakami N, Nakazato M (1999): Effect of lateral cerebroventricular injection of the appetite-stimulating neuropeptide, orexin and neuropeptide Y, on the various behavioral activities of rats. Brain Res 821:526-529.

Ida T, Nakahara K, Murakami T, Hanada R, Nkazato M, Murakami N (2000): Possible involvement of orexin in the stress reaction in rats. Biochem Biophys Res Commun 270:318-323. Kilduff TS, Peyron C (2000): The hypocretin/orexin ligand-receptor system: implications for sleep and sleep disorders. Trends Neurosci 23:359-365. Kunii K, Yamanaka A, Nambu T, Matsuzaki K, Goto K, Sakurai T (1999): Orexins/hypocretins regulate drinking behavior. Brain Res 842:256-261. Lin L, Faraco J, Li R, Kadotani H, Rogers W, Lin X, de Jong PJ, Nishino S, Mignot E (1999): The sleep disorder canine narcolepsy is caused by a mutation in the hypocretin (orexin) receptor 2 gene. Cell 98:365-376. Marcus JN, Aschkenasi CJ, Lee CE, Chemelli RM, Saper CB, Yanagisawa M, Elmquist JK (2001): J Comp Neurol 435:6-25.

Mignot E, Young T, Lin L, Finn L, Palta M (1998): Reduction of REM sleep latency associated with HLADQBl*0602 in normal adults. Lancet 351 (9104):727. Mitsuma T, Hirooka Y, Mori Y, Kayama M, Adachi K, Rhue N, Ping J, Nogimori T (1999): Effects of orexin A on thyrotropin-releasing hormone and thyrotropin secretion in rats. Horm Metab Res 31:606-609. Moriguchi T, Sakurai T, Nambu T, Yanagisawa M, Goto K (1999): Neurons containing orexin in the lateral hypothalamic area of the adult rat brain are activated by insulin-induced acute hypoglycemia. Neurosci Lett 264:101-104.

Nakamura T, Uramura K, Nambu T, Yada T, Goto K, Yanagisawa M, Sakurai T (2000): Orexin-induced hyperlocomotion and stereotypy are mediated by the dopaminergic system. Brain Res 873:181-187. Nambu T, Sakurai T, Mizukami K, Hosoya Y, Yanagisawa M, Goto K (1999): Distribution of orexin neurons in the adult rat brain. Brain Res 827:243-260. Nauta WJH (1946): Hypothalamic regulation of sleep in rats. An experimental study. J Neurophysiol 9:285-316. Nishino S, Ripley B, Overeem S, Lammers GJ, Mignot E (2000): Hypocretin(orexin) deficiency in human narcolepsy. Lancet 355:39. Paxinos G, Watson C (1998): The Rat Brain in Stereotaxic Coordinates. San Diego: Academic Press. Peyron C, Tighe DK, van den Pol AN, de Lecea L, Heller HC, Heller JG, Sutcliffe JG, Kilduff TS (1998): Neurons containing hypocretin (orexin) project to multiple neuronal systems. J Neurosci 18:9996-10015. Peyron C, Faraco J, Rogers W, Ripley B, Overeem S, Charnay Y, Nevsimaloya S, Aldrich M, Reynolds D, Albin R, Li R, Hungs M, Pedrazzoli M, Padigaru M, Kucherlapati M, Fan J, Maki R, Lammers GJ, Bouras C, Kucherlapati R, Nishino S, Mignot E (2000): A mutation in a case of early onset narcolepsy and a generalized absence of hypocretin peptides in human narcoleptic brains. Nat Med 6:991-997. Piper D, Upton N, Smith MI, Hunter AJ (2000): The novel brain neuropeptide, orexin-A, modulates the sleep-wake cycle of rats. Eur J Neurosci 12:726-730. Pu S, Jain MR, Kalra PS (1998): Orexins, a novel family of hypothalamic neuropeptides, modulate pituitary luteinizing hormone secretion in an ovarian steroid-dependent manner. Rept 78:133-136. Qu D, Ludwig DS, Gammeltoft S, Piper M, Pelleymounter MA, Cullen MJ, Mathes WF, Przypek R, Kanarek R, Maratos-Flier E (1996): A role for melanin-concentrating hormone in the central regulation of feeding behavior. Nature 380:243-247.

Reid MS, Tafti M, Nishino S, Sampathkkumaran R, Siegel JM, Mignot E (1996): Local administration of dopaminergic drugs into the ventral tegmental area modulates cataplexy in the narcoleptic canine. Brain Res 733:83-100.

Risold PY, Griffond B, Kilduff TS, Sutchiffe JG, Fellmann D (1999): Preprohypocretin (orexin) and prolactin-like immunoreactivity are coexpressed by neurons of the rat lateral hypothalamic area. Neurosci Lett 259:153-156. Sakurai T, Amemiya A, Ishii M, Matsuzaki I, Chemelli RM, Tanaka H, Williams SC, Richardson JA, Kozlowski GP, Wilson S, Arch JR, Buckingham RE, Haynes AC, Carr SA, Annan RS, Mcnulty DE, Liu WS, Terrett JA, Elshourbagy NA, Bergsma DJ, Yanagisawa M (1998): Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behaviour. Cell 92:573-585. Sakurai T, Moriguchi T, Furuya K, Kajiwara N, Nakamura T, Yanagisawa M, Goto K (1999): Structure and function of human prepro-orexin gene. J. Biol Chem 274:17771-17776. Samson WK, Gosnell B, Chang JK, Resch ZT, Murphy TC (1999): Cardiovascular regulatory actions of the hypocretins in brain. Brain Res 831:248-253.

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Saper CB, Swanson LW, Cowan WM (1979): An autoradiographic study of the efferent connections of the lateral hypothalamic area in the rat. J Comp Neurol 183:689-706. Thannkikal TC, Moore RY, Nienhuis R, Ramanathan L, Gulyani S, Aldrich M, Cornford M, Siegel JM (2000): Reduced number of hypocretin neurons in human narcolepsy. Neuron 27:469-474. Touzani K, Ferssiwi A, Velley L (1990): Localization of lateral hypothalamic neurons projecting to the medial part of the parabrachial area of the rat. Neurosci Lett 114:17-21. Trivedi P, Yu H, Douglas J, MacNeil LHT, der Ploeg V, Guan XM (1998): Distribution of orexin receptor mRNA in the rat brain. FEBS Lett 438:71-75. Van den Pol AN (1999): Hypothalamic hypocretin (orexin): Robust innervation of the spinal cord. J Neurosci 19:3171-3182. Villalobos J, Ferssiwi A (1987a): The differential ascending projections from the anterior, central and posterior regions of the lateral hypothalamic area: an autoradiographic study. Neurosci Lett 81:89-94. Villalobos J, Ferssiwi A (1987b): The differential descending projections from the anterior, central and posterior regions of the lateral hypothalamic area: an autoradiographic study. Neurosci Lett 81:95-99. Willie JT, Chemelli RM, Sinton CM, Yanagisawa M (2001): To eat or to sleep? Orexin in the regulation of feeding and wakefulness. Annu Rev Neurosci 24:429-458. Winn P, Tarbuck A, Dunnett SB (1984): Ibotenic acid lesions of the lateral hypothalamus comparison with the electrolytic lesion syndrome. Neuroscience 12:225-240. Yamanaka A, Sakurai T, Katsumoto T, Yanagisawa M, Goto K (1999): Chronic intracerebroventricular administration of orexin-A to rats increases food intake in daytime, but has no effect on body weight. Brain Res 849:248-252. Yamanaka A, Kunii K, Nambu T, Tsujino N, Sakai A, Matsuzaki I, Miwa Y, Goto K, Sakurai A (2000): Orexin-induced food intake involves neuropeptide Y pathway. Brain Res 859:404-409. Yamanaka A, Tsujino N, Funahashi H, Honda K, Guan JL, Wang QP, Tominaga M, Goto K, Shioda S, Sakurai T (2002): Orexins activate histaminergic neurons via the orexin 2 receptor. Biochem Biophys Res Commun 290:1237-1245.

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CHAPTER VI

Neurotensin receptors in the central nervous system PHILIPPE SARRET AND ALAIN BEAUDET

1. INTRODUCTION Like many other neuropeptides, the tridecapeptide neurotensin (NT) is a messenger of intercellular communication working as a neurotransmitter or neuromodulator in the brain (Nemeroff et al., 1982) and as a local hormone in the periphery (Fernstrom et al., 1980). Several pharmacological, morphological, and neurochemical data suggest that NT is involved in many physiological processes in the central nervous system (CNS) as well as in the gastrointestinal tract. Both central and peripheral modes of action of NT imply as a first step the recognition of the peptide by a specific receptor located on the plasma membrane of the target cell. The activated ligand-receptor complex then transduces the extracellular signal inside the cell. In this chapter, we will first review the discovery of NT and briefly discuss its synthesis, degradation, distribution, and biological effects in the CNS. We will then focus on the characterization and distribution of NT receptors and of their mRNA in adult and developing mammalian brain.

2. DISCOVERY OF NT

2.1. NEUROTENSIN AND RELATED PEPTIDES In the course of purifying substance P from bovine hypothalamic extracts, Carraway and Leeman (1973) detected a second bioactive factor that eluted before substance P activity in ion-exchange chromatograms. This fraction produced vasodilatation, hypotension, and increased vascular permeability in rat but, unlike substance P, did not induce salivation. The bioactivity was named 'neurotensin' because of its localization in neural tissue and hypotensive properties (Carraway and Leeman, 1973). Exploiting its vasoactivity to monitor purification procedures, Carraway and Leeman isolated NT in 1975 and determined its sequence: pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH (Carraway and Leeman, 1975a,b). Comparison of the primary structures of NT isolated from diverse vertebrates (Table 1) indicates that the carboxyl-terminal hexapeptide sequence is conserved, whereas the amino terminus exhibits more phylogenetic variability. Accordingly, structureactivity studies demonstrated that the six C-terminal amino acids of NT are responsible for

Handbook of Chemical Neuroanatomy, Vol. 20: Peptide Receptors, Part H R. Quirion, A. Bj0rklund and T. H0kfelt, editors 92003 Elsevier Science B.V. All rights reserved.

323

Reference

Sequence

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Species

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2

3

4

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6

7

8

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Hammer et al., 1980; Kislauskis et al., 1988; Dobner et al., 1987; Carraway and Leeman, 1975a,b Carraway and Bhatnagar, 1980 Shaw et al., 1992 Shaw et al., 1986 Shaw et al., 1991 Rodriguez-Bello et al., 1993

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Ch. VI

r~

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324

TABLE 1 . Primary structure of neurotensin in vertebrates and related peptides

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P. Sarret and A. Beaudet

Neurotensin receptors in the central nervous system

Ch. VI

its biological activity (Granier et al., 1982). A number of peptides with structural similarities to the carboxyl-terminus of NT, including the mammalian peptide neuromedin-N (which originates from the same precursor molecule as NT; see belor (Minamino et al., 1984), its avian counterpart LANT-6 (Carraway and Ferris, 1983), and the amphibian octapeptide xenopsin (Araki et al., 1973; Carraway et al., 1982a,b) have been isolated (Table 1). However, little is known about their biological role. 2.2. STRUCTURE OF THE NEUROTENSIN/NEUROMEDIN N GENE The neurotensin/neuromedin-N (NT/NN) gene has been isolated and sequenced in the dog (Dobner et al., 1987), rat (Kislauskis et al., 1988) and human (Bean et al., 1992). It is highly conserved between species, especially in the region encoding NT and NN. The rat NT/NN gene consists of a 10.2-kilobase segment containing four exons and three introns, the fourth exon encoding both NT and NN (Kislauskis et al., 1988). Elements involved in the regulation of NT/NN mRNA expression are located in the upstream 200-bp flanking region of the rat gene. In this region, several cis-regulatory elements function cooperatively to integrate environmental stimuli into a concerted transcriptional response: an AP1 sequence, two nearby cyclic AMP response elements (CRE), a glucocorticoid regulatory element (GRE), and a sequence identical to the human c-jun gene autoregulatory element (Kislauskis and Dobner, 1990; Dobner et al., 1992; Evers et al., 1995). Interestingly, the GRE identified in the rat gene is not present in the human (Bean et al., 1992). The transcription of the rat NT/NN gene gives rise to two mRNA species (1.0 and 1.5 kb) that are differentially expressed in tissues. These two species show a difference in the length of their 3' untranslated region, due to the existence of two consensus polyadenylation signals (Kislauskis et al., 1988). Whereas the 1.0 kb greatly predominates in intestine (Kislauskis et al., 1988) and anterior pituitary (Jones et al., 1989), approximately equal amounts of 1.0 and 1.5 kb are expressed in the CNS (Kislauskis et al., 1988). 2.3. TRANSLATIONAL AND POST-TRANSLATIONAL PROCESSING OF THE NT/NN PRECURSOR The NT/NN gene encodes a 169 amino acid precursor (pro-NT) which contains a single copy of NT and of its analog NN (Fig. 1). In addition, a sequence resembling NN (NN-like) occurs in the central region of the NT/NN precursor (Fig. 1). The post-translational processing of the precursor depends on four Lys-Arg doublets that represent potential sites of proteolytic cleavage (Fig. 1). Prohormone convertases PC1, PC2, and PC5A have all been shown to possess the ability to cleave the NT precursor in vitro (Rov~re et al., 1996; Barbero et al., 1998), and their cellular co-localization with NT in rat brain and gut suggests that they are also involved in the processing of pro-NT in vivo (Villeneuve et al., 2000a,b). Differential cleavage of these dibasic sites has been shown to yield different combinations of maturation products, depending on the tissues in which the NT/NN precursor is being processed. In the gut, the two most C-terminal basic residues are processed preferentially, giving rise to equivalent amounts of NT and of a larger biologically active peptide referred to as 'Big NN' (Fig. 1; Carraway and Mitra, 1990; Carraway et al., 1991). In the brain, the three most C-terminal dibasic sequences are extensively processed, thus releasing roughly equimolar amounts of NT and NN, albeit with some regional variations (Kitabgi et al., 1991; de Nadai et al., 1994; Woulfe et al., 1994).

325

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P. Sarret and A. Beaudet

Fig. 1. Schematic representation of the rat NT precursor (pro-NT/NN) and of the maturation products detected in tissues in which this pro-peptide is expressed. Rat prepro-NT/NN is 169 amino acid long and starts with a 22-residue signal peptide. The positions of the four Lys-Arg (KR) residues are shown with putative maturation products resulting from their cleavage. In the brain, pro-NT/NN processing gives rise to NT and NN. In the gut, processing leads mainly to the formation of NT and of a large peptide carrying the NN sequence at its C-terminus (Big-NN). In the adrenal glands, NT, large NN, and a large peptide ending with the NT sequence (Big-NT) are the major maturation products (Carraway et al., 1992, 1993; Kitabgi et al., 1992).

2.4. DEGRADATION OF NEUROTENSIN AND NEUROMEDIN N In neurons, NT and NN are both stored in axon terminals and are released in a Ca2+-dependent manner (Bissette and Nemeroff, 1995). As both peptides appear to be co-localized within the same vesicles and hence are presumably co-released (Villeneuve et al., 2000b), their differential degradation in the extracellular space governs the relative concentration at which they will reach and activate their receptors on target cells. Both NT and NN are inactivated through the dual action of endo- and aminopeptidases. In the brain, NT transmission is terminated through the action of neutral endopeptidase 24.11 (enkephalinase, Kerr and Kenny, 1974), metallo-endopeptidase 24.15 (thimet oligopeptidase, Orlowski et al., 1983), and metallo-endopeptidase 24.16 (neurolysin, Checler et al., 1986a). Other exo- and endopeptidases that will not be described here participate in the further degradation of the breakdown products generated by the action of these metalloendopeptidases (Barelli et al., 1989). The mode of degradation of NN is different from that of NT. NN is catabolized by aminopeptidases B and M (for review, see Checler, 1994), which efficiently remove the NHz-terminal Lys residue. In brain tissue, the reported half-life of NT is approximately 15 min, about 2.5 times longer than that of NN (Checler et al., 1986b,c). Furthermore, there is evidence for regional variations in the catabolism of NT and NN in both brain and gut, probably due to differential expression of the various endopeptidases (Checler 326

Neurotensin receptors in the central nervous system

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et al., 1991). This differential inactivation may in turn affect the physiological responses to NT and NN in a tissue-specific manner. The involvement of peptidases in the inactivation of both NT and NN is confirmed by the potentiation of hypothermic and analgesic effects of these peptides by specific peptidase inhibitors. For instance, it was found that thiorphan, a potent and specific inhibitor of enkephalinase, markedly potentiated the hypothermic and analgesic effects of intracerebroventricularly injected NT (Coquerel et al., 1986, 1988). It was also demonstrated that bestatin, a specific aminopeptidase inhibitor did not affect NT-induced responses, but increased the hypothermic and analgesic effects of centrally injected NN (Coquerel et al., 1988; Dubuc et al., 1988).

3. DISTRIBUTION OF NT IN THE CNS

Neurotensin is widely, yet selectively distributed throughout the CNS (for review, see Uhl, 1982). In general, the regional localization of NT in the brain of different mammalian species, such as rat, calf, and monkey parallels that found in man (for review, see Emson et al., 1985a). By radioimmunoassay, NT immunoreactivity was found to be mainly concentrated in the amygdala, hypothalamus, brainstem and spinal cord (Carraway and Leeman, 1976; Kobayashi et al., 1977; Emson et al., 1982). By immunohistochemistry, high densities of NT-immunoreactive nerve cell bodies and processes were detected throughout the brain and spinal cord. In particular, dense networks of NT-positive nerve cell bodies and/or axonal fibers were observed in the preoptic area, nucleus accumbens, bed nucleus of the stria terminalis, medial and lateral septal nuclei, basal forebrain, claustrum, central, basolateral and medial amygdaloid nuclei, subparafascicular and gustatory nuclei of the thalamus, paraventricular, periventricular and arcuate hypothalamic nuclei, lateral hypothalamus, substantia nigra, ventral tegmental area, periaqueductal gray, raphe nuclei, dorsal tegmentum of the pons, nucleus of the solitary tract, and substantia gelatinosa of the spinal trigeminal nucleus and dorsal horn of the spinal cord (Kataoka et al., 1979; Seybold and Elde, 1980, 1982; Cooper et al., 1981; Hunt et al., 1981; Emson et al., 1982; Jennes et al., 1982; Hara et al., 1982; Kahn et al., 1982; Goedert et al., 1983; H6kfelt et al., 1984; Difiglia et al., 1984; Jakeman et al., 1989; Woulfe et al., 1994). 4. CENTRAL EFFECTS OF NT Numerous behavioral effects of centrally administered NT have been reported in mammals (for review, see Brown and Miller, 1982; Kitabgi et al., 1985; Kitabgi and Nemeroff, 1992; Rost~ne and Alexander, 1997). We shall only briefly review here the most salient features of effects induced by this peptide in the CNS. Several reports have demonstrated that central injections of NT induce hypothermia, antinociception, and modulation of dopamine (DA) transmission. Intracisternal, intracerebroventricular, and intracerebral injection of NT in the anterior hypothalamus, the ventral thalamus, or the medial preoptic area, all produce a dose-dependent decrease in body temperature of rodents (Bissette et al., 1976; Nemeroff et al., 1977, 1980; Martin and Naruse, 1982; Kalivas et al., 1982a, 1985). The hypothermic action of NT is most pronounced in animals kept at low temperature (Chandra et al., 1981; Bissette et al., 1982). Intracisternal and intracerebroventricular administration of NT also decrease reactivity to painful stimuli (Clineschmidt and McGuffin, 1977; Osbahr et al., 1981; Hylden and Wilcox, 1983; Pazos et 327

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P. Sarret and A. Beaudet

al., 1984). Similar antinociceptive effects are observed after local microinjection of NT into the periaqueductal gray, pontine reticular formation and raphe magnus (Kalivas et al., 1982a,b; Urban and Smith, 1993). These antinociceptive effects of NT are insensitive to treatment with naloxone, indicating that they are opioid-independent (Clineschmidt and McGuffin, 1977; Osbahr et al., 1981). NT has several pharmacological properties in common with atypical neuroleptics. Like these drugs, it potentiates barbiturate- and ethanol-induced sedation (Nemeroff et al., 1977; Frye et al., 1981; Luttinger et al., 1981, 1982a, 1983), induces muscle relaxation (Osbahr et al., 1979; Jolicoeur et al., 1981; Snijders et al., 1982), antagonizes increases in locomotor activity produced by amphetamine and indirectly acting dopamine agonists (Ervin et al., 1981; Luttinger et al., 1981; Kalivas et al., 1984; Elliott and Nemeroff, 1986; Wagstaff et al., 1994), and produces hypothermia (Bissette et al., 1976). Intracerebroventricular administration of NT induces a dose-dependent suppression of food consumption in food-deprived rats (Luttinger et al., 1982b; Levine et al., 1983; Hoebel, 1985; Abiko and Takamura, 2001). These anorexigenic effects of NT are also observed after its injection into the nucleus of the solitary tract (de Beaurepaire and Suaudeau, 1988), the paraventricular nucleus of the hypothalamus (Stanley et al., 1983), the ventral tegmental area (Cador et al., 1986; Hawkins, 1986), and the substantia nigra (Vaughn et al., 1990). The satiating effects of centrally administrated NT are specific for feeding since intrahypothalamic or intrategmental injections of the peptide do not modify fluid intake in water-deprived animals (Luttinger et al., 1982b; Stanley et al., 1983; Baker et al., 1989). Central administration of NT evokes an extended latency in the appearance of deep sleep (Castel et al., 1989). More recently, intracerebral injection of NT was found to modulate both cortical activity and sleep-wake states through its action on basal forebrain cholinergic neurons (Cape et al., 2000; see below). Finally, NT was shown to modulate the secretion of various hormones from the hypothalamus and pituitary (for review, see Rost~ne and Alexander, 1997). Specifically, it was found to affect the release of corticotropin-releasing hormone (CRH; Nussdorfer et al., 1992), gonadotropin-releasing hormone (GnRH; Alexander et al., 1989), somatostatin (SRIF; Sheppard et al., 1979), and growth hormone-releasing hormone (GHRH, Niimi et al., 1991) from the hypothalamus, and to regulate the release of prolactin (PRL, McCann et al., 1982), growth hormone (GH, McCann and Vijayan, 1992), and luteinizing hormone (Motta and Martini, 1981) from the pituitary. 5. NT RECEPTORS IN MAMMALIAN CNS Central effects of NT and its analogues are mediated by the activation of at least three different receptor subtypes, identified as NTS1 (also called NTRH, NT1 or NTR1), NTS2 (also called NTRL, NT2 or NTR2), and NTS3 (also called NT3, NTR3 or gp95/sortilin) (for reviews, see Vincent et al., 1999; Tyler-McMahon et al., 2000; Binder et al., 2001; Mazella, 2001). A summary of the properties of these receptors and a list of available agonists and antagonists are provided in Table 2. 5.1. IDENTIFICATION OF NT BINDING SITES The existence of specific NT binding sites in mammalian CNS was first demonstrated using radioreceptor binding assays (Kitabgi et al., 1977; Lazarus et al., 1977; Uhl et al., 1977). 328

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Fig. 2. Characterization of NT binding in rat brain membranes and tissue sections in the presence or absence of the NTS2 ligand, levocabastine. (A) Membranes (0.1 mg) were incubated with increasing concentrations of 125I-labeled Tyr3-NT in the presence (open symbols) or absence (closed symbols) of 1 IxM levocabastine. Data are presented as Scatchard plots. B and F, bound and free concentrations of labeled ligand. (B) Chemical structure of the NTS2-specific ligand, levocabastine. (C,D) Autoradiographic distribution of [125I-Tyr3]NT binding in rat brain. Sagittal sections were incubated with 8 nM [125I-Tyr3]NT (100 ci/mmol), in the absence (C) or in the presence of 1 txM levocabastine (D). (C) In the absence of levocabastine, areas of intense [125I-Tyr3]NT labeling are superimposed over a relatively dense, diffuse background. (D) In the presence of levocabastine, the same areas of selective labeling are evident over a considerably reduced background. Note that 125I-Tyr3-NTlabeling in cerebellar cortex is totally displaced by levocabastine, indicating that it can be entirely accounted for by NTS2. Scale bar: 2.5 mm.

The properties of these specific binding sites were later characterized in brain homogenates, synaptosomes, synaptic membranes, brain tissue sections, N1E-115 neuroblastoma cells, NG108-15 hybrid cells, HT-29 human colonic adenocarcinoma cells and mouse primary neuronal cultures (Kitabgi et al., 1980; Young and Kuhar, 1981; Quirion et al., 1982; Uhl, 1982; Mazella et al., 1983; Goedert et al., 1984a; Nakagawa et al., 1984; Poustis et al., 1984; Mills et al., 1988; Turner et al., 1990; Mazella et al., 1991). Analysis of tritiated NT ([3H]NT) binding to synaptic membranes from guinea pig, calf, and human brain revealed a reversible, saturable, high-affinity interaction with a single class of receptors as evidenced by a linear Scatchard plot (Kitabgi et al., 1980, 1985; Goedert et al., 1984a; Kanba et al., 1986; Mills et al., 1988). Subsequent studies using radio-iodinated ligands with a higher specific activity delineated two distinct classes of NT binding sites in synaptic membranes from rat (Mazella et al., 1983), guinea pig (Sadoul et al., 1984a), and human (Sadoul et al., 1984b) brain. As illustrated in Fig. 2A, the binding of [125I-Tyr3]NT to rat brain synaptic membranes yields a curvilinear Scatchard plot that can be resolved into two independent components: a low-affinity binding site (Kd = 2-4 nM) with high capacity and a high affinity binding site (Kd = 0.1-0.3 nM) with relatively low capacity (Mazella et al., 1983). 329

NT receptor subtypes

NTSl (NTRH)

NTS2 (NTRL)

NTS3 (gp95/sortilin)

Receptor classification

G-protein coupled receptor (7 TM)

G-protein coupled receptor (7 TM)

Type I receptor ( 1 TM)

Cloned

rNTSl (Tanaka et al., 1990)

rNTS2 (Chalon et al., 1996)

mNTS1 (Genbank: 3551524) hNTSl (Vita et al., 1993)

mNTS2 (Mazella et al., 1996) hNTS2 (Vita et al., 1998)

rNTS3 (Lin et al., 1997; Moms et al., 1998) mNTS3 (Navarro et al., 2001) hNTS3 (Mazella et al., 1998)

Receptor size

rNTSl and mNTS1 = 424 aa hNTSl = 418 aa

rNTS2 and mNTS2 = 416 aa Variant isoform (282 aa, 5 TM) hNTS2 = 410 aa

mNTS3 = 825 aa hNTS3 = 833 aa

Molecular weight

50 and 60 kDa

45 kDa

100 kDa

Gene characteristics

hNTS 1 gene localized to the long arm (20q 13) of chromosome 20 Tetranucleotide repeat polymorphism located < 3 kb from the gene

mNTS2 gene mapped at 6 cm from the centromere on chromosome 12 Two splice variants derived from a single gene

hNTS3 gene mapped to the proximal short arm of chromosome 1 Two sortilin mRNA transcripts 3.5 and 8.0 kb expressed in human CNS

Kd = 0.1-0.3 nM (rat brain synaptic membranes)

Kd = 2 4 nM (rat brain synaptic membranes)

B,, = 13 fmol/mg (adult rat brain homogenate)

B,,, = 170 fmol/mg (adult rat brain homogenate)

Kd = 0.3 nM (CHAPS-solubilized human brain receptors) Kd = 17 nM (CHAPS-solubilized cloned hNTS3 in COS-7 transfected cells) B,,, = 150 fmol/mg (CHAPS-solubilized human brain receptors)

Maximal binding capacity (Bmax)

t

GTP sensitivity

+

Naf sensitivity

IC50 = 15 mM (Asp- 1 13)

1C.j" = 200 mM

?

Internalization

65-70% of total binding tl/2 = 7 min C-terminal tail (TLY)

60% of total binding t1,2=10 min intracellular loop 3 (13)

75% of total binding t l p = 5 min

+

+

Recycling

?

V237

?

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Levocabastine sensitivity

P s

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TABLE 2. Characteristics of the NT receptor subtypes

0

a

3 R

?

NT receptor subtypes

NTSl (NTRH)

NTS2 (NTRL)

NTS3 (gp95/sortilin)

Signal transduction

Phospholipase C (IP3 and Ca2+ mobilization), cGMP, CAMP,MAPKs and Krox-24 gene induction

Phospholipase C (oocytes) IP formation and Ca2+ mobilization, arachidonic acid release, MAPKs

Modulation of NTS1-mediated IP turnover and MAPKs

Cellular localization

Mainly neurons

Neurons and astrocytes

Neurons and glia

Agonists

NT

NT (receptors expressed in oocytes and cerebellar granular cells) SR48692 and SR142948A (receptors expressed in oocytes and CHO cells) Levocabastine (receptors expressed in oocytes and cerebellar granule cells)

NT?

NN xenopsin

Antagonists

SR48692

NT and levocabastine (receptors expressed in CHO cells)

SR142948A Physiological implications

NT-induced turning behavior

NT-induced analgesia

Modulation of dopamine transmission

NT-induced hypothermia?

NT-induced cell growth Effects of NT blocked by SR48692 and SR142948A

Effects of NT not blocked by SR48692 Effects of NT blocked by SR142948A

Sorting of luminal protein from the TGN to late endosomes Scavenger for multiple ligands (uptake and degradation) NT-induced cell growth NT-induced cell growth blocked by SR48692

Neurotensin receptors in the central nervous system

TABLE 2. (continued)

The table summarizes the pharmacological and biochemical properties, agonists, antagonists and the physiological implications of the three cloned NT receptors according to Kitabgi et al. (1985), Rostkne and Alexander (1997), Vincent (1992, 1995), Betancur et al. (1997), Hermans and Maloteaux (1998). Vincent et a]. (1999), Binder et al. (2001) and Mazella (2001). Abbreviations: rNTS, rat NTS; mNTS, mouse NTS; hNTS, human NTS; aa, amino acids; TM, transmembrane domain; NT, neurotensin; NN, neuromedin N; IP, inositol phosphate; MAPK, mitogen-activated protein kinases; TGN, trans-Golgi network.

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These two sites may also be distinguished by their differential sensitivity to the antihistamine, levocabastine (Fig. 2B). This compound, by itself devoid of NT-like activity, entirely blocks the binding of [lZSI-Tyr3]NT to the low-affinity binding site (NTS2) without affecting its binding to the high-affinity site (NTS1; Fig. 2) (Schotte et al., 1986; Schotte and Leysen, 1989; Kitabgi et al., 1987). However, only rat, mouse, and hamster NTS2 sites are sensitive to levocabastine. This drug has no effect on NT binding in guinea pig, cat, dog or human brain (Schotte et al., 1986; Kitabgi et al., 1987). In 1988, Mazella and coworkers detected, in an attempt to solubilize and purify mouse NT receptors, a NT binding protein with an apparent molecular mass of 100 kDa (Mazella et al., 1988). This 100-kDa NT binding protein, which is now known to correspond to the NTS3, was further purified after solubilization from mouse and human brain by affinity chromatography (Mazella et al., 1989; Zstirger et al., 1994). 5.2. NT RECEPTOR SUBTYPES Ultimate confirmation of the existence of the NTS 1, NTS2 and NTS3 subtypes was provided by their cloning. In the early 1990s, the NTS1 was isolated by screening a cDNA library from rat brain using the oocyte expression technique (Tanaka et al., 1990). Analysis of the deduced amino acid sequence of the cloned rat NTS 1 receptor reveals that it is composed of 424 residues. The primary sequence has a predicted molecular weight of 47,052 and displays seven stretches of hydrophobic amino acids corresponding to seven potential transmembrane domains (Fig. 3). Scatchard analysis of [3H]NT/[125I]NT binding to the recombinant NTS1 receptor transfected in mammalian cells revealed a specific, high-affinity association with a Ko of 0.2 nM, i.e. close to that reported for the high-affinity binding site in rat brain synaptic membranes (for review, see Kitabgi et al., 1985; Vincent, 1992, 1995). The resistance of the binding to levocabastine confirmed that the cloned receptor corresponded to the high-affinity site. Subsequently, the human NTS 1 receptor was cloned from the adenocarcinoma cell line HT29. It consists of 418 amino acids and shares 84% homology with rat NTS1 (Vita et al., 1993). The NTS1 receptor has been linked to a variety of signaling cascades, including formation of cGMP and cAMP, stimulation of phosphatidylinositol turnover and calcium mobilization, and activation of mitogen-activated protein kinases (Table 2) (for review see, Vincent, 1995; Vincent et al., 1999; Hermans and Maloteaux, 1998). Association of NT to the NTS1 receptor induces internalization of receptor-ligand complexes into NTSl-expressing cells via clathrin-coated pits (Chabry et al., 1994, 1995; Hermans et al., 1994; Nouel et al., 1997; Vandenbulcke et al., 2000). Biochemical and cell imaging evidence suggests that internalized receptors are not recycled to the plasma membrane but rather targeted to lysosomes for degradation (Chabry et al., 1993; Botto et al., 1998; Hermans et al., 1997; Vandenbulcke et al., 2000). The internalization process is dependent on the integrity of the C-terminal intracytoplasmic tail (Hermans et al., 1996; Najimi et al., 2002) and more particularly, on the Thr-422 and Tyr-424 residues at the extremity of the tail (Chabry et al., 1995). The levocabastine-sensitive, low affinity NT receptor (NTS2) was cloned from rat, mouse and human brain using a strategy based on sequence homology with the known NTS 1 receptor (Chalon et al., 1996; Mazella et al., 1996; Vita et al., 1998). The rat and mouse NTS2 (416 amino acids) are slightly longer than their human counterpart (410 amino acids). NTS2 also exhibits the structural features of a G-protein-coupled receptor (Fig. 3). The sequence similarity between this second receptor and the high-affinity NTS1 receptor is 64% in rat. An alternative splicing of the NTS2 primary transcript leads to the production of a truncated 332

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Fig. 3. Schematic representation of NTS 1, NTS2, and NTS3 receptor subtypes. NTS1 and NTS2 belong to the G-protein coupled receptor family. NTS3 is a single transmembrane domain receptor. Amino acids exclusive to NTS1 are represented in black. Invariant residues between NTS1 and NTS2 receptors are indicated in white. Sequences differing between NTS1 and NTS2 (El, i3 and i4) are hatched. The NTS3 is comprised of an N-terminal signal peptide (SP), a (1-44) propeptide (P) released by furin cleavage (FS), a cysteine-rich domain (crd), a transmembrane domain, and three internalization/sorting signals in the cytoplasmic tail (PS).

receptor (vNTS2). In the mouse, this shorter receptor (282 amino acids) only contains five putative transmembrane domains (Botto et al., 1997a). The function of this NTS2 receptor variant is unknown. In contrast to NTS 1, little is known of the pathways involved in NTS2 signaling (Table 2). NT, levocabastine, and the NTS 1 antagonist SR48692 (see below) were all found to induce an inward calcium-activated chloride current in Xenopus oocytes transfected with cDNA encoding the mouse NTS2 receptor (Mazella et al., 1996; Botto et al., 1997b). By contrast, SR48692, but neither NT nor levocabastine, was capable of activating classical secondmessenger systems (including Ca 2+ mobilization, inositol phosphate production, or mitogenactivated protein kinase phosphorylation) in mammalian cells transfected with human NTS2 (Vita et al., 1998). Furthermore, the effects of SR48692 were blocked by NT, suggesting that the endogenous peptide could act as a competitive antagonist at NTS2 sites (Vita et al., 1998). However, studies on rat cerebellar granule cells in culture recently showed that both NT and levocabastine could act as agonists on endogenously expressed NTS2 (Sarret et al., 2002). Accordingly, NTS2 receptors were found to efficiently internalize upon NT binding, a property usually associated with activation by agonists (Botto et al., 1998; Sarret et al., 2002). In contrast to the NTS 1, internalized NTS2 receptors are recycled back to the plasma 333

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membrane (Botto et al., 1998). The tyrosine-237 in the third intracellular loop seems to be critical for the recycling of the mouse NTS2 receptor (Martin et al., 2002a). The NTS3 receptor was only recently cloned (Mazella et al., 1998). It has an apparent molecular weight of 100 kDa and is 100% homologous to the sorting protein gp95/sortilin (Petersen et al., 1997). Unlike the other two NT receptors, NTS3/gp95/sortilin is not a 7 transmembrane domain G-protein-coupled receptor, but a single transmembrane domain protein (Table 2). This protein is 833 amino acids long and contains an N-terminal signal peptide, a cleavage site for furin, a long cysteine-rich luminal domain, and a short intracytoplasmic domain in addition to its transmembrane segment (Fig. 3; Marcusson et al., 1994; Mazella et al., 1998). The mouse NTS3 (825 amino acids) is slightly shorter than its human counterpart (Navarro et al., 2001). Although the NTS3 was originally reported to be mainly localized in the Golgi apparatus and intracellular vesicles (Morris et al., 1998; Nielsen et al., 2001), recent biochemical and imaging studies have shown it to be addressed to the cell surface in a variety of cell lines (Chabry et al., 1993; Navarro et al., 2001; Dal Farra et al., 2001). As the other two NT receptors, NTS3 is efficiently internalized subsequent to NT binding (Navarro et al., 2001). Little is known about the functional and signaling properties of the NTS3 receptor except that it may be involved in the growth response of human cancer cells to NT (Dal Farra et al., 2001) and can modulate NTS 1-elicited responses in HT29 cells (Martin et al., 2002b). 5.3. NT AGONISTS AND ANTAGONISTS As for any other peptide, the development of potent and selective NT agonists and antagonists is critical for defining the interaction of NT with its receptors, investigating the role of the various receptor subtypes in mediating the central actions of NT and, in the long run, developing novel therapeutic avenues.

5.3.1. Agonists Since the 8-13 fragment of NT is sufficient for receptor activation, a variety of NT(8-13) analogues have been designed to obtain degradation-resistant NT agonists (Gilbert et al., 1989; Lugrin et al., 1991; Dubuc et al., 1992; Cusack et al., 1993). Thus, a series of pseudopeptide analogues of NT was produced by systematically replacing the five peptide bonds in NT(8-13) with CH2NH (reduced) bonds, which are not cleaved by peptidases (Lugrin et al., 1991). All of these analogues were synthesized with a free amino terminus (H derivatives) or with a N-terminal tert-butyloxycarbonyl group (BOC derivatives) to deter the action of aminopeptidases (Lugrin et al., 1991; Labbr-Julli6 et al., 1994). Some of these NT analogues were indeed found to produce potent and long-lasting NT-like effects after administration in vivo or in vitro (al-Rodhan et al., 1991; Labbr-Julli6 et al., 1994). Another series of NT analogues was developed to investigate receptor-ligand interactions. Some of these analogues were found to exhibit species selectivity (human versus rat), stereoselectivity (L-isomers exhibited 200-700-fold greater potency than D-isomers), and superagonism (picomolar affinity at human and rat receptors) (Cusack et al., 1995, 1996). The importance of the Tyr residue in position 11 of the NT molecule and the orientation of a steric bulk in this position were also investigated. These studies indicated that the NT(8-13) binding site of the human NTS 1 was slightly smaller than that of the rat NTS 1 (Pang et al., 1996). Therefore, the human receptor preferentially binds molecules that contain an amino acid larger than Tyr and smaller than naphthylalanine in position 11, in contrast to the rat receptor (Cusack et al., 1995, 2000). 334

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NT analogues with the ability to cross the blood-brain barrier have also been recently developed (for review, see McMahon et al., 2002). The first two of these were the hexapeptide compounds (Me)Arg-Lys-Pro-Trp-tert-Leu-Leu-Oet and (Me)Arg-LysPro-Trp-tert-Leu-Leu, referred to as NT-1 and NT-2, respectively (Tokumura et al., 1990, 1993). These analogues cause the same behavioral changes as centrally injected NT when administered in vivo, including hypothermia and antinociception (Machida et al., 1993). More recently, Tyler et al. also produced a series of hexapeptide analogues of NT(8-13) (NT66L, NT67L and NT69L) that cross the blood-brain barrier very efficiently and exhibit strong agonistic properties at the NTS 1 receptor in vivo (Tyler et al., 1999).

5.3.2. Antagonists A better understanding of the central effects of endogenous NT can only be provided by the development of potent and selective antagonists (for review, see Gully et al., 1995; Betancur et al., 1997; Rost~ne et al., 1997). [D-Trpll]NT, a NT analogue in which Tyr ll is replaced by [D-Trp11], was the first NT derivative shown to be endowed with antagonistic properties (Quirion et al., 1980; Rioux et al., 1980). A tripeptide derivative was later developed that also exhibited antagonistic effects (Cusack et al., 1993). However, these peptide antagonists have several drawbacks, including high biodegradability, short duration of action, and poor blood-brain barrier penetration. A number of non-peptide antagonists were developed to circumvent these shortcomings, including Pfizer's UK-73,093 (Snider et al., 1992), Merck's L-734836 (Chakravarty et al., 1993), and Parke-Davis' PD-156425 (Kesten et al., 1994). The most potent and selective non-peptide NT antagonist developed so far, however, is Sanofi's SR48692 (Gully et al., 1993; Maffrand et al., 1993). This compound is orally active, crosses the blood-brain barrier, and has a long-lasting action. It also displays a much higher affinity for NTS 1 (IC50 = 5.6 nM) than for NTS2 (IC50 = 300 nM) and NTS3 (IC50 > 1 txM), which makes it a good tool for differentiating the contribution of NTS 1 from that of other NT receptors to the central effects of NT (Table 2; Gully et al., 1993; Mazella et al., 1996; Navarro et al., 2001). Given intraperitoneally or orally, SR48692 suppresses the turning behavior induced by unilateral injection of NT in mouse neostriatum (Gully et al., 1993; Poncelet et al., 1994). It also antagonizes the locomotor activity (but not the DA release in the accumbens) evoked by NT injection into the ventral tegmental area (Steinberg et al., 1994). However, it does not inhibit the hypothermic and analgesic responses to intracerebroventricularly injected NT in mice and rats, suggesting that these effects may not be mediated through NTS1 (Dubuc et al., 1994; Pugsley et al., 1995). A follow-up compound, SR142948A, was recently introduced which is more soluble but less selective than SR48692 in that it recognizes both NTS1 and NTS2 receptors with high affinity (IC50 = 1-4 nM) (Gully et al., 1997). Like SR48692, this new antagonist inhibits the NT-mediated turning behavior in mice and has no effect on DA release evoked by NT injection into the ventral tegmental area (Gully et al., 1997). However, unlike SR48692, SR142948A blocks both the hypothermic and analgesic effects induced by centrally administered NT, suggesting that the latter effects may be mediated by the NTS2 receptor subtype (Gully et al., 1997).

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5.4. LOCALIZATION OF NT RECEPTOR SUBTYPES

5.4.1. Methods of study Several approaches have been used to study the distribution of NT receptors in the CNS at the regional, cellular and subcellular levels. The existence of regional differences in the distribution of NT binding sites in mammalian CNS was first revealed by radioligand binding studies in calf and rat brain homogenates. These early studies showed [3H]NT binding to be higher in the midbrain, hypothalamus, and cingulate cortex than in other cortical areas, brainstem, or cerebellum (Lazarus et al., 1977; Uhl and Snyder, 1977). However, it is only with the application of autoradiographic binding techniques that a comprehensive picture of brain NT receptor distribution began to emerge. The first autoradiographic maps of NT binding in mammalian CNS were generated using [3H]NT as radioligand (Young and Kuhar, 1979, 1981; Quirion et al., 1982; Uhl, 1982; Goedert et al., 1984a; K6hler et al., 1985; Palacios et al., 1988). Later studies replaced [3H]NT with [125I]NT to gain greater sensitivity and, by resorting to liquid emulsion coating techniques, to provide us with a first appraisal of the receptors' cellular localization (Sadoul et al., 1984b; Sarrieau et al., 1985; Herve et al., 1986; Moyse et al., 1987; Kessler et al., 1987; Palacios et al., 1988; Dana et al., 1989; Szigethy et al., 1990a,b). Furthermore, in several of these later studies, non-radioactive levocabastine was added to the iodinated ligand to ensure the selective visualization of NTS 1 receptors or to determine, by subtraction, the distribution of NTS2 sites (Schotte et al., 1986; Kitabgi et al., 1987; Nicot et al., 1994a,b). Given the similarity between the distribution of specifically bound [3H]NT and that of [125I]NT bound in the presence of levocabastine, it can, retrospectively, be surmised that [3H]NT binding experiments largely reflected the distribution of NTS1 receptors. Although the possibility that some of the binding sites labeled with either [3H]NT or [125I]NT might have corresponded to the NTS3 receptor cannot be formally excluded, this possibility remains very unlikely given the close correspondence between autoradiographic labeling patterns and the distribution of NTS 1 receptor proteins observed by immunohistochemistry (see below). Furthermore, the affinity of the NTS3 sites for NT is too low for these sites to be picked up by the subnanomolar concentrations of radioactive ligands used in autoradiographic studies (Navarro et al., 2001). Subsequently, the cloning of NT receptors allowed the generation of mRNA probes to investigate the distribution of their mRNA by Northern blotting, RT-PCR, and in situ hybridization. It also made it possible to raise antibodies against specific peptide sequences of the receptors for their immunocytochemical visualization at light and electron microscopic levels. Because little is still known on the distribution of the NTS3 receptor subtype (or, for that matter, on its role) in mammalian CNS, we will concentrate here on the distribution of the NTS 1 and NTS2 receptor subtypes, as revealed by autoradiographic, in situ hybridization and, in the case of NTS 1, immunohistochemical studies.

5.4.2. NTS1 receptors 5.4.2.1. Distribution of NTS1 binding sites

As can be seen in film autoradiograms and in low magnification photomicrographs of emulsion-processed sections, the distribution of specifically labeled NTS1 binding sites is relatively widespread, but regionally selective (Fig. 4). At high magnification of emulsion336

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dipped sections, the labeling is seen to be either cellular, i.e. concentrated over nerve cell bodies, or diffuse, i.e. distributed throughout the neuropil. The islands of Calleja, anterodorsal thalamic nucleus (Fig. 5), vertical limb of the diagonal band of Broca, substantia innominata, dorsal motor nucleus of the vagus, lateral reticular nucleus, or nucleus raphe pallidus provide good (but not exclusive) examples of regions in which NTS1 labeling is predominantly localized over neuronal perikarya (Kessler et al., 1987; Moyse et al., 1987). Accordingly, all of these regions were later found to contain numerous nerve cell bodies expressing NTS1 mRNA and/or NTS1 receptor proteins by in situ hybridization and immunohistochemistry, respectively. Other regions exhibit only diffuse autoradiographic labeling which by immunohistochemistry was found to correspond to NTS 1-immunoreactive axons and axon terminals. The best example of this type of labeling pattern is observed in the caudate-putamen (Fig. 4B). Most regions, however, show a mix of perikaryal and diffuse, i.e. of axonal and/or dendritic labeling, making it difficult to precisely assess the cellular distribution of the autoradiographically labeled binding sites. Telencephalon

NTS1 binding sites are relatively widespread in the rat neocortex (Fig. 4A-G; Table 3). They are most prevalent in the cingulate, insular, perirhinal, entorhinal, and retrosplenial areas (Table 3; Young and Kuhar, 1981; Quirion et al., 1982; Moyse et al., 1987). However, whereas in the pregenual anteromedial cingulate cortex they predominate in layer I, in the pregenual and supragenual anterodorsal cingulate and insular cortices, they are concentrated in layer VI (Fig. 4A,B; Moyse et al., 1987). In the perirhinal and entorhinal cortex, NTS1 binding sites predominate over layers I-III (Fig. 4F), whereas in the retrosplenial cortex, they are found over the outermost aspect of layer I and across layer II (Figs. 4F and 5A; Moyse et al., 1987). The laminar distribution of NTS 1 binding sites in the retrosplenial cortex (Fig. 5A) conforms with the pattern of anterograde degeneration observed after lesioning the anterior dorsal thalamic nucleus (Domesick, 1972). Conversely, the distribution of labeled binding sites in the anterior dorsal nucleus of the thalamus (Fig. 5B) corresponds to the distribution of neurons showing retrograde changes in the same area following lesion of the retrosplenial cortex (Moyse et al., 1987), suggesting that NTS1 binding sites are present both pre- and postsynaptically on thalamoretrosplenial neurons. By and large, there is uneven correspondence between the distribution of NT immunoreactivity and that of NTS 1 binding sites in these cortical regions, except for the olfactory, entorhinal, and cingulate cortices (Emson et al., 1985a). In the monkey, high densities of NTS1 binding sites were reported in deep cortical layers (laminae IV and V) of the neocortex and throughout the cingulate cortex (Fig. 6A) while in the human, high levels of NTS1 labeling were observed in the frontal, temporal, cingulate, insular, and entorhinal cortices with varying laminar distributions in the different cortical regions (Quirion et al., 1987; Sarrieau et al., 1985; Wolf et al., 1994, 1995). The highest concentrations of [125I]NT binding sites, as assessed by densitometric analysis of film autoradiograms, were measured in the paraolfactory gyrus (Brodmann area 25), cingulate gyrus (Brodmann area 32), and entorhinal cortex (Brodmann area 28) (Sarrieau et al., 1985). Within the latter, NTS1 receptors were most prevalent over the cell clusters of layer II, throughout the area's rostrocaudal extent (Wolf et al., 1994). NTS 1 receptor binding densities are moderate in the rat caudate-putamen, with the densest labeling found dorsolaterally (Fig. 4B; Table 3), where the least amount of NT immunoreactivity has been localized (Quirion et al., 1982; Zahm and Heimer, 1988). Early excitotoxic

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Fig. 4. (G). Comparative distribution of ['251]neurotensinbinding sites (left) and mRNA encoding NTSl receptors (right) in the forebrain and midbrain of the adult rat. Autoradiograms of coronal sections arranged from rostra1 (AJi') to caudal (G,G'). For detection of NT binding sites, sections were incubated with 0.1 nM monoiodo ["'I-Tyr? lneurotensin (2000 ci/rnmol), fixed with glutaraldehyde, and autoradiographically processed using liquid emulsion (Moyse et al., 1987); Darkfield: in situ hybridization was performed using "S-sense or 3sS-antisense rat NTSl receptor riboprobes and film autoradiography (Alexander and Leeman, 1998); Brightfield: in situ hybridization images kindly provided by Dr. M.J. Alexander. See Section 7 for anatomical identification.

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TABLE 3. Regional distribution of NTS1 receptors in the adult rat brain Structure

[125I]NT binding a

mRNA levels b

NTR immunoreactivity c Cell bodies and dendrites

Axons and nerve terminals

++ +/++

+

Telencephalon Cerebral cortex Frontal cortex Layer II-III Layer IV Layer V Layer VI Parietal cortex Layer II-III Layer IV Layer V Cingulate cortex Layer II-IV Endopiriform cortex Layer VI Insular cortex Layer VI Perirhinal cortex Layer I-III Layer IV-V Layer VI Entorhinal cortex Retrosplenial cortex Layer I Layer II-III Basal ganglia Caudate putamen Nucleus accumbens Olfactory tubercle Islands of Calleja Basal forebrain Medial septum Diagonal band of Broca Vertical limb Horizontal limb Magnocellular preoptic area Substantia innominata Nucleus basalis Lateral septum Bed nucleus of the stria terminalis Amygdala Posterior cortical nucleus Basomedial nucleus Central nucleus Lateral nucleus Hippocampal formation Pre-, parasubiculum CA1, CA2, CA3 Dentate gyrus

342

+

+/++

++ +

+ ++ +/++

+++

++ + ++ ++

++

++

+++

+/++

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

++/+++ +

++

++ +

++

++ +++

++ ++

+

+/-

++

+

+

++++

+

++++

++

++++ ++++

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

+ +

++/+++ ++/+++

+/+/-

+++ +++

++++ ++ ++ ++

++ + ++/+++ +

++

++ + +/+

++++ + ++

+/++ + + (CA3)

+++ + +

++

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

++++ ++++

Neurotensin receptors in the central nervous system

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TABLE 3 (continued) Structure

[125I]NT binding a

mRNA levels b

NTR immunoreactivity c Cell bodies and dendrites

Diencephalon Thalamus Anterior dorsal thalamic nucleus Paraventricular thalamic nucleus Reticular thalamic nucleus Hypothalamus Median eminence Suprachiasmatic nucleus Periventricular nucleus Paraventricular nucleus Lateral hypothalamic area Lateral mammillary nucleus Subthalamus Zona incerta Epithalamus Medial habenula Mesencephalon Substantia nigra pars compacta Substantia nigra pars reticulata Ventral tegmental area Interpeduncular nucleus Interfascicular nucleus Periaqueductal gray Dorsal raphe nucleus Laterodorsal tegmental nucleus Superior colliculus

§247247247

_l§247247 §

Axons and nerve terminals

§247247 §247247 §247

§247247 +/++ +/++ §247247

_l§247247 ++ + §247

§247 § ++ ++ §247

§

+-t-

§

§247247

§247247

§247

§

§247247

+/++/+++

++

+

+++

§247 §247 § +§ §247247247 ++

§247247

+§247 §247247 +§247 §247247 +/§247 § §247

§247

§ §247247

Ports

Pontine nuclei Reticulotegmental nucleus Ventral tegmental nucleus Median raphe nucleus

Medulla Medial vestibular nucleus Dorsal cochlear nucleus Retrofacial nucleus Linear nucleus of the medulla Raphe pallidus nucleus Inferior olive Dorsal motor nucleus of the vagus Nucleus of the solitary tract External cuneate nucleus Lateral reticular nucleus

§247247247 -l-t-§247 §247 § §247 §247 +++ §247247 +§ +++ §247247247 §247247 § ++

a Density of [125I]NT binding sites as per Moyse et al. (1987): +, <3 fmol/mg protein; § 3-6 fmol/mg protein; + + + , 6-9 fmol/mg protein; + + + + , >9 fmol/mg protein. b NTR mRNA levels as per Alexander and Leeman (1998): +, low yet above background; + § moderate; § 2 4 7 2 4 7 high level; + § 2 4 7 2 4very 7 high. c NTR immunoreactivity as per Boudin et al. (1996): § limit of detection; § low signal; § 2 4 7moderate signal; § high signal; § 2 4 7 2 4 7very 2 4 7high signal.

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Fig. 5. Comparison between the distribution of radiolabeled NT binding sites (darkfield; left) (Moyse et al., 1987) and NTS1 immunoreactivity (brightfield; right) (Boudin et al., 1996) in the granular retrosplenial cortex (A,A') and the anterodorsal (AD) thalamic nucleus (B,B'). In both regions, the labeling patterns are strikingly similar. The immunoreactivity is mainly associated with nerve cell bodies in the AD and with axon terminals in the retrosplenial cortex. Within the latter, labeled axons are mainly evident in the outer molecular layer (I) and layers II-III, but are also seen to form columnar arrangements bridging these two zones, sm, stria medullaris; AV, anteroventral thalamic nucleus; AD, anterodorsal thalamic nucleus. Scale bars: 60 Ixm in A,A'; 180 txm in B,B'.

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Fig. 6. Autoradiographic distribution of [125I]NTbinding sites in sections of monkey brain. (A) In coronal sections, high densities of [125I]NT binding sites are found in the substantia nigra, hippocampal formation (H), caudate nucleus (C), putamen (P), and deep cortical layers. (B-E) Sections through the neostriatum (B,C) and substantia nigra (D,E) of normal (B,D) and 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine(MPTP)-treated (C,E) monkeys. [125I]NT binding is markedly decreased in the substantia nigra, pars compacta (SNC), the caudate and the putamen in MPTP-treated (C,E) monkeys as compared to normal controls (B,D). Photomicrographs kindly provided by Dr. R. Quirion (for technical details, see Quirion et al., 1987).

lesion studies of neuronal perikarya in the striatum, combined with 6-hydroxydopamine (6-OHDA) lesions of DA axon terminals suggested that over 50% of the NT binding sites in the rat striatum were associated with intrinsic neurons whereas 30% were localized to nigrostriatal axon terminals (Goedert et al., 1984b). The remaining 20% were attributed to presynaptic NT receptors on the terminals of cortical afferents. However, subsequent studies showed that virtually all of the levocabastine-resistant, NTS1 binding sites in the striatum were associated with DA nigral afferents (Palacios and Kuhar, 1981; Quirion, 1983; Quirion et al., 1985; Herve et al., 1986). Despite its high content in endogenous NT, the nucleus accumbens displays a relatively sparse dispersion of high affinity NT binding sites (Fig. 4B; Table 3), the majority of which were determined by selective lesion studies to be associated with DA nerve endings (Schotte and Leysen, 1989). In the rat, NT labeling in the striatum spares traversing fibers of the internal capsule, but does not appear to be affected by striosomal compartmentation (Fig. 4B; Moyse et al., 1987). By contrast, in the cat (Goedert et al., 1983, 1984c) and monkey (Waters et al., 1987) striatum, the distribution of [3H]NT binding sites was found to coincide with that of AChE-rich matrix tissue surrounding striosomes. In the cat striatum, this distribution of NT binding was inversely related to that of NT immunoreactivity, which is the highest in striosomes (Goedert et al., 1984b). In the monkey, moderate to high levels of NTS 1 binding 345

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were observed in the rostral caudate, putamen (Fig. 6A), and claustrum (Waters et al., 1987; Quirion et al., 1987). In the human brain, the highest levels of NT binding sites were observed in the caudate and claustrum with lower levels in the putamen (Quirion et al., 1987). As in the monkey, patches of low receptor densities aligned with the AChE-poor striosomes while regions of moderate receptor density corresponded with the AChE-rich matrix zone (Faull et al., 1989a). In addition, annular regions of high receptor density were reported in register with the AChE-negative border zone lying between striosome and matrix compartments (Faull et al., 1989a). In the basal forebrain, NTS1 binding sites are especially conspicuous in the olfactory bulb, islands of Calleja, medial and lateral septum, vertical and horizontal limbs of the diagonal band of Broca, and bed nucleus of the stria terminalis, in good agreement with the presence of NT-containing fibers and cells in these areas (Fig. 4B; Table 3) (Young and Kuhar, 1981; Quirion et al., 1982; Emson et al., 1985a; Moyse et al., 1987). In addition, selective concentrations of NTS1 binding sites are observed over numerous large nerve cell bodies in the magnocellular preoptic area, substantia innominata, and nucleus basalis of Meynert which, in the rat, is located along the medial edge of the globus pallidus (Fig. 4B,C; Szigethy and Beaudet, 1987). In monkey brain, the presence of NTS1 binding sites was noted in the olfactory bulb, olfactory tubercle, and claustrum (Fig. 6A; Quirion et al., 1987), while in the postmortem human brain, labeled NTS1 receptors were detected not only in the claustrum (Quirion et al., 1987; Palacios et al., 1991; Szigethy et al., 1990b), but also throughout the bed nucleus of the stria terminalis, islands of Calleja, olfactory tubercle, medial septum, diagonal band nucleus and nucleus basalis of Meynert (Szigethy et al., 1990b). Moderate to high densities of NT binding sites are evident in the posterior cortical nucleus as well as in the basomedial, central, and lateral nuclei of the rat amygdaloid complex (Fig. 4C,D,E; Quirion et al., 1982; Moyse et al., 1987). Curiously, whereas the highest concentrations of NTS1 receptors are detected in the posterior cortical nucleus, the highest densities of NT-immunoreactive neurons and axonal varicosities are found in the central nucleus, and most notably in its medial subdivision (Woulfe et al., 1994; Emson et al., 1985a). High concentrations of NT binding sites were also reported in various subdivisions of the monkey and human amygdaloid complex (Sarrieau et al., 1985; Quirion et al., 1987; Szigethy et al., 1990b; Lantos et al., 1996). In the hippocampus, a moderate density of NTS 1 binding sites is apparent over the lateral entorhinal area, pre- and parasubiculum, ventral subiculum, molecular layer of the ventral dentate gyrus, and induseum griseum (Fig. 4D,E) (Young and Kuhar, 1981; Quirion et al., 1982; Moyse et al., 1987). K6hler et al. (1985) found a high concentration of [3H]NT binding sites in the entorhinal region of the hippocampus in the rat, monkey and human brain (K6hler et al., 1985, 1987). In contrast, Sarrieau et al. (1985) observed only low amounts of binding in the human hippocampus, with the exception of the subiculum, where moderate levels were described. Diencephalon The anterior dorsal nucleus of the thalamus shows amongst the highest densities of NTS1 binding sites in the rat brain (Figs. 4C and 5B; Table 3; Moyse et al., 1987). Considerably lower NTS 1 receptor levels have been reported within the paraventricular nucleus, pretectal area, ventral lateral geniculate body, and nucleus of the optic tract. Accordingly, only low to moderate densities of NT axons have been localized by immunohistochemistry in these regions (Jennes et al., 1982; Emson et al., 1985a; Woulfe et al., 1992). Dense concentrations

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of radiolabeled NTS 1 binding sites were reported by most authors throughout the rostrocaudal extent of the zona incerta (Young and Kuhar, 1981; Quirion et al., 1982; Moyse et al., 1987). In the epithalamus, NTS 1 binding sites are concentrated in the medial aspect of the medial habenula, where they overlay densely packed neuronal perikarya (Figs. 4D and 7A). In the monkey, only low levels of NT binding were observed in most thalamic nuclei (Quirion et al., 1987). In the hypothalamus, moderate to high densities of NTS1 receptors are visible in the anterior, peri- and paraventricular, arcuate, dorsomedial, posterior, premammillary, and mammillary nuclei (Fig. 4C,D; Young and Kuhar, 1981; Quirion et al., 1982; Moyse et al., 1987). However, the highest concentrations by far are observed in the suprachiasmatic nucleus, particularly within its ventromedial segment (Fig. 4C). Likewise, dense concentrations of NTS 1 binding sites were reported in the suprachiasmatic nucleus of the monkey (Quirion et al., 1987). In humans, only a moderate density of NT binding was found in the hypothalamus (Sarrieau et al., 1985; Quirion et al., 1987). Mesencephalon

Among the highest concentrations of NT binding sites detected in rat brain are found at the level of the substantia nigra, pars compacta and ventral tegmental area, including the interfascicular nucleus (Fig. 4E,F; Table 3; Young and Kuhar, 1981; Quirion et al., 1982; Moyse et al., 1987). The demonstration that levocabastine exerts a negligible effect on the density of these binding sites confirmed that they correspond to NTS1 receptors (Szigethy and Beaudet, 1989). A modest density of labeled NTS1 receptors was also found in the substantia nigra, pars reticulata (Szigethy and Beaudet, 1989). High resolution autoradiography demonstrated that these mainly originated from large dendrites radiating from cell bodies in the compacta. This dense concentration of NTS 1 receptors in the ventral midbrain is congruent with the high concentrations of NT-immunoreactive nerve cell bodies, axons, and axon terminals documented in this region (Goedert et al., 1984c; Emson et al., 1985a). Most conspicuous, however, is the similarity between the distribution of these labeled binding sites and that of the cell bodies of origin and dendritic arborizations of nigrostriatal, mesocortical, and mesolimbic DA neurons. As we will see below, a large body of evidence has indeed demonstrated a close association between DA neurons and NTS 1 receptors in this region of the brain. Similarly, high concentrations of NTS 1 receptors were autoradiographically labeled in the substantia nigra of cat (Goedert et al., 1984c), monkey (Fig. 6A; Quirion et al., 1987) and human brain (Sadoul et al., 1984b; Uhl et al., 1984; Sarrieau et al., 1985; Quirion et al., 1987). Moderate levels of autoradiographically labeled NTS1 receptors are also evident in the interpeduncular nucleus and periaqueductal gray (Fig. 4E,F). In the dorsal raphe nucleus, labeled receptor densities are relatively modest, even though high concentrations of NTimmunoreactive fibers, terminals and perikarya were reported in this nucleus (Jennes et al., 1982; Emson et al., 1985a). Finally, high densities of NTS 1 binding sites are observed in the superior colliculus of both rat and monkey (Fig. 4F; Young and Kuhar, 1981; Quirion et al., 1982, 1987; Moyse et al., 1987). eons

NT binding sites in the pons are prevalent over the pontine nuclei (Fig. 4G), the reticulotegmental nucleus, the dorsal tegmental nucleus, the laterodorsal and pedunculopontine tegmental nuclei, and the nucleus raphe medianus (Fig. 4G; Young and Kuhar, 1981; Quirion et al., 1982; Moyse et al., 1987; Kessler et al., 1987). Most of these regions also reportedly contain relatively high levels of NT-immunoreactive axons (Hunt et al., 1981; Seybold and 347

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Elde, 1982; Jennes et al., 1982; H6kfelt et al., 1984; Emson et al., 1985a). Conversely, despite the presence of high densities of NT-immunoreactive fibers in the dorsal parabrachial nucleus, this area seems to contain only low levels of NTS 1 binding sites.

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Medulla In the medulla, the highest concentrations of NTS 1 binding sites are observed over the dorsal motor nucleus of the vagus nerve as well as over the nucleus tractus solitarius (Young and Kuhar, 1981; Quirion et al., 1982; Kessler et al., 1987). However, whereas in the former the labeled receptors are mainly concentrated over neuronal perikarya, in the latter, they are diffusely distributed throughout the neuropil (Kessler and Beaudet, 1989). Transection experiments have indicated that a proportion of NT binding sites in these two nuclei is associated with vagal afferents (Kessler and Beaudet, 1989). Modest densities of NT receptors are also associated with the medial vestibular and dorsal cochlear nuclei, the lateral reticular nucleus, the external cuneate nucleus, the nucleus linearis caudalis, the retrofacial nucleus, and the nucleus raphe pallidus (Young and Kuhar, 1981; Quirion et al., 1982; Kessler et al., 1987). In the brainstem of the monkey, moderate to high levels of NTS1 binding were found in the nucleus of the solitary tract, area postrema and median raphe (Quirion et al., 1987). In the human brainstem, low levels of NT binding sites were detected throughout with the exception of the nucleus of the spinal trigeminal nerve (Sarrieau et al., 1985). Spinal cord High concentrations of NTS1 receptors were detected by autoradiography in the substantia gelatinosa of the rat dorsal horn as well as in the spinal trigeminal nucleus (Young and Kuhar, 1981; Kar and Quirion, 1995). The persistence of radiolabeled NTS1 in the dorsal horn following dorsal rhizotomy suggests that these receptors are unlikely to be concentrated on terminals of primary afferents in this region (Ninkovic et al., 1981; Emson et al., 1985a). In the human spinal cord, the highest density of NTS 1 receptors was likewise localized in lamina II of the dorsal horn, and most prominently in the deeper inner segment IIi. A moderate density of labeled receptors was also detected in laminae I, III, VII, and IX (Faull et al., 1989b). 5.4.2.2. Distribution of NTS1 mRNA The cloning of the NTS1 receptor subtype made it possible to investigate its expression in brain and periphery. Expression of NTS1 mRNA was frst examined by Northern blotting, using sequence-specific 32p-labeled cDNA probes. These Northern blots revealed a single hybridized band with an estimated mRNA size of approximately 3.8 kb in the adult rat brain (Fig. 16A; Tanaka et al., 1990; Mazella et al., 1996). A transcript of comparable size was detected in homogenates from human brain (Vita et al., 1993). Subsequently, the regional distribution of NTS 1 receptor transcripts was analyzed by RT-PCR in rat brain using NTSl-specific oligonucleotides (Mendez et al., 1997). The highest levels of NTS1 mRNA were detected in the hypothalamus and ventral midbrain. Intermediate levels of expression

<...._._

Fig. 7. Comparative distribution of autoradiographically labeled [125I]NT binding sites (A) and NTS1 mRNAhybridizing cells (B) in coronal sections through the habenula of the rat. Darkfield. (A) [125I]NT-labeled binding sites are restricted to the medial part of the medial habenula. (B) The most intense NTS 1 mRNA labeling is detected in the ventral part of the medial habenula (MHv) although scattered, less intensely labeled cells are also apparent in the dorsal part of the medial habenula (MHd). Note that the lateral habenula (LH) also contains scattered cells exhibiting a range of labeling densities. In situ hybridization image kindly provided by Dr. M.J. Alexander (for technical details, see Alexander and Leeman, 1998). Scale bar: 100 Ixm 349

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were measured in the prefrontal cortex and striatum. Only traces were found in the cerebellum (Mendez et al., 1997). However, the most comprehensive data on the distribution NTS1 mRNA in mammalian CNS were provided by in situ hybridization analyses using either oligonucleotide (Elde et al., 1990; Sato et al., 1992; Nicot et al., 1994a, 1995; Azzi et al., 1996) or mRNA probes (Alexander and Leeman, 1998).

Telencephalon Within the rat cerebral cortex, moderate to dense NTS 1 expression is observed in the frontal, parietal, cingulate, endopiriform, insular, perirhinal, and entorhinal cortex (Fig. 4A'-G' and Table 3; Alexander and Leeman, 1998). Scattered NTS1 mRNA containing cells are also visible in the retrosplenial cortex (Table 3; Fig. 4F'). According to a comprehensive study by Alexander and Leeman (1998), NTS1 mRNA-expressing cells are more numerous and more intensely labeled within cortical areas considered to be transitional between isocortex and alocortex than in isocortical areas proper. Also, whereas in the former NTS1 mRNA labeled cells are evident in both superficial and deep layers (with the exception of the retrosplenial cortex where labeled cells are limited to layer II), in isocortex, they are usually found in the deeper layer, adjacent to the corpus callosum. Virtually no NTS1 mRNA expression is detected within the neostriatum (Table 3). A few, lightly labeled cells are observed within the nucleus accumbens (Table 3). By contrast, intensely labeled cells were reported within the pallidum, in the position of the large cholinergic neurons of the rodent equivalent of the nucleus basalis of Meynert (Fig. 4C'; Alexander and Leeman, 1998). In the basal forebrain, NTS 1 mRNA-expressing cells are evident in all subdivisions of the septal region, including the lateral and medial septal nuclei, the nucleus of the diagonal band of Broca, the septofimbrial nucleus, and the bed nucleus of the stria terminalis (Fig. 4B',C'; Table 3; Elde et al., 1990; Nicot et al., 1994a; Azzi et al., 1996; Alexander and Leeman, 1998). Within the lateral septum, labeled cells were observed throughout the intermediate, ventral and dorsal parts of the nucleus (Alexander and Leeman, 1998). In the medial septum and adjoining diagonal band, intensely labeled cells are distributed in a pattern characteristic of basal forebrain cholinergic neurons (Fig. 4B',C'). In the bed nucleus of the stria terminalis, NTS 1 mRNA labeling is most prominent in the oval nucleus (Fig. 4B'). Lightly to moderately labeled NTS 1 mRNA-expressing cells are also observed in the mitral and granule cell layers of the main olfactory bulb, as well as within the deep layers of the tenia tecta, the dorsal part of the endopiriform nucleus, and the islands of Calleja (Fig. 4B'). NTS1 mRNA is evident throughout the amygdaloid complex (Fig. 4D',E'; Table 3). The labeling is reportedly most prominent within the central, cortical, intercalated, and medial nuclei (Alexander and Leeman, 1998). In the hippocampus, numerous NTS1 mRNAexpressing cells were detected throughout the parasubiculum, the presubiculum and the pyramidal cell layer of the ventral, but not the dorsal subiculum (Alexander and Leeman, 1998). A few moderately to heavily labeled cells were also observed in non-pyramidal layers of CA3. Diencephalon In the thalamus, NTS 1 mRNA is prominent in the medial habenula and zona incerta (Nicot et al., 1994a, 1995; Azzi et al., 1996; Alexander and Leeman, 1998). Within the habenula, most of the labeled cells are concentrated within the medial tier, the lateral habenula containing only scattered labeled cells (Fig. 7B). In the zona incerta, moderately to densely labeled cells are apparent throughout most of the structure (Fig. 4D'). Moderately labeled cells are 350

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also visible throughout the anterodorsal nucleus of the thalamus (Fig. 4C'). Finally, low to moderate NTS1 expression was observed in a number of other thalamic nuclei including the medial and lateral geniculate nuclei, the suprageniculate nucleus and several intralaminar nuclei (Alexander and Leeman, 1998). In the hypothalamus, high concentrations of NTS 1 mRNA-expressing cells were originally reported within the suprachiasmatic nucleus, dorsomedial hypothalamic nucleus, supramammillary nucleus and dorsal premammillary nucleus (Fig. 4C',Dt; Table 3; Elde et al., 1990; Nicot et al., 1994a, 1995; Azzi et al., 1996). Alexander and Leeman (1998), using sensitive RNA probes, additionally detected NTS 1 mRNA-expressing cells throughout the rostrocaudal extent of the arcuate nucleus, within most parts of the descending and parvicellular divisions of the paraventricular nucleus, and within the magnocellular paraventricular and supraoptic nuclei. Cells containing NTS1 mRNA were also reported in the anterior and posterior hypothalamic nuclei, as well as in the medial preoptic nucleus, particularly in its medial part. Finally, NTS 1-expressing cells were detected in the ventral lateral part of the ventral medial hypothalamic nucleus as well as within the lateral hypothalamic area, particularly at tuberal levels (Alexander and Leeman, 1998). Mesencephalon In the mesencephalon, as expected from autoradiographic binding studies, intensely labeled NTS 1 mRNA-expressing cells are evident throughout the substantia nigra, pars compacta as well as within the ventral tegmental area and interfascicular nucleus (Table 3; Fig. 4E',F'; Elde et al., 1990; Nicot et al., 1994a, 1995; Azzi et al., 1996; Alexander and Leeman, 1998). Scattered, moderately labeled cells are also apparent in the pars reticulata of the substantia nigra (Fig. 4E'). Cells expressing NTS1 mRNA were also detected in the periaqueductal gray matter, deep layers of the superior colliculus, pretectal region and nucleus of the optic tract (Alexander and Leeman, 1998). Lightly to heavily labeled cells were also observed in the dorsal and median raphe nuclei, lateral dorsal tegmental nucleus, and pedunculopontine nucleus. eons

In the pons, moderately to heavily labeled NTS 1 mRNA-expressing cells are abundant in the pontine nuclei, the reticular tegmental nucleus and the median raphe nucleus (Fig. 4F',G'). No in situ hybridization data are available on the distribution of NTS 1 mRNA-expressing cells in the medulla or spinal cord. 5.4.2.3. Distribution of NTS1 receptor proteins

The regional distribution of NTS1 immunoreactivity was studied using antibodies directed against sequences in the third intracytoplasmic loop (Boudin et al., 1996) and N-terminal extracellular domain (Fassio et al., 2000) of the cloned rat NTS 1 receptor. By and large, both antibodies gave rise to similar distributional patterns. In turn, the distribution of immunoreactive NTS1 showed striking similarities with that of [125I]NT binding sites documented by high-resolution autoradiography (Table 3; Fig. 5). Thus, NTS 1 immunoreactivity was prominent in regions such as the islands of Calleja, diagonal band of Broca, magnocellular preoptic nucleus, pre- and parasubiculum, suprachiasmatic nucleus of the hypothalamus, anterodorsal nucleus of the thalamus, and substantia nigra, all of which had been found to contain high concentrations of radiolabeled NT binding sites. Furthermore, region to region superimposition of immunohistochemical and autoradiographic staining showed near perfect overlap of 351

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the two labeling patterns in most of these areas, as illustrated here for the anterodorsal nucleus of the thalamus and the retrosplenial cortex (Fig. 5). NTS 1 immunoreactivity was mainly concentrated within nerve cell bodies and dendrites (Table 3; Boudin et al., 1996; Fassio et al., 2000). With rare exceptions, regions containing NTimmunoreactive nerve cell bodies were the same as those found to exhibit high concentrations of perikaryal [125I]NT binding or NTS1 mRNA. Examples of these include the islands of Calleja, the diagonal band of Broca, the anterodorsal nucleus of the thalamus (Fig. 5) or the medial habenula (Fig. 7). Conversely, areas in which NTS1 immunoreactivity was mostly concentrated within neuronal processes, including distal dendrites, axons, and axon terminals, matched those in which radiolabeled NTS1 binding sites were diffusely distributed over the neuropil. These include the insular and endopiriform cortices, the lateral septum, the neostriatum and the bed nucleus of the stria terminalis. Telencephalon In cerebral cortex, the cellular distribution and laminar patterning of NTS 1 immunostaining varies markedly according to cytoarchitectonic areas (Table 3). In frontal and parietal cortices, the label is mainly confined to pyramidal cell bodies in layers II-III and V, with dendrites extending into layer IV (Fig. 8B). In the anterior cingulate, endopiriform, and insular cortices, NTS 1 immunostaining is predominantly axonal and concentrated in layer VI. In the perirhinal cortex, terminal labeling is present in layers I-III and VI and scattered nerve cell bodies are detected in layers IV and V. In the entorhinal cortex, only scattered, moderately to weakly NTS 1-immunoreactive nerve cell bodies are evident. Finally, in the retrosplenial cortex, NTS 1 immunoreactivity is found in both nerve cell bodies and axon terminals within the outermost aspect of molecular layer I, and in layers II-III (Fig. 5A'). In the neostriatum, NTS 1 immunoreactivity is detected within heterogeneously distributed axon terminals (Table 3). A few weakly labeled perikarya may be observed in the ventral tier of the structure. The nucleus accumbens exhibits a few scattered, moderately labeled neurons as well as a few axons in the vicinity of the anterior commissure. In the basal forebrain, NTS 1-immunoreactive perikarya are evident throughout the medial septum, islands of Calleja (Fig. 8A), nucleus of the diagonal band of Broca (Fig. 8C), and magnocellular preoptic nucleus (Table 3). NTSl-immunoreactive multipolar neurons are also visible throughout the substantia innominata, from the magnocellular preoptic nucleus, rostrally, to the medial edge of the globus pallidus, caudally. The lateral septum, as well as the bed nucleus of the stria terminalis, contains a dense network of NTS 1-immunoreactive axons, but only small and scattered immunoreactive cell bodies. In the amygdala, NTSl-immunoreactive perikarya, dendrites and axon terminals are apparent in the posterior cortical nucleus (Table 3). Only weak to moderate terminal staining was reported in the central, basomedial, and lateral nuclei (Table 3). In the hippocampus, the pre- and parasubiculum and, to a lesser extent, the subiculum contain numerous NTSl-immunoreactive nerve cell bodies, dendrites and axon terminals (Table 3). Labeled dendrites are particularly numerous at the border between the pre- and parasubiculum. Throughout the CA1, and CA2 and CA3 fields of Ammon's horn, the pyramidal layer exhibits scattered immunoreactive perikarya. A few moderately labeled nerve cell bodies were also detected in the granule cell layer of the dentate gyrus as well as in the hilus (Boudin et al., 1996).

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Fig. 8. Distribution of NTS1 immunoreactivity in the rat telencephalon (Boudin et al., 1996). (A) Islands of Calleja (ICj). The immunoreactivity is evident over perikarya and dendrites of granule cells. (B) Parietal cortex. NTSl-immunoreactive nerve cell bodies are detected within layers II-III and V, with labeled processes extending across layer IV. (C) Vertical limb of the diagonal band of Broca (VDB). Intensively immunoreactive nerve cell bodies are detected amongst a dense network of labeled dendrites. Tu, olfactory tubercle. Scale bars: 200 ~tm in A; 75 Ixm in B; 100 Ixm in C. Diencephalon In the thalamus, N T R i m m u n o r e a c t i v i t y is m o s t prevalent in the anterior dorsal n u c l e u s w h e r e it is present in both p e r i k a r y a and processes (Fig. 5B'; Table 3). A dense n e t w o r k of intensely labeled b e a d e d fibers was also found in the paraventricular nucleus. In the reticular n u c l e u s of 353

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the thalamus, moderately labeled nerve cell bodies, arranged in clusters of four to five amidst a very lightly stained neuropil, have been observed between the myelinated fiber bundles. Immunoreactive cell bodies and dendrites were also seen in the nucleus of the optic tract. In the hypothalamus, a moderate to dense network of NTR-immunoreactive terminals is apparent throughout both the medial and lateral subdivisions (Table 3). In the medial hypothalamus, these terminals form a uniform field, seemingly oblivious of nuclear boundaries except in the median eminence where they are clearly confined to the outer layer. Superimposed over this terminal labeling, intensely labeled perikarya and processes are prominent throughout the ventral tier of the suprachiasmatic nucleus. Small, intensely labeled neurons were also observed amongst immunoreactive axons within the periventricular nucleus, the parvocellular part of the anterior paraventricular nucleus, and the lateral mammillary nucleus (Table 3). In the lateral hypothalamic area, immunoreactive fibers were detected ventrally, surrounding and occasionally abutting scattered, medium-sized, NTS 1-positive nerve cell bodies. In the subthalamus, NTR immunoreactivity is essentially present in the zona incerta (Table 3). In the ventral part, the immunoreactivity is predominantly associated with perikarya, whereas in the dorsal part, it is mainly associated with a dense dendritic network. In the epithalamus, NTS1 immunoreactivity is concentrated within cell bodies and processes throughout the medial habenula. Neuronal staining was also observed over scattered neurons in the lateral habenula. Mesencephalon In the midbrain, intensely immunostained NTS 1-immunoreactive perikarya and dendrites are evident throughout the substantia nigra, pars compacta and, to a lesser extent, in the ventral tegmental area and interfascicular nucleus, extending caudally into the nucleus raphe linearis caudalis (Table 3). Discrete, intensely immunoreactive cell bodies were also detected in the substantia nigra, pars reticulata, and pars lateralis. Weakly to moderately labeled NTSl-immunoreactive perikarya and dendrites were observed in the periaqueductal gray, immediately dorsally and ventrally to the aqueduct as well as within the dorsal raphe and laterodorsal tegmental nuclei (Table 3). Scattered immunoreactive axons were also detected throughout the laterodorsal tegmentum and within the rostral half of the dorsal tegmental nucleus. Finally, immunoreactive perikarya and dendrites were detected in the optic nerve and intermediate layers, but not in the zonal superficial layer, of the superior colliculi. Pons

In the pons, intense NTS 1 immunolabeling is apparent throughout the pontine nuclei, where it pervades both perikarya and neuropil (Table 3). Heavily stained NTSl-immunoreactive perikarya were also observed within the reticulotegmental nucleus. More dorsally, a compact group of intensely labeled neurons was observed within the ventral tegmental nucleus and more sparsely and weakly labeled cells were detected within the median raphe nucleus (Table 3). Medulla Within the rostral medulla, NTS 1-immunoreactive nerve cell bodies are apparent throughout the medial vestibular and dorsal cochlear nuclei, prominent over light neuropil staining (Table 3). Two groups of strongly labeled perikarya stand out conspicuously over the weak background labeling of the parvocellular reticular formation: the ventralmost corresponds to the retrofacial segment of the ambiguous nucleus and the dorsalmost to the linear nucleus

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Fig. 9. Distribution of NTS 1 immunoreactivity in the rat medulla (Boudin et al., 1996). (A) Dorsal motor nucleus of the vagus (nX) and nucleus of the solitary tract (NTS). NTS 1 immunolabeling is associated with perikarya and dendrites in the dorsal motor nucleus of the vagus and with axon terminals in the nucleus of the solitary tract. (B) Retrofacial (Rf) and linear (Li) medullary nuclei. In both nuclei, NTS 1 immunoreactivity is selectively concentrated over nerve cell bodies. (C,D) Nucleus raphe pallidus (RE C) and external cuneate nucleus (ECu, D). In both nuclei, immunoreactive perikarya are embedded in a dense meshwork of labeled dendrites. Cu, cuneate nucleus; Sp5, nucleus of the spinal trigeminal tract. Scale bars: 200 gm in A; 250 Ixm in B,D; 50 ~tm in C.

of the medulla (Fig. 9B). Medially, a few m o d e r a t e l y labeled but profusely arborized neurons are apparent within the nucleus raphe pallidus (Fig. 9C). Ventrally, intensely i m m u n o r e a c t i v e perikarya and dendrites are evident in the inferior olivary nucleus, medially, and paragigantocellular nucleus, laterally. In the caudal medulla, N T R i m m u n o r e a c t i v i t y is most p r o m i n e n t within the dorsal motor nucleus of the vagus and nucleus of the solitary tract (Fig. 9A). W h e r e a s in the former it is p r e d o m i n a n t l y associated with perikarya and dendrites, in the latter it is exclusively associated with terminal arbors (Fig. 9A). A few m o d e r a t e l y labeled nerve cell bodies, surrounded by a dense network of labeled dendrites, are also apparent in the external cuneate nucleus, dorsolaterally (Fig. 9D) and in the lateral reticular nucleus, ventrally.

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Spinal cord In the spinal cord, a very dense plexus of NTS 1-immunoreactive fibers was detected in the substantia gelatinosa (layer II). In addition, immunolabeled nerve cell bodies were observed in the deep part of layer II (Fassio et al., 2000). 5.4.2.4. Identification of NTSl-expressing systems It is striking that both autoradiographic and immunohistochemical studies concur in demonstrating an association of NTS 1 receptors with both the cell bodies of origin and the terminal arborizations of specific projection systems. We mentioned above the labeling of nerve cell bodies in the anterodorsal nucleus of the thalamus and terminal labeling characteristic of this nucleus' pattern of arborization in the retrosplenial cortex. Similarly, the patchy distribution of NTS1 terminal labeling in the interpeduncular nucleus conforms to that of projections from the medial habenula which, in turn, shows intense perikaryal NTS1 labeling by receptor autoradiography, in situ hybridization and immunocytochemistry. Other examples of NTS 1-expressing systems include the nigrostriatal, mesocortical, and mesolimbic projection systems, the basocortical projection system (from the basal forebrain to frontal and parietal cortices), and the projection of nodose ganglion cells to the nucleus of the tractus solitarius through the vagus nerve (Fig. 15; Moyse et al., 1987; Kessler and Beaudet, 1989; Boudin et al., 1996). These labeling patterns suggest that neurons that express NTS1 receptors target these receptors to both their somatodendritic and axonal domains and, therefore, that NT may act both pre- and postsynaptically upon the same neurons. Several of these NTS 1 projection systems have been identified in terms of their neurotransmitter contents and results obtained so far suggest that NTS 1 may play a predominant role in the regulation of a restricted number of neurotransmitter pathways critical for cognition, memory, locomotion, circadian rhythms and the sleep-waking cycle.

Association of NTS1 receptors with midbrain DA pathways We saw that among the highest concentrations of NTS1 binding sites, NTS1 mRNA, and NTS1 immunoreactivity were detected over nerve cell bodies of the substantia nigra and ventral tegmental area. These cell bodies were first suggested to correspond to DA cells on the basis of the substantial decrease in NT binding observed in the midbrain tegmentum following local injection of the cytotoxic drug, 6-hydroxydopamine in the rat (Palacios and Kuhar, 1981; Quirion et al., 1982, 1985; Goedert et al., 1984b; Herve et al., 1986). In monkeys, treatment with the neurotoxin, 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP), which selectively destroys mesencephalic DA cells, similarly produced a significant loss of NT binding sites in the substantia nigra (Fig. 6; Waters et al., 1987; Quirion et al., 1987; Goulet et al., 1999). Direct evidence for the localization of NTS1 receptors on the perikarya and dendrites of midbrain DA cells was provided by combined autoradiographic and immunohistochemical data which showed a selective association of NTS1 receptors with approximately 95% and 90% of tyrosine hydroxylase-immunoreactive neurons in the rat substantia nigra (SN) and ventral tegmental area (VTA), respectively (Fig. 10; Szigethy and Beaudet, 1989). Direct association of NTS1 receptors with tyrosine hydroxylase-immunoreactive neurons was also observed in primary cultures of rat embryonic mesencephalic cells (Dana et al., 1991; Brouard et al., 1992). More recently, fluorescent NT (fluo-NT) was reported to internalize selectively, in a NTS 1-dependent manner, in tyrosine-hydroxylase-immunoreactive neurons in slices from the rat substantia nigra (Faure et al., 1995a). Lesion studies suggest that NTS1 receptors are not only associated with the somato356

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Fig. 10. Autoradiographic detection of [125I]NT-labeled binding sites (A; darkfield) and tyrosine hydroxylaseimmunoreactivity (B; brightfield) in adjacent 5-txm-thick sections of the rat ventral midbrain. Radiolabeled NT binding sites in A are selectively accumulated over areas showing tyrosine hydroxylase-stained DA cells in the substantia nigra (pars compacta), ventral tegmental area, and interfascicular nucleus in B (Szigethy and Beaudet, 1989). See Section 7 for anatomical identification.

dendritic arbor of DA cells in the ventral midbrain, but also with their terminal fields in the caudate-putamen, nucleus accumbens, and prefrontal cortex in rat, mouse, and monkey (Fig. 6) (Palacios and Kuhar, 1981; Herve et al., 1986; Quirion, 1983; Quirion et al., 1985, 1987; Goulet et al., 1999; Tanji et al., 1999). However, whereas virtually all NTS1 receptors in the rat caudate putamen appear to be presynaptically associated with DA terminals, in the nucleus accumbens and prefrontal cortex, they reportedly are both presynaptically associated with DA axons and postsynaptically associated with intrinsic neurons. Given that a significant proportion of VTA DA neurons are documented to co-express NT in the rat (H6kfelt et al., 1984; Seroogy et al., 1987) (but seemingly not in human, see Berger et al., 1992), it is likely that a fraction of NTS 1 receptors present in the VTA actually 357

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corresponds to autoreceptors. Indeed, dual immunocytochemical studies demonstrated an association of NTS 1 receptors with NT-immunoreactive terminals of presumptive VTA origin in the rat nucleus accumbens (Pickel et al., 2001). Combined [125I]NT autoradiography and NT immunohistochemistry also demonstrated the presence of NTS 1 autoreceptors in cultures from rat hypothalamus (Bachelet et al., 1997). The anatomical investment of nigrostriatal, mesocortical and mesolimbic DA systems by NTS 1 receptors is consistent with the reported presence of NT-immunoreactive axon terminals throughout the ventral midbrain as well as in the territories of projection of DA cells in the neostriatum, frontal cortex and nucleus accumbens (Jennes et al., 1982; H6kfelt et al., 1984; Woulfe et al., 1992). However, electron microscopic dual labeling studies in the SN and VTA demonstrated that while there is a dense network of NT-immunoreactive terminals interspersed among DA neurons throughout these two regions, only a small proportion of these nerve terminals establish synaptic contacts with DA perikarya or dendrites, suggesting that NT reaches its receptors through diffusion in the extracellular space, outside of synaptic junctions (Woulfe and Beaudet, 1992). There is an abundant literature on the physiological implications and pathophysiological consequences of the selective expression of NTS 1 receptors by central DA neurons. Numerous reviews have appeared on this topic, which is beyond the scope of the present chapter (Nemeroff et al., 1982; Kitabgi et al., 1989; Binder et al., 2001; Kinkead and Nemeroff, 2002). Suffice it to recall here that there is now solid evidence for a selective modulation of central DA transmission by NT. Consistent with animal data, substantial decreases in the density of NTS 1 receptors have been reported in the substantia nigra of patients with Parkinson's disease, leading to speculation that NT might be implicated in the physiopathology of this disorder (Sadoul et al., 1984c; Uhl et al., 1984, 1986; Chinaglia et al., 1990). The association of NTS1 receptors with mesocortical and mesolimbic DA systems has also led to the hypothesis that altered NT function might contribute to the pathogenesis of schizophrenia and other psychoses (Nemeroff, 1986; Emson et al., 1985b). Indeed, central administration of NT produces effects similar to those elicited by atypical antipsychotics, and lowered NT levels (Garver et al., 1991; Sharma et al., 1997) as well as reduced number of NT binding sites (Wolf et al., 1995; Lahti et al., 1998) have been reported in the brain of schizophrenics (for references, see Section 4). However, although a link has been reported between schizophrenia and the gene encoding NTS1 (Lee et al., 1999), association studies failed to demonstrate a role for this gene in susceptibility to schizophrenia (Austin et al., 2000). Association of NTS1 receptors with basal forebrain cholinergic cells The presence of high [125I]NT binding densities over nerve cell bodies in the position of basal forebrain cholinergic neurons from the medial septum and diagonal band of Broca, rostrally, to the substantia innominata and nucleus basalis, caudally, was first reported by Moyse et al. (1987), using high resolution autoradiography. Further studies confirmed that the observed labeling concerned NTS 1 receptors as it was unaffected by the addition of the NTS2 ligand, levocabastine (Szigethy et al., 1988). Dual labeling studies carried out on adjacent 5-~m-thick sections demonstrated that a large proportion of these [125I]NT-labeled neurons were in register with acetylcholinesterase-positive neurons (Fig. 11) which, in this region of the brain, selectively correspond to cholinergic cells (Szigethy and Beaudet, 1987). Further evidence for a selective association of NTS 1 receptors with cholinergic neurons in this region was provided by the demonstration of in vitro and in vivo internalization of fluo-NT in choline acetyltransferase-immunopositive neurons in guinea pig and rat brain, respectively (Faure et al., 1995b,c; Cape et al., 2000).

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Fig. 11. Low magnification photomicrographs of two adjacent 10-~m-thick coronal sections through the forebrain of a rat with unilateral ibotenic acid lesion of the nucleus basalis magnocellularis (NBM) (Szigethy and Beaudet, 1989). Sections are processed for either acetylcholinesterase (ACHE) pharmacohistochemistry (A, brightfield) or [125I]NT autoradiography (B, darkfield). Moderate to high [125I]NT binding densities are detected in the caudateputamen (CPu), the anterodorsal thalamic nucleus (AD), the suprachiasmatic nucleus (SCh), and the substantia innominata (SI). Ibotenic acid abolishes virtually all AchE staining (A, asterisk) and [125I]NT binding (B) in the ipsilateral NBM (intact cells in the contralateral side indicated by arrows). See Section 7 for anatomical identification. 359

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NTS 1 receptors are likely expressed not only on the somata and dendrites of basal forebrain cholinergic cells, but also on their axonal arborizations in cerebral cortex. Indeed, NT was found to increase acetylcholine release in a tetrodotoxin-independent manner (i.e. by action on axon terminals) in slices from rat frontal cortex (Lapchak et al., 1993). This presynaptic effect was no longer observed following cytotoxic lesion of basal forebrain neurons with ibotenic acid (Fig. 11), indicating that it was exerted upon axons originating from basal forebrain cholinergic cells (Lapchak et al., 1993). Studies in postmortem human brain suggest that in human, as in the rat, NTS1 receptors are highly expressed on basal forebrain cholinergic neurons (Szigethy et al., 1990b). The loss of these neurons documented in the brain of patients with Alzheimer's disease (Whitehouse et al., 1982) likely accounts for the massive decrease in NT receptors observed in the hippocampus of patients with this condition (Jansen et al., 1990). Immunohistochemical studies reveal a dense network of NT-immunoreactive axon terminals throughout the rat basal forebrain (Jennes et al., 1982; Woulfe and Beaudet, 1992; Morin et al., 1996). Retrograde tract tracing studies indicate that this NT innervation originates from both forebrain (predominantly the lateral septum and nucleus accumbens) and midbrain (namely the VTA and dorsal raphe nucleus) nuclei, indicating that basal forebrain cholinergic neurons are subjected to both ascending and descending NT regulatory influences (Morin and Beaudet, 1998). Within the caudal basal forebrain, i.e. in the posterior substantia innominata and ventral and caudal aspects of the globus pallidus, NT axons form dense plexuses in amongst cholinergic cells. By contrast, rostrally, NT fibers are concentrated in the lateral septum and anterior substantia innominata whereas cholinergic neurons are located in the medial septum, diagonal band of Broca and magnocellular preoptic nucleus. In these regions, only a few NT axonal varicosities are observed in close proximity to cholinergic neurons, suggesting that endogenously released NT must diffuse in the extracellular space to reach its receptive targets (Morin et al., 1996). In keeping with their massive NTS1 investment, basal forebrain cholinergic cells show robust responses to exogenous NT application. In vitro, local application of NT in slices of the basal forebrain produces, via a direct mechanism, a membrane potential depolarization in neurons electrophysiologically and anatomically identified as cholinergic (Alonso et al., 1994). Most significantly, NT promotes an oscillatory bursting behavior that can shape into complex spindle-like sequences (Alonso et al., 1994). In vivo, intracerebral injection of NT into the basal forebrain diminishes delta EEG activity associated with slow wave sleep and stimulates gamma and theta activity together with waking and paradoxical sleep states by inducing bursting discharges in choline acetyltransferase-immunoreactive cells (Cape et al., 2000). It is interesting to note that within the rat caudal brainstem, cell bodies found to express NTS 1 receptors were almost all detected in regions documented to contain high concentrations of acetylcholinesterase. Unlike in the basal forebrain, however, only a few of these cholinesterase-staining cells are actually cholinergic. These include the dorsal motor nucleus of the vagus, the retrofacial nucleus, the laterodorsal tegmental nucleus, and the pedunculopontine tegmental nucleus (Kessler et al., 1987; Boudin et al., 1996). The link, if any, between the expression of NTS 1 receptors and that of acetylcholinesterase remains to be explored. Association of NTS1 receptors with vasoactive polypeptide (VIP)-containing neurons in the suprachiasmatic nucleus Early studies in the rat reported on the similarity between the distribution of [125I]NT binding and that of VIP immunoreactivity within the suprachiasmatic nucleus (SCN) of the hypothalamus (Moyse et al., 1987). This observation prompted a dual labeling study, which

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demonstrated precise overlap between autoradiographically labeled NTS 1 receptors and VIPimmunoreactive neurons throughout the SCN (Fig. 12; Fran~ois-Bellan et al., 1992). NTS1 receptors in the SCN are not confined to VIP-immunoreactive cells, however, since their density decreases following cytotoxic lesion of serotonin neurons with 5,7-dihydroxytryptamine, suggesting that a contingent is presynaptically associated with serotoninergic axons originating from the midbrain raphe (Fran~ois-Bellan et al., 1992). The high concentration of NTS1 receptors observed in the SCN is puzzling, as only a sparse NT innervation has been detected within this nucleus by immunohistochemistry (Jennes et al., 1982). Endogenously released NT would therefore have to diffuse from neighboring hypothalamic areas to reach its receptive targets within the SCN. The SCN is known to play a critical role in the generation of circadian rhythms, including those governing the patterns of luteinizing hormone (LH) secretion. The association of NTS 1 receptors with VIP neurons may be particularly significant in this regard, since these receptors were found to be down-regulated by gonadal steroids (Moyse et al., 1988). It is likely, therefore, that NT-VIP interactions are implicated in the regulation of the phasic surge of LH by gonadal steroids. For a thorough review of the role of NT in neuroendocrine regulations (see Rostbne and Alexander, 1997). 5.4.2.5. Subcellular distribution of NTS1 receptors in the rat CNS

The subcellular distribution of NTS1 receptors has been examined in the SN, VTA, and nucleus basalis magnocellularis by high resolution autoradiography following incubation of rat brain slices with [125I]NT (Dana et al., 1989; Szigethy et al., 1990a; Boudin et al., 1998), and in the SN, globus pallidus, ventral pallidum, and nucleus accumbens by immunocytochemistry (Boudin et al., 1998; Fassio et al., 2000; Pickel et al., 2001). When used in the same region (the SN), these two technical approaches gave rise to very similar results (Figs. 13 and 14). In brief, in all areas examined so far, NTS 1 receptors were found to be predominantly associated with neurons, although a small proportion of labeled receptors was observed over astrocytes (Dana et al., 1989; Boudin et al., 1998; Pickel et al., 2001; see also Trudeau, 2000). In the SN, VTA, and nucleus basalis, labeled receptors were mainly observed over perikarya and dendrites, whereas in the nucleus accumbens, they were mostly associated with axon terminals. Accordingly, a proportion of labeled receptors in both the SN and VTA were found inside myelinated and unmyelinated axons, indicating that they were axonally transported. Further evidence for both anterograde and retrograde axonal transport of NTS1 receptors was provided by vagal ligation experiments which showed a pile up of autoradiographically labeled receptors on both sides of the suture (Fig. 15; Kessler and Beaudet, 1989). Predictably, a significant proportion of both immunoreactive and radiolabeled NTS1 receptors was found in association with neuronal plasma membranes. In the SN/VTA and nucleus basalis, they were mainly associated with the plasma membrane of DA and cholinergic cells, respectively (Dana et al., 1989; Szigethy et al., 1990a; Boudin et al., 1998), whereas in the nucleus accumbens, they were mainly observed over the membrane of axon terminals (Pickel et al., 2001). Although a few of these membrane-associated receptors were observed over synaptic specializations (Fig. 14A,B), the vast majority was localized to extrasynaptic membrane segments (Figs. 13 and 14C,D). Furthermore, in experiments in which NTS1 receptors and NT-immunoreactive terminals were co-localized, the NTS 1 receptors were not seen within the synapses established by NT-immunoreactive axons (Pickel et al., 2001). Similarly, in the SN and VTA, only a small proportion of NT-immunoreactive terminals 361

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Fig. 12. Distribution of VIP-immunoreactive cells (A; brightfield) and of autoradiographically labeled [125I]NT binding sites (B; darkfield) in adjacent 5 Ixm-thick sections of the rat suprachiasmatic nucleus (SCh). Superimposition of immunohistochemical and autoradiographic images shows that within labeled areas, zones of dense VIP-immunoreactive cell clustering are in direct register with zones of intense [125I]NT labeling. Also note that [125I]NT labeling is more intense in the medial than in the lateral portion of the ventral SCh. 3V, third ventricle; ChO, optic chiasm (Franqois-Bellan et al., 1992). Scale bar: 200 p~m.

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Fig. 13. Electron microscopic distribution of autoradiographically (A) and immunolabeled (B) NTS1 receptors in the rat ventral midbrain. (A) Dendritic shaft from a section through the ventral tegmental area (VTA) autoradiographically labeled with [125I]NT. Two silver grains are located over the cytoplasm (asterisk) whereas three others are associated with the plasma membrane (arrowheads) (Dana et al., 1989). (B) Dendritic NTS1 receptors detected by immunogold electron microscopy in the substantia nigra, pars compacta. One of the labeled dendrites (D1) shows only intracellular labeling whereas the other (D2) is labeled predominantly on its plasma membrane (arrowheads). Note the presence of a gold particle opposite a myelinated axon (ma). (Boudin et al., 1998). Scale bar: 0.5 gm.

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Fig. 14. Immunolabeled NTS1 receptor proteins (A,C) and radiolabeled NT binding sites (B,D) associated with dendritic plasma membranes in rat substantia nigra (Boudin et al., 1998). In the top panels, gold particles (A) as well as autoradiographic grains (B) are detected over axodendritic contacts. (A) An asymmetric synaptic specialization is clearly visible at the site of contact (black arrowhead). Note the presence of labeled intracellular receptors associated with an endocytotic vesicle (white arrowhead). (B) Two silver grains are visible at the site of contact. Note that the abutting terminal exhibits the same morphological features as the immunoreacted one in A. In the bottom panels, gold particles (C) and silver grains (D) associated with dendritic plasma membranes are detected opposite thin astroglial leaflets. Scale bars: 0.5 ~m.

were found to contact N T S l - e n d o w e d DA perikarya or dendrites, the majority contacting non-DA elements (Woulfe and Beaudet, 1992). These results again suggest that NT reaches its receptive targets through diffusion in the extracellular space, an interpretation supported by the fact that NTS 1 receptors were found to be more or less homogeneously distributed along perikarya and dendrites, as opposed to being concentrated opposite afferent axon terminals (Fig. 13; Dana et al., 1989; Szigethy et al., 1990a; Boudin et al., 1998). Somewhat surprisingly, an important fraction of both immunoreactive and radiolabeled NTS1 receptors were found intracellularly, suggesting that neurons expressing NTS1 receptors hold large receptor reserves. An important fraction of these intracellular receptors are associated with the endoplasmic reticulum and Golgi apparatus, indicating that they correspond to receptors in the course of synthesis and glycosylation. Others were associated with vesicular elements of various sizes and shapes, many of which had the ultrastructural characteristics of endosomes (Fig. 15C). This finding is consistent with the results of in vitro and in vivo experiments which have demonstrated that both presynaptic (Castel et al., 1990; Nguyen et al., 2002) and postsynaptic (Fig. 15B; Mazella et al., 1991; Vanisberg et al., 1991; Chabry et al., 1993; Faure et al., 1995b,c) NTS 1 receptors internalize upon ligand stimulation.

5.4.2.6. Ontogenic developmental pattern Biochemical, in situ hybridization, receptor autoradiographic, and immunohistochemical studies all concur in demonstrating that in the rat brain, the NTS1 receptor is expressed 364

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Fig. 15. (A) [125I]NT binding visualized by light microscopic autoradiography in rat ligated vagus nerve. Intense [125I]NT labeling is apparent on both sides of the nerve crush indicating that NT receptors are transported both anterogradely and retrogradely along the nerve. Note that the pile up is more intense on the proximal than on the distal side (DV) and that no labeling is present over the superior laryngeal nerve (SLN), which is included in the ligature (Kessler and Beaudet, 1989). Scale bar: 200 t~m. (B) Confocal microscopic image of a cat stellate ganglion cell labeled in vitro with Nc~-Bodipy-NT(2-13) (fluo-NT). The internalized fluorescent ligand forms numerous hot spots distributed throughout the cytoplasm of the cell (unpublished data). Scale bar: 10 Ixm. (C) Electron micrograph of a NTS 1-immunoreactive dendrite in rat substantia nigra. The labeled dendrite receives a symmetrical synaptic contact from an unlabeled terminal (arrowhead). Immunogold particles are detected in association with the outer membrane of an endosomal vesicle (white arrow) and of an endocytic profile (black arrow) (Boudin et al., 1998). N, nucleus. Scale bar: 0.5 ~m.

early in development (before birth), peaks at around postnatal day 10, and progressively decreases thereafter until adulthood (Fig. 16B; Kiyama et al., 1987; Schotte and Laduron, 1987; Sato et al., 1992; Hermans et al., 1993; Mazella et al., 1996; L6p6e-Lorgeoux et al., 1999). Comparable developmental patterns are observed in the mouse, rabbit, and human brain, albeit with variable peaks of expression, both in terms of levels and timing of overexpression (Fig. 16B; Lobo and Parnavelas, 1988; Mailleux et al., 1990; Zstirger et al., 365

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Fig. 16. Comparative developmental patterns of NTS1 and NTS2 receptors in mammalian brain. (A) Blot hybridization analysis of poly(A+) RNAs isolated from rat (r) and mouse (m) brains at ages 7, 15, 35 days, carried out using NTS 1- and NTS2-selective probes. (B) Comparative developmental patterns of NTS 1 and NTS2 binding sites in rat, mouse, rabbit, and human brain (Zstirger et al., 1992). The maximal binding capacity of NTS1 (closed symbols) and NTS2 (open symbols) binding sites was determined from Scatchard plots as described in Fig. 2.

1992). Thus, maximal NTS 1 binding is already observed at birth in the rabbit and is measured at approximately day 8 in the mouse and at day 25 in the human (Fig. 16B). Furthermore, peak binding levels are considerably higher in these three species than in the rat (Fig. 16B; Schotte and Laduron, 1987; Zstirger et al., 1992). This early expression and postnatal overexpression of NTS 1 receptors is regionally selective. Thus, in the rat, neurons containing NTS 1 m R N A are detected within the first weeks of life in cortical layers II-VI, including pyramidal and 366

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non-pyramidal cells in layer V (Fig. 17B,D; Sato et al., 1992). Similarly, [125I]NT binding is observed over all cortical layers excluding molecular layer I (Palacios et al., 1988). After postnatal day 21, the number of NT1 mRNA-expressing cortical cells, as well as the density of cortical [125I]NT labeling, decreases markedly to be almost undetectable except in the retrosplenial and perirhinal cortices where residual NTS1 receptor expression is detected throughout adulthood (Nicot et al., 1994a; Alexander and Leeman, 1998). The overexpression of NTS1 receptors documented during the first days of life parallels that of their endogenous ligand (Kiyama et al., 1987, 1991; Palacios et al., 1988; Mailleux and Vanderhaeghen, 1988; Mailleux et al., 1990; Sato et al., 1991, 1992; Hermans et al., 1993). Thus, in rat CNS, NT was found by radioimmunoassay (Bissette et al., 1984; L6p6e-Lorgeoux et al., 2000) and immunohistochemistry (Hara et al., 1982; Kiyama et al., 1991) to be expressed very abundantly during the first two postnatal weeks, after which its concentration rapidly decreased down to adult levels. As for NTS1 receptors, this overexpression is regionally selective, as it affects predominantly cells located in layers II-III and VI of the retrosplenial and cingulate cortices, the pyramidal cell layer of the subiculum and hippocampal CA1, and the mitral cell layer of the olfactory bulb (Hara et al., 1982; Kiyama et al., 1991; Sato et al., 1991). Yet, postnatal blockade of NTS 1 receptors with the selective antagonist SR48692 did not modify their density or distribution in cerebral cortex, caudate-putamen, or midbrain, suggesting that NT is not critical for the establishment of the NTS 1 developmental pattern (L6p6e-Lorgeoux et al., 2000). Nonetheless, the regionally selective increase in NTS 1 receptor densities observed in the perinatal period suggests that this receptor subtype plays a critical role in brain, and particularly cortical, development. Immunoblotting experiments using two different antibodies recognizing distinct sequences of the NTS 1 receptor revealed the presence of two differentially glycosylated forms of NTS 1 receptor proteins in developing rat brain: one migrating at 54 and the other at 52 kDa. Whereas the 54-kDa form was expressed from birth to adulthood, the 52-kDa form was detected only at 10 and 15 days postnatal (Boudin et al., 2000). Furthermore, by immunohistochemistry, the 54-kDa form was found within both nerve cell bodies and neuronal processes (Fig. 17C), whereas the 52-kDa form was confined to perikarya and proximal dendrites. Topographically, both forms of the receptor were predominantly expressed in cerebral cortex and dorsal hippocampus, in keeping with binding and in situ hybridization data (Fig. 17A,C). However, whereas the 52-kDa form extensively colocalized with the Golgi marker a-mannosidase II, the 54-kDa form was predominantly located along plasma membranes, suggesting that the transitorily expressed 52-kDa protein corresponds to an immature, incompletely glycosylated and largely intracellular form of the NTS 1 receptor and that the 54-kDa protein corresponds to a mature, fully glycosylated, and largely membrane-associated form (Boudin et al., 2000). It is unclear, however, whether the immature, 52-kDa isoform plays a specific developmental role or is more readily detected in the young than in the adult merely because of higher expression and, presumably, higher turnover of the receptor in the former.

5.4.3. NTS2 receptors 5.4.3.1. Distribution of NTS2 binding sites

Early autoradiographic binding studies, based on the displacement of specific [125I]NT, [3H]NT, or [3H]SR142948A binding by levocabastine, reported a widespread, albeit selective distribution of levocabastine-sensitive NTS2 receptor sites in adult rat brain (Fig. 2; Kitabgi et al., 1987; Schotte et al., 1986, 1988; Schotte and Laduron, 1987; Betancur et al., 1998). 367

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A comparative semi-quantitative analysis of the localization of NTS2 binding sites obtained by this indirect, subtractive approach using [125I]NT or [3H]SR142948A as radioligands, is provided in Table 4. In the absence of levocabastine, there is an excellent correlation between the regional distribution of the two ligands. The addition of levocabastine induces a widespread reduction of [125I]NT and [3H]SR142948A binding in most brain areas, indicating that NTS2 receptors are widely distributed throughout the rat CNS (Table 4; Schotte et al., 1986; Kitabgi et al., 1987). NTS2 binding sites are highly concentrated in the granular layer of the olfactory bulb and throughout most of the cerebral cortex, including the prefrontal, frontoparietal, parietal, temporal, and occipital cortices (Fig. 2; Table 4). High NTS2 binding densities are also detected in the globus pallidus, the dorsal subiculum and CA1 subfield of Ammon's horn, the bed nucleus of the stria terminalis, the olfactory tubercle, some thalamic nuclei (particularly the pretectal and lateral geniculate nuclei), the superior colliculus, and the red nucleus (Fig. 2; Table 4). The cerebellar cortex stands out as the only structure which shows high concentrations of NTS2 binding sites (over both molecular and granule cell layers) in the absence of any significant NTS 1 binding (Fig. 2; Table 4). Moderate NTS2 binding is observed in the nucleus accumbens, the amygdaloid complex, the lateral and medial habenular nuclei, the zona incerta, the pars compacta of the substantia nigra, the ventral tegmental area, and the nucleus raphe magnus. It is noteworthy that no or only low levels of NTS2 binding sites are detected in the cingulate and retrosplenial cortex, the caudate-putamen, the septohippocampal nucleus, the dentate gyrus of the hippocampus, and most of the hypothalamus (Table 4; Schotte et al., 1986; Kitabgi et al., 1987; Betancur et al., 1998; Sarret et al., 2002). More recently, direct autoradiographic labeling of NTS2 receptors was performed using [3H]levocabastine in the adult rat brain (Asselin et al., 2001). In conformity with the results obtained by indirect autoradiographic approaches, [3H]levocabastine-labeled NTS2 binding sites were diffusely distributed throughout the brain (Asselin et al., 2001). High densities of [3H]levocabastine binding sites were observed in the frontal, parietal, temporal, occipital and insular cortices, the septohippocampal nucleus, the amygdaloid complex, the medial thalamus, the mammillary bodies, and the superior colliculi. Moderate labeling was detected in the nucleus accumbens, the caudate-putamen, the olfactory tubercle, the septum, the interpeduncular nucleus, the periaqueductal gray matter, the dorsal raphe, the vestibular complex, and the cerebellum. A few structures, including the substantia nigra and ventral tegmental area, were almost entirely devoid of NTS2 binding sites (Asselin et al., 2001). Despite the overall similarity between the patterns of NTS2 distribution obtained by these two approaches, a few discrepancies may be noted. Thus, in areas such as the cingulate cortex, the caudate-putamen, the septohippocampal nucleus, and the hypothalamus, NTS2 labeling was observed using [3H]levocabastine, whereas no competition of [125I]NT binding

~m

Fig. 17. Comparative distribution of NTS1 receptor proteins (A,C) and mRNA (B,D), as detected by immunohisto-

chemistry and in situ hybridization, respectively, in the brain of neonatal rats. (A) NTS 1 immunostaining is evident within layers II-III and V, but is most prominent in layer VI of the frontoparietal cortex (Boudin et al., 2000). (B) NTS 1 mRNA hybridization is evident over the same cortical layers in a corresponding darkfield autoradiogram (Sato et al., 1992). (C) In parietal cortex, immunoreactive cell bodies (white arrows) are visible in layers II, III and V. In addition, neuronal processes, including apical dendrites arising from pyramidal cells (black arrows), span all cortical layers (Boudin et al., 2000). In an autoradiogram from the same region, NTS1 hybridization signal predominates over layers II-III (Sato et al., 1992). See Section 7 for anatomical identification. Scale bars: 1 mm in A,B; 50 Ixm in C,D. 369

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TABLE 4. Distribution of levocabastine-sensitive NTS2 receptor sites in adult rat brain Brain region

Telencephalon Olfactory bulb External plexif0rm layer Cerebral cortex Prefrontal cortex Frontal cortex Frontoparietal cortex Layers I-II Layers IIi-IV Layers V-VI Parietal cortex Layers I-II Layers III-IV Layers V-VI Cingulate cortex Occipital cortex Dorsal endopiriform cortex Agranular insular cortex Dorsal peduncular cortex Temporal cortex Layers I-II Layers III-IV Layers V-VI Perirhinal area Retrosplenial cortex Rhinal sulcus Basal ganglia Caudate putamen Nucleus accumbens, shell Nucleus accumbens, core Globus pallidus Basal forebrain Lateral septal nucleus, dorsal part Septohippocampal nucleus Olfactory tubercle Amygdala Central anaygdaloid nucleus Posteromedial cortical amygdaloid nucleus Hippocampal formation Field CA1 of Ammon's horn Pyramidal layer Molecular layer Field CA3 of Ammon's horn Pyramidal layer Dorsal subiculum Dentate gyms, dorsal part Dentate gyrus, ventral part Diencephalon Thalamus Pretectal nucleus Lateral geniculate nucleus

370

[125I]NT binding

[3H]SR142948A

Control

Levocabastine (1 I~M)

45.2 4- 2.6

51.4 + 4.2

6.7 • 0.9 16.44-1.3

4.7 + 0.9 12.44-0.4

6.7 4- 0.5 10.9 4-1.0 8.5 4- 0.7

3.3 4- 0.3 3.1 + 0.2 3.3 4- 0.3

7.3 • 0.3 7.8 4- 0.5 7.3 4- 0.3 84.3 4- 9.7 8.0 4- 0.5 76.4 4- 5.4 68.1 4- 7.9 1034-16.3

2.6 4- 0.2 2.8 4- 0.2 2.9 4- 0.2 86.3 4- 7.6 5.0 4- 0.5 48.3 4- 2.9 44.1 4- 3.7 75.64-3.8

4.5 4- 0.5 5.2 4- 0.5 5.0 4- 0.3 103.4 4-13.4 44.94-7.1 32.4 4- 3.6

2.9 4- 0.2 2.9 4- 0.2 2.9 4- 0.2 96.24-2.1 35.24-2.5 26.8 4- 5.4

24.4 4- 0.8 21.8 4- 0.8 23.1 4-1.3 5.0 4- 0.3

Control

Levocabastine (1 txM)

13.44-0.7

7.84-0.2

12.94-0.4

5.7 -t-0.4

46.4 4-1.9

45.0 4- 0.9

35.5 4- 0.8 53.2 4-1.4 66.24-1.3 15.5 4- 0.4

32.2 4- 0.6 39.7 4- 3.5 53.84-1.9 7.1 4- 0.4

73.94-2.0 21.84-0.6

6 3 + 1.5 14.04-0.8

21.5 4- 0.8 14.7 4- 0.4 15.5 4-0.4 2.9 4- 0.5

18.0 4- 0.9 18.3 4- 0.6 16.7 4-0.6

14.9 4- 0.4 11.6 4- 0.5 10.8 4-0.4

47.5 4-0.8 87.8 4-19.7 66.9 4- 4.7

42.8 4-2.1 84.4 4- 7.1 21.7 4- 2.8

42.04-1.7 41.4 4-1.9

32.64-2.0 38.5 4- 0.7

54.24-3.7 76.9 4- 8.4

39.04-0.8 67.2 4- 5.0

35.1 4-2.3 64.3 4- 2.6

21.7 4-0.6 56 4-1.8

7.3 + 0.3 7.1 4- 0.7

4.3 + 0.2 3.8 4- 0.2

6.9 4- 0.5 4.7 4-0.3 8.7 4- 0.7 68.9 + 5.2

5.9 + 0.5 2.1 4-0.2 10.0 + 0.9 59.2 4- 5.2

59 4- 3.1

54 4- 2.2

12.6 4-1.6 9.2 + 0.7

4.7 4-1.6 3.8 4- 0.2

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TABLE 4 (continued) Brain region

Hypothalamus Subthalamus Zona incerta Epithalamus Medial habenular nucleus Lateral habenular nucleus

Midbrain Substantia nigra, pars compacta Substantia nigra, pars reticulata Ventral tegmental area Red nucleus Superior colliculus Layer I Layer II Layer III Dorsal raphe Cerebellum

[125I]NT binding

[3H]SR142948A

Control

Levocabastine (1 lxM)

Control

Levocabastine (1 ixM)

10.2 4- 0.5

10.6 4- 0.9

67.6 4- 2.5

55.4 4-1.3

55 4- 0.9

39.7 4- 3.2

~~o ~ 5.8 50.4 4- 2.5

80.6 4- 2.9 33.6 4-1.7

74.9 4- 3.3 38.04-1.4

63 4- 3.5 28.14-0.7

1004- 4.2 41.6 4- 2.1 91.6 4- 7.1 4.0 4- 0.3

86.1 4-10.9 34.9 4- 2.9 71.8 4- 7.9 2.8 4- 0.3

1004- 7.5 22.5 4- 0.9 105 4- 7.5

79.6 4- 7.5 19.6 4-1.0 68.6 4- 6.8

28.74-0.9

16.7•

34.6 4- 2.4 20.9 4-1.4 39.64-3.1 14.4 4 - 1 . 7

19.2 4- 2.1 10.4 4-1.6 14.74-1.9 12.64-1.6

40 4- 2.4

3.5 -4-1.2

[125I]Neurotensin and [3H]SR142948A specific binding to NTS1 and NTS2 receptors is determined on rat brain sections by autoradiography in the presence (1 txM) or absence (control) of levocabastine. The occlusion of NTS2 binding sites by levocabastine allows the detection of NTS1 receptors. The difference between binding in the absence and in the presence of levocabastine represents binding to NTS2 receptors. Binding is cletermined by densitometry on film autoradiograms and is expressed relative to NT or SR142948A in the substantia nigra, pars compacta taken as 100. Modified from Schotte et al. (1986), Kitabgi et al. (1987) and Betancur et al. (1998). by levocabastine was apparent. These discrepancies are probably due to the fact that the concentrations of [125I]NT used in the indirect studies (0.1 nM) are too low to label all NTS2 receptors, as these have a relatively poor affinity for NT (Kd = 2 - 4 nM). The ubiquitous distribution of NTS2 binding sites in rat brain is in stark contrast with the highly regionally selective distribution of NTS 1 receptors. Accordingly, the correspondence between the distribution of NTS2 binding sites correlates considerably less than that of NTS 1 receptors with the distribution of NT-immunoreactive axon terminals. This lack of correlation led a number of authors to propose that NT may not be the only endogenous ligand to act upon NTS2 receptors (Sarret et al., 1998; Richard et al., 2001).

5.4.3.2. Distribution of NTS2 mRNA The cloning of the NTS2 receptor both confirmed its existence as a distinct molecular entity and provided tools to investigate its expression in m a m m a l i a n brain. Expression of NTS2 m R N A was first examined by blot hybridization analysis (Fig. 16A). Northern blotting using sequence-specific 32p-labeled cDNA probes for the NTS2 receptor revealed two alternatively spliced transcripts of 1.6 and 1.4 kb in rat brain (Chalon et al., 1996). As previously described, the two protein products of these transcripts, NTS2 and vNTS2 differ in their sequences, NTS2 being 134 amino acids longer than vNTS2. In mouse brain, Northern blot analysis using probes which recognize both isoforms revealed a single transcript of 1.8 kb (Mazella et al., 1996), however, more sensitive RT-PCR approaches revealed that both transcripts 371

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Fig. 18. (A) Confocal microscopic image of fluo-NT-labeled cultured astrocytes. After 60 min incubation with 20 nM fluo-NT at 37~ large and irregular fluorescent hot spots are visible throughout the cell surface (arrowheads) (Nouel et al., 1999). Scale bar -- 10 Ixm. (B) Expression of NTS2 mRNA in cultured astrocytes (Glia) and 21-day-old cerebellar granule cells in culture (Cgc) as compared to whole adult rat brain. Two bands of 620 and 439 bp in size are detected by RT-PCR in whole brain and cerebellar granule cells. The large band corresponds to the unspliced form of the rat NTS2 receptor and the 439 bp band to the spliced variant (vNTS2). Only the larger band is present in glial extracts. (C,D) Dual immunohistochemical labeling of glial fibrillary acidic protein (GFAP, C) and in situ hybridization of NTS2 mRNA (D) performed on sections from a stab-lesioned rat brain. (C) The borders of the lesion (L) exhibit a massive proliferation of GFAP-immunoreactive cells (arrowheads). (D) In darkfield, NTS2 hybridization is evident at the level of glial immunoreactivity (arrowheads) (Nouel et al., 1999). Scale bars: 10 ~m in A,B; 180 Ixm in C,D.

were also present in this species (Fig. 18B; Botto et al., 1997a). Using NTS2-specific oligonucleotides, mRNA for the two NTS2 receptor variants was detected by RT-PCR in the olfactory bulb, the neocortex, the striatum, the hypothalamus and the cerebellum of both rat and mouse brain (Chalon et al., 1996; Botto et al., 1997a). By contrast, the long form of the receptor is selectively expressed in the mesencephalon, whereas the truncated vNTS2 form is selectively expressed in the spinal cord (Chalon et al., 1996; Botto et al., 1997a). Interestingly, only the long form of the receptor is detected in cortical glial cells in culture, whereas both forms are present in cultured cerebellar granule cells, suggesting that the shorter, variant isoform of the receptor may be selectively neuronal (Fig. 18B). The distribution of NTS2 mRNA was investigated by in situ hybridization histochemistry in both mouse and rat brain, using probes which did not discriminate between the two spliced forms (Sarret et al., 1998; Walker et al., 1998; L6p6e-Lorgeoux et al., 1999). In 372

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conformity with the results of autoradiographic binding studies, film and light microscopic autoradiograms of adult rat or mouse brain sections hybridized with either 33p-labeled riboprobes or 35S-labeled oligonucleotides revealed a widespread distribution of mRNA encoding NTS2 (Sarret et al., 1998; Walker et al., 1998). This distribution, as observed in serial coronal and sagittal mouse brain sections, is illustrated in Figs. 19 and 20. Quantitative data, as obtained from densitometric analysis of film autoradiograms, are summarized in Table 5. Telencephalon High NTS2 expression levels are evident throughout all major olfactory relay nuclei including the olfactory bulb, the anterior olfactory nucleus, and the prepiriform and piriform cortices (Figs. 19-21). Within the olfactory bulb, NTS2 mRNA is highly concentrated in the granular and mitral cell layers and is also present in moderate amounts in the glomerular layer. By contrast, only weak labeling is evident over the external plexiform layer, in accordance with the low levels of [125I]NT levocabastine-sensitive binding sites detected in this layer (Table 4). In the anterior olfactory nucleus, the label is detected over a subpopulation of small, ovoid neurons (Fig. 21A); in the piriform cortex, it is selectively distributed over neurons in layer II. Together with the piriform cortex, the retrosplenial and entorhinal cortices show the highest expression of NTS2 mRNA in the mouse brain (Table 5). As in the piriform cortex, the labeling in the retrosplenial cortex predominates over neurons in layer II (Fig. 21B). All neocortical areas exhibit moderate to high NTS2 hybridization signal. Laminar differences are less obvious than in paleocortex, save for a relative sparing of molecular layer I. NTS2expressing cells are numerous across all other cortical layers and include both small (in layers II-III) and large (in layers V-VI) pyramids as well as smaller neurons of the interneuronal type (Fig. 21C). In the basal ganglia, sparsely distributed spiny type I neurons show moderate NTS2 expression (Figs. 19C and 20A). NTS2 hybridizing cells are both more numerous and more intensely labeled ventrally, within the nucleus accumbens (Figs. 19C and 20A). No hybridization signal is observed in the globus pallidus. Within the basal forebrain, moderate to dense NTS2 expression is evident in the medial septal nucleus, diagonal band of Broca, bed nucleus of the stria terminalis and magnocellular preoptic nucleus (Figs. 19C and 20A; Table 5). By contrast, the lateral septal nucleus, the substantia innominata, and the ventral pallidum display only low to undetectable levels of NTS2 mRNA. In the hippocampal formation, intense NTS2 hybridization is present over both the pyramidal cell layer of the CA1-CA3 subfields of Ammon's horn and in the granule cell layer of the dentate gyms (Fig. 19D,E). A few moderately NTS2-hybridizing neurons are also observed in strata radiatum and lacunosum moleculare of the hippocampus (Fig. 21D). NTS2 mRNA is also abundant in the subiculum. All nuclei of the amygdaloid complex, including the basomedial, basolateral, medial, and cortical nuclei exhibit intense NTS2 hybridization (Fig. 19D,E). Diencephalon

Very high NTS2 expression is evident in the medial habenula (Figs. 19D and 21F). By contrast, the lateral habenula is virtually label-free (Table 5). Within the thalamus, moderate NTS2 expression is observed in the reticular and parafascicular nuclei, as well as in the medial and lateral geniculate nuclei (Figs. 19A,D and 20A). Moderate NTS2 hybridization is also apparent throughout the zona incerta (Fig. 19D). 373

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Fig. 20. Regional and cellular localization of NTS2 receptors in the cerebellar cortex of the rodent brain. (A) Film autoradiogram of a sagittal mouse brain section hybridized for NTS2 mRNA using an antisense 33p-labeled riboprobe. Note the intense labeling over the granule cell layer. (B) Section from the cerebellar cortex of adult mouse brain hybridized for NTS2 mRNA and processed according to standard dipping techniques. Darkfield. (C) At high magnification, NTS2 mRNA is found in a subpopulation of cerebellar granule cells. By contrast, Purkinje cells are virtually devoid of hybridization signal (arrowheads). (D) Confocal microscopic image of a fluo-NT-labeled cerebellar granule cell cultured from neonatal rat. After 10-min incubation with 20 nM Na-Bodipy-NT(2-13) at 37~ internalized ligand is visible throughout the cytoplasm of soma and processes (arrowheads). N, nucleus. See Section 7 for anatomical identification. Scale bars: 0.5 mm in B; 25 txm in C; 5 Ixm in D. A restricted n u m b e r of hypothalamic nuclei show intense and highly selective N T S 2 hybridization signal. These include the suprachiasmatic, supraoptic, dorsomedial, ventromedial and arcuate nuclei (Figs. 19 and 20A). Moderate to dense hybridization is also visible within m a m m i l l a r y and s u p r a m a m m i l l a r y nuclei (Fig. 19E).

Mesencephalon In the mesencephalon, substantial amounts of N T S 2 m R N A are detected in the superior and inferior colliculi as well as throughout the periaqueductal gray matter and nucleus raphe dorsalis. Moderate hybridization is also evident over the red nucleus and interpeduncular nucleus (Fig. 19F and Table 5). In the substantia nigra, moderately to intensely NTS2hybridizing cells are detected in the pars compacta whereas only a few weakly labeled cells are visible in the pars reticulata. Only low levels of N T S 2 m R N A are detected in the ventral tegmental area (Table 5).

Fig. 19. Distribution of mRNA encoding NTS2 receptors in adult mouse brain. Film autoradiograms of serial sagittal and coronal sections incubated with 33p-sense and 33p-antisense riboprobes, as described in detail elsewhere (Sarret et al., 1998). Labeling densities are color coded according to standard low (in blue) to high (red) density gradient. For abbreviations, see Section 7. Scale bars: 5 mm in coronal sections; 2 mm in sagittal sections. 375

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TABLE 5. Regional distribution of NTS2 mRNA in adult mouse brain Structure mRNA levels a

Telencephalon Cerebral cortex Prepiriform cortex Piriform cortex Cingulate cortex Retrosplenial cortex Entorhinal cortex Perirhinal cortex Other neocortical areas Basal ganglia Caudate-putamen Nucleus accumbens Olfactory systems Olfactory bulb (granular layer) Anterior olfactory nucleus Basal forebrain Olfactory tubercle Medial septal nucleus Lateral septal nucleus Diagonal band of Broca Bed nucleus of the stria terminalis Magnocellular preoptic nucleus Amygdaloid complex Basomedial amygdaloid nucleus Basolateral amygdaloid nucleus Cortical amygdaloid nucleus Medial amygdaloid nucleus Hippocampal formation Fields CA1-3 of Ammon's horn Dentate gyrus Subiculum

91 • 0.25 98 • 1.50 62 -4-0.99 78 + 0.99 74 + 1.27 54 + 1.10 68 + 0.61 36 + 1.32 40 4- 0.77 93 4-1.06 92 4-1.45 94 4- 0.94 38 4-0.78 19 4- 0.69 78 + 0.84 72 4-1.21 59 4- 0.58 85 • 0.89 80 4- 0.93 83 4-0.89 88 4-0.96 96 4-1.29 98 4- 0.73 94 4- 0.87

Diencephalon Thalamus Reticular nucleus Parafascicular nucleus Medial geniculate nucleus Lateral geniculate nucleus Epithalamus Medial habenular nucleus Lateral habenular nucleus Subthalamus Zona incerta Hypothalamus Anterior hypothalamic area Suprachiasmatic nucleus Supraoptic nucleus Paraventricular nucleus Arcuate nucleus Ventromedial nucleus Dorsomedial nucleus Supramammillary nucleus Mammillary nucleus

376

614- 0.8 70 4-1.5 65 4-2.1 54 4- 2.1 89 4- 0.78 214-0.2 63 + 0.84 18 4-1.73 82 + 1.65 76 4-1.55 22 4- 0.6 87 4- 0.65 81 4- 0.4 85 4-1.38 57 4- 0.87 64 4- 0.85

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TABLE 5 (continued) Structure mRNA levels a

Mesencephalon Substantia nigra, pars compacta Substantia nigra, pars reticulata Ventral tegmental area Red nucleus Interpeduncular nucleus Superior colliculus Periaqueductal gray (including raphe dorsalis) Inferior colliculus

51 4- 0.81 32 4-0.40 21 + 0.45 69 4- 0.74 46 4- 0.40 78 4-1.63 69 4- 0.59 76 4-1.76

Pons Pontine nuclei Superior olivary nucleus Principal sensory trigeminal nucleus Motor trigeminal nucleus

79 4- 2.05 70 4- 0.55 58 4-1.50 63 4- 0.97

Medulla Vestibular complex Ventral cochlear nucleus Nucleus raphe magnus Gigantocellular reticular nucleus, pars alpha Lateral paragigantocellular nucleus Lateral reticular nucleus Inferior olivary nucleus Nucleus of the solitary tract Dorsal motor nucleus of the vagus

60 4- 0.85 62 4- 0.85 46 4- 0.95 51 4-1.07 53 4-1.65 30 4-1.10 28 4- 2.55 34 4-0.85 18 4- 0.30

Cerebellum Cerebellar cortex (granule cell layer)

96 4-1.24

a Arbitrary optical density units. Scale: 0-100. Background levels (subtracted) 10-15. Adapted from Sarret et al. (1998).

Pons

In the pons, the highest levels of NTS2 hybridization are found in the pontine nuclei. Moderate to dense hybridization is also evident in the superior olivary nucleus as well as in the motor and main sensory nuclei of the trigeminal nerve (Fig. 19F; Table 5). Medulla and cerebellum

Within the medulla, moderate levels of NTS2 mRNA hybridization are observed in the vestibular complex and nucleus of the solitary tract, dorsally, and in the ventral cochlear nucleus and throughout the rostral ventral medulla, ventrally (Table 5). In the latter, large NTS2-hybridizing cells are apparent amongst unlabeled ones within the gigantocellular reticular nucleus, pars alpha and in the lateral paragigantocellular nucleus (Fig. 21E). Numerous ovoid, densely hybridizing cells are also evident within the nucleus raphe magnus, where they clearly stand out against the adjacent pontine reticular formation (Fig. 21G). Only low NTS2 hybridization signal is measurable in the inferior olivary nucleus, the dorsal motor nucleus of the vagus, and the nucleus of the spinal trigeminal tract (Table 5). Intense NTS2 hybridization is evident throughout the granule cell layer of the mouse cerebellar cortex (Fig. 20A-C). At high magnification, a few NTS2-hybridizing neurons are also detected in the molecular layer. Purkinje cells are virtually devoid of hybridization signal (Fig. 20C). 377

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Spinal cord Only weak NTS2 hybridization is detected over the substantia gelatinosa of the cervical spinal cord. Likewise, labeling in other laminae of the spinal gray matter is barely above background levels.

Species differences in NTS2 expression By and large, there is a good correlation between the distribution of NTS2 mRNA described here in the mouse brain (from Sarret et al., 1998) and that reported for the rat brain based on in situ hybridization of 35S-labeled oligonucleotides (Walker et al., 1998; L6p6e-Lorgeoux et al., 1999). There are, however, a number of differences between the two set of studies that may be accounted for by either technical or interspecies variability. Thus, in the rat, NTS2 hybridization was consistently observed over the white matter, namely at the level of the cingulum, forceps minor and genu of the corpus callosum, and internal capsule whereas such white matter labeling was never observed in the mouse. Also, a number of gray matter areas, including the external plexiform layer of the olfactory bulb, lateral habenular nucleus, paraventricular nucleus of the hypothalamus, and ventral tegmental area showed high NTS2 mRNA levels in the rat while being virtually devoid of labeling in the mouse. Conversely, the amygdaloid complex as well as number of medullary structures (i.e. the nucleus raphe magnus, lateral paragigantocellular and gigantocellular reticular nucleus) displayed NTS2 hybridization in the mouse but not in the rat. At the cellular level, intense NTS2 expression was detected in ependymal cells in rat, but not in mouse brain. In cerebellar cortex, NTS2 mRNA was mainly concentrated in granule cells in the mouse, but selectively distributed over Purkinje cells in the rat. In the latter case, the observed difference may be only technical since rat cerebellar granule cells in culture were found to abundantly express NTS2 receptors (Figs. 18B and 20D; Sarret et al., 2002).

5.4.3.3. Distribution of NTS2 receptor proteins In spite of the recent cloning of two splice variants of NTS2, no antibodies have yet been raised against this receptor subtype. Therefore, the regional and cellular distribution of NTS2 receptor protein remains to be investigated.

5.4.3.4. Identification of NTS2-expressing systems Cellular localization of NTS2 As stated above, in situ hybridization studies clearly demonstrate that the expression of NTS2 in rodent brain is predominantly neuronal. Further evidence for the expression of functional

Fig. 21. Cellular localization of NTS2 mRNA in the adult mouse brain. Brightfield photomicrographs of emulsioncoated slides hybridized with 33p-labeled probes (Sarret et al., 1998). (A) Anterior olfactory nucleus: the labeling is detected over a subpopulation of small, ovoid neurons (arrowheads). (B) Retrosplenial cortex: silver grains are predominantly distributed over layer II neurons. (C) Frontoparietal cortex: numerous NTS2 hybridizing neurons are evident in layers II-III (arrowheads). (D) Hippocampal formation: the bulk of the label is evident over a subpopulation of pyramidal cells. (E) Lateral paragigantocellular nucleus: large labeled cells (arrowheads) are apparent amongst unlabeled ones (arrows). (F) Medial habenular nucleus: numerous, intensively labeled neurons are detected amid unlabeled ones. Note that ependymal cells located medially to the nucleus are label-free (arrowheads). (G) Raphe magnus: numerous ovoid densely labeled cells are labeled throughout the nucleus. See Section 7 for anatomical identifcation. Scale bars: 50 Ixm in A,D,F; 30 ~m in B; 60 ~m in C,G; 75 txm in E. 379

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NTS2 receptors in neurons was recently provided by studies in rat cerebellar granule cells in culture, which demonstrated both cell surface NTS2 binding and ligand-induced NTS2 internalization in these cells (Fig. 20D; Sarret et al., 2002). The similarity between the autoradiographic distribution of NTS2 mRNA and that of levocabastine-sensitive NT binding sites suggests that NTS2 receptors may be largely somatodendritic. Indeed, cultured cerebellar granule cells were found to express NTS2 receptors throughout their somatodendritic arbor (Fig. 20D). However, current data do not exclude the possibility that NTS2-expressing neurons also target their receptors presynaptically, to axon terminals located in other brain areas. Indeed, recent results from our laboratory suggest that a proportion of NTS2 receptors present in the molecular layer of the rat cerebellar cortex may be localized presynaptically on parallel fibers (Sarret et al., 2002). Despite the overall dissimilarity between the distribution of NTS1 and NTS2 receptors, there are regions in which the two overlap, including the entorhinal and retrosplenial cortices, the anterior olfactory nucleus, the medial septum, the diagonal band of Broca and magnocellular preoptic nucleus, the reticular thalamic nucleus, the zona incerta, the substantia nigra, the suprachiasmatic nucleus of the hypothalamus and the pontine nuclei. This dual localization suggests that in some brain regions, NTS1 and NTS2 may be concurrently expressed by the same neurons. In addition to being abundantly expressed by neurons, NTS2 receptors are also clearly expressed in glial cells. Levocabastine-sensitive NT binding sites were first proposed to be associated with glia based on their widespread distribution, pattern of ontogenic development (Schotte and Laduron, 1987), and reappearance after local destruction of neurons with kainic acid (Schotte et al., 1988). The association of NTS2 receptors with glial cells was confirmed by RT-PCR and binding studies on astrocytes grown in culture from postnatal rat cerebral cortex (Fig. 18; Nouel et al., 1997, 1999). However, this glial expression appears to be highly regionally selective since no NTS2 expression was detected in glia of mesencephalic (Trudeau, 2000) or cerebellar (Sarret et al., 2002) origin. Formal demonstration of NTS2 expression by glial cells in the adult rat brain was provided by combined GFAP immunohistochemistry and NTS2 mRNA in situ hybridization (Nouel et al., 1999). These studies showed that while in steady-state conditions, only a small proportion of GFAP-immunopositive astrocytes express NTS2, following acute cortical lesions, there is a marked enhancement of NTS2 expression within GFAP-immunopositive reactive astrocytes in the neighborhood of the lesion (Fig. 18C,D). This up-regulation of NTS2 mRNA during reactive gliosis suggests that NTS2 receptors may play a role in regulating the astroglial response to injury. NTS2-expressing neural pathways A striking feature of the distribution of NTS2 mRNA is its association with virtually every single sensory afferent system in the CNS. Olfactory systems are the most conspicuous in this regard as they show exceedingly high NTS2 expression levels within all major olfactory relay nuclei including the olfactory bulb, the anterior olfactory nucleus and the piriform cortex. Less intense, but equally congruent hybridization signals are detected throughout the auditory system from the ventral cochlear and superior olivary nuclei, caudally, to the inferior colliculus, medial geniculate body and temporal cortex, rostrally. High hybridization signals are likewise detected in the visual system, including the suprachiasmatic nucleus, lateral geniculate body and occipital cortex and, to a lesser extent, within the somatosensory system, including the principal nucleus of the trigeminal nerve (but not the gracile and/or cuneiform nuclei).

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The transmission of pain information is strongly influenced by the activity of supraspinal structures. Several studies have shown that electrical stimulation of periaqueductal gray matter (PAG) produces intense analgesia in the rat (Mayer and Price, 1989). Superior colliculi, which receive pain stimuli from the spinal column through the anterodorsal somatosensory pathways, have also been reported to be involved in nociceptive processing in several electrophysiological studies (McHaffie et al., 1989). Brainstem structures implicated in descending antinociceptive systems, including the PAG, nucleus raphe magnus, gigantocellular reticular nucleus, pars alpha, lateral paragigantocellular nucleus, and superior colliculi all exhibit high NTS2 mRNA expression (Sarret et al., 1998; Walker et al., 1998). High levels of NTS2 receptor binding sites were also reported in some of these structures, including the PAG and superior colliculi (Table 4; Asselin et al., 2001). By contrast, there is a conspicuous lack of NTS2 expression within ascending nociceptive pathways and, most notably, within the substantia gelatinosa of the spinal cord and spinal trigeminal nucleus. The high density of NTS2 in supraspinal antinociceptive structures is consistent with the results of pharmacological studies which have implicated this receptor subtype in the mediation of NT-induced analgesia. Indeed, the NT antagonist SR142948A (Gully et al., 1997), which recognizes both high and low affinity NT receptors, blocks NT-induced analgesia, whereas the NTSl-specific SR48692 does not (Gully et al., 1993; Dubuc et al., 1994; Labb6-Julli6 et al., 1994). The highly NTS2-selective levocabastine was also reported to inhibit completely (Dubuc et al., 1999a), or partially (Tyler et al., 1998b), NT-induced analgesia. Furthermore, injection of NTS2 antisense oligonucleotides was found to markedly inhibit NT-induced antinociception (Dubuc et al., 1999b). While providing strong evidence for the involvement of NTS2 in mediating supraspinal analgesia, these results do not exclude the participation of other NT receptor subtypes, including NTS 1. Indeed, recent studies using knockdown strategies have suggested that NTS 1 may be implicated in spinal antinociception (Tyler et al., 1998c; Pettibone et al., 2002), which would be congruent with the presence of high concentrations of NTS 1, but of low levels of NTS2 receptors in the substantia gelatinosa of the nucleus caudalis and dorsal horn of the spinal cord. Only negligible amounts of NTS2 mRNA and NTS2 binding sites are detected in regions such as the preoptic area, anterior and posterior hypothalamus and spinal trigeminal nucleus, in which microinjections of NT had previously been found to produce hypothermia (Bissette et al., 1976; Kalivas et al., 1982a). These observations are consistent with the finding that NT-induced hypothermia is insensitive to the administration of the NTS1/NTS2 antagonist SR142948A (Gully et al., 1997). Furthermore, administration of NTS2 antisense oligonucleotides is without effect on the NT-induced hypothermic response, suggesting that the latter is mediated through a receptor distinct from the NTS2 (Dubuc et al., 1999b). Accordingly, the NT analogues Boc-[~Pl2,13]NT(8-13) and Boc-[Lys8-9,Nalll]NT(8-13), which exhibit equipotency for binding to the NTS2 and for inducing analgesia, differ by a factor of 25 in their potency for inducing hypothermia (Dubuc et al., 1999b). In addition, Tyler and coworkers identified an analogue of the NT(8-13) fragment (substitution of D-orn9), that causes hypothermic but not analgesic responses when microinjected into the periaqueductal gray of the rat (Tyler et al., 1998a). Whether NT elicits hypothermia through the NTS3 receptor or an as yet unidentified receptor subtype remains to be determined. In contrast to what is observed in the case of NTS 1, there is relatively poor complimentarity between the distribution of NTS2 binding sites and/or mRNA and that of NT-containing axon terminals immunohistochemically detected in rodent brain (Jennes et al., 1982; Inagaki et al., 1983; Woulfe et al., 1994). Nonetheless, a number of the regions expressing high densities of NTS2 mRNA and/or binding sites have been documented to contain high concentrations of 381

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NT-immunoreactive axon terminals, including the olfactory bulb, anterior olfactory nucleus, olfactory tubercle, piriform, cingulate and retrosplenial cortices, nucleus accumbens, bed nucleus of the stria terminalis, zona incerta, mammillary nuclei, superior colliculi, PAG, nuclei raphe magnus and paragigantocellularis. However, some of the regions that show the highest densities of NTS2 binding sites and/or concentrations of NTS2 mRNA, such as the neocortical and cerebellar cortices, suprachiasmatic and ventromedial hypothalamic nuclei, hippocampus, medial habenula, pontine nuclei, motor nucleus of the trigeminal nerve, and vestibular and cochlear nuclei have been described as containing only sparse, if any, NT-immunoreactive fibers (Jennes et al., 1982). This discrepancy has led us (Sarret et al., 1998) and others (Richard et al., 2001) to propose that NT may not be the sole endogenous ligand to act upon NTS2 receptors. 5.4.3.5. Ontogeny of NTS2 receptors

Receptor autoradiographic, Northern blot and in situ hybridization studies all concur demonstrating that in the rodent brain, NTS2 receptors are absent at birth, appear late during development, and do not reach their maximal levels until adulthood (Figs. 16 and 22; Schotte and Laduron, 1987; Zstirger et al., 1992; Mazella et al., 1996; Sarret et al., 1998; Lrpre-Lorgeoux et al., 1999). Using levocabastine to selectively inhibit [3H]NT binding to low-affinity sites in rat brain homogenates, Schotte and Laduron (1987) demonstrated that NTS2 receptors were not detectable before postnatal day 10 (P10) and reached a maximal value at P30 (Fig. 16B). Comparable NTS2 developmental patterns were reported in other species, including in human and mouse brain (Fig. 16B; Zstirger et al., 1992). No NTS2 binding sites were detected in rabbit brain (Fig. 16B; Zsiirger et al., 1992). The ontogenetic profile of NTS2 mRNA is similar to that reported for NTS2 binding sites (Schotte and Laduron, 1987; Zstirger et al., 1992). In the mouse brain, NTS2 mRNA is first detected by Northern blot (Fig. 16A; Mazella et al., 1996) and in situ hybridization (Sarret et al., 1998) during the second postnatal week (Fig. 22). From then on, NTS2 mRNA levels increase progressively and uniformly to reach adult levels between the fourth and eighth postnatal week (Fig. 22). In the early phase of development, NTS2 hybridization is confined to areas which exhibit the highest concentrations of NTS2 mRNA in the adult (e.g. pyramidal cell layer of the hippocampus, piriform cortex, medial habenula; Fig. 22E,F). Other regions, such as the neocortex, amygdaloid complex, ventromedial and dorsomedial nuclei of the hypothalamus, and periaqueductal only start expressing NTS2 mRNA later in development (Fig. 22). In rat brain, the development of NTS2 mRNA reportedly varies between cerebral gray matter, where it shows a continuous increase from P5 to P30, and myelinated fiber tracts, over which it shows a marked increase between P5 and P10, a peak at P15, and a plateau or slight decrease thereafter (Lrp6e-Lorgeoux et al., 1999). This differential developmental pattern was interpreted as reflecting different ontogenic maturation of NTS2 between neural and glial cells (Lrpre-Lorgeoux et al., 1999). The developmental pattern of NTS2 expression is markedly different from that of NTS 1 as reviewed above. This difference suggests that in contrast to NTS1, NTS2 receptors are unlikely to be involved in putative developmental effects of NT. In fact, the increase in cortical NTS2 levels parallels the concurrent decrease of both NTS 1 and endogenous NT, suggesting a shift towards NTS2 for adult NT functional patterns.

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Fig. 22. Film autoradiograms illustrating the distribution of NTS2 mRNA in mouse brain at different stages of postnatal development: 7 days (A,B), 14 days (C,D), 1 month (E,F), and adulthood (G,H). Coronal sections taken at the level of the neostriatum (A,C,E,G) and hippocampal formation (B,D,F,H). The hybridization signal is first detected at postnatal day 14 and increases gradually thereafter. Labeling densities are color coded according to standard low (in blue) to high (red) density gradient. Scale bar: 5 mm.

6. SUMMARY AND CONCLUSIONS In summary, three distinct NT receptor subtypes have been cloned to date: the first two, NTS 1 and NTS2, are both classical, seven transmembrane domain, G-protein-coupled receptors; the 383

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third, NTS3, is a type I, single transmembrane domain protein, the first of its kind to be identified as a neuropeptide receptor. All available data point to NTS1 as playing a predominant role in the transduction of NT's effects in mammalian CNS. It binds NT with subnanomolar affinity, as expected from a neuropeptide receptor; its distribution is selective and shows good overall correspondence with the distribution of NT axon terminals; its blockade by selective antagonists or gene knockdown counter a wide variety of the documented actions of exogenous NT. A salient feature of the distribution of NTS1 receptors is their selective association with the somatodendritic and axonal arbors of chemically defined neuronal systems. This association accounts for many of the reported central effects of NT. Thus, their association with striatonigral, mesocortical and mesolimbic DA systems accounts for the regulation by NT of DA-mediated behaviors, including locomotion, cognition and reward. It also explains the neuroleptic-like effects of centrally administered NT. Their association with basal forebrain cholinergic cells accounts for the effects of NT on the sleep-wake cycle and memory, which have been tied to the activation of basocortical and septohippocampal cholinergic systems, respectively. Their association with DA and VIPergic hypothalamic neurons is likely responsible for many of the documented effects of NT in the regulation of cyclical hypothalamopituitary functions. Finally, the dramatic overexpression of NTS1 receptors observed in the perinatal period suggests that this receptor subtype may play a critical role in neural development. The contribution of NTS2 to the mediation of central NT functions is not as clearly established as that of NTS1. In fact, the relatively low-affinity of NTS2 receptors for NT, together with their widespread distribution in the brain and their lack of correlation with NT terminal fields, suggest that NT may not be the prime endogenous ligand for these receptor sites. Nonetheless, both the high concentrations of NTS2 receptors detected over brainstem antinociceptive descending pathways and the action of selective NTS2 agonists and antisense oligonucleotides suggest that NTS2 plays a role in the mediation of NT's supraspinal analgesic effects. A striking feature of NTS2 is its association with glia. The documented up-regulation of this receptor within reactive astrocytes points to a role in the regulation of inflammatory responses and/or neuronal regeneration. There is still little information on the distribution and function of NTS3 receptors and the putative implication of this molecule in the central effects of NT. In fact, it is unclear if this protein, the function of which is clearly not restricted to NT neurotransmission, acts as a receptor by itself or necessitates interaction with either NTS 1 or NTS2 to fulfill this role. Clearly, a precise knowledge of this receptor's distribution in the brain and the development of selective agonists/antagonists and of knockdown models will be necessary to help resolve these issues.

7. ABBREVIATIONS I II III IV V VI 384

layer layer layer layer layer layer

1 2 3 4 5 6

Neurotensin receptors in the central nervous system

3V ACAd ACAv Acb AD AV AON ARH AF APir APT BMA BST CA1-3 C cc

CEA1 Cg ChO CoA COAp CPu CSm Cu DB DG DMH DV ECT ECu ENTm Ent EPd fa Fc Gia GL GP H

HF IA ic IC ICj IF ILA IP LH

Ch. VI

third ventricle anterior cingulate area, dorsal part anterior cingulate area, ventral part accumbens nucleus anterodorsal tt~alamic nucleus anteroventral tl~alamic nucleus anterior olfactory nucleus arcuate hypothalamic nucleus amygdaloid fissure, amygdaloid piriforln transition anterior pretectal area basomedial amygdaloid nucleus bed nucleus of the stria terminalis fields CA1-3 of Ammon's horn caudate corpus callosum central nucleus amygdala, lateral part cingulate cortex optic chiasm cortical amygdaloid nucleus cortical nucleus amygdala, posterior part caudate-putamen central superior nucleus raphe, medial part cuneate nucleus diagonal band of Broca dentate gyms dorsomedial hypothalamic nucleus vagus nerve distal ectorhinal area external cuneate nucleus entorhinal area, medial part entorhinal cortex endopiriform nucleus, dorsal part corpus callosum, anterior forceps folia cerebelli gigantocellular reticular nucleus, pars alpha granule cell layer globus pallidus hippocampus hippocampal fissure intercalated nuclei amygdala internal capsule inferior colliculus island of Calleja interfascicular nucleus infralimbic area interpeduncular nucleus lateral habenula 385

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LHA Li LPGi LS LSd MA MEApd MCPO MHb MHd MHv ML MN MOp MOs MPNm MRN MS N NDB nX NOT NTS OB or

ORBv P PAG PAR PERI PG PIN PH Pir PL PN Pn POST pP

PPN PRE Py ra

RD RL Rf RF RMg 386

P. Sarret and A. Beaudet

lateral hypothalamic area linear medullary nucleus lateral paragigantocellular nucleus lateral septal nucleus lateral septum nucleus, dorsal part magnocellular preoptic nucleus medial nucleus amygdala, posterodorsal part magnocellular preoptic nucleus medial habenular nucleus medial habenula, dorsal part medial habenula, ventral part molecular layer mammillary nucleus primary motor area secondary motor areas medial preoptic nucleus, medial part mesencephalic reticular nucleus medial septal nucleus neocortex nucleus of the diagonal band dorsal motor nucleus of the vagus nucleus of the optic tract nucleus of the solitary tract olfactory bulb stratum area of Ammon's horn orbital area, ventral part putamen periaqueductal gray parasubiculum perirhinal area pontine gray pineal gland posterior hypothalamic nucleus piriform cortex prelimbic area pontine nucleus paranigral nucleus postsubiculum prepiriform cortex pedunculopontine nucleus presubiculum stratum pyramidale of Ammon's horn stratum radiatum nucleus raphe dorsalis rostral linear nucleus raphe retrofacial medullary nucleus rhinal fissure raphe magnus

Neurotensin receptors in the central nervous system

RP RR RS RSPv Rt SC SCh SI SLN sm

SNC SNR SO SON Sp5 SPFp SuM TEv TTd Th TRN Tu 4V VCP VDB Ve VMH VMHvl VTA ZI

Ch. VI

nucleus raphe pallidus mesencephalic reticular nucleus retrosplenial cortex retrosplenial area, ventral part reticular thalamic nucleus superior colliculus suprachiasmatic nucleus substantia innominata superior laryngeal nerve stria medullaris substantia nigra, compact part substantia nigra, reticular part supraoptic nucleus superior olivary nucleus nucleus of the spinal trigeminal tract subparafascicular thalamic nucleus, parvicellular part supramammillary nucleus ventral temporal association areas taenia tecta, dorsal part thalamus tegmental reticular nucleus, pontine gray olfactory tubercle 4th ventricle ventral cochlear nucleus, posterior part vertical limb of the diagonal band of Broca vestibular nucleus ventromedial hypothalamic nucleus ventromedial hypothalamic nucleus, ventrolateral part ventral tegmental area zona incerta

8. ACKNOWLEDGEMENTS

The authors would like to thank Dr. Mark J. Alexander for providing us with NTS 1 in situ hybridization autoradiograms of the rat brain and Dr. R6mi Quirion for supplying us with autoradiograms of [125I]NT binding sites in the monkey brain. We are also grateful to Dr. Thomas Stroh for his critical reading of the manuscript and to Naomi Takeda for secretarial work. Personal studies referred to in the text were supported by Grant MT-7366 from the Canadian Institutes of Health Research (CIHR) to A.B.P.S. is the recipient of a fellowship from the Fonds de la Recherche en Sant6 du Qu6bec (FRSQ).

9. REFERENCES Abiko T, Takamura Y (2001): Syntheses of neurotensin (nt) analogues and their comparative anorectic effect on food intake in rats. Curr Pharm Des 8:461-468. al-Rodhan NR, Richelson E, Gilbert JA, McCormick DJ, Kanba KS, Pfenning MA, Nelson A, Larson EW, Yaksh

387

Ch.

VI

P. S a r r e t a n d A. B e a u d e t

TL (1991): Structure-antinociceptive activity of neurotensin and some novel analogues in the periaqueductal gray region of the brainstem. Brain Res 557:227-235. Alexander MJ, Leeman SE (1998): Widespread expression in adult rat forebrain of mRNA encoding high-affinity neurotensin receptor. J Comp Neurol 402:475-500. Alexander MJ, Mahoney PD, Ferris CF, Carraway RE, Leeman SE (1989): Evidence that neurotensin participates in the central regulation of the preovulatory surge of luteinizing hormone in the rat. Endocrinology 124:783-788. Alonso A, Faure MP, Beaudet A (1994): Neurotensin promotes oscillatory bursting behavior and is internalized in basal forebrain cholinergic neurons. J Neurosci 14:5778-5792. Araki K, Tachibana S, Uchiyama M, Nakajima T, Yasuhara T (1973): Isolation and structure of a new active peptide 'Xenopsin' on the smooth muscle, especially on a strip of fundus from a rat stomach, from the skin of Xenopus laevis. Chem Pharm Bull (Tokyo) 21:2801-2804. Asselin ML, Dubuc I, Coquerel A, Costentin J (2001): Localization of neurotensin NTS2 receptors in rat brain, using. NeuroReport 12:1087-1091. Austin J, Buckland P, Cardno AG, Williams N, Spurlock G, Hoogendoorn B, Zammit S, Jones G, Sanders R, Jones L, McCarthy G, Jones S, Bray NJ, McGuffin P, Owen MJ, O'Donovan MC (2000): The high affinity neurotensin receptor gene (NTSR1): comparative sequencing and association studies in schizophrenia. Mol Psychiatry 5:552-557. Azzi M, Boudin H, Mahmudi N, Pelaprat D, Rostbne W, Berod A (1996): In vivo regulation of neurotensin receptors following long-term pharmacological blockade with a specific receptor antagonist. Brain Res Mol Brain Res 42:213-221. Bachelet CM, Scarceriaux V, Rost~ne W, Pelaprat D (1997): Evidence for neurotensin autoreceptors and relationship of neurotensin and its receptors with tyrosine hydroxylase-positive neurons in rat primary hypothalamic cultures. Eur J Neurosci 9:1483-1487. Baker JD, Hawkins MF, Baumeister AA, Nagy M (1989): Microinjection of neurotensin into the CNS induces hyperdipsia in the rat. Pharmacol Biochem Behav 33:7-10. Barbero P, Rovbre C, De Bie I, Seidah N, Beaudet A, Kitabgi P (1998): PC5-A-mediated processing of proneurotensin in early compartments of the regulated secretory pathway of PC5-transfected PC12 cells. J Biol Chem 273:25339-25346. Barelli H, Ahmad S, Kostka P, Fox JA, Daniel EE, Vincent JP, Checler F (1989): Neuropeptide-hydrolysing activities in synaptosomal fractions from dog ileum myenteric, deep muscular and submucous plexi. Their participation in neurotensin inactivation. Peptides 10:1055-1061. Bean AJ, Dagerlind A, H6kfelt T, Dobner PR (1992): Cloning of human neurotensin/neuromedin N genomic sequences and expression in the ventral mesencephalon of schizophrenics and age/sex matched controls. Neuroscience 50:259-268. Berger B, Gaspar P, Verney C (1992): Colocalization of neurotensin in the mesocortical dopaminergic system. Restricted regional and laminar distribution in rat, lack of colocalization in human. Ann N Y Acad Sci 668:307310. Betancur C, Azzi M, Rostbne W (1997): Nonpeptide antagonists of neuropeptide receptors: tools for research and therapy. Trends Pharmacol Sci 18:372-386. Betancur C, Canton M, Burgos A, Labeeuw B, Gully D, Rostbne W, Pelaprat D (1998): Characterization of binding sites of a new neurotensin receptor antagonist, [3H]SR 142948A, in the rat brain. Eur J Pharmaco1343:67-77. Binder EB, Kinkead B, Owens MJ, Nemeroff CB (2001): Neurotensin and dopamine interactions. Pharmacol Rev 53:453-486. Bissette G, Nemeroff CB (1995): The neurobiology of neurotensin. In: Bloom FE, Kupfer DJ (Eds), Neuropsychopharmacology: The fourth Generation of Progress. New York: Raven Press, pp. 573-582. Bissette G, Nemeroff CB, Loosen PT, Prange AJ, Lipton MA (1976): Hypothermia and intolerance to cold induced by intracisternal administration of the hypothalamic peptide neurotensin. Nature 262:607-609. Bissette G, Luttinger D, Mason GA, Hernandez DE, Loosen PT (1982): Neurotensin and thermoregulation. Ann N Y Acad Sci 400:268-282. Bissette G, Richardson C, Kizer JS, Nemeroff CB (1984): Ontogeny of brain neurotensin in the rat: a radioimmunoassay study. J Neurochem 43:283-287. Botto JM, Sarret P, Vincent JP, Mazella J (1997a): Identification and expression of a variant isoform of the levocabastine-sensitive neurotensin receptor in the mouse central nervous system. FEBS Lett 400:211-214. Botto JM, Guillemare E, Vincent JP, Mazella J (1997b): Effects of SR 48692 on neurotensin-induced calciumactivated chloride currents in the Xenopus oocyte expression system: agonist-like activity on the levocabastinesensitive neurotensin receptor and absence of antagonist effect on the levocabastine insensitive neurotensin receptor. Neurosci Lett 223:193-196. 388

Neurotensin receptors in the central nervous system

Ch. VI

Botto JM, Chabry J, Sarret R Vincent JP, Mazella J (1998): Stable expression of the mouse levocabastinesensitive neurotensin receptor in HEK 293 cell line: binding properties, photoaffinity labeling, and internalization mechanism. Biochem Biophys Res Commun 243:585-590. Boudin H, Pelaprat D, Rost~ne W, Beaudet A (1996): Cellular distribution of neurotensin receptors in rat brain: immunohistochemical study using an antipeptide antibody against the cloned high affinity receptor. J Comp Neurol 373:76-89. Boudin H, Pelaprat D, Rost~ne W, Pickel VM, Beaudet A (1998): Correlative ultrastructural distribution of neurotensin receptor proteins and binding sites in the rat substantia nigra. J Neurosci 18:8473-8484. Boudin H, Lazaroff B, Bachelet CM, Pelaprat D, Rost~ne W, Beaudet A (2000): Immunologic differentiation of two high-affinity neurotensin receptor isoforms in the developing rat brain. J Comp Neurol 425:45-57. Brouard A, Pelaprat D, Dana C, Vial M, Lhiaubet AM, Rost~ne W (1992): Mesencephalic dopaminergic neurons in primary cultures express functional neurotensin receptors. J Neurosci 12:1409-1415. Brown DR, Miller RJ (1982): Neurotensin. Br Med Bull 38:239-245. Cador M, Kelley AE, Le Moal M, Stinus L (1986): Ventral tegmental area infusion of substance P, neurotensin and enkephalin: differential effects on feeding behavior. Neuroscience 18:659-669. Cape EG, Manns ID, Alonso A, Beaudet A, Jones BE (2000): Neurotensin-induced bursting of cholinergic basal forebrain neurons promotes gamma and theta cortical activity together with waking and paradoxical sleep. J Neurosci 20:8452-8461. Carraway R, Leeman SE (1973): The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalami. J Biol Chem 248:6854-6861. Carraway R, Leeman SE (1975a): The amino acid sequence of a hypothalamic peptide, neurotensin. J Biol Chem 250:1907-1911. Carraway R, Leeman SE (1975b): The synthesis of neurotensin. J Biol Chem 250:1912-1918. Carraway R, Leeman SE (1976): Radioimmunoassay for neurotensin, a hypothalamic peptide. J Biol Chem 251:7035-7044. Carraway R, Bhatnagar YM (1980): Isolation, structure and biologic activity of chicken intestinal neurotensin. Peptides 1:167-174. Carraway R, Ruane SE, Kim HR (1982a): Distribution and immunochemical character of neurotensin-like material in representative vertebrates and invertebrates: apparent conservation of the COOH-terminal region during evolution. Peptides 3:115-123. Carraway R, Ruane SE, Feurle GE, Taylor S (1982b): Amphibian neurotensin (NT) is not xenopsin (XP): dual presence of NT-like and XP-like peptides in various amphibia. Endocrinology 110:1094-1101. Carraway RE, Ferris CF (1983): Isolation, biological and chemical characterization, and synthesis of a neurotensinrelated hexapeptide from chicken intestine. J Biol Chem 258:2475-2479. Carraway RE, Mitra SP (1990): Differential processing of neurotensin/neuromedin N precursor(s) in canine brain and intestine. J Biol Chem 265:8627-8631. Carraway RE, Mitra SP, Paradise C (1991): Characterization of large neuromedin-N using antisera towards regions of the neurotensin/neuromedin-N precursor. Peptides 12:601-607. Carraway RE, Mitra SE Spaulding G (1992): Posttranslational processing of the neurotensin/neuromedin-N precursor. Ann N Y Acad Sci 668:1-16. Carraway RE, Mitra SP, Duke GE (1993): A common precursor to neurotensin and LANT6 and its differential processing in chicken tissues. Peptides 14:1245-1251. Castel MN, Stutzmann JM, Lucas M, Lafforgue J, Blanchard JC (1989): Effects of ICV administration of neurotensin and analogs on EEG in rats. Peptides 10:95-101. Castel MN, Malgouris C, Blanchard JC, Laduron PM (1990): Retrograde axonal transport of neurotensin in the dopaminergic nigrostriatal pathway in the rat. Neuroscience 36:425-430. Chabry J, Gaudriault G, Vincent JP, Mazella J (1993): Implication of various forms of neurotensin receptors in the mechanism of internalization of neurotensin in cerebral neurons. J Biol Chem 268:17138-17144. Chabry J, Labb6-Julli6 C, Gully D, Kitabgi P, Vincent JE Mazella J (1994): Stable expression of the cloned rat brain neurotensin receptor into fibroblasts: binding properties, photoaffinity labeling, transduction mechanisms, and internalization. J Neurochem 63:19-27. Chabry J, Botto JM, Nouel D, Beaudet A, Vincent JP, Mazella J (1995): Thr-422 and Tyr-424 residues in the carboxyl terminus are critical for the internalization of the rat neurotensin receptor. J Biol Chem 270:2439-2442. Chakravarty PK, Chen A, Naylor EM, Ransom RW, Schneck KA and Clineschmidt B.V. (1993): Phthalazinone biphenyl acylsulfonamides: A new class of non-peptide neurotensin antagonists. In: Proceedings of the 206th American Chemical Society National Meeting, August 22-27, Chicago, IL. Washington DC: American Chemical Society, p. MEDI 198.

389

Ch.

VI

P. S a r r e t a n d A. B e a u d e t

Chalon P, Vita N, Kaghad M, Guillemot M, Bonnin J, Delpech B, Le Fur G, Ferrara P, Caput D (1996): Molecular cloning of a levocabastine-sensitive neurotensin binding site. FEBS Lett 386:91-94. Chandra A, Chou HC, Chang C, Lin MT (1981): Effects of intraventricular administration of neurotensin and somatostatin on thermoregulation in the rat. Neuropharmacology 20:715-718. Checler F (1994): The Prepro-neurotensin/Neuromedin N Gene: Structural Organization, Regulation of Expression and Post-transcriptional Events, Vol. 1. Portland: Portland Press, pp. 133-160. Checler F, Vincent JP, Kitabgi P (1986a): Purification and characterization of a novel neurotensin-degrading peptidase from rat brain synaptic membranes. J Biol Chem 261:11274-11281. Checler F, Vincent JR Kitabgi P (1986b): Neuromedin N: high affinity interaction with brain neurotensin receptors and rapid inactivation by brain synaptic peptidases. Eur J Pharmacol 126:239-244. Checler F, Mazella J, Kitabgi P, Vincent JP (1986c): High-affinity receptor sites and rapid proteolytic inactivation of neurotensin in primary cultured neurons. J Neurochem 47:1742-1748. Checler F, Dauch P, Masuo Y, Vincent JP (1991): Neurotensin and neuromedin N are differently metabolized in ventral tegmental area and nucleus accumbens. J Neurochem 56:1320-1328. Chinaglia G, Probst A, Palacios JM (1990): Neurotensin receptors in Parkinson's disease and progressive supranuclear palsy: an autoradiographic study in basal ganglia. Neuroscience 39:351-360. Clineschmidt BV, McGuffin JC (1977): Neurotensin administered intracisternally inhibits responsiveness of mice to noxious stimuli. Eur J Pharmaco146:395-396. Cooper PE, Fernstrom MH, Rorstad OP, Leeman SE, Martin JB (1981): The regional distribution of somatostatin, substance P and neurotensin in human brain. Brain Res 218:219-232. Coquerel A, Dubuc I, Menard JF, Kitabgi P, Costentin J (1986): Naloxone-insensitive potentiation of neurotensin hypothermic effect by the enkephalinase inhibitor thiorphan. Brain Res 398:386-389. Coquerel A, Dubuc I, Kitabgi P, Costentin J (1988): Potentiation by thiorphan and bestatin of the naloxoneinsensitive analgesic effects of neurotensin and neuromedin N. Neurochem Int 12:361-366. Cusack B, Richelson E, Pang YE Zaidi J, Kozikowski AP (1993): Pharmacological studies on novel neurotensin mimetics: discovery of a pharmacologically unique agent exhibiting concentration-dependent dual effects as antagonist and agonist. Mol Pharmacol 44:1036-1040. Cusack B, McCormick DJ, Pang YP, Souder T, Garcia R, Fauq A, Richelson E (1995): Pharmacological and biochemical profiles of unique neurotensin 8-13 analogs exhibiting species selectivity, stereoselectivity, and superagonism. J Biol Chem 270:18359-18366. Cusack B, Groshan K, McCormick DJ, Pang YP, Perry R, Phung CT, Souder T, Richelson E (1996): Chimeric rat/human neurotensin receptors localize a region of the receptor sensitive to binding of a novel, species-specific, picomolar affinity peptide [published erratum appears in J Biol Chem 1996 Sep 6;271(36):22280]. J Biol Chem 271:15054-15059. Cusack B, Chou T, Jansen K, McCormick DJ, Richelson E (2000): Analysis of binding sites and efficacy of a species-specific peptide at rat and human neurotensin receptors. J Pept Res 55:72-80. Dal Farra C, Sarret P, Navarro V, Botto JM, Mazella J, Vincent JP (2001): Involvement of the neurotensin receptor subtype NTR3 in the growth effect of neurotensin on cancer cell lines. Int J Cancer 92:503-509. Dana C, Vial M, Leonard K, Beauregard A, Kitabgi P, Vincent JP, Rost~ne W, Beaudet A (1989): Electron microscopic localization of neurotensin binding sites in the midbrain tegmentum of the rat. I. Ventral tegmental area and the interfascicular nucleus. J Neurosci 9:2247-2257. Dana C, Pelaprat D, Vial M, Brouard A, Lhiaubet AM, Rost~ne W (1991): Characterization of neurotensin binding sites on rat mesencephalic cells in primary culture. Brain Res Dev Brain Res 61:259-264. de Beaurepaire R, Suaudeau C (1988): Anorectic effect of calcitonin, neurotensin and bombesin infused in the area of the rostral part of the nucleus of the tractus solitarius in the rat. Peptides 9:729-733. de Nadai F, Rov~re C, Bidard JN, Cuber JC, Beaudet A, Kitabgi P (1994): Post-translational processing of the neurotensin/neuromedin N precursor in the central nervous system of the rat-I. Biochemical characterization of maturation products. Neuroscience 60:159-166. Difiglia M, Aronin N, Leeman SE (1984): Ultrastructural localization of immunoreactive neurotensin in the monkey superficial dorsal horn. J Comp Neuro1225:1-12. Dobner PR, Barber DL, Villa-Komaroff L, McKiernan C (1987): Cloning and sequence analysis of cDNA for the canine neurotensin/neuromedin N precursor. Proc Natl Acad Sci USA 84:3516-3520. Dobner PR, Kislauskis E, Bullock BP (1992): Cooperative regulation of neurotensin/neuromedin N gene expression in PC12 cells involves AP-1 transcription factors. Ann N Y Acad Sci 668:17-29. Domesick VB (1972): Thalamic relationships of the medial cortex in the rat. Brain Behav Evol 6:457-483. Dubuc I, Nouel D, Coquerel A, Menard JF, Kitabgi P, Costentin J (1988): Hypothermic effect of neuromedin N in mice and its potentiation by peptidase inhibitors. Eur J Pharmacol 151:117-121. 390

N e u r o t e n s i n r e c e p t o r s in the c e n t r a l n e r v o u s s y s t e m

Ch.

VI

Dubuc I, Costentin J, Doulut S, Rodriguez M, Martinez J, Kitabgi P (1992): JMV 449: a pseudopeptide analogue of neurotensin-(8-13) with highly potent and long-lasting hypothermic and analgesic effects in the mouse. Eur J Pharmacol 219:327-329. Dubuc I, Costentin J, Terranova JP, Barnouin MC, Soubrie P, Le Fur G, Rostbne W, Kitabgi P (1994): The nonpeptide neurotensin antagonist, SR 48692, used as a tool to reveal putative neurotensin receptor subtypes. Br J Pharmacol 112:352-354. Dubuc I, Remande S, Costentin J (1999a): The partial agonist properties of levocabastine in neurotensin-induced analgesia. Eur J Pharmacol 381:9-12. Dubuc I, Sarret E Labb6-Julli6 C, Botto JM, Honore E, Bourdel E, Martinez J, Costentin J, Vincent JP, Kitabgi P, Mazella J (1999b): Identification of the receptor subtype involved in the analgesic effect of neurotensin. J Neurosci 19:503-510. Elde R, Schalling M, Ceccatelli S, Nakanishi S, H6kfelt T (1990): Localization of neuropeptide receptor mRNA in rat brain: initial observations using probes for neurotensin and substance P receptors. Neurosci Lett 120:134-138. Elliott PJ, Nemeroff CB (1986): Repeated neurotensin administration in the ventral tegmental area: effects on baseline and D-amphetamine-induced locomotor activity. Neurosci Lett 68:239-244. Emson PC, Goedert M, Williams B, Ninkovic M, Hunt SP (1982): Neurotensin: regional distribution, characterization, and inactivation. Ann N Y Acad Sci 400:198-215. Emson PC, Goedert M, Mantyh PW (1985a): Neurotensin-containing neurons. In: Bj6rklund A, H6kfelt T (Eds), Handbook of Chemical Neuroanatomy. Vol 4, GABA and Neuropeptides in the CNS, Part I. Amsterdam: Elsevier, pp. 355-405. Emson PC, Horsfield PM, Goedert M, Rossor MN, Hawkes CH (1985b): Neurotensin in human brain: regional distribution and effects of neurological illness. Brain Res 347:239-244. Ervin GN, Birkemo LS, Nemeroff CB, Prange AJ (1981): Neurotensin blocks certain amphetamine-induced behaviours. Nature 291:73-76. Evers BM, Wang X, Zhou Z, Townsend CM, McNeil GP, Dobner PR (1995): Characterization of promoter elements required for cell-specific expression of the neurotensin/neuromedin N gene in a human endocrine cell line. Mol Cell Biol 15:3870-3881. Fassio A, Evans G, Grisshammer R, Bolam JP, Mimmack M, Emson PC (2000): Distribution of the neurotensin receptor NTS 1 in the rat CNS studied using an amino-terminal directed antibody. Neuropharmacology 39:14301442. Faull RL, Dragunow M, Villiger JW (1989a): The distribution of neurotensin receptors and acetylcholinesterase in the human caudate nucleus: evidence for the existence of a third neurochemical compartment. Brain Res 488:381-386. Faull RL, Villiger JW, Dragunow M (1989b): Neurotensin receptors in the human spinal cord: a quantitative autoradiographic study. Neuroscience 29:603-613. Faure ME Nouel D, Beaudet A (1995a): Axonal and dendritic transport of internalized neurotensin in rat mesostriatal dopaminergic neurons. Neuroscience 68:519-529. Faure ME Labb6-Julli6 C, Cashman N, Kitabgi E Beaudet A (1995b): Binding and internalization of neurotensin in hybrid cells derived from septal cholinergic neurons. Synapse 20:106-116. Faure ME Alonso A, Nouel D, Gaudriault G, Dennis M, Vincent JP, Beaudet A (1995c): Somatodendritic internalization and perinuclear targeting of neurotensin in the mammalian brain. J Neurosci 15:4140-4147. Fernstrom MH, Carraway RE, Leeman SE (1980): Neurotensin. In: Martini L, Ganong WF (Eds), Frontiers in Neuroendocrinology, Vol. 6. New York: Raven Press, pp. 103-127. Fran~ois-Bellan AM, Bosler O, Tonon MC, Wei LT, Beaudet A (1992): Association of neurotensin receptors with VIP-containing neurons and serotonin-containing axons in the suprachiasmatic nucleus of the rat. Synapse 10:282-290. Frye GD, Luttinger D, Nemeroff CB, Vogel RA, Prange AJ, Breese GR (1981): Modification of the actions of ethanol by centrally active peptides. Peptides 2:99-106. Garver DL, Bissette G, Yao JK, Nemeroff CB (1991): Relation of CSF neurotensin concentrations and antipsychotic treatment in schizophrenia and schizoaffective disorder. Am J Psychiatry 148:484-488. Gilbert JA, McCormick DJ, Pfenning MA, Kanba KS, Enloe LJ, Moore A, Richelson E (1989): Neurotensin(8-13): comparison of novel analogs for stimulation of cyclic GMP formation in neuroblastoma clone N1E-115 and receptor binding to human brain and intact N1E-115 cells. Biochem Pharmacol 38:3377-3382. Goedert M, Mantyh PW, Hunt SP, Emson PC (1983): Mosaic distribution of neurotensin-like immunoreactivity in the cat striatum. Brain Res 274:176-179. Goedert M, Pittaway K, Williams BJ, Emson PC (1984a): Specific binding of tritiated neurotensin to rat brain membranes: characterization and regional distribution. Brain Res 304:71-81. 391

Ch. VI

P. Sarret and A. Beaudet

Goedert M, Pittaway K, Emson PC (1984b): Neurotensin receptors in the rat striatum: lesion studies. Brain Res 299:164-168.

Goedert M, Mantyh PW, Emson PC, Hunt SP (1984c): Inverse relationship between neurotensin receptors and neurotensin-like immunoreactivity in cat striatum. Nature 307:543-546. Goulet M, Morissette M, Grondin R, Falardeau E Bedard PJ, Rostbne W, Di Paolo T (1999): Neurotensin receptors and dopamine transporters: effects of MPTP lesioning and chronic dopaminergic treatments in monkeys. Synapse 32:153-164. Granier C, van Rietschoten J, Kitabgi P, Poustis C, Freychet P (1982): Synthesis and characterization of neurotensin analogues for structure/activity relationship studies. Acetyl-neurotensin-(8-13) is the shortest analogue with full binding and pharmacological activities. Eur J Biochem 124:117-124. Gully D, Canton M, Boigegrain R, Jeanjean F, Molimard JC, Poncelet M, Gueudet C, Heaulme M, Leyris R, Brouard A et al. (1993): Biochemical and pharmacological profile of a potent and selective nonpeptide antagonist of the neurotensin receptor. Proc Natl Acad Sci USA 90:65-69. Gully D, Jeanjean F, Poncelet M, Steinberg R, Soubrie P, Le Fur G, Maffrand JP (1995): Neuropharmacological profile of non-peptide neurotensin antagonists. Fundam Clin Pharmacol 9:513-521. Gully D, Labeeuw B, Boigegrain R, Oury-Donat F, Bachy A, Poncelet M, Steinberg R, Suaud-Chagny ME Santucci V, Vita N, Pecceu F, Labb6-Julli6 C, Kitabgi E Soubrie P, Le Fur G, Maffrand JP (1997): Biochemical and pharmacological activities of SR 142948A, a new potent neurotensin receptor antagonist. J Pharmacol Exp Ther 280:802-812.

Hammer RA, Leeman SE, Carraway R, Williams RH (1980): Isolation of human intestinal neurotensin. J Biol Chem 255:2476-2480.

Hara Y, Shiosaka S, Senba E, Sakanaka M, Inagaki S, Takagi H, Kawai Y, Takatsuki K, Matsuzaki T, Tohyama M (1982): Ontogeny of the neurotensin-containing neuron system of the rat: immunohistochemical analysis. I. Forebrain and diencephalon. J Comp Neurol 208:177-195. Hawkins MF (1986): Central nervous system neurotensin and feeding. Physiol Behav 36:1-8. Hermans E, Maloteaux JM (1998): Mechanisms of regulation of neurotensin receptors. Pharmacol Ther 79:89-104. Hermans E, Jeanjean AP, Laduron PM, Octave JN, Maloteaux JM (1993): Postnatal ontogeny of the rat brain neurotensin receptor mRNA. Neurosci Lett 157:45-48. Hermans E, Octave JN, Maloteaux JM (1994): Receptor mediated internalization of neurotensin in transfected Chinese hamster ovary cells. Biochem Pharmacol 47:89-91. Hermans E, Octave JN, Maloteaux JM (1996): Interaction of the COOH-terminal domain of the neurotensin receptor with a G protein does not control the phospholipase C activation but is involved in the agonist-induced internalization. Mol Pharmaco149:365-372. Hermans E, Vanisberg MA, Geurts M, Maloteaux JM (1997): Down-regulation of neurotensin receptors after ligand-induced internalization in rat primary cultured neurons. Neurochem Int 31:291-299. Herve D, Tassin JP, Studler JM, Dana C, Kitabgi P, Vincent JP, Glowinski J, Rost~ne W (1986): Dopaminergic control of 125I-labeled neurotensin binding site density in corticolimbic structures of the rat brain. Proc Natl Acad Sci USA 83:6203-6207. Hoebel BG (1985): Brain neurotransmitters in food and drug reward. Am J Clin Nutr 42:1133-1150. H6kfelt T, Everitt BJ, Theodorsson-Norheim E, Goldstein M (1984): Occurrence of neurotensinlike immunoreactivity in subpopulations of hypothalamic, mesencephalic, and medullary catecholamine neurons. J Comp Neurol 222:543-559.

Hunt SP, Kelly JS, Emson PC, Kimmel JR, Miller RJ, Wu JY (1981): An immunohistochemical study of neuronal populations containing neuropeptides or gamma-aminobutyrate within the superficial layers of the rat dorsal horn. Neuroscience 6:1883-1898. Hylden JL, Wilcox GL (1983): Antinociceptive action of intrathecal neurotensin in mice. Peptides 4:517-520. Inagaki S, Shinoda K, Kubota Y, Shiosaka S, Matsuzaki T, Tohyama M (1983): Evidence for the existence of a neurotensin-containing pathway from the endopiriform nucleus and the adjacent prepiriform cortex to the anterior olfactory nucleus and nucleus of diagonal band (Broca) of the rat. Neuroscience 8:487-493. Jakeman LB, Reier PJ, Bregman BS, Wade EB, Dailey M, Kastner RJ, Himes BT, Tessler A (1989): Differentiation of substantia gelatinosa-like regions in intraspinal and intracerebral transplants of embryonic spinal cord tissue in the rat. Exp Neurol 103:17-33. Jansen KL, Faull RL, Dragunow M, Synek BL (1990): Alzheimer's disease: changes in hippocampal N-methylD-aspartate, quisqualate, neurotensin, adenosine, benzodiazepine, serotonin and opioid receptors: an autoradiographic study. Neuroscience 39:613-627. Jennes L, Stumpf WE, Kalivas PW (1982): Neurotensin: topographical distribution in rat brain by immunohistochemistry. J Comp Neurol 210:211-224.

392

Neurotensin receptors in the central nervous system

Ch. VI

Jolicoeur FB, Barbeau A, Rioux F, Quirion R, St-Pierre S (1981): Differential neurobehavioral effects of neurotensin and structural analogues. Peptides 2:171-175. Jones PM, Ghatei MA, Steel J, O'Halloran D, Gon G, Legon S, Burrin JM, Leonhardt U, Polak JM, Bloom SR (1989): Evidence for neuropeptide Y synthesis in the rat anterior pituitary and the influence of thyroid hormone status: comparison with vasoactive intestinal peptide, substance P, and neurotensin. Endocrinology 125:334-341. Kahn D, Hou-Yu A, Zimmerman EA (1982): Localization of neurotensin in the hypothalamus. Ann N Y Acad Sci 400:117-131.

Kalivas PW, Jennes L, Nemeroff CB, Prange AJ (1982a): Neurotensin: topographical distribution of brain sites involved in hypothermia and antinociception. J Comp Neurol 210:225-238. Kalivas PW, Gau BA, Nemeroff CB, Prange AJ (1982b): Antinociception after microinjection of neurotensin into the central amygdaloid nucleus of the rat. Brain Res 243:279-286. Kalivas PW, Nemeroff CB, Prange AJ (1984): Neurotensin microinjection into the nucleus accumbens antagonizes dopamine-induced increase in locomotion and rearing. Neuroscience 11:919-930. Kalivas PW, Nemeroff CB, Miller JS, Prange AJ (1985): Microinjection of neurotensin into the ventral tegmental area produces hypothermia: evaluation of dopaminergic mediation. Brain Res 326:219-227. Kanba KS, Kanba S, Okazaki H, Richelson E (1986): Binding of [3H]neurotensin in human brain: properties and distribution. J Neurochem 46:946-952. Kar S, Quirion R (1995): Neuropeptide receptors in developing and adult rat spinal cord: an in vitro quantitative autoradiography study of calcitonin gene-related peptide, neurokinins, mu-opioid, galanin, somatostatin, neurotensin and vasoactive intestinal polypeptide receptors. J Comp Neurol 354:253-281. Kataoka K, Mizuno N, Frohman LA (1979): Regional distribution of immunoreactive neurotension in monkey brain. Brain Res Bull 4:57-60. Kerr MA, Kenny AJ (1974): The purification and specificity of a neutral endopeptidase from rabbit kidney brush border. Biochem J 137:477-488. Kessler JP, Beaudet A (1989): Association of neurotensin binding sites with sensory and visceromotor components of the vagus nerve. J Neurosci 9:466-472. Kessler JP, Moyse E, Kitabgi P, Vincent JP, Beaudet A (1987): Distribution of neurotensin binding sites in the caudal brainstem of the rat: a light microscopic radioautographic study. Neuroscience 23:189-198. Kesten S, Akunne H, Johnson SJ, Wise LD, Wustrow DJ (1994): Novel small molecule neurotensin antagonists. In: 24th Natl Med Chem Symposium. June 21-25, 1994, Salt Lake City, UT. Kinkead B, Nemeroff CB (2002): Neurotensin: an endogenous antipsychotic?. Curr Opin Pharmacol 2:99-103. Kislauskis E, Dobner PR (1990): Mutually dependent response elements in the cis-regulatory region of the neurotensin/neuromedin N gene integrate environmental stimuli in PC12 cells. Neuron 4:783-795. Kislauskis E, Bullock B, McNeil S, Dobner PR (1988): The rat gene encoding neurotensin and neuromedin N. Structure, tissue-specific expression, and evolution of exon sequences. J Biol Chem 263:4963-4968. Kitabgi P, Nemeroff CB (1992): The neurobiology of neurotensin. Ann N Y Acad Sci 668:1-374. Kitabgi P, Carraway R, Van Rietschoten J, Granier C, Morgat JL, Menez A, Leeman S, Freychet P (1977): Neurotensin: specific binding to synaptic membranes from rat brain. Proc Natl Acad Sci USA 74:1846-1850. Kitabgi P, Poustis C, Granier C, Van Rietschoten J, Rivier J, Morgat JL, Freychet P (1980): Neurotensin binding to extraneural and neural receptors: comparison with biological activity and structure-activity relationships. Mol Pharmacol 18:11-19.

Kitabgi P, Checler F, Mazella J, Vincent JP (1985): Pharmacology and biochemistry of neurotensin receptors. Rev Clin Basic Pharm 5:397-486. Kitabgi P, Rost~ne W, Dussaillant M, Schotte A, Laduron PM, Vincent JP (1987): Two populations of neurotensin binding sites in murine brain: discrimination by the antihistamine levocabastine reveals markedly different radioautographic distribution. Eur J Pharmacol 140:285-293. Kitabgi P, Herve D, Studler JM, Tramu G, Rost~ne W, Tassin JP (1989): Neurotensin/dopamine interactions, Encephale. 15 Spec No:91-94.

Kitabgi P, Masuo Y, Nicot A, Berod A, Cuber JC, Rost~ne W (1991): Marked variations of the relative distributions of neurotensin and neuromedin N in micropunched rat brain areas suggest differential processing of their common precursor. Neurosci Lett 124:9-12. Kitabgi P, De Nadai F, Rov~re C, Bidard JN (1992): Biosynthesis, maturation, release, and degradation of neurotensin and neuromedin N. Ann N Y Acad Sci 668:30-42. Kiyama H, Inagaki S, Kito S, Tohyama M (1987): Ontogeny of [3H]neurotensin binding sites in the rat cerebral cortex: autoradiographic study. Brain Res 428:303-306. Kiyama H, Sato M, Emson PC (1991): Ontogeny of neurotensin immunoreactivity and mRNA in the rat central

393

Ch. VI

P. Sarret and A. Beaudet

nervous system. In: Bji3rklund A, H6kfelt T, Tohyama M (Eds), Handbook of Chemical Neuroanatomy. Vol. 10. Ontogeny of Transmitters and Peptides in the CNS. Amsterdam: Elsevier, pp. 339-430. Kobayashi RM, Brown M, Vale W (1977): Regional distribution of neurotensin and somatostatin in rat brain. Brain Res 126:584-588.

K6hler C, Radesater AC, Hall H, Winblad B (1985): Autoradiographic localization of [3H]neurotensin-binding sites in the hippocampal region of the rat and primate brain. Neuroscience 16:577-587. K6hler C, Radesater AC, Chan-Palay V (1987): Distribution of neurotensin receptors in the primate hippocampal region: a quantitative autoradiographic study in the monkey and the postmortem human brain. Neurosci Lett 76:145-150. Labb6-Julli6 C, Dubuc I, Brouard A, Doulut S, Bourdel E, Pelaprat D, Mazella J, Martinez J, Rost~ne W, Costentin Jet al. (1994): In vivo and in vitro structure-activity studies with peptide and pseudopeptide neurotensin analogs suggest the existence of distinct central neurotensin receptor subtypes. J Pharmacol Exp Ther 268:328-336. Lahti RA, Cochrane EV, Roberts RC, Conley RR, Tamminga CA (1998): [3H]Neurotensin receptor densities in human postmortem brain tissue obtained from normal and schizophrenic persons. An autoradiographic study. J Neural Transm 105:507-516.

Lantos TA, Palkovits M, Rost~ne W, Berod A (1996): Neurotensin receptors in the human amygdaloid complex. Topographical and quantitative autoradiographic study. J Chem Neuroanat 11:209-217. Lapchak PA, Araujo DM, Pasinetti G, Hefti F (1993): Differential alterations of cortical cholinergic and neurotensin markers following ibotenic acid lesions of the nucleus basalis magnocellularis. Brain Res 613:239-246. Lazarus LH, Brown MR, Perrin MH (1977): Distribution, localization and characteristics of neurotensin binding sites in the rat brain. Neuropharmacology 16:625-629. Lee YS, Han JH, Kim HB, Joo YH, Yang BH (1999): An association study of neurotensin receptor gene polymorphism with schizophrenia. Schizophr Res 36:92. L6p6e-Lorgeoux I, Betancur C, Rost~ne W, Pelaprat D (1999): Differential ontogenetic patterns of levocabastinesensitive neurotensin NT2 receptors and of NT1 receptors in the rat brain revealed by in situ hybridization. Brain Res Dev Brain Res 113:115-131.

L6p6e-Lorgeoux I, Betancur C, Souaze F, Rost~ne W, Berod A, Pelaprat D (2000): Regulation of the neurotensin NT(1) receptor in the developing rat brain following chronic treatment with the antagonist SR 48692. J Neurosci Res 60:362-369. Levine AS, Kneip J, Grace M, Morley JE (1983): Effect of centrally administered neurotensin on multiple feeding paradigms. Pharmacol Biochem Behav 18:19-23. Lin BZ, Pilch PF and Kandror KV (1997): Sortilin is a major protein component of Glut4-containing vesicles. J Biol Chem 272:24145-24147.

Lobo DH, Parnavelas JG (1988): Ontogeny of neurotensin receptor binding sites in the rat visual cortex. Neurosci Lett 93:152-157. Lugrin D, Vecchini F, Doulut S, Rodriguez M, Martinez J, Kitabgi P (1991): Reduced peptide bond pseudopeptide analogues of neurotensin: binding and biological activities, and in vitro metabolic stability. Eur J Pharmacol 205:191-198.

Luttinger D, Nemeroff CB, Mason GA, Frye GD, Breese GR, Prange AJ (1981): Enhancement of ethanol-induced sedation and hypothermia by centrally administered neurotensin, beta-endorphin and bombesin. Neuropharmacology 20:305-309. Luttinger D, Frye GD, Bissette G (1982a): Effects of neurotensin on the actions of barbiturates and ethanol. Ann N Y Acad Sci 400:259-267.

Luttinger D, King RA, Sheppard D, Strupp J, Nemeroff CB, Prange AJ (1982b): The effect of neurotensin on food consumption in the rat. Eur J Pharmacol 81:499-503. Luttinger D, Frye GD, Nemeroff CB, Prange AJ (1983): The effects of neurotensin, beta-endorphin, and bombesin on ethanol-induced behaviors in mice. Psychopharmacology 79:357-363. Machida R, Tokumura T, Tsuchiya Y, Sasaki A, Abe K (1993): Pharmacokinetics of novel hexapeptides with neurotensin activity in rats. Biol Pharm Bull 16:43-47. Maffrand JP, Gully D, Boigegrain R, Soubrie P, Kitabgi P, Rost~ne W, Le Fur G (1993): Neurotensin receptor agonists and antagonists. Drug Fut 18:1137-1141. Mailleux P, Vanderhaeghen JJ (1988): Transient neurotensin in the human inferior olive during development. Brain Res 456:199-203.

Mailleux P, Pelaprat D, Vanderhaeghen JJ (1990): Transient neurotensin high-affinity binding sites in the human inferior olive during development. Brain Res 508:345-348. Marcusson EG, Horazdovsky BF, Cereghino JL, Gharakhanian E, Emr SD (1994): The sorting receptor for yeast vacuolar carboxypeptidase Y is encoded by the VPS 10 gene. Cell 77:579-586.

394

Neurotensin receptors in the central nervous system

Ch. VI

Martin GE, Naruse T (1982): Differences in the pharmacological actions of intrathecally administered neurotensin and morphine. Regul Pept 3:97-103. Martin S, Vincent JP, Mazella J (2002a): Recycling ability of the mouse and the human neurotensin type 2 receptors depends on a single tyrosine residue. J Cell Sci 115:165-173. Martin S, Navarro V, Vincent JP and Mazella J (2002b): Neurotensin receptor-1 and -3 complex modulates the cellular signaling of neurotensin in the HT29 cell line. Gastroenterology, in press. Mayer DJ, Price DD (1989): The neurobiology of pain. In: Snyder-Machler L, Robinson A (Eds), Clinical Electrophysiology: Electrotherapy and Electrophysiology. Baltimore, MD: Williams and Wilkins, pp. 139-202. Mazella J (2001): Sortilin/neurotensin receptor-3: a new tool to investigate neurotensin signaling and cellular trafficking?. Cell Signal 13:1-6. Mazella J, Poustis C, Labbe C, Checler F, Kitabgi P, Granier C, van Rietschoten J, Vincent JP (1983): Monoiodo[Trpl 1]neurotensin, a highly radioactive ligand of neurotensin receptors. Preparation, biological activity, and binding properties to rat brain synaptic membranes. J Biol Chem 258:3476-3481. Mazella J, Chabry J, Kitabgi P, Vincent JP (1988): Solubilization and characterization of active neurotensin receptors from mouse brain. J Biol Chem 263:144-149. Mazella J, Chabry J, Zstirger N, Vincent JP (1989): Purification of the neurotensin receptor from mouse brain by affinity chromatography. J Biol Chem 264:5559-5563. Mazella J, Leonard K, Chabry J, Kitabgi P, Vincent JP, Beaudet A (1991): Binding and internalization of iodinated neurotensin in neuronal cultures from embryonic mouse brain. Brain Res 564:249-255. Mazella J, Botto JM, Guillemare E, Coppola T, Sarret P, Vincent JP (1996): Structure, functional expression, and cerebral localization of the levocabastine-sensitive neurotensin/neuromedin N receptor from mouse brain. J Neurosci 16:5613-5620. Mazella J, Zstirger N, Navarro V, Chabry J, Kaghad M, Caput D, Ferrara P, Vita N, Gully D, Maffrand JP, Vincent JP (1998): The 100-kDa neurotensin receptor is gp95/sortilin, a non-G-protein-coupled receptor. J Biol Chem 273:26273-26276. McCann SM, Vijayan E (1992): Control of anterior pituitary hormone secretion by neurotensin. Ann N Y Acad Sci 668:287-297. McCann SM, Vijayan E, Koenig J, Krulich L (1982): The effects of neurotensin on anterior pituitary hormone secretion. Ann N Y Acad Sci 400:160-171. McHaffie JG, Kao CQ, Stein BE (1989): Nociceptive neurons in rat superior colliculus: response properties, topography, and functional implications. J Neurophysiol 62:510-525. McMahon BM, Boules M, Warrington L, Richelson E (2002): Neurotensin analogs indications for use as potential antipsychotic compounds. Life Sci 70:1101-1119. Mendez M, Souaze F, Nagano M, Kelly PA, Rost~ne W, Forgez P (1997): High affinity neurotensin receptor mRNA distribution in rat brain and peripheral tissues. J Mol Neurosci 9:93-102. Mills A, Demoliou-Mason CD, Barnard EA (1988): Characterization of neurotensin binding sites in intact and solubilized bovine brain membranes. J Neurochem 50:904-911. Minamino N, Kangawa K, Matsuo H (1984): Neuromedin N: a novel neurotensin-like peptide identified in porcine spinal cord. Biochem Biophys Res Commun 122:542-549. Morin AJ, Beaudet A (1998): Origin of the neurotensinergic innervation of the rat basal forebrain studied by retrograde transport of cholera toxin. J Comp Neurol 391:30-41. Morin AJ, Tajani M, Jones BE, Beaudet A (1996): Spatial relationship between neurotensinergic axons and cholinergic neurons in the rat basal forebrain: a light microscopic study with three-dimensional reconstruction. J Chem Neuroanat 10:147-156. Morris NJ, Ross SA, Lane WS, Moestrup SK, Petersen CM, Keller SR, Lienhard GE (1998): Sortilin is the major 110-kDa protein in GLUT4 vesicles from adipocytes. J Biol Chem 273:3582-3587. Motta M, Martini L (1981): Neurotensin inhibits LH release. Proc Soc Exp Biol Med 168:62-64. Moyse E, Rost~ne W, Vial M, Leonard K, Mazella J, Kitabgi P, Vincent JP, Beaudet A (1987): Distribution of neurotensin binding sites in rat brain: a light microscopic radioautographic study using monoiodo [125I]Tyr3neurotensin. Neuroscience 22:525-536. Moyse E, Miller MM, Rost~ne W, Beaudet A (1988): Effects of ovariectomy and estradiol replacement on the binding of 125I-neurotensin in rat suprachiasmatic nucleus. Neuroendocrinology 48:53-60. Najimi M, Gailly P, Maloteaux JM, Hermans E (2002): Distinct regions of C-terminus of the high affinity neurotensin receptor mediate the functional coupling with pertussis toxin sensitive and insensitive G-proteins. FEBS Lett 512:329-333. Nakagawa Y, Higashida H, Miki N (1984): A single class of neurotensin receptors with high affinity in

395

Ch. VI

P. Sarret and A. Beaudet

neuroblastoma X glioma NG108-15 hybrid cells that mediate facilitation of synaptic transmission. J Neurosci 4:1653-1661. Navarro V, Martin S, Sarret P, Nielsen MS, Petersen CM, Vincent J, Mazella J (2001): Pharmacological properties of the mouse neurotensin receptor 3. Maintenance of cell surface receptor during internalization of neurotensin. FEBS Lett 495:100-105.

Nemeroff CB (1986): The interaction of neurotensin with dopaminergic pathways in the central nervous system: basic neurobiology and implications for the pathogenesis and treatment of schizophrenia. Psychoneuroendocrinology 11:15-37. Nemeroff CB, Bissette G, Prange AJ, Loosen PT, Barlow TS, Lipton MA (1977): Neurotensin: central nervous system effects of a hypothalamic peptide. Brain Res 128:485-496. Nemeroff CB, Bissette G, Manberg PJ, Osbahr AJ, Breese GR and Prange AJ Jr. (1980): Neurotensin-induced hypothermia: evidence for an interaction with dopaminergic systems and the hypothalamic-pituitary-thyroid axis. Brain Res 195:69-84. Nemeroff CB, Hernandez DE, Luttinger D, Kalivas PW, Prange AJ (1982): Interactions of neurotensin with brain dopamine systems. Ann N Y Acad Sci 400:330-344. Nguyen HMK, Cahill CM, McPherson PS, Beaudet A (2002): Receptor-mediated internalization of [3H]Neurotensin in synaptosomal preparations from rat neostriatum. Neuropharmacology 42:1089-1098. Nicot A, Rost~ne W, Berod A (1994a): Neurotensin receptor expression in the rat forebrain and midbrain: a combined analysis by in situ hybridization and receptor autoradiography. J Comp Neurol 341:407-419. Nicot A, Berod A, Gully D, Rowe W, Quirion R, de Kloet ER, Rost~ne W (1994b): Blockade of neurotensin binding in the rat hypothalamus and of the central action of neurotensin on the hypothalamic-pituitary-adrenal axis with non-peptide receptor antagonists. Neuroendocrinology 59:572-578. Nicot A, Rost~ne W, Berod A (1995): Differential expression of neurotensin receptor mRNA in the dopaminergic cell groups of the rat diencephalon and mesencephalon. J Neurosci Res 40:667-674. Nielsen MS, Madsen P, Christensen EI, Nykjaer A, Gliemann J, Kasper D, Pohlmann R, Petersen CM (2001): The sortilin cytoplasmic tail conveys Golgi-endosome transport and binds the VHS domain of the GGA2 sorting protein. EMBO J 20:2180-2190. Niimi M, Takahara J, Sato M, Kawanishi K (1991): Neurotensin and growth hormone-releasing factor-containing neurons projecting to the median eminence of the rat: a combined retrograde tracing and immunohistochemical study. Neurosci Lett 133:183-186. Ninkovic M, Hunt SE Kelly JS (1981): Effect of dorsal rhizotomy on the autoradiographic distribution of opiate and neurotensin receptors and neurotensin-like immunoreactivity within the rat spinal cord. Brain Res 230:111-119. Nouel D, Faure ME St Pierre JA, Alonso R, Quirion R, Beaudet A (1997): Differential binding profile and internalization process of neurotensin via neuronal and glial receptors. J Neurosci 17:1795-1803. Nouel D, Sarret E Vincent JP, Mazella J, Beaudet A (1999): Pharmacological, molecular and functional characterization of glial neurotensin receptors. Neuroscience 94:1189-1197. Nussdorfer GG, Malendowicz LK, Meneghelli V, Mazzocchi G (1992): Neurotensin enhances plasma adrenocorticotropin concentration by stimulating corticotropin-releasing hormone secretion. Life Sci 50:639-643. Orlowski M, Michaud C, Chu TG (1983): A soluble metalloendopeptidase from rat brain. Purification of the enzyme and determination of specificity with synthetic and natural peptides. Eur J Biochem 135:81-88. Osbahr AJ, Nemeroff CB, Manberg PJ, Prange AJ (1979): Centrally administered neurotensin: activity in the Julou-Courvoisier muscle relaxation test in mice. Eur J Pharmacol 54:299-302. Osbahr AJ, Nemeroff CB, Luttinger D, Mason GA, Prange AJ (1981): Neurotensin-induced antinociception in mice: antagonism by thyrotropin releasing hormone. J Pharmacol Exp Ther 217:645-651. Palacios JM, Kuhar MJ (1981): Neurotensin receptors are located on dopamine-containing neurones in rat midbrain. Nature 294:587-589.

Palacios JM, Pazos A, Dietl MM, Schlumpf M, Lichtensteiger W (1988): The ontogeny of brain neurotensin receptors studied by autoradiography. Neuroscience 25:307-317. Palacios JM, Chinaglia G, Rigo M, Ulrich J and Probst A (1991): Neurotensin receptor binding levels in basal ganglia are not altered in Huntington's chorea or schizophrenia. Synapse 7:114-122. Pang YP, Cusack B, Groshan K, Richelson E (1996): Proposed ligand binding site of the transmembrane receptor for neurotensin(8-13). J Biol Chem 271:15060-15068. Pazos A, Lopez M, Florez J (1984): Different mechanisms are involved in the respiratory depression and analgesia induced by neurotensin in rats. Eur J Pharmacol 98:119-123. Petersen CM, Nielsen MS, Nykjaer A, Jacobsen L, Tommerup N, Rasmussen HH, Roigaard H, Gliemann J, Madsen P, Moestrup SK (1997): Molecular identification of a novel candidate sorting receptor purified from human brain by receptor-associated protein affinity chromatography. J Biol Chem 272:3599-3605.

396

Neurotensin receptors in the central nervous system

Ch. VI

Pettibone DJ, Hess JF, Hey PJ, Jacobson MA, Leviten M, Lis EV, Mallorga PJ, Pascarella DM, Snyder MA, Williams JB, Zeng Z (2002): The effects of deleting the mouse neurotensin receptor NTR1 on central and peripheral responses to neurotensin. J Pharmacol Exp Ther 300:305-313. Pickel VM, Chan J, Delle Donne KT, Boudin H, Pelaprat D, Rost~ne W (2001): High-affinity neurotensin receptors in the rat nucleus accumbens: subcellular targeting and relation to endogenous ligand. J Comp Neurol 435:142155. Poncelet M, Gueudet C, Gully D, Soubrie P, Le Fur G (1994): Turning behavior induced by intrastriatal injection of neurotensin in mice: sensitivity to non-peptide neurotensin antagonists. Naunyn Schmiedeberg's Arch Pharmacol 349:57-60. Poustis C, Mazella J, Kitabgi P, Vincent JP (1984): High-affinity neurotensin binding sites in differentiated neuroblastoma N1E115 cells. J Neurochem 42:1094-1100. Pugsley TA, Akunne HC, Whetzel SZ, Demattos S, Corbin AE, Wiley JN, Wustrow DJ, Wise LD, Heffner TG (1995): Differential effects of the nonpeptide neurotensin antagonist, SR 48692, on the pharmacological effects of neurotensin agonists. Peptides 16:37-44. Quirion R (1983): Interactions between neurotensin and dopamine in the brain: an overview. Peptides 4:609-615. Quirion R, Rioux F, Regoli D, St-Pierre S (1980): Selective blockade of neurotensin-induced coronary vessel constriction in perfused rat hearts by a neurotensin analogue. Eur J Pharmacol 61:309-312. Quirion R, Gaudreau P, St-Pierre S, Rioux F, Pert CB (1982): Autoradiographic distribution of [3H]neurotensin receptors in rat brain: visualization by tritium-sensitive film. Peptides 3:757-763. Quirion R, Chiueh CC, Everist HD, Pert A (1985): Comparative localization of neurotensin receptors on nigrostriatal and mesolimbic dopaminergic terminals. Brain Res 327:385-389. Quirion R, Welner S, Gauthier S, Bedard P (1987): Neurotensin receptor binding sites in monkey and human brain: autoradiographic distribution and effects of 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine treatment. Synapse 1:559-566. Richard F, Barroso S, Martinez J, Labb6-Julli6 C, Kitabgi P (2001): Agonism, inverse agonism, and neutral antagonism at the constitutively active human neurotensin receptor 2. Mol PharmacoI 60:1392-1398. Rioux F, Quirion R, Regoli D, Leblanc MA, St-Pierre S (1980): Pharmacological characterization of neurotensin receptors in the rat isolated portal vein using analogues and fragments of neurotensin. Eur J Pharmacol 66:273279. Rodriguez-Bello A, Kah O, Tramu G, Conlon JM (1993): Purification and primary structure of alligator neurotensin. Peptides 14:1055-1058. Rost~ne W, Azzi M, Boudin H, Lepee I, Souaze F, Mendez-Ubach M, Betancur C, Gully D (1997): Use of nonpeptide antagonists to explore the physiological roles of neurotensin. Focus on brain neurotensin/dopamine interactions. Ann N Y Acad Sci 814:125-141. Rost~ne WH, Alexander MJ (1997): Neurotensin and neuroendocrine regulation. Front Neuroendocrinol 18:115173. Rovbre C, Barbero P, Kitabgi P (1996): Evidence that PC2 is the endogenous pro-neurotensin convertase in rMTC 6-23 cells and that PC1- and PC2-transfected PC12 cells differentially process pro-neurotensin. J Biol Chem 271:11368-11375. Sadoul JL, Mazella J, Amar S, Kitabgi P, Vincent JP (1984a): Preparation of neurotensin selectively iodinated on the tyrosine 3 residue. Biological activity and binding properties on mammalian neurotensin receptors. Biochem Biophys Res Commun 120:812-819. Sadoul JL, Kitabgi P, Rost~ne W, Javoy-Agid F, Agid Y, Vincent JP (1984b): Characterization and visualization of neurotensin binding to receptor sites in human brain. Biochem Biophys Res Commun 120:206-213. Sadoul JL, Checler F, Kitabgi P, Rost~ne W, Javoy-Agid F, Vincent JP (1984c): Loss of high affinity neurotensin receptors in substantia nigra from parkinsonian subjects. Biochem Biophys Res Commun 125:395-404. Sarret P, Beaudet A, Vincent JP, Mazella J (1998): Regional and cellular distribution of low affinity neurotensin receptor mRNA in adult and developing mouse brain. J Comp Neurol 394:344-356. Sarret P, Gendron L, Kilian P, Nguyen HMK, Gallo-Payet N, Payet MD, Beaudet A (2002): Pharmacology and functional properties of NTS2 neurotensin receptors in cerebellar granule cells. J Biol Chem 277: in press. Sarrieau A, Javoy-Agid F, Kitabgi P, Dussaillant M, Vial M, Vincent JP, Agid Y, Rost~ne WH (1985): Characterization and autoradiographic distribution of neurotensin binding sites in the human brain. Brain Res 348:375380. Sato M, Kiyama H, Yoshida S, Saika T, Tohyama M (1991): Postnatal ontogeny of cells expressing preproneurotensin/neuromedin N mRNA in the rat forebrain and midbrain: a hybridization histochemical study involving isotope-labeled and enzyme-labeled probes. J Comp Neurol 310:300-315.

397

Ch. VI

P. Sarret and A. Beaudet

Sato M, Kiyama H, Tohyama M (1992): Different postnatal development of cells expressing mRNA encoding neurotensin receptor. Neuroscience 48:137-149. Schotte A, Laduron PM (1987): Different postnatal ontogeny of two [3H]neurotensin binding sites in rat brain. Brain Res 408:326-328. Schotte A, Leysen JE (1989): Further characterization of neurotensin binding in the rat brain: levocabastinedisplaceable neurotensin binding sites are not histamine-H1 receptors. Biochem Pharmacol 38:3891-3893. Schotte A, Leysen JE, Laduron PM (1986): Evidence for a displaceable non-specific [3H]neurotensin binding site in rat brain. Naunyn Schmiedeberg's Arch Pharmacol 333:400-405. Schotte A, Rost~ne W, Laduron PM (1988): Different subcellular localization of neurotensin-receptor and neurotensin-acceptor sites in the rat brain dopaminergic system. J Neurochem 50:1026-1031. Seroogy KB, Mehta A, Fallon JH (1987): Neurotensin and cholecystokinin coexistence within neurons of the ventral mesencephalon: projections to forebrain. Exp Brain Res 68:277-289. Seybold V, Elde R (1980): Immunohistochemical studies of peptidergic neurons in the dorsal horn of the spinal cord. J Histochem Cytochem 28:367-370. Seybold VS, Elde RP (1982): Neurotensin immunoreactivity in the superficial laminae of the dorsal horn of the rat: I. Light microscopic studies of cell bodies and proximal dendrites. J Comp Neurol 205:89-100. Sharma RE Janicak PG, Bissette G, Nemeroff CB (1997): CSF neurotensin concentrations and antipsychotic treatment in schizophrenia and schizoaffective disorder. Am J Psychiatry 154:1019-1021. Shaw C, Thim L, Conlon JM (1986): [Ser7]neurotensin: isolation from guinea pig intestine. FEBS Lett 202:187192. Shaw C, Murphy R, Thim L, Furness JB, Buchanan KD (1991): Marsupial possum neurotensin: a unique mammalian regulatory peptide exhibiting structural homology to the avian analogue. Regul Pept 35:49-57. Shaw C, McKay DM, Halton DW, Thim L, Buchanan KD (1992): Isolation and primary structure of an amphibian neurotensin. Regul Pept 38:23-31. Sheppard MC, Kronheim S, Pimstone BL (1979): Effect of substance P, neurotensin and the enkephalins on somatostatin release from the rat hypothalamus in vitro. J Neurochem 32:647-649. Snider RM, Pereira DA, Longo KP (1992): UK-73,093: A non-peptide neurotensin receptor antagonist. Biorg Med Chem Lett 2:1535-1540. Snijders R, Kramarcy NR, Hurd RW, Nemeroff CB, Dunn AJ (1982): Neurotensin induces catalepsy in mice. Neuropharmacology 21:465-468. Stanley BG, Hoebel BG, Leibowitz SF (1983): Neurotensin: effects of hypothalamic and intravenous injections on eating and drinking in rats. Peptides 4:493-500. Steinberg R, Brun P, Fournier M, Souilhac J, Rodier D, Mons G, Terranova JP, Le Fur G, Soubrie P (1994): SR 48692, a non-peptide neurotensin receptor antagonist differentially affects neurotensin-induced behaviour and changes in dopaminergic transmission. Neuroscience 59:921-929. Szigethy E, Beaudet A (1987): Selective association of neurotensin receptors with cholinergic neurons in the rat basal forebrain. Neurosci Lett 83:47-52. Szigethy E, Wenk GL, Beaudet A (1988): Anatomical substrate for neurotensin-acetylcholine interactions in the rat basal forebrain. Peptides 9:1227-1234. Szigethy E, Beaudet A (1989): Correspondence between high affinity 125I-neurotensin binding sites and dopaminergic neurons in the rat substantia nigra and ventral tegmental area: a combined radioautographic and immunohistochemical light microscopic study. J Comp Neurol 279:128-137. Szigethy E, Leonard K, Beaudet A (1990a): Ultrastructural localization of [125I]neurotensin binding sites to cholinergic neurons of the rat nucleus basalis magnocellularis. Neuroscience 36:377-391. Szigethy E, Quirion R, Beaudet A (1990b): Distribution of 125I-neurotensin binding sites in human forebrain: comparison with the localization of acetylcholinesterase. J Comp Neurol 297:487-498. Tanaka K, Masu M, Nakanishi S (1990): Structure and functional expression of the cloned rat neurotensin receptor. Neuron 4:847-854. Tanji H, Araki T, Fujihara K, Nagasawa H, Itoyama Y (1999): Alteration of neurotensin receptors in MPTP-treated mice. Peptides 20:803-807. Tokumura T, Tanaka T, Sasaki A, Tsuchiya Y, Abe K, Machida R (1990): Stability of a novel hexapeptide, (Me)Arg-Lys-Pro-Trp-tert-Leu-Leu-OEt, with neurotensin activity, in aqueous solution and in the solid state. Chem Pharm Bull (Tokyo) 38:3094-3098. Tokumura T, Machida R, Tsuchiya Y, Sasaki A, Abe K, Tanaka T, Saeki Y (1993): Pharmacokinetics of (Me)ArgLys-Pro-Trp-tert-Leu-Leu-OEt and (Me)Arg-Lys-Pro-Trp-tert-Leu-Leu administered intravenously to beagle dogs. J Pharm Sci 82:725-728.

398

Neurotensin receptors in the central nervous system

Ch. VI

Trudeau LE (2000): Neurotensin regulates intracellular calcium in ventral tegmental area astrocytes: evidence for the involvement of multiple receptors. Neuroscience 97:293-302. Turner JT, James-Kracke MR, Camden JM (1990): Regulation of the neurotensin receptor and intracellular calcium mobilization in HT29 cells. J Pharmacol Exp Ther 253:1049-1056. Tyler BM, Cusack B, Douglas CL, Souder T, Richelson E (1998a): Evidence for additional neurotensin receptor subtypes: neurotensin analogs that distinguish between neurotensin-mediated hypothermia and antinociception. Brain Res 792:246-252.

Tyler BM, Groshan K, Cusack B, Richelson E (1998b): In vivo studies with low doses of levocabastine and diphenhydramine, but not pyrilamine, antagonize neurotensin-mediated antinociception. Brain Res 787:78-84. Tyler BM, McCormick DJ, Hoshall CV, Douglas CL, Jansen K, Lacy BW, Cusack B, Richelson E (1998c): Specific gene blockade shows that peptide nucleic acids readily enter neuronal cells in vivo. FEBS Lett 421:280-284. Tyler BM, Douglas CL, Fauq A, Pang YP, Stewart JA, Cusack B, McCormick DJ, Richelson E (1999): In vitro binding and CNS effects of novel neurotensin agonists that cross the blood-brain barrier. Neuropharmacology 38:1027-1034. Tyler-McMahon BM, Boules M, Richelson E (2000): Neurotensin: peptide for the next millennium. Regul Pept 93:125-136. Uhl GR, Bennett JP, Snyder SH (1977): Neurotensin, a central nervous system peptide: apparent receptor binding in brain membranes. Brain Res 130:299-313. Uhl GR, Snyder SH (1977): Neurotensin receptor binding, regional and subcellular distributions favor transmitter role. Eur J Pharmaco141:89-91. Uhl GR (1982): Distribution of neurotensin and its receptor in the central nervous system. Ann N Y Acad Sci 400:132-149.

Uhl GR, Whitehouse PJ, Price DL, Tourtelotte WW, Kuhar MJ (1984): Parkinson's disease: depletion of substantia nigra neurotensin receptors. Brain Res 308:186-190. Uhl GR, Hackney GO, Torchia M, Stranov V, Tourtellotte WW, Whitehouse PJ, Tran V, Strittmatter S (1986): Parkinson's disease: nigral receptor changes support peptidergic role in nigrostriatal modulation. Ann Neurol 20:194-203. Urban MO, Smith DJ (1993): Role of neurotensin in the nucleus raphe magnus in opioid-induced antinociception from the periaqueductal gray. J Pharmacol Exp Ther 265:580-586. Vandenbulcke F, Nouel D, Vincent J, Mazella J, Beaudet A (2000): Ligand-induced internalization of neurotensin in transfected COS-7 cells: differential intracellular trafficking of ligand and receptor. J Cell Sci 113:2963-2975. Vanisberg MA, Maloteaux JM, Octave JN, Laduron PM (1991): Rapid agonist-induced decrease of neurotensin receptors from the cell surface in rat cultured neurons. Biochem Pharmaco142:2265-2274. Vaughn AW, Baumeister AA, Hawkins MF, Anticich TG (1990): Intranigral microinjection of neurotensin suppresses feeding in food deprived rats. Neuropharmacology 29:957-960. Villeneuve P, Seidah NG, Beaudet A (2000a): Immunohistochemical evidence for the implication of PC1 in the processing of proneurotensin in rat brain. NeuroReport 11:3443-3447. Villeneuve P, Lafortune L, Seidah NG, Kitabgi P, Beaudet A (2000b): Immunohistochemical evidence for the involvement of protein convertases 5A and 2 in the processing of pro-neurotensin in rat brain. J Comp Neurol 424:461-475.

Vincent JP (1992): Neurotensin receptors. Binding properties, transduction mechanisms, and purification. Ann N Y Acad Sci 668:90-100.

Vincent JP (1995): Neurotensin receptors: binding properties, transduction pathways, and structure. Cell Mol Neurobiol 15:501-512. Vincent JP, Mazella J, Kitabgi P (1999): Neurotensin and neurotensin receptors. Trends Pharmacol Sci 20:302-309. Vita N, Laurent P, Lefort S, Chalon P, Dumont X, Kaghad M, Gully D, Le Fur G, Ferrara P, Caput D (1993): Cloning and expression of a complementary DNA encoding a high affinity human neurotensin receptor. FEBS Lett 317:139-142.

Vita N, Oury-Donat F, Chalon P, Guillemot M, Kaghad M, Bachy A, Thurneyssen O, Garcia S, Poinot-Chazel C, Casellas P, Keane P, Le Fur G, Maffrand JP, Soubrie P, Caput D, Ferrara P (1998): Neurotensin is an antagonist of the human neurotensin NT2 receptor expressed in Chinese hamster ovary cells. Eur J Pharmacol 360:265-272.

Wagstaff JD, Bush LG, Gibb JW, Hanson GR (1994): Endogenous neurotensin antagonizes methamphetamineenhanced dopaminergic activity. Brain Res 665:237-244. Walker N, L6p6e-Lorgeoux I, Fournier J, Betancur C, Rost~ne W, Ferrara P, Caput D (1998): Tissue distribution and cellular localization of the levocabastine-sensitive neurotensin receptor mRNA in adult rat brain. Brain Res Mol Brain Res 57:193-200.

399

Ch. VI

P. Sarret and A. Beaudet

Waters CM, Hunt SP, Jenner E Marsden CD (1987): Localization of neurotensin receptors in the forebrain of the common marmoset and the effects of treatment with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Brain Res 412:244-253.

Whitehouse PJ, Price DL, Struble RG, Clark AW, Coyle JT, Delon MR (1982): Alzheimer's disease and senile dementia: loss of neurons in the basal forebrain. Science 215:1237-1239. Wolf SS, Hyde TM, Moody TW, Saunders RC, Weinberger DR, Kleinman JE (1994): Autoradiographic characterization of 125I-neurotensin binding sites in human entorhinal cortex. Brain Res Bull 35:353-358. Wolf SS, Hyde TM, Saunders RC, Herman MM, Weinberger DR, Kleinman JE (1995): Autoradiographic characterization of neurotensin receptors in the entorhinal cortex of schizophrenic patients and control subjects. J Neural Transm Gen Sect 102:55-65.

Woulfe J, Beaudet A (1992): Neurotensin terminals form synapses primarily with neurons lacking detectable tyrosine hydroxylase immunoreactivity in the rat substantia nigra and ventral tegmental area. J Comp Neurol 321:163-176.

Woulfe J, Lafortune L, de Nadai F, Kitabgi P, Beaudet A (1992): Immunohistochemical detection of proneurotensin/neuromedin N processing intermediates in rat brain. Ann N Y Acad Sci 668:368-371. Woulfe J, Lafortune L, de Nadai F, Kitabgi P, Beaudet A (1994): Post-translational processing of the neurotensin/neuromedin N precursor in the central nervous system of the r a t - II. Immunohistochemical localization of maturation products. Neuroscience 60:167-181. Young WSd, Kuhar MJ (1979): Neurotensin receptors: autoradiographic localization in rat CNS. Eur J Pharmacol 59:161-163. Young WSd, Kuhar MJ (1981): Neurotensin receptor localization by light microscopic autoradiography in rat brain. Brain Res 206:273-285.

Zahm DS, Heimer L (1988): Ventral striatopallidal parts of the basal ganglia in the rat: I. Neurochemical compartmentation as reflected by the distributions of neurotensin and substance P immunoreactivity. J Comp Neurol 272:516-535.

Zstirger N, Chabry J, Coquerel A, Vincent JP (1992): Ontogenesis and binding properties of high-affinity neurotensin receptors in human brain. Brain Res 586:303-310. Zstirger N, Mazella J, Vincent JP (1994): Solubilization and purification of a high affinity neurotensin receptor from newborn human brain. Brain Res 639:245-252.

400

Subject Index

AGRANULAR INSULAR CORTEX Delta Opioid receptors mRNA, 15 OX1R Immunoreactivity, 258 PreproOFQ mRNA, 111 ALPHA-MELANOCYTESTIMULATING-HORMONE, 249

ALZHEIMER'S DISEASE, 199, 200 AMYGDALA/AMYGDALOID COMPLEX Delta Opioid receptors Autoradiography, 9 mRNA, 15 125I-GALANIN Autoradiography, 216 GALR1 mRNA, 217 GALR3 mRNA, 227 GPR54 mRNA, 207 Kappa Opioid receptors Autoradiography, 9 Immunoreactivity, 19 mRNA, 16 Mu Opioid receptors Autoradiography, 8 Immunoreactivity, 17 mRNA, 14 NT Autoradiography, 346 Immunoreactivity, 327 NTS 1 receptor Immunoreactivity, 352 mRNA, 350

NTS2 receptor Autoradiography, 369 mRNA, 373 OFQ Immunoreactivity, 134 mRNA, 157 OREXIN Immunoreactivity, 249 ORL1 receptor Autoradiography, 149 Biological effects, 105 mRNA, 134, 160, 161 OX1R mRNA, 259 OXR2 mRNA, 259 PreproOFQ mRNA, 134, 156, 158 SLC-1 Immunoreactivity, 57, 69 mRNA, 59 ARCUATE NUCLEUS GALANIN Immunoreactivity, 197 GALP Immunoreactivity, 202 mRNA, 202 GALR2 mRNA, 223 NTS 1 receptor Autoradiography, 347 mRNA, 351 NTS2 receptor mRNA, 375 OFQ Immunoreactivity, 133 mRNA, 132, 158 OREXIN Immunoreactivity, 249

401

Subject Index ORL1 receptor Autoradiography, 149 mRNA, 133, 160, 161 OX1R Immunoreactivity, 260 OX2R mRNA, 258, 262 PreproOFQ mRNA, 157 SLC-1 Immunoreactivity, 69 AREA POSTREMA

Mu Opioid receptors Autoradiography, 8 Immunoreactivity, 19 mRNA, 15 OREXIN Immunoreactivity, 251 ORL1 receptor Autoradiography, 152 mRNA, 142 BASAL GANGLIA, 131

MCH Immunoreactivity, 42 NTS2 receptor mRNA, 373 ORL 1 receptor Autoradiography, 148 BASAL NUCLEUS OF MEYNERT

NTS 1 receptor mRNA, 350 OFQ Immunoreactivity, 130, 131 ORL 1 RECEPTOR Autoradiography, 149 mRNA, 131 PreproOFQ mRNA, 131 BED NUCLEUS OF THE OLFACTORY TRACT

125I-GALANIN Autoradiography, 215 GALR1 402

mRNA, 217 OFQ Immunoreactivity, 132, 134 ORL1 receptor Autoradiography, 149 mRNA, 132, 134, 138, 160 PreproOFQ mRNA, 132 BED NUCLEUS OF THE STRIA TERMINALIS

Delta Opioid receptors Autoradiography, 9 mRNA, 15 125I-GALANIN Autoradiography, 216 GALP Immunoreactivity, 202 GALR1 mRNA, 217 GALR3 mRNA, 227 Kappa Opioid receptors Autoradiography, 12 Immunoreactivity, 19 mRNA, 16 Mu Opioid receptors Autoradiography, 8 NT Immunoreactivity, 327 NTS 1 receptor Immunoreactivity, 352 mRNA, 350 NTS2 receptor Autoradiography, 369 mRNA, 373 OFQ Immunoreactivity, 130, 132, 134 mRNA, 157 ORL1 receptor Autoradiography, 149 mRNA, 132, 160, 161 OX1R mRNA, 259 OX2R mRNA, 259 PreproOFQ mRNA, 131,134, 157, 158

Subject Index BRAIN STEM

Delta Opioid receptors Immunoreactivity, 19 mRNA, 16 Kappa Opioid receptors mRNA, 17 MCH Immunoreactivity, 42 Mu Opioid receptors Autoradiography, 8 NT Immunoreactivity, 327 ORL 1 receptor mRNA, 161 CAUDATE-PUTAMEN

Delta Opioid receptors Immunoreactivity, 19 mRNA, 15 GALR3 mRNA, 225 Kappa Opioid receptors Autoradiography, 9 Immunoreactivity, 19 Mu Opioid receptors Autoradiography, 8 Immunoreactivity, 17 mRNA, 14 NTS 1 receptor Autoradiography, 337 NTS2 receptor Autoradiography, 369 ORL1 receptor Autoradiography, 148 OX1R Immunoreactivity, 259 SLC-1 Immunoreactivity, 57, 69 mRNA, 59 CEREBELLAR NUCLEI

Delta Opioid receptors mRNA, 16 Mu Opioid receptors mRNA, 15 OFQ Immunoreactivity, 110

OX1R Immunoreactivity, 263 CEREBELLUM/CEREBELLAR CORTEX

125I-GALANIN Autoradiography, 215 Kappa Opioid receptors Autoradiography, 12 mRNA, 17 GALR2 mRNA, 221,223 Mu Opioid receptors mRNA, 15 NTS 1 receptor mRNA, 350 NTS2 receptor Autoradiography, 369 mRNA, 377 OFQ Immunoreactivity, 110, 139 ORL1 receptor mRNA, 139 PreproOFQ mRNA, 139 SLC-1 Immunoreactivity, 72 mRNA, 59 CLAUSTRUM

Kappa Opioid receptors Autoradiography, 12 Immunoreactivity, 19 Mu Opioid receptors mRNA, 14 NT Immunoreactivity, 327 OFQ Immunoreactivity, 131 mRNA ORL1 receptor Autoradiography, 148 mRNA, 131,161 PreproOFQ mRNA, 131,157, 159 COCHLEAR NUCLEI

Delta Opioid receptors mRNA, 16 403

Subject Index Kappa Opioid receptors mRNA, 17 NT Autoradiography, 349 NTS 1 receptor Immunoreactivity, 354 NTS2 receptor mRNA, 377 OFQ Immunoreactivity, 140 ORL1 receptor mRNA, 141 PreproOFQ mRNA, 140 CORTEX/CEREBRAL CORTEX Delta Opioid receptors Autoradiography, 9 Immunoreactivity, 19 GALANIN Immunoreactivity, 196 GALR2 mRNA, 221 GALR3 mRNA, 225 Kappa Opioid receptors Immunoreactivity, 19 mRNA, 16 Mu Opioid receptors Autoradiography, 8 Immunoreactivity, 17 mRNA, 14 NT Autoradiography, 346 NTS 1 receptor Autoradiography, 337 Immunoreactivity, 352 mRNA, 350 NTS2 receptor Autoradiography, 369 mRNA, 373 OFQ Immunoreactivity, 122 OREXIN Immunoreactivity, 249 ORL1 receptor Autoradiography, 148 Immunoreactivity, 122 404

mRNA, 126, 127, 139, 160, 161 OX1R Immunoreactivity, 259 mRNA, 258 OX2R mRNA, 258 PreproOFQ mRNA, 111 PreproOREXIN mRNA, 248 SLC-1 Immunoreactivity, 57 mRNA, 59 CUNEATUS NUCLEUS Delta Opioid receptors mRNA, 16 Kappa Opioid receptors mRNA, 17 Mu Opioid receptors mRNA, 15 NT Autoradiography, 349 NTS 1 receptor Immunoreactivity, 355 ORL 1 receptor mRNA, 142 PreproOFQ mRNA, 141 DELTA OPIOID RECEPTORS, 5

Autoradiography, 9 Immunoreactivity, 19 mRNA, 15 DENTATE GYRUS

IE5I-GALANIN Autoradiography, 216 GALR2 mRNA, 222 GALR3 mRNA, 225 Kappa Opioid receptors Autoradiography, 12 Mu Opioid receptors Immunoreactivity, 17 NTS 1 receptor

Subject Index Immunoreactivity, 352 NTS2 receptor mRNA, 373 ORL1 receptor Autoradiography, 150 Biological effects, 105 mRNA, 160, 161 OX2R mRNA, 259 PreproOFQ mRNA, 158 SLC-1 Immunoreactivity, 69 DIAGONAL BAND OF BROCA

GALANIN Immunoreactivity, 196 125I-GALANIN Autoradiography, 216 GALR1 mRNA, 217 GALR2 mRNA, 221 GALR3 mRNA, 227 MCH Immunoreactivity, 42 Mu Opioid receptors mRNA, 14 NTS 1 receptor Autoradiography, 337 Immunoreactivity, 337, 351,352 mRNA, 337, 350 NTS2 receptor mRNA, 373 OFQ Immunoreactivity, 130 mRNA, 129 ORL1 receptor Autoradiography, 148 mRNA, 130, 160, 161 OX1R

Immunoreactivity, 259 mRNA, 259 OX2R mRNA, 259 PreproOFQ mRNA, 129, 158

SLC-1 Immunoreactivity, 69 DIENCEPHALON

GALANIN Immunoreactivity, 197 125I-GALANIN Autoradiography, 216 GALR1 mRNA, 217 GALR2 mRNA, 223 GALR3 mRNA, 227 MCH Immunoreactivity, 42 NTS 1 receptor Autoradiography, 346 Immunoreactivity, 353 mRNA, 350 NTS2 receptor mRNA, 373 PreproOFQ mRNA, 156 DORSAL MOTOR NUCLEUS OF THE VAGUS NERVE

Delta Opioid receptors mRNA, 16 GALR1 mRNA, 219 Kappa Opioid receptors Immunoreactivity, 21 mRNA, 17 Mu Opioid receptors mRNA, 15 NTS 1 receptor Autoradiography, 337, 349 Immunoreactivity, 337, 355 mRNA, 337 NTS2 receptor mRNA, 377 ORL1 receptor mRNA, 142 OX1R mRNA, 262 OX2R mRNA, 262 405

Subject Index PreproOFQ mRNA, 141 DORSAL ROOT GANGLIA (DRG)

GALANIN Autoradiography, 230 mRNA, 230 GMAP, 202 Mu Opioid receptors Immunoreactivity, 17 OX1R Immunoreactivity, 263 ENDOPIRIFORM NUCLEUS

Kappa Opioid receptors Autoradiography, 12 Mu Opioid receptors mRNA, 14 NTS 1 receptor mRNA, 350 OFQ Autoradiography, 148 Immunoreactivity, 131 ORL1 receptor Autoradiography, 148 mRNA, 131,161 PreproOFQ mRNA, 131 ENTOPEDUNCULAR NUCLEUS

Mu Opioid receptors Autoradiography, 8 Immunoreactivity, 17 OFQ Immunoreactivity, 131 ORL 1 receptor Autoradiography, 149 mRNA, 131 PreproOFQ mRNA, 131 FACIAL MOTOR NUCLEUS

Delta Opioid receptors Immunoreactivity, 19 FUNDUS STRIATI

406

OFQ Immunoreactivity, 130 mRNA, 129 PreproOFQ mRNA, 129 GALANIN

Antagonists, 200 Cognition and memory, 198 Discovery, 195, 196 Feeding, 197 Galanin-like receptors, 207 Genetic manipulations, 201 Immunoreactivity, 249 Sensory transmission/nociception, 199 Related peptides : GMAP, 201 GALP, 202 Subtypes: 203 GALR1 204 GALR2 205 GALR3 205, 198 Therapeutic implications, 199, 200 GLIAL CELLS

GALR, 228, 229 GLOBUS PALLIDUS

Delta Opioid receptors Autoradiography, 9 mRNA, 15 Kappa Opioid receptors Autoradiography, 12 Immunoreactivity, 19 mRNA, 16 Mu Opioid receptors Autoradiography, 8 Immunoreactivity, 17 mRNA, 14 NTS 1 receptor Immunoreactivity, 352 NTS2 receptor Autoradiography, 369 OFQ Immunoreactivity, 131 ORL1 receptor Autoradiography, 148

Subject Index mRNA, 131 OX1R Immunoreactivity, 259 OX2R mRNA, 259 PreproOFQ mRNA, 131 SLC-1 Immunoreactivity, 69 GRACILIS NUCLEI Kappa Opioid receptors mRNA, 17 Mu Opioid receptors mRNA, 15

HABENULA/HABENULAR NUCLEI/HABENULO INTERPEDUNCULAR COMPLEX

Delta Opioid receptors Autoradiography, 9 125I-GALANIN Autoradiography, 216 GALR1 mRNA, 218 GALR2 mRNA, 223 GALR3 mRNA, 225, 227 Kappa Opioid receptors Autoradiography, 12 mRNA, 16 Mu Opioid receptors Autoradiography, 8 Immunoreactivity, 17 mRNA, 14 NTS 1 receptor Autoradiography, 347 Immunoreactivity, 354 mRNA, 350 NTS2 receptor Autoradiography, 369 mRNA, 373 ORL1 receptor mRNA, 160, 161 SLC-1 Immunoreactivity, 69

mRNA, 59 H I P P O C A M P U S / HIPPOC AMPAL FORMATION

Delta Opioid receptors Autoradiography, 9 Immunoreactivity, 19 mRNA, 15 GALANIN Immunoreactivity, 196 125I-GALANIN Autoradiography, 207, 215, 216 GALR1 mRNA, 217 GALR2 mRNA, 221 GALR3 mRNA, 225 Kappa Opioid receptors Autoradiography, 12 Immunoreactivity, 19 mRNA, 16 MCH Immunoreactivity, 42 Mu Opioid receptors Autoradiography, 8 Immunoreactivity, 17, 19 mRNA, 14 NTS 1 receptor Autoradiography, 346 Immunoreactivity, 352 mRNA, 350 NTS2 receptor Autoradiography, 369 mRNA, 373 OFQ Immunoreactivity, 130, 135 mRNA, 130 OREXIN Immunoreactivity, 249 ORL1 receptor Autoradiography, 150 mRNA, 110, 131,135, 160, 161 OX1R Immunoreactivity, 259 mRNA, 258, 259 OX2R mRNA, 259 407

Subject Index

PreproOFQ mRNA, 135, 159 SLC-1 Immunoreactivity, 57, 69 mRNA, 59 HUMAN BRAIN, 155 MCH Immunoreactivity, 41 NT Autoradiography, 346, 347, 349 NTS 1 receptor Autoradiography, 346, 347, 349 OFQ mRNA, 163, 165 ORL1 receptor mRNA, 156, 164, 165 PreproOFQ mRNA, 161,163 SLC-1 Gene expression, 74 HYPOCRETIN, See OREXIN HYPOGLOSSAL NUCLEUS Delta Opioid receptors Immunoreactivity, 19 mRNA, 16 Kappa Opioid receptors mRNA, 17

HYPOTHALAMUS / n YPOTHALAMUS NUCLEI Delta Opioid receptors Autoradiography, 9 mRNA, 15 GALANIN Immunoreactivity, 196 125I-GALANIN Autoradiography, 216 GALP Immunoreactivity, 202 mRNA, 202 GALR1 mRNA, 218 GALR2 mRNA, 221,223 GALR3 408

mRNA, 225, 227 GMAP Immunoreactivity, 201 GPR54 mRNA, 207 Kappa Opioid receptors Autoradiography, 9, 12 Immunoreactivity, 19 mRNA, 16 MCH Immunoreactivity, 41, 42 Mu Opioid receptors Autoradiograhy, 8 Immunoreactivity, 17, 19 mRNA, 14 NT Immunoreactivity, 327 NTS 1 receptor Autoradiography, 347 Immunoreactivity, 351,354 mRNA, 349, 351 NTS2 receptor mRNA, 375 OFQ Immunoreactivity, 109, 110, 130, 132, 133 mRNA, 158 OREXIN Immunoreactivity, 248, 249 ORL1 receptor Autoradiography, 149 Immunoreactivity, 106 mRNA, 132, 133, 160, 161 OX1R mRNA, 258, 260 OX2R mRNA, 258, 260, 262 PreproOFQ mRNA, 130, 132, 133, 156, 157, 158 PreproOREXIN mRNA, 247 SLC-1 Immunoreactivity, 57 mRNA, 59

Subject Index IMMUNE SYSTEM

Opioid effects, 3 INFERIOR COLLICULUS

Delta Opioid receptors Autoradiography, 9 mRNA, 15 GALANIN Immunoreactivity, 197 125I-GALANIN Autoradiography, 216 GALR1 mRNA, 219 Kappa Opioid receptors Autoradiography, 12 Immunoreactivity, 21 Mu Opioid receptors Immunoreactivity, 18 mRNA, 14 NTS2 receptor mRNA, 375 OFQ Immunoreactivity, 138 ORL1 receptor mRNA, 139, 160 PreproOFQ mRNA, 137 SLC-1 Immunoreactivity, 72 mRNA, 59 INFUNDIBULUM / I NFUND IB ULAR STEM, INFUNDIBULUM STALK

GALP Immunoreactivity, 202 Kappa Opioid receptors mRNA, 16 Mu Opioid receptors Autoradiography, 8 INTERPEDUNCULAR

NUCLEUS/INTERPEDUNCULAR COMPLEX

Delta Opioid receptors Immunoreactivity, 19 mRNA, 15 Kappa Opioid receptors Autoradiography, 9, 12

mRNA, 16 Mu Opioid receptors mRNA, 14 NTS 1 receptor Autoradiography, 347 NTS2 receptor Autoradiography, 369 mRNA, 375 OFQ Immunoreactivity, 138 mRNA, 158 ORL 1 receptor mRNA, 139, 160, 161 OX1R Immunoreactivity, 262 PreproOFQ mRNA, 137, 158, 159 SLC-1 Immunoreactivity, 74 ISLANDS OF CALLEJA

125I-GALANIN Autoradiography, 223 GALR3 mRNA, 225 NTS 1 receptor Autoradiography, 337 Immunoreactivity, 337, 351,352 mRNA, 337, 350 OFQ Immunoreactivity, 130 mRNA, 129 ORL1 receptor mRNA, 130 PreproOFQ mRNA, 129 SLC-1 Immunoreactivity, 64 KAPPA OPIOID RECEPTORS, 7

Autoradiography, 9 Immunoreactivity, 19 mRNA, 16 LOCUS COERULUS

Delta Opioid receptors 409

Subject Index Immunoreactivity, 19 GALANIN Immunoreactivity, 197 125I-GALANIN Autoradiography, 216 GALR1 mRNA, 219 GALR2 mRNA, 223 GALR3 mRNA, 228 Kappa Opioid receptors Autoradiography, 12 mRNA, 16 Mu Opioid receptors Immunoreactivity, 18, 19 mRNA, 15 OFQ Immunoreactivity, 140 OREXIN Immunoreactivity, 249 ORL1 receptor Autoradiography, 151 mRNA, 141 OX1R Immunoreactivity, 262 mRNA, 258, 262 PreproOFQ mRNA, 140 SLC-1 Immunoreactivity, 72 MAMMILLARY NUCLEUS/MAMM. COMPLEX, MAMM. PEDUNCLE GALR2 mRNA, 223 Kappa Opioid receptors mRNA, 16 Mu Opioid receptors Immunoreactivity, 17 NTS 1 receptor Autoradiography, 347 NTS2 receptor mRNA, 375 OFQ Immunoreactivity, 133 mRNA, 158

410

ORL1 receptor Autoradiography, 149 mRNA, 133, 161 PreproOFQ mRNA, 133, 137 MEDIAN EMINENCE GALP Immunoreactivity, 202 mRNA, 202 Kappa Opioid receptors Autoradiography, 9, 12 mRNA, 16 MCH Immunoreactivity, 42 NTS 1 receptor Immunoreactivity, 354 OFQ Immunoreactivity, 133 ORL 1 receptor mRNA, 134 PreproOFQ mRNA, 133

MEDULLA/MEDULLARY RETICULAR FORMATION Delta Opioid receptors Immunoreactivity, 19 GALANIN Immunoreactivity, 196 125I-GALANIN Autoradiography, 215 GALR2 mRNA, 223 GALR3 mRNA, 228 Mu Opioid receptors Autoradiography, 8 mRNA, 15 NTS 1 receptor Autoradiography, 349 Immunoreactivity, 354 NTS2 receptor mRNA, 377 OFQ Immunoreactivity, 141 ORL1 receptor mRNA, 142, 160

Subject Index

OX2R mRNA, 262 PreproOFQ mRNA, 141 MELANIN-CONCENTRATING HORMONE (MCH), 31,248

Binding sites, 51 Degradation by peptidases, 45 Discovery, 31-33 Feeding, food intake, 35, 38, 48-50, 85 MCH2 receptor subtype, 52-54 Pharmacology, 56 Signalling, 55 Physiological secretion, 45 PRO-MCh Gene, 36 Receptor, see also SLC-1, 50 Regulation of the HPA, 46 Reproductive functions, 47 SLC-1 receptor subtype, 51-52 Pharmacology, 55 Signalling, 54 Vigilance, 80 Water balance, 47 MESENCEPHALON

Delta Opioid receptors mRNA, 15, 16 GALANIN Immunoreactivity, 197 125I-GALANIN Autoradiography, 216 GALR1 mRNA, 219 GALR2 mRNA, 223 GALR3 mRNA, 227 Kappa Opioid receptors Immunoreactivity, 21 mRNA, 16 Mu Opioid receptors mRNA, 14 NTS 1 receptor Autoradiography, 347 Immunoreactivity, 354 mRNA, 351 NTS2 receptor

mRNA, 375 OFQ Immunoreactivity, 138 ORL1 receptor Autoradiography, 150 mRNA, 138 PreproOFQ mRNA, 137 METENCEPHALON

OFQ Immunoreactivity, 140 ORL1 receptor Autoradiography, 151 mRNA, 141 PreproOFQ mRNA, 140 MONKEY BRAIN

NT Autoradiography, 346, 347 NTS 1 receptor Autoradiography, 345, 346, 347, 349 MORRIS SWIM MAZE, 198 MU OPIOID RECEPTORS, 5, 198

Autoradiography, 8 Immunoreactivity, 17 mRNA, 12 MYELENCEPHALON

MCH Immunoreactivity, 42 OFQ Immunoreactivity, 141 ORL1 receptor Autoradiography, 152 mRNA, 141 PreproOFQ mRNA, 141 NEOCORTEX

Delta Opioid receptors Autoradiography, 9 125I-GALANIN 411

Subject Index Autoradiography, 207, 215 GALR2 mRNA, 222 Kappa Opioid receptors Autoradiography, 12 MCH Immunoreactivity, 42 NTS 1 receptor Autoradiography, 337 OFQ Immunoreactivity, 122 mRNA, 157 ORL1 receptor Autoradiography, 148 mRNA, 126, 160, 161 PreproOFQ mRNA, 111,157, 158, 159 NEUROMEDIN N, 325,326, 327 NEUROTENSIN RECEPTORS

Agonists, 334 Antagonists, 335 Discovery, 323 Feeding, 328 NTS 1 association with midbrain DA pathways, 356-358 with basal forebrain cholinergic cells, 358-360 with VIP neurons in the suprachiasmatic nucleus, 360-361 Ontogeny, 364-367, 382 Sleep, 328 Structure, 325 Subcellular distribution of NTS 1 receptors, 361,364 Subtypes: NTS 1, NTS2, NTS3, 332-334 NOCICEPTION

ORL1 Receptor, 105 NT, 327 NUCLEUS ACCUMBENS Delta Opioid receptors Autoradiography, 9 412

mRNA, 15 125I-GALANIN Autoradiography, 216 GALR1 mRNA, 217 GALR3 mRNA, 225 Kappa Opioid receptors Autoradiography, 9, 12 Immunoreactivity, 19 mRNA, 16 Mu Opioid receptors Autoradiography, 8 Immunoreactivity, 17 mRNA, 14 NT Autoradiography, 345 Immunoreactivity, 327 NTS 1 receptor Immunoreactivity, 352 mRNA, 350 NTS2 receptor Autoradiography, 369 mRNA, 373 OFQ Immunoreactivity, 129 mRNA, 129 ORL1 receptor Autoradiography, 148 mRNA, 130 OX2R mRNA, 258 PreproOFQ mRNA, 129 SLC-1 Immunoreactivity, 69 NUCLEUS AMBIGUS Delta Opioid receptors Immunoreactivity, 19 mRNA, 16 Mu Opioid receptors Autoradiography, 8 Immunoreactivity, 18, 19 mRNA, 15 OFQ Immunoreactivity, 141 ORL1 receptor

Subject Index mRNA, 142 OX2R mRNA, 262 SLC-1 Immunoreactivity, 72 NUCLEUS OF THE SOLITARY TRACT/NUCLEUS TRACTUS SOLITARIUS

Delta Opioid receptors Autoradiography, 9 Immunoreactivity, 19 mRNA, 16 GALANIN Immunoreactivity, 197 125I-GALANIN Autoradiography, 216 GALR1 mRNA, 219 Kappa Opioid receptors Immunoreactivity, 21 mRNA, 17 Mu Opioid receptors Autoradiography, 8 Immunoreactivity, 18, 19 mRNA, 15 NT Immunoreactivity, 327 NTS 1 receptor Immunoreactivity, 355 NTS2 receptor mRNA, 377 OFQ Immunoreactivity, 141 ORL 1 receptor Autoradiography, 152 mRNA, 142 OX1R mRNA, 262 OX2R mRNA, 262 SLC-1 Immunoreactivity, 72 mRNA, 59 OLFACTORY BULB Delta Opioid receptors

Autoradiography, 9 Immunoreactivity, 19 mRNA, 15 125I-GALANIN Autoradiography, 215 GALR1 mRNA, 217 GALR2 mRNA, 221 Kappa Opioid receptors Autoradiography, 12 mRNA, 16 Mu Opioid receptors Immunoreactivity, 17 mRNA, 14 NTS 1 receptor mRNA, 350 NTS2 receptor Autoradiography, 369 mRNA, 373 ORL1 receptor Autoradiography, 148 mRNA, 127, 160 PreproOFQ mRNA, 156 OLFACTORY NUCLEUS Delta Opioid receptors mRNA, 15 125I-GALANIN Autoradiography, 216 GALR1 mRNA, 217 GALR2 mRNA, 221 Kappa Opioid receptors mRNA, 16 Mu Opioid receptors Autoradiography, 8 Immunoreactivity, 17 NTS2 receptor mRNA, 373 OFQ Immunoreactivity, 129 mRNA, 129 ORL1 receptor Autoradiography, 148 mRNA, 130, 160, 161

413

Subject Index OX1R Immunoreactivity, 258 PreproOFQ mRNA, 129 OLFACTORY TUBERCULE Delta Opioid receptors Autoradiography, 9 mRNA, 15 GALR2 mRNA, 221 GALR3 mRNA, 225 Kappa Opioid receptors Autoradiography, 9, 12 Immunoreactivity, 19 mRNA, 16 MCH Immunoreactivity, 41 NTS2 receptor Autoradiography, 369 OFQ Immunoreactivity, 130 mRNA, 129, 157 ORL1 receptor Autoradiography, 148 OX1R Immunoreactivity, 258 OX2R mRNA, 258 PreproOFQ mRNA, 129, 157, 158 SLC-1 Immunoreactivity, 64 mRNA, 59 OLIVARY COMPLEX/oLIVARY NUCLEUS

Delta Opioid receptors Immunoreactivity, 19 mRNA, 16 Kappa Opioid receptors mRNA, 17 Mu Opioid receptors Immunoreactivity, 19 OREXIN RECEPTORS Biology, 247

414

Discovery, 245 Features of Orexin system in mammals, 247-249 Feeding, 250, 313 Sleep, 251, 313 Structures of OREXIN-A and -B, 245, 246 Vigilance, 251, 313 Water intake, 251, 313 ORL1 RECEPTOR Autonomic and physiologic function, 173 Colocalization with Mu receptor, 166 with Kappa receptor, 166 with Delta receptor, 167 Learning and memory, 169 Limbic hypothalamic-pituary-adremal axis (L-HPA), 168 Motor system, 169 Pain perception, 172 Reinforcement and reward, 170 Sexual behavior, 171 Special sensory systems, 173 ORPHANIN FQ, 109 Autonomic and physiologic function, 173 Immunochemistry, 110 Learning and memory, 169 Limbic hypothalamic-pituary-adremal axis (L-HPA) 168 Motor system, 169 Pain perception, 172 Reinforcement and reward, 170 Sexual behavior, 171 Special sensory systems, 173

PALLIDUM Delta Opioid receptors mRNA, 15 Kappa Opioid receptors Immunoreactivity, 19 mRNA, 16 Mu Opioid receptors Immunoreactivity, 17

Subject Index NTS 1 receptor mRNA, 350 OFQ Immunoreactivity, 130 mRNA, 129 ORL1 receptor mRNA, 130, 160 PreproOFQ mRNA, 129, 158 SLC-1 Immunoreactivity, 69 PARABRACHIAL NUCLEUS

Delta Opioid receptors Immunoreactivity, 19 mRNA, 16 GALANIN Immunoreactivity, 197 125I-GALANIN Autoradiography, 216 GALR1 mRNA, 219 GALR3 mRNA, 227, 228 Kappa Opioid receptors Autoradiography, 12 mRNA, 16 MCH Immunoreactivity, 42 Mu Opioid receptors Autoradiography, 8 Immunoreactivity, 18 mRNA, 15 NT Immunoreactivity, 348 NTS 1 receptor Autoradiography, 348 OFQ Immunoreactivity, 140 ORL1 receptor Autoradiography, 151 mRNA, 139, 141 PreproOFQ mRNA, 140 PARASUBICULUM

Delta Opioid receptors mRNA, 15

Kappa Opioid receptors mRNA, 16 NTS 1 receptor Autoradiography, 346 Immunoreactivity, 351,352 mRNA, 350 SLC-1 Immunoreactivity, 69 PARAVENTRICULAR NUCLEUS

GALR2 mRNA, 223 MCH Immunoreactivity, 41, 42 NTS 1 receptor Autoradiography, 346, 347 Immunoreactivity, 353, 354 mRNA, 351 OFQ Immunoreactivity, 133 mRNA, 158 ORL1 receptor Autoradiography, 149, 150 mRNA, 133, 160, 161 OX1R Immunoreactivity, 260 mRNA, 260 OX2R mRNA, 258, 260 PreproOFQ mRNA, 133, 158 PERIAQUEDUCTAL GRAY

Delta Opioid receptors Autoradiography, 9 Immunoreactivity, 19 mRNA, 15 GALANIN Immunoreactivity, 197 125I-GALANIN Autoradiography, 216 GALR1 mRNA, 219 GALR2 mRNA, 223 GALR3 mRNA, 227 Kappa Opioid receptors 415

Subject Index Autoradiography, 12 Immunoreactivity, 21 mRNA, 16 MCH Immunoreactivity, 42 Mu Opioid receptors Immunoreactivity, 18 mRNA, 14 NT Immunoreactivity, 327 NTS 1 receptor Autoradiography, 347 Immunoreactivity, 354 mRNA, 351 NTS2 receptor Autoradiography, 369 mRNA, 375 OX1R Immunoreactivity, 262 mRNA, 262 OX2R mRNA, 262 PERIFORNICAL NUCLEUS OFQ Immunoreactivity, 133 ORL 1 receptor Autoradiography, 149, 150, PreproOFQ mRNA, 133 PITUITARY/PITUARY GLAND GALANIN mRNA, 203 GMAP Immunoreactivity, 202 ORL1 receptor mRNA, 134 PreproOFQ mRNA, 133 PONS GALANIN Immunoreactivity, 196 125I-GALANIN Autoradiography, 215 GALR2 mRNA, 223

416

GALR3 mRNA, 228 GMAP Immunoreactivity, 201 GPR54 mRNA, 207 MCH Immunoreactivity, 42 NT Autoradiography, 347 Immunoreactivity, 327 NTS 1 receptor Immunoreactivity, 354 mRNA, 351 NTS2 receptor mRNA, 377 ORL1 receptor mRNA, 160 OX2R mRNA, 262 SLC-1 mRNA, 59 PREOPTIC NUCLEUS GALR1 mRNA, 218 GALR3 mRNA, 227 MCH Immunoreactivity, 41 NTS 1 receptor Immunoreactivity, 351,352 NTS2 receptor mRNA, 373 OFQ Immunoreactivity, 132 ORL1 receptor Autoradiography, 149 mRNA, 132, 161 OX1R Immunoreactivity, 259 OX2R mRNA, 262 PreproOFQ mRNA, 132 SLC-1 Immunoreactivity, 69

Subject Index PREPRO-OREXIN, 246, 247 PRESUBICULUM

Delta Opioid receptors mRNA, 15 Kappa Opioid receptors mRNA, 16 NTS 1 receptor Autoradiography, 346 Immunoreactivity, 351,352 mRNA, 350 SLC-1 Immunoreactivity, 69 PRETECTAL NUCLEI

Mu Opioid receptors Autoradiography, 8 PRODYNORPHIN

Comparison with Kappa receptor, 166 PROENKEPHALIN

Comparison with Delta receptor, 167 PROOPIOMELANOCORTIN

Comparison with the Mu receptor, 166 RAPHE NUCLEI Delta Opioid receptors Immunoreactivity, 19 mRNA, 16 GALANIN Immunoreactivity, 197 125I-GALANIN Autoradiography, 216 GALR1 mRNA, 219 Kappa Opioid receptors Autoradiography, 12 mRNA, 16 Mu Opioid receptors Autoradiography, 8 Immunoreactivity, 18 mRNA, 14 NT Autoradiography, 347, 349 Immunoreactivity, 327, 347

NTS 1 receptor Autoradiography, 337, 347 Immunoreactivity, 337, 354, 355 mRNA, 337, 351 NTS2 receptor Autoradiography, 369 mRNA, 375, 377 OFQ Immunoreactivity, 138, 142 OREXIN Immunoreactivity, 249 ORL1 receptor mRNA, 138, 141,142 OX1R mRNA, 258, 262 OX2R mRNA, 262 PreproOFQ mRNA, 137, 140 SLC-1 Immunoreactivity, 74 RED NUCLEUS Delta Opioid receptors Immunoreactivity, 19 NTS2 receptor Autoradiography, 369 mRNA, 375 OFQ Immunoreactivity, 138 ORL1 receptor mRNA, 138, 139, 161 PreproOFQ mRNA, 137 SLC-1 Immunoreactivity, 72 RETICULAR NUCLEI/PONTINE

RETICULAR NUCLEI/PONTINE RETICULAR FORMATION, PONTINE NUCLEI Delta Opioid receptors Immunoreactivity, 19 mRNA, 16 GALANIN Immunoreactivity, 197 GALR1 mRNA, 219 417

Subject Index GALR2 mRNA, 223 Kappa Opioid receptors mRNA, 16, 17 MCH Immunoreactivity, 41, 42 Mu Opioid receptors mRNA, 15 NT Autoradiography, 347, 349 NTS 1 receptor Autoradiography, 337 Immunoreactivity, 337, 354, 355 mRNA, 337 NTS2 receptor mRNA, 373, 377 OFQ Immunreactivity, 138, 140 mRNA, 158 ORL1 receptor Autoradiography, 152 mRNA, 139, 141,160 OX1R Immunoreactivity, 260, 262 PreproOFQ mRNA, 137, 140, 141,158 SLC-1 mRNA, 59 RI-IOMBENCEPI-IALON 125I-GALANIN Autoradiography, 216 GALR1 mRNA, 219 GALR2 mRNA, 223 GALR3 mRNA, 227 RHOMBOID NUCLEI Kappa Opioid receptors Immunoreactivity, 21 mRNA, 16 OX2R mRNA, 260 SENSORY NUCLEUS/sENSORY NUCLEUS OF THE

418

TRIGEMINAL/SENSORY TRIGEMINAL NUCLEUS Delta Opioid receptors mRNA, 16 Kappa Opioid receptors mRNA, 17 ORL1 receptor Autoradiography, 151 PreproOFQ mRNA, 140 SLC-1 Immunoreactivity, 72 SEPTUM/SEPTAL NUCLEUS Delta Opioid receptors mRNA, 15 GALANIN Immunoreactivity, 196 125I-GALANIN Autoradiography, 216 GALP Immunoreactivity, 202 GALR1 mRNA, 217 GALR3 mRNA, 225 GMAP Immunoreactivity, 201 Kappa Opioid receptors Autoradiography, 12 Immunoreactivity, 19 mRNA, 16 MCH Immunoreactivity, 42 Mu Opioid receptors Autoradiography, 8 Immunoreactivity, 17, 19 mRNA, 14 NT Immunoreactivity, 327 NTS 1 receptor Immunoreactivity, 352 mRNA, 350 NTS2 receptor Autoradiography, 369 mRNA, 373 OFQ Immunoreactivity, 109, 130

Subject Index

mRNA, 157 OREXIN Immunoreactivity, ORL1 receptor Autoradiography, mRNA, 131,160, OX1R Immunoreactivity, mRNA, 259 OX2R mRNA, 259 PreproOFQ mRNA, 130 SLC-1 Immunoreactivity,

249 148 161 259

69

SLC-1 RECEPTOR, 35 SPINAL CORD

Delta Opioid receptors Autoradiography, 9 Immunoreactivity, 19 mRNA, 16 GALANIN Immunoreactivity, 197 125I-GALANIN Autoradiography, 216 GALR1 mRNA, 219 GALR2 mRNA, 225 GALR3 mRNA, 228 GMAP Immunoreactivity, 201 Kappa Opioid receptors Autoradiography, 12 mRNA, 17 Immunoreactivity, 21 MCH Immunoreactivity, 42 Mu Opioid receptors Autoradiography, 8 Immunoreactivity, 18, 19 mRNA, 15 NT Immunoreactivity, 327

NTS 1 receptor Autoradiography, 349 Immunoreactivity, 356 NTS2 receptor mRNA, 379 OFQ Immunoreactivity, 143 ORL1 receptor Autoradiography, 152 Biological effects, 105 Immunoreactivity, 106 mRNA, 110, 143, 160 OX1R Immunoreactivity, 263 mRNA, 263 PreproOFQ mRNA, 110, 142, 157 SLC-1 Immunoreactivity, 74 SPINAL TRIGEMINAL NUCLEUS

Delta Opioid receptors Immunoreactivity, 19 mRNA, 16 Kappa Opioid receptors mRNA, 17 Mu Opioid receptors Autoradiography, 8 Immunoreactivity, 18, 19 NT Immunoreactivity, 327 OFQ Immunoreactivity, 142 ORL1 receptor Autoradiography, 152 mRNA, 141,142, 160 PreproOFQ mRNA, 141,157 STRIATUM/NEOSTRIATUM Delta Opioid receptors Autoradiography, 9 125I-GALANIN Autoradiography, 207, 216 Kappa Opioid receptors mRNA, 16 Mu Opioid receptors Immunoreactivity, 19 419

Subject Index NT Autoradiography, 345 NTS 1 receptor Immunoreactivity, 352 mRNA, 350 OFQ Immunoreactivity, 131 ORL1 receptor mRNA, 160 PreproOFQ mRNA, 131 SLC-1 Immunoreactivity, 69 SUBFORNICAL ORGAN

Mu Opioid receptors Autoradiography, 8 OREXIN Immunoreactivity, 251 SUBICULUM

Delta Opioid receptors mRNA, 15 125I-GALANIN Autoradiography, 216 Kappa Opioid receptors mRNA, 16 NTS 1 receptor Immunoreactivity, 352 mRNA, 350 NTS2 receptor mRNA, 373 ORL1 receptor mRNA, 160, 161 PreproOFQ mRNA, 159 SLC-1 Immunoreactivity, 69 SUBSTANTIA INNOMINATA

GALR1 mRNA, 217 NTS 1 receptor Autoradiography, 337 Immunoreactivity, 337, 352 mRNA, 337 OFQ Immunoreactivity, 130 420

ORL 1 receptor Autoradiography, 148 mRNA, 130 OX1R Immunoreactivity, 259 OX2R mRNA, 259 SLC-1 Immunoreactivity, 69 SUBSTANTIA NIGRA

Delta Opioid receptors Autoradiography, 9 Immunoreactivity, 19 mRNA, 15 GALANIN Immunoreactivity, 197 125I-GALANIN Autoradiography, 216 GALR1 mRNA, 219 GALR2 mRNA, 223 GALR3 mRNA, 227 Kappa Opioid receptors Autoradiography, 12 Immunoreactivity, 21 mRNA, 16 Mu Opioid receptors mRNA, 14 NT Autoradiography, 347 Immunoreactivity, 327 NTS 1 receptor Autoradiography, 347 Immunoreactivity, 351,354 mRNA, 351 NTS2 receptor Autoradiography, 369 mRNA, 375 OFQ Immunoreactivity, 131 mRNA, 157, 158 ORL 1 receptor Autoradiography, 148 mRNA, 131,160, 161

Subject Index OX1R mRNA, 262 PreproOFQ mRNA, 131,158, 159 SLC-1 Immunoreactivity, 72 SUBTHALAMIC NUCLEUS OFQ Immunoreactivity, 131 mRNA, 157 ORL1 receptor Autoradiography, 149 mRNA, 131 OX1R Immunoreactivity, 260 mRNA, 260 OX2R mRNA, 258 PreproOFQ mRNA, 131

SUPERIOR COLLICULUS Delta Opioid receptors Autoradiography, 9 mRNA, 15 125I-GALANIN Autoradiography, 216 GALR1 mRNA, 219 Kappa Opioid receptors Autoradiography, 12 Immunoreactivity, 21 mRNA, 16 Mu Opioid receptors Autoradiography, 8 Immunoreactivity, 18 mRNA, 14 NTS 1 receptor Autoradiography, 347 Immunoreactivity, 354 mRNA, 351 NTS2 receptor Autoradiography, 369 mRNA, 375 OFQ Immunoreactivity, 138 ORL1 receptor

Autoradiography, 150 mRNA, 139, 160, 161 PreproOFQ mRNA, 137, 157 SLC-1 mRNA, 59 SUPRACI-IIASMATIC NUCLEUS NTS 1 receptor Autoradiography, 347 Immunoreactivity, 351,354 mRNA, 351 NTS2 receptor mRNA, 375 OFQ Immunoreactivity, 133 ORL1 receptor Autoradiography, 149 OX1R Immunoreactivity, 260 PreproOFQ mRNA, 133 SLC-1 Immunoreactivity, 69 SUPRAOPTIC NUCLEUS GALR1 mRNA, 217 MCH Immunoreactivity, 42 Mu Opioid receptors Immunoreactivity, 17 NTS 1 receptor mRNA, 351 NTS2 receptor mRNA, 375 OFQ Immunoreactivity, 133 ORL1 receptor Autoradiograpyhy, 149 mRNA, 133, 160, 161 OX1R Immunoreactivity, 260 mRNA, 260 PreproOFQ mRNA, 133 421

Subject Index TEGMENTAL NUCLEI Delta Opioid receptors Immunoreactivity, 19 NT Autoradiography, 347 NTS 1 receptor Immunoreactivity, 354 OFQ Immunoreactivity, 140 OX1R mRNA, 262 TELENCEPHALON

Delta Opioid receptors mRNA, 15 GALANIN Immunoreactivity, 196 125I-GALANIN Autoradiography, 215 GALR1 mRNA, 217 GALR2 mRNA, 221 GALR3 mRNA, 227 Kappa Opioid receptors Immunoreactivity, 19 Mu Opioid receptors mRNA, 14 NTS 1 receptor Autoradiography, 337 Immunoreactivity, 352 mRNA, 350 NTS2 receptor mRNA, 373 ORL1 receptor Autoradiography, 149 OX1R mRNA, 258 OX2R mRNA, 258 PreproOFQ mRNA, 131 THALAMUS/THALAMUS NUCLEI Delta Opioid receptors Autoradiography, 9 mRNA, 15 422

GALANIN Immunoreactivity, 196 125I-GALANIN Autoradiography, 216 GALR1 mRNA, 217 GALR2 mRNA, 223 Kappa Opioid receptors Autoradiograhy, 9, 12 Immunoreactivity, 21 mRNA, 16 Mu Opioid receptors Autoradiography, 8 Immunoreactivity, 17, 18 mRNA, 14 NTS 1 receptor Autoradiography, 337, 346 Immunoreactivity, 337, 351,354 mRNA, 337, 350 NTS2 receptor Autoradiography, 369 mRNA, 373 OFQ Immunoreactivity, 109, 136 mRNA, 158 OREXIN Immunoreactivity, 249 ORL1 receptor Autoradiography, 150 mRNA, 136, 160, 161 OX1R mRNA, 260 OX2R mRNA, 260 PreproOFQ mRNA, 136, 159 SLC-1 Immunoreactivity, 57, 69 mRNA, 59 TRAPEZOID NUCLEUS

Delta Opioid receptors mRNA, 16 Kappa Opioid receptors mRNA, 17

Subject Index TRIGEMINAL MOTOR NUCLEUS Delta Opioid receptors Immunoreactivity, 19 mRNA, 16 PreproOFQ mRNA, 140 TRIGEMINAL NUCLEUS/SENSORY NUCLEUS OF TRIGEMINALNERVE Delta Opioid receptors mRNA, 16 GALR2 mRNA, 223 Kappa Opioid receptors Immunoreactivity, 21 mRNA, 17 NTS2 receptor mRNA, 377 OFQ Immunoreactivity, 140 ORL1 receptor Autoradiography, 151 OX2R mRNA, 262 PreproOFQ mRNA, 140 TUBER CINEREUM OFQ Immunoreactivity, 133 ORL1 receptor Autoradiography, 149 mRNA, 133 PreproOFQ mRNA, 133 TUBERAL MAGNOCELLULAR NUCLEI OFQ Immunoreactivity, 133 ORL1 receptor mRNA, 133 PreproOFQ mRNA, 133 VENTRAL TEGMENTAL AREA (VTA)/VENTROLATERAL TEG. NUCLEI

Delta Opioid receptors mRNA, 15 GALANIN Immunoreactivity, 197 GALR2 mRNA, 223 Kappa Opioid receptors mRNA, 16 Mu Opioid receptors mRNA, 14 NT Autoradiography, 347 Immunoreactivity, 327 NTS 1 receptor Immunoreactivity, 354 mRNA, 351 NTS2 receptor Autoradiography, 369 mRNA, 375 ORL1 receptor mRNA, 138, 139, 160, 161 OX1R mRNA, 262 OX2R mRNA, 262 PreproOFQ mRNA, 137, 158, 159 SLC-1 Immunoreactivity, 72 VESTIBULAR NUCLEI Delta Opioid receptors Immunoreactivity, 19 mRNA, 16 Kappa Opioid receptors mRNA, 17 NT Autoradiography, 349 NTS 1 receptor Immunoreactivity, 354 OFQ Immunoreactivity, 140 ORL1 receptor Autoradiography, 152 mRNA, 141, 142 PreproOFQ mRNA, 140

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