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Ciba Foundation Symposium 172 CORTICOTROPIN- RELEASING FACTOR A Wiley-Interscience Publication 1993 JOHN WILEY & SONS Chichester . New York . Brisbane . Toronto . Singapore

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Page 1: CORTICOTROPIN- RELEASING · The functional neuroanatomy of corticotropin-releasing factor 5 Discussion 2 1 J. A. Majzoub, R. Emanuel, G. Adler, C. Martinez, B. Robinson and G. Wittert

Ciba Foundation Symposium 172

CORTICOTROPIN- RELEASING

FACTOR

A Wiley-Interscience Publication

1993

JOHN WILEY & SONS

Chichester . New York . Brisbane . Toronto . Singapore

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Page 3: CORTICOTROPIN- RELEASING · The functional neuroanatomy of corticotropin-releasing factor 5 Discussion 2 1 J. A. Majzoub, R. Emanuel, G. Adler, C. Martinez, B. Robinson and G. Wittert

CORTICOTROPIN-RELEASING FACTOR

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The Ciba Foundation is an international scientific and educational charity. It was established in 1947 by the Swiss chemical and pharmaceutical company of ClBA Limited-now Ciba-Geigy Limited. The Foundation operates independently in London under English trust law.

The Ciba Foundation exists to promote international cooperation in biological, medical and chemical research. It organizes about eight international multidisciplinary symposia each year on topics that seem ready for discussion by a small group of research workers. The papers and discussions are published in the Ciba Foundation symposium series. The Foundation also holds many shorter meetings (not published), organized by the Foundation itself or by outside scientific organizations. The staff always welcome suggestions for future meetings.

The Foundation’s house at 41 Portland Place, London W1N 4BN, provides facilities for meetings of all kinds. Its Media Resource Service supplies information to journalists on all scientific and technological topics. The library, open five days a week to any graduate in science or medicine, also provides information on scientific meetings throughout the world and answers general enquiries on biomedical and chemical subjects. Scientists from any part of the world may stay in the house during working visits to London.

Page 5: CORTICOTROPIN- RELEASING · The functional neuroanatomy of corticotropin-releasing factor 5 Discussion 2 1 J. A. Majzoub, R. Emanuel, G. Adler, C. Martinez, B. Robinson and G. Wittert

Ciba Foundation Symposium 172

CORTICOTROPIN- RELEASING

FACTOR

A Wiley-Interscience Publication

1993

JOHN WILEY & SONS

Chichester . New York . Brisbane . Toronto . Singapore

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OCiba Foundation 1993

Published in 1993 by John Wiley & Sons Ltd Baffins Lane, Chichester West Sussex PO19 IUD, England

All rights reserved.

No part of this book may be reproduced by any means, or transmitted, or translated into a machine language without the written permission of the publisher.

Other Wiley Editorial Offices

John Wiley & Sons, Inc., 605 Third Avenue, New York, NY 10158-0012, USA

Jacaranda Wiley Ltd, G.P.O. Box 859, Brisbane, Queensland 4001, Australia

John Wiley & Sons (Canada) Ltd, 22 Worcester Road, Rexdale, Ontario M9W 1L1, Canada

John Wiley & Sons (SEA) Pte Ltd, 37 Jalan Pemimpin #05-04, Block B, Union Industrial Building, Singapore 2057

Suggested series entry for library catalogues: Ciba Foundation Symposia

Ciba Foundation Symposium 172 x+357 pages, 56 figures, 12 tables

Library of Congress Cataloging-in-Publication Data Corticotropin-releasing factor/ [editors, Derek J. Chadwick, Joan

Marsh, and Kate Ackrill] . p. cm.-(Ciba Foundation symposium; 172.)

"Symposium on Corticotropin-Releasing Factor, held at the Ciba

Includes bibliographical references and index. ISBN 0 471 93448 8 1. Corticotropin releasing hormone-Congresses.

Foundation, London, 10-12 March 1992."

I. Chadwick, Derek. 11. Marsh, Joan. 111. Ackrill, Kate. IV. Symposium on Corticotropin-Releasing Factor (1993: Ciba Foundation) QP572.C62C66 1992

V. Series.

612.8'262 - dc20 92-36532 CIP

British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library

ISBN 0 471 93448 8

Phototypeset by Dobbie Typesetting Limited, Tavistock, Devon. Printed and bound in Great Britain by Biddles Ltd, Guildford.

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Contents

Symposium on Corticotropin-Releasing Factor, held at the Ciba

Editors: Derek J. Chadwick, Joan Marsh (Organizers) and Kate Ackrill

The topic for this symposium was proposed by Dr Ashley Grossman

Foundation, London, 10-12 March 1992

W. Vale Introduction 1

P. E. Sawchenko, T. Imaki, E. Potter, K. KOV~CS, J. Imaki and W. Vale The functional neuroanatomy of corticotropin-releasing factor 5 Discussion 2 1

J. A. Majzoub, R. Emanuel, G. Adler, C. Martinez, B. Robinson and G. Wittert Second messenger regulation of mRNA for corticotropin- releasing factor 30 Discussion 43

P. M. Plotsky, K. V. Thrivikraman and M. J. Meaney Central and feedback regulation of hypothalamic corticotropin-releasing factor secretion 59 Discussion 75

D. E. Grigoriadis, J. A. Heroux and E. B. De Souza Characterization and regulation of corticotropin-releasing factor receptors in the central nervous, endocrine and immune systems Discussion 10 1

85

P. J. Lowry Corticotropin-releasing factor and its binding protein In human plasma 108 Discussion 1 15

A. Grossman, A. Costa, P. Navarra and S. Tsagarakis The regulation of 129 hypothalamic corticotropin-releasing factor release: in vitro studies

Discussion 143

V

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Contents VI

0. F. X. Almeida, A. H. S. Hassan and F. Holsboer Intrahypothalamic neuroendocrine actions of corticotropin-releasing factor Discussion 169

15 1

S. L. Lightman and M. S. Harbuz Expression of corticotropin-releasing factor mRNA in response to stress Discussion 181

173

General discussion A consideration of methodology 199

C. Rivier and S. Rivest Mechanisms mediating the effects of cytokines on neuroendocrine functions in the rat 204 Discussion 220

A. J. Dunn Infection as a stressor: a cytokine-mediated activation of the hypothalamo-pituitary-adrenal axis? 226 Discussion 239

L. A. Fisher Central actions of corticotropin-releasing factor on autonomic nervous activity and cardiovascular function 243 Discussion 253

E. T. Wei, G. C. Gao and H. A. Thomas Peripheral anti-inflammatory actions of corticotropin-releasing factor 258 Discussion 268

G . F. Koob, S. C. Heinrichs, E. M. Pich, F. Menzaghi, H. Baldwin, K. Miczek and K. T. Britton in behavioural responses to stress 277 Discussion 290

The role of corticotropin-releasing factor

M. J. Owens and C. B. Nemeroff The role of corticotropin-releasing factor in the pathophysiology of affective and anxiety disorders: laboratory and clinical studies 296 Discussion 308

K. von Werder and 0. A. Miiller The role of corticotropin-releasing factor in the investigation of endocrine diseases Discussion 3 33

317

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Contents

Final general discussion Functional anatomy 337 Regulation of CRF 338 Immune aspects 339 Clinical aspects 340 Future directions 341

vii

Index of contributors 342

Subject index 344

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Participants

G. Aguilera Endocrine Physiology Section, Developmental Endocrinology Branch, NICHD, NIH, Building 10 Room 10N262, Bethesda, MD 20892, USA

0. F. X. Almeida Department of Neuroendocrinology, Max Planck Institute for Psychiatry, Kraepelinstrasse 2- 10, D-8000 Munich 40, Germany

I. Assenmacher Department of Physiology, Laboratoire de Neurobiologie Endocrinologique, CNRS Unit 1 197, UniversitC de Montpellier 2, Place Eugene Bataillon, F-34095 Montpellier Cedex 5 , France

F. Berkenbosch Department of Pharmacology, Free University of Amsterdam, Van der Boechorststraat 7, NL-1081 BT, Amsterdam, The Netherlands

E. R. de Kloet Division of Medical Pharmacology, Center for Bio- Pharmaceutical Sciences, University of Leiden, PO Box 9503, 2300 RA Leiden, The Netherlands

E. B. De Souza Central Nervous System Diseases Research, The Du Pont Merck Pharmaceutical Company, Experimental Station E400/4358, PO Box 80400, Wilmington, DE 19880-0400, USA

A. J. Dunn Department of Pharmacology 8z Therapeutics, Louisiana State University Medical Center, 1501 Kings Highway, PO Box 33932, Shreveport, LA 71130-3932, USA

G. Fink MRC Brain Metabolism Unit, Department of Pharmacology, University of Edinburgh, 1 George Square, Edinburgh EH8 9JZ, UK

L. A. Fisher Department of Pharmacology, College of Medicine, Arizona Health Sciences Center, Tucson, AZ 85724, USA

P. W. Gold Clinical Neuroendocrinology Branch, NIMH, Building 10 Room 38231, Bethesda, MD 20892, USA

viii

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Participants ix

A. B. Grossman Department of Endocrinology, St Bartholomew’s Hospital, West Smithfield, London EClA 7BE, UK

G. F. Koob Department of Neuropharmacology, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037, USA

R. Le Feuvre Department of Physiological Sciences, School of Biological Sciences, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, UK

S. L. Lightman* Medical School, Charing Cross Hospital, Fulham Palace Road, London W6 8RF, UK

Neuroendocrinology Unit, Charing Cross & Westminster

P. J. Lowry Department of Biochemistry & Physiology, School of Animal and Microbial Sciences, AMS Building, University of Reading, PO Box 228, Whiteknights, Reading RG6 2AJ, UK

J. A. Majzoub Division of Endocrinology, Children’s Hospital, Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA

C. B. Nemeroff Laboratory of Neuropsychopharmacology, Department of Psychiatry, Emory University School of Medicine, PO Drawer AF, Atlanta, GA 30322, USA

F. Petraglia Department of Obstetrics & Gynaecology, University of Modena School of Medicine, Via Pozzo 71, 1-41100 Modena, Italy

P. M. Plotsky Stress Neurobiology Laboratory, Department of Psychiatry, Emory University School of Medicine, Atlanta, GA 30322, USA

C. Rivier The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 North Torrey Pines Road, PO Box 85800, San Diego, CA 92128, USA

P. E. Sawchenko Laboratory of Neuronal Structure & Function, The Salk Institute, 10010 North Torrey Pines Road, PO Box 85800, San Diego, CA 92186, USA

*Present address: University of Bristol, Department of Medicine, Bristol Royal Infirmary, Upper Mouldin Street, Bristol BS2, UK

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X Participants

R. Smith Endocrine Unit, John Hunter Hospital, Locked Bag No. 1, Newcastle Mail Centre, NSW 2310, Australia

E. Sternberg Clinical Neuroendocrinology Branch, NIMH, Building 10 Room 38231, Bethesda, MD 20892, USA

F. J. H. Tilders Department of Pharmacology, Free University of Amsterdam, Van der Boechorststraat 7, NL-1081 BT Amsterdam, The Netherlands

W. W. Vale The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 North Torrey Pines Road, PO Box 85800, San Diego, CA 92128, USA

K. von Werder Department of Medicine, Schlosspark-Klinik, Free University of Berlin, Heubnerweg 2, D-1000 Berlin 19, Germany

E. T. Wei 316 Warren Hall, School of Public Health, University of California, Berkeley, CA 94720, USA

M. H. Whitnall Department of Physiology, Armed Forces Radiobiology Research Institute, Bethesda, MD 20889-5 145, USA

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Introduction Wylie Vale

The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 100 10 North Torrey Pines Road, PO Box 85800, San Diego, CA 92128, USA

The concepts of the neural regulation of the pituitary, championed by Sir Geoffrey Harris of Oxford (1948) and others, culminated in 1955 in the experiments of Guillemin & Rosenberg (1955) and Saffran & Schally (1955) who provided direct evidence for the existence of the first hypothalamic releasing factor, corticotropin-releasing factor (CRF). This factor was hypothesized to be produced in the hypothalamus, especially under stressful circumstances, and to reach the anterior pituitary through the hypothalamic hypophysial portal system where it regulated the production of adrenocorticotropic hormone (corticotropin, ACTH). With the realization that ACTH was processed from a precursor, pro-opiomelanocortin, that included other biologically active peptides, P-endorphin and a- and P-melanotropins, the efforts to characterize CRF took on additional significance. However, the structure of this molecule was not determined until 1981 when our group reported the isolation, characterization, synthesis and in vitro and in vivo biological activities of ovine hypothalamic CRF, a 41 amino acid straight chain polypeptide (Vale et a1 1981). The presence in hypothalamic extracts of vasopressin, oxytocin, angiotensin I1 and catecholamines, which are weak ACTH releasers on their own but can act in synergy with CRF, complicated the purification of CRF, as did the fact that the 41 residue CRF did not separate from ACTH on gel filtration. The fractions in that molecular weight range were ignored by most workers, who attributed the activity of the fractions to ACTH. The development of radioimmunoassays for ACTH (Orth 1979) and a quantitative in vitro method for assaying hypophysiotropic substances (Vale et a1 1972) allowed us to determine CRF activity in the presence of ACTH and to focus on that zone, from which we ultimately isolated CRF using ion exchange chromatography and high performance liquid chromatography.

Rat CRF, which we subsequently isolated (Rivier et a1 1983), is identical to human CRF, the amino acid sequence of which was deduced by Numa’s group from its genomic DNA sequence (Shibahara et a1 1983); both differ from ovine CRF by seven residues. Mammalian CRFs are related to sauvagine isolated from frog skin (Montecucchi & Henshen 1981) and urotensin I1 purified from fish urophyses (Lederis et a1 1982). Until recently, it was considered likely that

1

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2 Vale

sauvagine and urotensin I1 were the CRF equivalents in frogs and fish, but peptides that are much closer to mammalian CRFs have now been characterized in frog and fish species (Okawara et al 1988, Stenzel-Poore et a1 1992). Whether there is a mammalian homologue of sauvagine or urotensin remains an open question.

Results from studies monitoring dynamic changes in portal blood and median eminence CRF and paraventricular mRNA levels, when considered with results from immunoneutralization and CRF receptor blocking experiments, support the key role of this peptide in the regulation of the pituitary-adrenal axis during basal and many stressful circumstances. We now realize, however, that the distribution and the actions of CRF go beyond its role as a hypothalamic releasing factor. CRF also has a number of neuroendocrine actions at the hypothalamic level, modulating secretion of growth hormone and suppressing the secretion of reproductive hormones and inducing fever and inhibiting appetite. CRF also has effects on the autonomic nervous system, stimulating the sympathetic nervous system and inhibiting the parasympathetic nervous system. The behavioural effects of CRF range from arousal to anxiety to fear and depression, depending on the dose and the context of the experiment. These findings and many of the papers presented here will reinforce the notion that CRF is an important integrator and coordinator of the endocrine, neuroendocrine, autonomic and behavioural responses to stress (Vale & Greer 1985).

In addition to the paraventricular nucleus, the expression of CRF in some other brain areas is altered by stress; these include Barrington’s micturition centre and some areas of the hippocampus, where stress increases CRF mRNA, and the olfactory bulb, where stress decreases CRF mRNA (Imaki et al1991). Stress does not affect the expression of CRF in most neurons; the roles of CRF in the extra-paraventricular CRF system have not been established.

CRF is a key integrator of interactions between the neuroendocrine and immune systems. Cytokines such as interleukin 1 produced by monocytes and macrophages in response to pathogens can stimulate the production of CRF and thereby ACTH and glucocorticoids, which then suppress immune functions. Thus, it has been argued that the hypothalamo-pituitary-adrenal (HPA) axis has been co-opted by the immune system for its own negative regulation, CRF has been found in a number of peripheral sites, including sites of inflammation (Chrousos & Gold 1992), where the peptide may stimulate cytokine production and exert pro-inflammatory actions. The same regulators appear to be interacting at many levels. Interleukin 1 modulates hypothalamic CRF production, CRF can modulate peripheral interleukin 1 production, and glucocorticoids have effects on the production of CRF as well as on its actions at the level of the pituitary and on inflammatory processes. This concept of multiple-level interaction is an instructive one, and it stresses that CRF is not a circulating hormone-it is largely a locally acting hormone.

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Introduction 3

One of the most interesting of CRF’s paracrine or autocrine actions is in the placenta, where it plays a role in hormonogenesis and perhaps in the regulation of uterine contractility. CRF is produced in large quantities by the primate placenta, so much so that in the primate during pregnancy, especially during the later stages, CRF can be measured in the blood. A CRF-binding protein (CRF-BP) produced by the human liver can bind to and inactivate CRF. We collaborated with Phil Lowry in the cloning and characterization of this protein in humans and rats (Potter et a1 1991) and have found that it is present in the brain of several species where it may interact with and modulate CRF-dependent systems.

Other issues that we will be discussing at length relate to the pathophysiological effects of CRF. The HPA axis appears to be activated in a variety of circumstances, including in response to stressors and in affective disorders, such as depression and alcoholism, in anorexia nervosa and in shy children, shy kittens and subordinate baboons. What is the common denominator in this activation of the HPA axis? Do all these conditions involve increases in CRF production? If CRF is increased in anorexia nervosa, for example, is the increase a state or trait phenomenon? Is CRF production increased as a consequence of the underlying condition, or is CRF somehow fundamentally involved in causing the circumstance, or, perhaps more likely, does it exacerbate the process? It is difficult to approach most of these questions without the proper tools. With experimental animals one can make lesions, one can collect tissue samples to measure mRNA and peptide levels and one can use antibodies for passive immunization; however, our repertoire is limited for human studies and we are forced to conduct mainly correlative studies. One of the tools that would be most useful in investigating the clinical significance of CRF is improved antagonists. The present generation of peptide CRF antagonists is insufficiently potent for parenteral administration in human beings; they have, however, been used to explore and establish the importance of CRF as a regulator of the endocrine and autonomic nervous systems and in behaviour. Over the next two and a half days we will be discussing a variety of issues concerning the anatomy, the molecular biology, the regulation, the physiology and the pathophysiology of this important peptide.

References

Chrousos GP, Gold PW 1992 The concept of stress and stress system disorders: overview of physical and behavioral homeostasis. JAMA (J Am Med Assoc) 267:1244-1252

Guillemin R, Rosenberg B 1955 Humoral hypothalamic control of anterior pituitary: study with combined tissue cultures. Endocrinology 57599-607

Harris GW 1948 Neural control of pituitary gland. Physiol Rev 28:139-179 Imaki T, Nahon JL, Rivier C , Sawchenko PE, Vale W 1991 Differential regulation of

corticotropin-releasing factor mRNA in rat brain cell types by glucocorticoids and stress. J Neurosci 11585-599

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4 Vale

Lederis K, Letter A, McMaster D, Moore G 1982 Complete amino acid sequence of urotensin I, a hypotensive and corticotropin-releasing neuropeptide from Catostomus. Science (Wash DC) 218:162-164

Montecucchi PC, Henshen A 1981 Amino acid composition and sequence analysis of sauvagine, a new active peptide from the skin of Phylomedusa sauvagei. Int J Pept Protein Res 18:113-120

Okawara Y, Morley SD, Burzio LO, Zwiers H, Lederis K, Richter D 1988 Cloning and sequence analysis of cDNA for corticotropin-releasing factor precursor from the teleost fish Catostomus commersoni. Proc Natl Acad Sci USA 85:8439-8443

Orth D 1979 Adrenocorticotropic hormone. In: Jaffe BM, Behrman HR (eds) Methods of hormone radioimmunoassay, 2nd edn. Academic Press, New York, p 245-284

Potter E, Behan DP, Fischer WH, Linton EA, Lowry PJ, Vale WW 1991 Cloning and characterization of the cDNAs for human and rat corticotropin releasing factor-binding proteins. Nature (Lond) 349:423-426

Rivier J , Spiess J, Vale W 1983 Characterization of rat hypothalamic corticotropin- releasing factor. Proc Natl Acad Sci USA 80:4851-4855

Saffran M, Schally AV 1955 Release of corticotropin by anterior pituitary tissue in vitro. Can J Biochem Physiol 33:408-415

Shibahara S, Morimoto Y, Furutani Y et a1 1983 Isolation and sequence analysis of the human corticotropin-releasing factor precursor gene. EMBO (Eur Mol Biol Organ)

Stenzel-Poore MP, Heldwin KA, Stenzel P, Lee S, Vale WW 1992 Characterization of the genomic corticotropin-releasing factor (CRF) gene from Xenopus laevis: two members of the CRF family exist in amphibians. Mol Endocrinol 6:1716-1724

Vale W, Grant G, Amoss M, Blackwell R, Guillemin R 1972 Culture of enzymatically dispersed pituitary cells: functional validation of a method. Endocrinology 91 :562-572

Vale W, Greer M (eds) 1985 Corticotropin-releasing factor. (Proc Kroc Found Conf on CRF, Santa Barbara, CA, February 1984) Fed Proc 44(1) Part 2

Vale W, Spiess J , Rivier C, Rivier J 1981 Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and &endorphin. Science (Wash DC) 213:1394-1397

J 2:775-779

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The functional neuroanatomy of co rt i cot r o p i n - re I easi n g factor P. E. Sawchenko, T. Irnaki, E. Potter, K. Kovacs, J. lrnaki and W. Vale

Laboratory of Neuronal Structure and Function, The Salk Institute for Biological Studies and The Clayton Foundation Laboratories for Peptide Biology California Division, PO Box 85800, San Diego, CA 92186, USA

Abstract. Descriptions of the central distribution of corticotropin-releasing factor (CRF) have been taken as generally supporting the proposition that this neuropeptide is involved in the mediation of complementary neuroendocrine, autonomic and behavioural responses to stress. The hypothalamic paraventricular nucleus (PVN) is recognized as the principal source of CRF in hypophysial portal plasma; CRF mRNA and peptide expression in parvocellular neurosecretory neurons are regulated negatively by adrenal steroids and positively by many stressors. Consistent with the latter, the hypophysiotropic zone of the PVN receives a rich, and biochemically differentiated, afferent supply that provides visceral, somatic and special sensory systems with access to the ‘CRF neuron’. Within the PVN, CRF is also expressed, and differentially regulated, in oxytocinergic magnocellular neurosecretory neurons and in autonomic-related projection neurons. CRF expression in at least some extrahypothalamic cell groups (olfactory bulb, Barrington’s nucleus) is responsive to certain stressful stimuli, but not to perturbations of the steroid environment. Refinement of our understanding of the central distribution of CRF has been provided by the recognition that most CRF antisera cross-react with an amidated dipeptide encoded by the melanin- concentrating hormone precursor, and by the likelihood that some central sites of CRF peptide expression may be muted or masked by the presence of a CRF- binding protein (CRF-BP). The CRF-BP is expressed prominently in the telencephalon, where it is co-localized with CRF in some neurons, and in anterior pituitary corticotrophs.

1993 Corticotropin-releasing factor. Wiley, Chichesier (Ciba Foundation Symposium 172) p 5-29

The 41-residue peptide corticotropin-releasing factor (CRF) is acknowledged as the principal hypophysiotropic factor driving stress-induced adreno- corticotropic hormone (ACTH) secretion. There also exists a consensual identification of the paraventricular nucleus of the hypothalamus (PVN) as the principal seat of the parvocellular neurosecretory neurons responsible for delivering C R F to the hypophysial portal vasculature and initiating the stress cascade (Antoni 1986, Sawchenko & Swanson 1989). In addition, however, CRF

5

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6 Sawchenko et al

is among the more widely distributed of the neuroendocrine peptides in the central nervous system. On the basis of CRF’s ability to act centrally to mobilize autonomic and behavioural mechanisms that ostensibly complement its neuroendocrine role in an organism’s adaptation to stress (see Koob et al 1993 and Fisher 1993, this volume), the proposition remains viable that CRF expressed in different cell groups and acting at different targets may function in a unified manner to achieve integrated multi-system responses to stress. It is against this backdrop that we summarize here some recent developments that have served to clarify the manner in which CRF-expressing systems in the rat brain are organized to respond to challenges posed by perturbations in the internal or external environments.

The paraventricular nucleus

The paraventricular nucleus of the hypothalamus (PVN) was first convincingly implicated as the source of hypophysiotropic CRF activity in the lesion studies of Makara and colleagues (1981). Soon after the isolation of the CRF peptide (Vale et a1 198 l), immunohistochemical localization work confirmed the existence of a substantial complement of CRF-immunoreactive neurons (of the order of 2000 per side in colchicine-treated animals) centred in the dorsal aspect of the medial parvocellular subdivision of this nucleus (e.g. Swanson et al 1983; see Fig. 1). Evidence supporting the PVN as the principal source of CRF in portal plasma includes the observation that CRF-immunoreactive neurons identified as projecting to the median eminence are overwhelmingly concentrated at this locus (Kawano et a1 1988). In addition, lesions of the PVN, or its projections to the median eminence, markedly reduce the number of CRF- immunoreactive terminals in the external lamina of the median eminence and hypophysial portal CRF titres (Antoni et a1 1983, Plotsky & Vale 1984). Finally, in keeping with acknowledged negative feedback control of hypothalamic CRF activity (Keller-Wood & Dallman 1984), CRF mRNA and peptide levels in the parvocellular neurosecretory zone of the PVN, and peptide content of the median eminence and hypophysial portal plasma, are prominently up-regulated in response to corticosteroid withdrawal (Young et al 1986, Plotsky & Sawchenko 1987, Swanson & Simmons 1989).

It is important to point out that the central limb of the hypothalamo-pituitary- adrenal (HPA) axis is by no means a simple, or closed, system. The hypophysiotropic CRF neuron has the capacity to express a number of additional neuropeptides (Table l), some of which, such as arginine vasopressin, are capable of interacting with CRF to stimulate corticotropin secretion (Rivier & Vale 1983). Moreover, other hypothalamic cell types, notably magnocellular neurosecretory neurons (e.g. Holmes et al 1986), may participate prominently in modifying the output of the axis by the virtue of their capacity to deliver to the anterior lobe peptides which can act directly on corticotrophs. The vascular route(s) by

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Neuroanatomy of CRF 7

TABLE 1 neuron a

Co-localization of neuroactive peptides within the parvocellular CRF

Extent of Peptide co-localization

Angiotensin I1 Extensive Cholecystokinin Extensive Enkephalin Extensive Neurotensin Slight PHI Slight

Minimal conditions

Colchicine + adrenalectomy Colchicine + adrenalectomy Colchicine Colchicine Colchicine

ACTH secretagogue

Weak Weak No No No

Vasopressin Extensive Adrenalectomy Moderate VIP Slight Colchicine Weak

"Modified from Sawchenko & Swanson 1989. Results summarized here are derived from Kiss et al 1984, Sawchenko et al 1984b, Lind et al1985, Mezey et al1986, Sawchenko 1987b, Ceccatelli et al 1989. PHI, peptide histidine-isoleucine (an amidated neuropeptide with His at the C-terminus and Ile at the N-terminus); VIP, vasoactive intestinal peptide.

which this may occur, and the mechanism(s) by which such effects may be integrated with the recognized outputs of the parvocellular and magnocellular neurosecretory systems, remain to be fully characterized.

Despite the likelihood that hypophysiotropic cells account for the bulk of CRF expression in the PVN under most conditions, even within this single cell nucleus additional anatomically and functionally distinct cell types are capable of expressing CRF (Fig. 1). Relatively small subsets of oxytocin-containing magnocellular neurosecretory neurons have been shown to be capable of expressing CRF peptide in the colchicine-treated rat (Sawchenko et a1 1984a). In response to particular systemic challenges, such as salt loading, however, CRF gene expression and peptide levels in the magnocellular system are markedly enhanced (Young 1986, Dohanics et a1 1990, Imaki et a1 1992), suggesting that the capacity to produce CRF is a common, and probably universal, attribute of magnocellular oxytocinergic neurons. The PVN also harbours another major visceromotor cell type which gives rise to long, intracerebral projections whose targets include structures associated with the control of the autonomic nervous system; the terminal fields of this projection system include medullary parasympathetic (vagal) and spinal sympathetic preganglionic neurons (Swanson & Kuypers 1980). These autonomic-related projections of the PVN have been recognized as being biochemically heterogeneous, with small subsets sharing biochemical phenotypes (but not connectivities) with larger, adjoining populations of neurosecretory neurons (Sawchenko & Swanson 1982). Among the largest of the chemically specified subsets yet defined is one displaying CRF immunoreactivity, which accounts for roughly 7% of the total complement of PVN neurons that give rise to long intracerebral projections (Sawchenko 1987a). From available estimates of the number of oxytocin-containing magnocellular

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8 Sawchenko et al

FIG. 1. CRF immunostaining in the hypothalamic paraventricular nucleus (PVN). Bright-field photomicrographs showing CRF-immunoreactive neurons in untreated (Normal), adrenalectomized (ADX) and colchicine-pretreated rats. A Nissl-stained section through a comparable level is shown for reference. In normal animals, the relatively few CRF-immunoreactive cells detected are principally in the dorsal aspect of the medial parvocellular subdivision of the nucleus (mp,). In response to removal of steroid feedback (ADX), the number and staining intensity of immunoreactive neurons increases preferentially in this same subdivision. Non-specific enhancement of perikaryal staining by colchicine reveals a more expansive distribution of cells with the capacity to express the peptide; this is not limited to the hypophysiotropic zone, but includes cells in the magnocellular division (pm) and autonomic-related projection neurons also (dp, mp,). AHA, anterior hypothalamic area; dp, dorsal parvocellular part; fx, fornix; mp,, dorsal medial parvocellular part; mp,, ventral medial parvocellular part; pm, posterior magnocellular part; pv, periventricular part; ZI, zona incerta.

neurosecretory neurons and CRF-containing parvocellular neurosecretory and autonomic-related projection neurons it appears that CRF is capable of being expressed in roughly one third of the some 10 000 cells that cofistitute the PVN as defined by Swanson S'z Kuypers (1980). Regulatory influences on CRF expression in these distinct visceromotor compartments of the PVN are not exerted uniformly across cell types, but differentially, and on the basis of connectivity and functional associations. For example, unlike parvocellular neurosecretory neurons, neither magnocellular neurosecretory (Sawchenko et al1984b) nor autonomic-related projection neurons (Sawchenko 1987a, Swanson

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Neuroanatomy of CRF 9

& Simmons 1989) display marked alterations in CRF gene and peptide expression in response to adrenalectomy.

One of the major recent developments in our understanding of the workings of the hypophysiotropic CRF system has been the recognition that any number of disparate stress paradigms are capable of modifying indices of the synthesis (mRNA levels), as well as the release, of CRF (e.g. Lightman & Young 1988, Harbuz & Lightman 1989, Herman et al 1989, T. Imaki et al 1991). This provides a counterpoint to the negative regulation by corticosteroids in suggesting that the principal positive drive that initiates and maintains the output of the HPA axis under challenged conditions is provided by stress itself. Presumably, these effects are mediated through modality-specific arrays of neural inputs that convey relevant sensory information to the PVN.

Afferent control

In keeping with the great diversity of stimuli and sensory modalities that are capable of supporting a response to stress (e.g. Feldman 1985), the hypophysio- tropic zone of the PVN is known to receive a rich afferent innervation (Fig. 2). As summarized elsewhere (Sawchenko & Swanson 1985), these afferent sources may be grouped into four major classes. Firstly, a series of largely, but not exclusively, catecholaminergic pathways (Cunningham & Sawchenko 1988, Cunningham et al 1990) is in a position to relay visceral sensory information gated through the nucleus of the solitary tract (NTS). The NTS is the principal central recipient of primary interoceptive inputs conveyed by the vagus and glosso- pharyngeal nerves, which innervate broad territories of the thoracic and abdominal viscera. Secondly, a series of interconnected cell groups constituting the lamina terminalis (the rostra1 margin of the third ventricle), which lie outside the blood-brain barrier, have been implicated as transducers of information carried by blood-borne macromolecules (such as angiotensin 11) and ions (reflect- ing the osmotic composition of blood) (Gross 1987). Thirdly, nearly all cell groups in the hypothalamus and preoptic area send projections into the hypo- physiotropic zone of the PVN, providing substrates for a broad-based integration of the central drive on the HPA axis with other neuroendocrine, autonomic and behavioural regulatory mechanisms that rest under hypothalamic control (Sawchenko & Swanson 1983). Finally, a number of cell groups in the limbic region of the telencephalon, including portions of the septal, amygdaloid and hippocampal complexes, are generally thought to exert tonic inhibitory influences on neuroendocrine functioning, and, more specifically, to be potential sites through which corticosteroid feedback effects on the HPA axis may be exerted (Kovacs & Makara 1988). These limbic structures themselves give rise to only very sparse projections to the PVN, but do provide convergent inputs to structures such as the bed nucleus of the stria terminalis, which, in turn, projects prominently to the hypophysiotropic zone of the PVN (Sawchenko & Swanson 1983).

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Neuroanatomy of CRF 11

Collectively, these reasonably well characterized pathways provide substrates through which potent interoceptive and blood-borne influences on the HPA axis may be exerted, and enable these influences to be coordinated with the activities of other hypothalamic control systems. The means by which other sensory modalities may gain access to the PVN are far less well understood, because the PVN is not known to receive any substantial direct projections from the cerebral cortex or the sensory thalamus. Recent axonal transport studies focusing on afferents from the thalamus, midbrain and pons have revealed the existence of pathways in a position suitable for serving such roles (Fig. 2). Results from retrograde and anterograde tracing experiments (Levin et a1 1987) were consistent in revealing projections to the parvocellular division of the PVN arising from: (1) the pedunculopontine and laterodorsal tegmental nuclei, predominantly cholinergic cell groups that receive substantial somatic sensory inputs from the spinal cord (Rye et a1 1987); (2) a series of mesencephalic cell groups (posterior intralaminar, peripeduncular and parvocellular part of the subparafascicular nuclei) that receive projections from auditory relays in the midbrain and thalamus (LeDoux et a1 1985); (3) the intergeniculate leaflet, a thalamic cell group that receives a direct retinal input (Pickard 1985), and (4) the mesencephalic and pontine central grey, a complex structure best known for its role in the processing of central autonomic and nociceptive information (Depaulis & Bandler 1990). Though the functional roles of these pathways in modifying CRF release by hypophysiotropic neurons remain to be examined, these results do broaden our understanding of the range of afferents and sensory systems with the potential to impinge on the central limb of the HPA axis.

Although we now have a reasonable understanding of the range of central pathways that may influence the hypophysiotropic CRF neuron, a major task that remains is to link these directly to function-that is, to specify the kind@) of stress-related information that may be carried by each. We (Kovacs & Sawchenko 1992) have recently used a variety of in situ assays to evaluate the circuits that may mediate the coordinate changes in CRF expression in the magnocellular and parvocellular neurosecretory systems seen in the salt-loading paradigm alluded to above. First, localization of FOS, a product of the cellular intermediate early gene c-fos, which is now widely used as an index of functional activation (Morgan & Curran 1991), implicated lamina terminalis-associated structures as potential mediators of the effects of the salt-loading stress on neuroendocrine neurons. This possibility was evaluated by making discrete unilateral knife cuts intended to sever lamina terminalis-associated outputs directed towards the endocrine hypothalamus (Fig. 3). These reversed the effect of salt-loading on CRF mRNA expression in both the magnocellular and parvocellular compartments of the PVN on the ipsilateral side. Cuts rostra1 to the anterior limit of the third ventricle, as well as others intended to more selectively eliminate influences of the median preoptic nucleus and/or the subfornical organ, were ineffective, implicating the region of the vascular organ

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12 Sawchenko et at

4

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Neuroanatomy of CRF 13

of the lamina terminalis as a mediator of the coordinate effects of hyperosmolality on the magnocellular and parvocellular neurosecretory systems.

Extrahypothalamic systems

Detailed surveys (e.g. Swanson et a1 1983) and a recent review (Sawchenko & Swanson 1989) of the extra-paraventricular distribution of CRF immunoreactivity can be found elsewhere. Here, we shall summarize some recent findings that have bearing on the validity and limitations of the available immunohisto- chemical results, and on the notion, voiced above, that components of the non- endocrine CRF system may be involved in complementary autonomic and behavioural responses to stress.

Stress-induced modification of CRF expression is not limited to hypophysio- tropic, or even neuroendocrine, neurons. Intermittent foot-shock stress, for example, promotes a rapid up-regulation of mRNA for CRF in Barrington’s nucleus (T. Imaki et a1 1991), a pontine cell group that is pivotally involved in the autonomic control of micturition (de Groat et a1 1979), and a down- regulation in the olfactory bulb, the principal relay for a sensory modality that governs much of rodent behaviour. The effects of this stress paradigm on CRF mRNA are seen acutely in these extrahypothalamic cell groups, whereas those in the PVN are delayed, presumably as a result of the negative feedback effects of increased glucocorticoid. Consistent with this, CRF mRNA levels in the bulb and pons are not responsive to steroid withdrawal (T. Imaki et a1 1991); hypophysiotropic neurons remain the only site where CRF is known to be regulated positively by stress and negatively by glucocorticoids.

FIG. 3. Lamina terminalis mediation of coordinate effects of hyperosmolality on CRF mRNA in magnocellular and parvocellular neurosecretory neurons. A: Schematic drawing of a mid-sagittal section through the rat diencephalon illustrating interconnections among the subfornical organ (SFO), median preoptic nucleus (MePO) and the vascular organ of the lamina terminalis (OVLT) and their projections to the hypothalamic paraventricular nucleus (PVH) and supraoptic nucleus (SO). Numbered lines indicate places where discrete knife cuts were made. Salt-loading results in a down-regulation of CRF mRNA in parvocellular neurosecretory neurons, and an up-regulation in magnocellular oxytocinergic cells, such that the distribution of CRF mRNA is virtually indistinguishable from that of oxytocin. B: Dark-field photomicrograph through the paraventricular nucleus in a salt-loaded animal that received a unilateral knife cut (1) designed to isolate the PVH from lamina terminalis afferents. The contralateral control side (Contra) shows the predominantly magnocellular (m) distribution of CRF message that is characteristic of salt-loaded animals. The lesioned (Ipsi) side shows a largely parvocellular (p) distribution characteristic of intact rats. Cuts 2, 3 and 4 did not modify the CRF mRNA response to salt-loading, implicating afferents from the ventral lamina terminalis as mediators of these effects. ac, anterior commissure; AP, anterior lobe of the pituitary; cc, corpus callosum; IL, intermediate lobe of the pituitary; me, median eminence; och, optic chiasm; PP, posterior lobe of the pituitary; vhc, ventral hippocampal commissure.

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14 Sawchenko et al

A more tightly defined physiological challenge, the increased plasma osmolality associated with salt-loading, results in a reduction in CRF transcripts in Barrington’s nucleus, and a marked increase in magnocellular neurosecretory cells, accompanied by a paradoxical reduction in CRF mRNA in parvocellular neurosecretory neurons (Young 1986, Imaki et al 1992). Though their physiological relevance remains to be proven, these responses may each be construed as representing an adaptation to promote antidiuresis (Imaki et a1 1992).

Observations such as these define possibilities as to how CRF in disparate sites may function in an integrated manner to effect adaptive responses to stress. As a consequence, it becomes important to consider factors that bear upon the validity of the probes used for localization of CRF, and influences that may modify CRF dynamics within and beyond the neuroendocrine system.

Antiserum specificity and the melanin-concentrating hormone

Many antisera against ratlhuman CRF (rat and human CRF have the same primary structure) stain a large population of cells in the lateral hypothalamic area and zona incerta that are also immunopositive for several neuroendocrine peptides, including the melanotropic peptides a-melanocyte-stimulating hormone (MSH) and melanin-concentrating hormone (MCH) (Fellmann et a1 1987, Kawano et a1 1988), the latter of which has been proposed as a putative corticotropin-inhibiting factor (Baker et a1 1985). With the cDNA cloning of the rat MCH precursor (Nahon et a1 1989), it became evident that rat CRF, a-MSH and a newly discovered neuropeptide (neuropeptide EI, or NEI, an amidated neuropeptide with Glu at its C-terminus and Ile at its N-terminus) encoded by the MCH precursor all contain amidated aliphatic amino acids at their C-termini (Fig. 4). Immunohistochemical competition studies revealed that of the competing peptides tested (Table 2) only CRF effectively blocked staining in the PVN; NEI and peptides containing an aliphatic amide were roughly equally effective in blocking immunostaining in the dorsolateral hypothalamus. Thus, the apparent immunostaining for rat CRF (and a-MSH) in the dorsolateral hypothalamus, but not in the PVN (or arcuate nucleus), can be explained by cross-reactivity with this epitope of the NEI peptide (Nahon et al 1989). The widespread finding of rat CRF and a-MSH immunoreactivity in the dorsolateral hypothalamus is thus probably spurious. Because this epitope is ostensibly highly immunogenic, in studies to localize CRF in perikarya and axons this potential artifact needs to be controlled. It is of interest that peptide HI (PHI), a neuropeptide that has been co-localized with CRF in the hypophysiotropic zone of the PVN (Ceccatelli et a1 1989), also contains a C-terminal bulky aliphatic amide.

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Neuroanatomy of CRF 15

NGE NEI

71 1 - 1

L A ~ A M ~ R K L M-

Rat preproMCH(109-144) K G P A V F P-N G V E N T E W T Q E K R- D m E N S A K F C

RaVhuman CRF (24-41) a-MSH (1-13) * s v s M ~ M F R W G KIIIV.

FIG. 4. Cross-reactivity of CRF antisera with epitopes encoded by the melanin- concentrating hormone (MCH) precursor. The sequence of a portion of the MCH precursor that encodes the putative neuropeptides NEI and NGE is aligned with portions of the rat CRF and a-MSH sequences. Shading represents sequence similarities among two or more of these molecules, whose immunoreactivities had been reported to coexist in dorsolateral hypothalamic neurons. Solid squares indicate amidation sites. All three peptides have amidated aliphatic (proline or isoleucine) amino acids at their C-termini.

Novel sites of CRF expression and the CRF-binding protein

Hybridization histochemical methods have provided sensitive tools with which to establish the chemical phenotype of neurons. Although these approaches have provided independent confirmation of most consensus sites of CRF expression as gleaned from immunohistochemical studies, some unexpectedly exuberant, and other, quite novel, sites of cellular CRF expression have been suggested (e.g. Imaki et al 1989, J. Imaki et a1 1991, Smith et a1 1991). The olfactory bulb, for example, has typically been reported to contain no more than a few widely scattered CRF-immunoreactive cells and fibres. In contrast, hybridization histochemical localization of CRF mRNA revealed a widespread distribution within most major cell types in the bulb (Imaki et a1 1989). Regional Northern analysis indicated that CRF mRNA extracted from the olfactory bulb was similar

TABLE 2 Minimum effective concentrations (&I) of peptides required to block CRF immunostaiainga in the rat dorsolateral and paraventricular hypothalamus

Dorsolateral Paraventricular Peptide hypothalamus nucleus

Rat CRF

NEI NGE Val-NH, Ile-NH, Pro-Ile-NH, Pro-Val-NH,

a-MSH < 1.4 140.0 7.0

> 140.0 > 140.0 > 140.0 c 1.4 7.0

< 1.4 > 140.0 > 140.0 > 140.0 > 140.0 > 140.0 > 140.0 > 140.0

aThe anti-rat CRF antiserum C70 was used for immunostaining. MSH, melanocyte-stimulating hormone. NEI, neuropeptide EI; NGE, neuropeptide GE; in these amidated neuropeptides the letters represent the amino acids (one-letter codes) at the C-terminus and N-terminus (see Fig. 4).

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16 Sawchenko et al

in size to, but, surprisingly, considerably more abundant than, that extracted from whole hypothalamus. Attempts to confirm this using immunohistochemistry with traditional middle- to carboxy terminal-directed antisera were unsuccessful. Other anti-CRF sera, however, including one raised against a synthetic N-terminal fragment, CRF (1 -21), revealed a distribution of CRF-immuno- positive cells in the olfactory bulb, along with their axonal projections, that was fully compatible with the hybridization results (Imaki et a1 1989). This apparent discrepancy could arise from an unusual post-translational processing of nascent CRF precursor in the bulb. Alternatively, authentic CRF-41 peptide may exist in the bulb but be bound or sequestered in such a manner that particular epitopes on the peptide are rendered inaccessible to immunoglobulins.

Lowry (1993) summarizes elsewhere in this volume the isolation and characterization of a protein from the plasma of pregnant women which is capable of binding CRF and reversibly neutralizing its biological activity. Rat and human cDNAs encoding this CRF-binding protein (CRF-BP) have been isolated, and found to be expressed in the brain (Potter et a1 1991). Recently, immunohistochemical and hybridization histochemical methods have been used to map the sites of CRF-BP expression in the rat brain, and to compare these with the distribution of CRF (Potter et al 1992; see Fig. 5) . Intriguingly, the loci of CRF-BP expression include the olfactory bulb and very nearly all the novel sites of CRF expression alluded to above,

Results from both labelling approaches indicated a predominantly telencephalic distribution of the binding protein. This includes all major cortical fields, as well as some subcortical limbic system structures. Of particular interest here was the finding that central relays for several sensory modalities, including the olfactory bulb, were prominent sites of CRF-BP gene and protein expression. This raises the possibility that CRF peptide may be masked by the presence of the binding protein in at least some cell groups in the central nervous system. Dual immunostaining studies did indeed reveal some limited co-localization of binding protein and peptide. Quite unexpectedly, CRF-BP immunoreactive terminal fields were found in close association with several CRF-immunoreactive cell groups in the forebrain, suggesting that the binding protein may, at some select loci, be released from terminals in a position where it could modify local synaptic, autocrine or paracrine actions of CRF.

What is the potential for the CRF-BP to serve as a modifier of CRF’s activity in the neuroendocrine system? The PVN, as well as other hypothalamic neurosecretory cell groups, contained no more than a few scattered cells showing CRF-BP immunoreactivity or expressing CRF-BP mRNA. The binding protein is, however, broadly expressed in anterior pituitary corticotrophs, suggesting a site at which the binding protein could, via mechanisms that are as yet completely obscure, modulate CRF’s actions at the pituitary level.

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Neuroanatomy of CRF

A. CRF Binding Protein

17

\ :..

B. CRF

FIG. 5 . Schematic drawing of mid-sagittal sections through the rat brain to compare the cellular localizations of CRF-binding protein (A) and CRF (B) immunoreactivities in the rat. Major known areas of overlap (i.e. co-localization) include the olfactory bulb, amygdala, bed nucleus of the stria terminalis, and several cell groups in the brainstem reticular core. Note also that the binding protein is expressed in anterior pituitary corticotrophs, the principal endocrine targets of CRF in hypophysial portal blood.

Discussion

In the relatively short interval since the isolation of CRF, its central representation has become one of the better characterized peptidergic systems in the brain. The hypophysiotropic CRF pathway, and the nature of major neural and hormonal regulatory influences upon it, are understood to a reasonable first approximation. Here, the challenge for the immediate future will be to determine the broader context in which the neuroendocrine CRF system operates. What is the relative importance of CRF versus other secretagogues, and of the parvocellular versus magnocellular neurosecretory systems, in imparting the situation-specific drive for ACTH secretion? What are the pathways and neurotransmitter systems by which relevant events are conveyed

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18 Sawchenko et al

from the sensorium to the endocrine hypothalamus, and how is integration across multiple converging input and output channels achieved?

Because of CRF’s demonstrated capacity to act centrally to elicit autonomic and behavioural activation, responses that are ostensibly complementary to its principal neuroendocrine effects, aspects of the central CRF system provide some of the more compelling examples of how a single biologically active molecule may participate at disparate sites and in different systems within an organism to achieve a unified and adaptive response to stress. However, CRF is often only one of several neuroactive substances contained within an individual cell group, or even within individual neurons within a given cell group, and we are limited by a dearth of analyses at the cellular level which address the role and relative importance of CRF as a transmitter/modulator in central pathways. Finally, further progress in this area will need to take into account the possibility of modulation by the CRF-BP, which can affect the biological actions of CRF in plasma and the pituitary, and, if only by virtue of its distribution, has significant potential to exert similar effects in the brain. We are at an early stage in understanding the biology of this protein, and further study of its regulation in response to stress and perturbations in the corticosteroid environment, and of the nature of its relationship (if any) to the still elusive CRF receptor, will be required before we can fully appreciate its role in sculpting the central and neuroendocrine actions of CRF.

Acknowledgements

The work from our laboratories that was summarized here was supported by USPHS grants NS-21182, HL-35137 and DK-26741, and was conducted in part by the Foundation for Medical Research. P. E. S. and W. V. are Investigators of the Foundation for Medical Research.

References

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Antoni FA, Palkovits M, Makara G, Linton EA, Lowry PJ, Kiss JZ 1983 Immunoreactive corticotropin-releasing hormone in the hypothalamoinfundibular tract. Neuro- endocrinology 36:415-423

Baker BI, Bird DJ, Buckingham JC 1985 Salmonid melanin-concentrating hormone inhibits corticotrophin release. J Endocrinol 106:RS-RS

Ceccatelli S, Eriksson M, Hokfelt T 1989 Distribution and coexistence of corticotropin- releasing factor-, neurotensin-, enkephalin-, cholecystokinin-, galanin- and vasoactive intestinal polypeptidelpeptide histidine isoleucine-like peptides in the parvocellular part of the paraventricular nucleus. Neuroendocrinology 49:309-323

Cunningham ET Jr, Sawchenko PE 1988 Anatomical specificity of noradrenergic inputs to the paraventricular and supraoptic nuclei of the rat hypothalamus. J Comp Neurol 274~60-76