Neural Integration of Physiological Mechanisms and Behaviour: J.A.F. Stevenson Memorial Volume 9781487578466

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NEURAL INTEGRATION OF PHYSIOLOGICAL MECHANISMS AND BEHAVIOUR

J.A.F. Stevenson Memorial Volume

Neural Integration of Physiological Mechanisms and Behaviour edited by Gordon J. Mogenson and Franco R. Calaresu (with the editorial assistance of Blanche Box) University of Toronto Press Toronto and Buffalo

© University of Toronto Press 1975 Toronto and Buffalo Printed in Canada

Reprinted in 2018 Library of Congress Cataloging in Publication Data

Main entry under title: Neural integration of physiological mechanisms and behaviour. Includes bibliographies. 1. Biological control systems . 2. Neurophysiology. 3. Psychology, Physiological. 4. Stevenson, James Alexander Franklin, 1918-1971. 1. Stevenson, James Alexander Franklin, 1918-1971. 11. Mogenson, Gordon J. , 1931- ed. 111. Calaresu , Franco R. , 1931- ed . [DNLM : 1. Behavior. 2. Neurophysiology . 3. Psycho-physiology . WLl02 M696n) QP356.N48 1975 596' .01 ' 88 74-79003 ISBN 0-8020-2165-4 ISBN 978-1-4875-7914-2 (paper)

Senior Authors and their Affiliation

Professor E.F. Adolph, Department of Physiology, The University of

Rochester, School of Medicine and Dentistry, Rochester, New York 14642, USA Professor B. Andersson, Fysiologiska Institutionen I, Karolinska Institutet,

S-104-01 Stockholm 60, Sweden

Professor J.R. Brobeck, Department of Physiology , School of Medicine,

University of Pennsylvania, Philadelphia, Pa. 19174, USA

Professor M.R. Covian, Department of Physiology, School of Medicine and

Dentistry, University of Sao Paulo, Ribeirao Preto, Sao Paulo, Brazil Professor Alan Epstein, Leidy Laboratory of Biology, University of Penn-

sylvania, Philadelphia, Pa. 19174,

USA

Dr J.T. Fitzsimons, Physiological Laboratory, Cambridge, England,

CB23EG Professor E. Fonberg, Department of Neurophysiology, Nencki Institute of

Experimental Biology, 3 Pasteur Street, Warsaw 22, Poland

Professor Claude Fortier, Department of Physiology, Faculty of Medicine,

Laval University, Quebec I0e, Canada Professor M.J. Fregly, Department of Physiology, The J . Hillis Miller

Health Center, University of Florida, Gainesville, Florida 32601, USA Dr J.P. Girvin, Department of Physiology, University of Western Ontario,

London, Ontario Professor E. Hall, Department of Anatomy, University of Ottawa, Ottawa, Ontario, KIN 6N5 Dr Charles L. Hamilton, Department of Physiology, University of Penn-

sylvania, Philadelphia, Pa. 19104, USA Professor J .D. Hardy, Director of Epidemiology and Physiology, John B.

Pierce Foundation Lab., New Haven, Conn. 06519,

USA

Professor G.R. Hervey, Department of Physiology, The University of

Leeds, Leeds, England, LS2 9JT Dr C.H. Hockman, School of Basic Medical Sciences, University of Illinois, College of Medicine, 1205 West California St, Urbana, Illinois 61801, USA *Dr G.C. Kennedy, Dunn Nutritional Laboratory, University of Cambridge

and Medical Research Council, Milton Road, Cambridge, England CB4 IXJ Professor J. Le Magnen, Lab. de Physiologie, College of France, 11, Place

Marcelin Berthelot, Paris ye, France

Professor R.D. Lisk, Department of Biology, Princeton University, Prince-

ton, New Jersey 08540, USA Professor F .C. MacIntosh, Department of Physiology, McGill University,

Montreal, Quebec Professor G .J. Mogenson, Departments of Physiology and Psychology,

University of Western Ontario, London, Ontario

Dr Peter J. Morgane, The Worcester Foundation for Experimental Biol-

ogy, Shrewsbury, Mass. 01545, USA Professor Y. Oomura, Chairman, Department of Physiology, Kanazawa

University, Kanazawa, Japan Dr M. Russek, Department of Physiology, Escue la N acional de Ciencias

Biologicas, Mexico 17, DF Professor K.N. Sharma, Department of Physiology, St John's Medical College, Bangalore 34, India Professor M.J. Wayner, Brain Research Lab., Syracuse University , Syra-

cuse , New York, USA Editors

Professor F .R. Calaresu, Department of Physiology, University of Western

Ontario, London, Ontario Professor G.J. Mogenson, Department of Physiology, University of Western Ontario, London, Ontario

*Deceased

Generous financial assistance towards publication of this book was provided by the University of Western Ontario

Acknowledgment

This book is published under the sponsorship of the International Commission on the Physiology of Food and Fluid Intake, an agency of the International Union of Physiological Sciences. Jacques LeMagnen, France, Chairman Alan N. Epstein, USA, Secretary Miguel R. Covian, Brazil James T. Fitzsimons, England Elzbieta Fonberg, Poland Gordon J. Mogenson, Canada Yutaka Oomura, Japan Georges Peters, Switzerland Mauricio Russek, Mexico Kamal Sharma, India

Preface

This collection of articles contributed by scientific colleagues and friends is dedicated to the memory of Professor James A.F. Stevenson and to the view, which he practised and taught, that physiology studies the characteristics and performance of biological systems at all levels of organization from the cellular to the organismic . Living organisms rely on dynamic processes by which essential materials are obtained from the environment and waste products are eliminated. Living cells require an appropriate milieu for survival and, in the course of evolution, mechanisms have been developed for both the exchange of materials between the organism and the external environment and for the maintenance, in a variable environment, of conditions compatible with life. Survival depends upon the functioning of the alimentary, respiratory, circulatory, and excretory systems; these systems in tum are under nervous and endocrine control. Many physiological regulations also involve external or behavioural responses characterized by movement of the total organism in its external environment and by interaction with and manipulation of external objects. These external responses are under the control of the brain and are co-ordinated with the internal responses. In the experimental analysis of physiological systems the mechanisms responsible for certain responses are identified and characterized, but the

x Preface final goal of physiology is to demonstrate how the dynamic properties of organisms arise from the properties of process at the lower level. The multilevel character of physiological systems is usually not considered in any one specific experiment; the system is studied in isolation. However, after obtaining fragmentary evidence about different levels of organization, a final synthesis must be made in order to attempt an explanation of the overall function of the whole animal. James Stevenson had a unique gift for seeing physiological mechanisms in perspective and insisted that students and colleagues attempt to show how their specific, experimental findings fitted with the general 'wisdom of the body.' It was difficult to escape his curiosity and interest in what one was doing and his attempts to suggest experimental approaches that would bring even the most highly specialized areas of research into the context of physiological adaptation of the whole animal. The articles in this book consider a variety of integrated physiological regulations. It begins with contributions by MacIntosh and Brobeck, who remember James Stevenson as a scientist and friend, and by Adolph, who gives a historical account of how physiological regulations became the subject of experimental investigation. The anatomical and chemical foundations for the existence of physiological control systems in the brainstem, hypothalamus, and limbic system are presented by Morgane, who cautions us about assigning specific physiological functions to 'brain centres.' The anatomical connections between the hypothalamus and limbic system are reviewed by Hall. James Stevenson's interests, like the subject of physiological regulations, were very broad, but his major contributions were in the fields of energy and water balance and the control of food and water intake . For this reason several articles deal with these topics. Le Magnen proposes an integrated approach to energy balance and Hervey shows how control theory can be used in the analysis of sophisticated systems that control energy exchange. Epstein and co-workers review the evidence for brain glucoreceptors and Russek presents evidence for the existence of hepatic glucoreceptors. Fonberg describes her work on the role of the amygdala and its functional relationships with the hypothalamus in the control of food intake. The multiple signals that are utilized by the brain to monitor energy balance and to initiate feeding are considered by Sharma and co-workers and Hamilton considers the influence of environmental and body temperature on feeding behaviour. The next four articles deal with the central mechanisms that control water and salt balance . Andersson summarizes the evidence for central receptors for sodium and their primary role in thirst and Fitzsimons re-

Preface xi views recent investigations of angiotensin as a possible thirst hormone. Mogenson considers the available information regarding the electrical activity of neural structures associated with water intake and Covian and co-workers review the complex role played by the hypothalamus and limbic system in controlling sodium balance. The final chapters of the book are other examples of integrated physiological regulations. Hockman and Duffin review what is known about the neural control ofrespiration. Hardy describes some new findings about the role ofprostaglandins as possible central transmitters in temperature regulation . Fregly considers the integrated hormonal control of body temperature and Kennedy discusses the relation between appetite and growth and the development of endocrine glands in the young animal. Lisk looks at the reciprocal interaction between the nervous and endocrine systems in the control of sexual behaviour and Fortier reviews some of the exciting new developments in the field of neuroendocrinology. Wayner considers the question of the specificity of the behavioural regulations that contribute to adaptation of the animal and to homeostasis . In the final chapter Girvin reviews what has been learned in the clinic about the hypothalamus and limbic system, structures of the brain which have been shown in experimental studies to make important contributions to physiological regulations and behaviour. Tumours and ablations of these structures influence visceral and endocrine functions, ingestive, sexual, and other motivated behaviour, and emotional expression . Many of the observations are similar to those obtained in studies using experimental animals. We have not attempted a comprehensive treatment of the control of physiological regulations by the nervous and endocrine systems, which is impossible in a single volume, but rather the presentation of a variety of examples of contemporary developments in this field. The leitmotiv of the book, which we have tried to communicate to the contributors, is the complexity of physiological regulations and the dangers of considering them in isolation. We want to thank the contributors who gracefully agreed to modify their essays to comply with space limitations. Special thanks is due to Dr Douglas Bocking for his interest and support. Our thanks also go to Miss Anne Baxter and Miss Rebecca Woodside for help with the various phases in the preparation of the book and to Dr R. Weick for his assistance. G .J. Mogenson F.R. Calaresu

Contents

Preface ix James Alexander Franklin Stevenson, 1918-1971 F .C. MacIntosh 3 On attaining a clear perspective John R. Brobeck 7 Seven discoveries of physiological regulations E.F. Adolph 11 Anatomical and neurobiochemical bases of the central nervous control of physiological regulations and behaviour P.J . Morgane 24 The anatomy of the limbic system Elizabeth Hall 68 Current concepts in energy balance J . Le Magnen 95 The problem of energy balance in the light of control theory G.R. Hervey 109 Current hypotheses in the control of feeding behaviour Mauricio Russek 128

xiv Contents The glucoprivic control of food intake and the glucostatic theory of feeding behaviour Alan N. Epstein, Stylianos Nicolaidis, and Richard Miselis 148 The amygdala and ingestive behaviour Elzbieta Fonberg 169 Feeding and temperature C.L. Hamilton 186 Electrophysiological monitoring of multilevel signals related to food intake K. N. Sharma, S. Dua-Sharma, and Harry L. Jacobs 194 The central control of water and salt balance Bengt Andersson 213 Endocrine mechanisms in the control of water intake J . T. Fitzsimons 226 Electrophysiological studies of the mechanisms that initiate ingestive behaviours with special emphasis on water intake G .J. Mogenson 248 Central control of salt balance M. R. Covian, J. Antunes-Rodrigues, C.G. Gentil, W.A. Saad, L.A. Camargo, and C.R. Silva Neto 267 Forebrain mechanisms in the control of respiration Charles H. Hockman and James Duffin 283 Control of body temperature James D. Hardy 294 Hormonal interactions in body temperature regulation Melvin J. Fregly 308 Some aspects of the relation between appetite and endocrine development in the growing animal Gordon C. Kennedy 326 Integrative functions of hypothalamic and limbic systems in the control of female sexual behaviour Robert D. Lisk 339 New frontiers in neuroendocrinology Claude Fortier 362 Contribution of electrophysiological techniques to the understanding of central control systems Yutaka Oomura, Mutsuyuki Sugimori, Tsutomu Nakamura, and Yasuyuki Yamada 375 Lateral preoptic / lateral hypothalamic / brain stem motor control system and adjunctive behaviour Matthew J. Wayner 396

Contents xv Clinical correlates of hypothalamic and limbic system function J.P. Girvin 412 Index 435

NEURAL INTEGRATION OF PHYSIOLOGICAL MECHANISMS AND BEHAVIOUR

James Alexander Franklin Stevenson,1918-1971 F.C. MacIntosh

This volume has been prepared in honour of J.A.F. Stevenson by colleagues who shared his scientific interests and were his friends . Jim Stevenson had a wider range of interests and a wider range of friendships than most scientists, and he enjoyed his interests and his friendships , but he did not enjoy them passively. He had no use for inertia in any context. It was clear to him that there were many things that ought to be done ; it would be interesting as well as useful to do them; the right way of doing them had to be discussed in some detail; indeed vehement argument would clarify the issues, and might even have to be provoked, if that was the way to get things moving. This facet of Jim Stevenson, the sharp-edged one , was quite often on display, and some people who were only slightly acquainted with him never saw the other facets , which were no less characteristic of him . For his equipment included wit , a listening ear, a capacity for being infected by other people's enthusiasms , a large reserve of patience that could be called on in case of need, and a great fund of kindness and sympathy , available even for the wrong-headed . Jim Stevenson was born on 15 March 1918 in Nanton, a small Alberta town where his father was both high school principal and Presbyterian minister. His mother too was a university graduate and a fine teacher. Later the family lived in other parts of Canada, including the province of Quebec,

4 F.C. MacIntosh for which Jim always retained a special affection. He took pride in being a Scot by descent and a son of the manse, but especially in being a Canadian. In later life he was to give his best efforts to activities that brought Canadians from different parts of the country together. Entering McGill University at age 16, he took bachelor's and master's degrees in Psychology before choosing medical school, from which he graduated in 1942. After his year of internship he was enrolled as a captain in the Royal Canadian Army Medical Corps; he served for two years as a nutritional adviser and retired with the rank of major. By that time he had no doubt about his vocation for research, and when a suitable fellowship was offered to him he seized the opportunity to work at Yale - first in Physiological Chemistry under C.N.H. Long and J.R. Brobeck, and after that in Physiology as an Assistant Professor under J.F. Fulton. The Yale researchers had been deeply involved for some time in studying the hypothalamic mechanisms that are related to food and water intake - a theme on which much of Stevenson's own research was centred from that time onward. In 1950 Stevenson joined the tiny Department of Physiology at the University of Western Ontario, and in the next year he succeeded F.R. Miller as Professor and Head . A period of expansion for the basic sciences in Canadian medical schools was soon to begin; and over the twenty years of Stevenson's leadership, and largely through his exertions, the Physiology Department at Western increased greatly in size and strength. New teaching and research programmes were developed, partly in collaboration with clinical departments and the Science and Dentistry faculties. As the years passed Stevenson's capacities were increasingly recognized outside the medical school. He played a major part in developing a new constitution for the University; and in 1970, though he was still deeply engaged in the teaching and research activities of the Physiology Department, it seemed a natural step for him to accept the Deanship of the Faculty of Graduate Studies, the post he held at his death a year later. His scientific contributions had been recognized earlier, by frequent invitations to participate in international conferences and in the preparation of monographs, as well as by his election in 1968 to Fellowship in the Royal Society of Canada. His contributions now find recognition again, through the willingness of so many distinguished scholars to contribute to this commemorative volume. Within the Canadian community of medical and biological scientists few people were as widely known or as influential as Jim Stevenson. At one time or another he held most of the senior posts in the societies to which he

J.A.F. Stevenson 5 belonged: Secretary and President of the Canadian Physiological Society; Honorary Associate Secretary of the Canadian Federation of Biological Societies; President of the Nutrition Society of Canada, the Canadian Society for Clinical Investigation, the Canadian Society of Clinical Chemists, and the Biological Council of Canada; and finally Vice-President of sc1rnc, the Association of the Scientific, Engineering and Technical Community of Canada. He was for three years co-editor of the Canadian Journal of Biochemistry and Physiology; then for five years editor of its successor, the new Canadian Journal of Physiology and Pharmacology. He became a member of the Council of the Royal Society of Canada, and he served on several committees of the American Physiological Society. All of these varied assignments he handled with his usual mixture of determination, gusto, and commonsense. His colleagues on boards or councils sometimes found they were expected to do more work than they had bargained for, but his successors found their affairs in better order. Jim Stevenson enjoyed being a member of these organs of the scientific establishment, not out of any wish to be conspicuous, but because he liked to puncture complacency and he liked starting new ventures; these two incentives in combination were hard for him to resist. In recent years Jim Stevenson had become involved in a number of international scientific efforts. He helped to promote the series of international conferences on the Regulation of Food and Water Intake, and served as secretary of two of the conferences. He was elected in 1969 to the Council of the International Union of Physiological Sciences, having already become the editor of the IUPS Newsletter. On 23 July 1971, Jim Stevenson died without warning in Zurich, while on his way with his wife Joan and his daughter Catherine to Munich, where the Council of IUPS was to meet the next day. He left three other daughters, Penelope, Alexandra, and Margaret, and a son, Skai. The news of his death was heard with sorrow during the following days at the plenary session of the xxv International Congress of the Physiological Congress of the Physiological Sciences and at the 1v International Congress on the Regulation of Food and Water Intake. His memory will stay fresh in the minds of his friends and colleagues, and they will be grateful to those who have honoured it by contributing to this volume. Jim Stevenson never lost his capacity for judging research quality or for being excited by new research findings. As his responsibilities outside the laboratory grew he became less concerned with the minutiae of his own research programme, but he remained in firm control of its strategy and

6 F.C. MacIntosh overall tactics . He switched his attention quickly and cheerfully from national or international business to problems at the laboratory bench. It was somehow fitting that so much of his research had to do with the hypothalamus, that organ of remarkable versatility, sensitive to so many inputs and a central link in so many control systems. That his work in the laboratory was significant and will stimulate further research is apparent from the articles in this volume . Perhaps he did even more by his work outside the laboratory.

On attaining a clear perspective

John R. Brobeck

What Jim Stevenson asked for at conferences, symposia, and even at large national meetings was a broader perspective in the minds of all of us studying controls, regulations, neuroendocrinology, behaviour and the functions of the hypothalamus and other parts of the brainstem. It troubled him that laboratories devoted to temperature regulation do not follow closely the work on releasing factors; that neuroendocrinologists are not particularly interested in behaviour; that physiological psychologists overlook metabolic controls; and so on through all permutations and combinations of the subspecialities of anatomical, physiological, neural, behavioural, endocrine, and biochemical science pertaining to the ability of higher animals to stay alive. No doubt he sensed this need for correlation for integration as Sherrington used the word - because his own diversified scientific education enabled him to see interrelations that others were likely to overlook. He studied particular mechanisms, but he thought also about their relation to general problems and principles. His preparation for science was unusual. For example, the influence of his Presbyterian parents remained with him throughout his life. 'A child of the manse' was his expression - more likely from his mother's usage than his father's. In his studies at McGill University his first formal preceptor in science was a psychologist, Professor William Dunlop Tait, born in Nova

8 John R. Brobeck Scotia in 1879, a graduate student in psychology at Harvard, and head of the department at McGill from 1924. Under his supervision in 1938 Stevenson presented his master's thesis on the topic , 'The measurement of scientific aptitude in the field of student personnel work .' From this training in psychology Stevenson entered medical school, completed his internship, enrolled in the Royal Canadian Medical Corps, and as a nutrition officer collaborated with Dr Robert Kark and with Dr John S.L. Browne (Stevenson et al . , 1945, 1946). Browne was born in London, England, in 1904 and received his medical education at McGill; his research activities have been in clinical investigation, particularly endocrinology and metabolism. It was Browne who arranged for Stevenson to come to Yale, where he not only came under the influence ofC.H .N. Long, but acquired a new circle of friends and collaborators such as Knud Lundbaek (from Denmark), Jane Russell and Alfred Wilhelmi, and many others (Bondy, 1968), including the writer who collaborated with Long for some 12 years . In the two years that Stevenson worked under Long's supervision the vistas of endocrinology were added to the backgrounds in psychology, medicine, and nutrition he had already acquired. In this period, moreover, Stevenson began to study animals with hypothalamic lesions , and thus embarked upon what became a major part of his scientific career. After completing his fellowship with Long, he was invited by John F. Fulton to join a research group in the Laboratory of Physiology studying functions of the frontal lobes of monkeys , chimpanzees , and baboons. His colleagues were Jose Delgado, Robert Livingston, Paul MacLean, Karl Pribram, H .E. Rosvold, Patrick D. Wall, and others (Fulton, 1951). The neural control of metabolic processes was Stevenson's particular responsibility . He was well equipped for this type of investigation, and it gave strong impetus to his growing interest in the nervous system. In this period the dominant intellectual forces in the Laboratory of Physiology were the neurosurgical techniques developed by Harvey Cushing, a painstaking and thorough neurological examination, and the physiological concepts of Sir Charles Sherrington (MacLean, 1960). Both Cushing and Sherrington had had prepotent influence in the training of Fulton, and from him their ideas were diffused throughout all of his Laboratory at Yale . One other important feature of Stevenson's training, however, has not been noted in any of the appreciations I have seen. He and other junior members of Fulton's laboratory became indebted to a man most of them never knew, Stephen Walter Ranson of the Institute of Neurology of Northwestern University. Now that stereotaxic instruments are so commonly used, it may be impossible to depict the status of research upon the

On attaining a clear perspective 9 brain between 1930 and 1940. Fulton, Keller, Kennard, and others, trained essentially in neurosurgery, were removing, incising, or undercutting all accessible portions of cerebrum and cerebellum, or stimulating and recording under direct observation in the fashion ofDusser de Barenne, McCulloch, and their disciples. By contrast, Ranson with the aid of Joseph Hinsey, W.R. Ingram, and H.W. Magoun was beginning to explore the functions of the hypothalamus and brainstem via the Horsley-Clarke technique (Hinsey, 196 I). Graduate students in Ranson' s Institute in the late 1930s knew personally every individual in the world who was able to do stereotaxic surgery. They had all come to Chicago to learn: S.C. Wang came from Peking; Marcel Monnier from Geneva; George Alexander from Edinburgh; Teizo Ogawa from Tokyo; and Robert Gaupp from Freiburg. This is a complete listing of those from outside the us until 1939 (Anon ., 1943). In the same period Kendall Corbin had come from Stanford, Robert Pitts from NYU, and John Brookhart from Michigan. All of the rest of us were or had been Ranson's graduate students. The Laboratory of Physiology at Yale had a Horsley-Clarke instrument, a duplicate of Ranson' s latest model. It served for operations upon the rats that Tepperman, Lundbaek, Strominger, Cort and I studied, including those of Stevenson's classic paper (1949) on the effect of hypothalamic lesions upon water and energy exchange of rats. It later served also for the experiments by Ruch, Patton, and others who operated upon monkeys. In the volume we offer as tribute to our friend, Jim Stevenson, I discern three kinds of intellectual effort. E.F. Adolph has traced the first, the concept of regulations as it developed from Lavoisier through Adolph's own work. Second, there is Sherrington's theory of'The integrative action of the nervous system,' that one absorbed in classes, conferences, discussions, at the operating table, and practically by osmosis in Fulton's department at Yale. Third, there is the sometimes overlooked fact that most of us, including Stevenson, could not have pursued the research interests we attempted if Ranson (or perhaps someone else) had not rediscovered stereotaxic procedures. Stevenson's career in science involved a synthesis of the different kinds of training he had received, and more than that, of the points of view of the several disciplines he represented. The work of his associates, friends and disciples testifies to the wide scope of his influence upon our science. REFERENCES Anonymous. 1943. A list of graduate students, research assistants, fellows and colleagues of Professor Ranson . Pub/. Inst. Neuro/., Northwestern Univ . Med. School , Chicago, Ill., Vol. 15

10 John R. Brobeck Bondy, P.K. 1968. Cyril Norman Hugh Long. YaleJ. Biol. Med., 41 :95-8 Fulton, J. F. 1951. Frontal Lobotomy and Affective Behaviour. New York: W.W. Norton Co. Hinsey,J.C. 1961. Pharos, Jan.:13-23 MacLean, P.D. 1960. John F. Fulton (1899-1960). Yale J. Biol. Med., 33:85-93 Stevenson, J.A.F. 1949. Recent Prag . Harm. Res., 4:363-94 Stevenson, J.A.F., Schenker, V., and Browne, J .S.L. 1945. J . Canad. Med. Services, 2:345-58 Stevenson, J.A.F., Whittaker, Joan, and Kark, R. 1946. Brit. Med. J., 2:45-7

Seven discoveries of physiological regulations

E.F. Adolph

The aim of this chapter is to survey the concept that physiological activities are internally regulated. For this purpose I recount experiments by seven different investigators which substantiate the concept. Physiological regulations designate those arrangements and activities by means of which an organism's processes are modulated: this way or that, fast or slow. They result from patterns of control or self-management. Many of the patterns favour survival of the individual, and presumably have persisted through natural selection among individuals. Obviously an organism is not a jumble of independent processes and events; there is co-ordination among regulated activities. Such interrelations provide the wherewithal oflife. These self-contained integrations may be regarded as the most special property ofliving beings as compared with non-living systems . If all the internal regulations should suddenly cease, decompositions of great variety would result. If more than a few minutes passed, destruction of life would become irreversible. That fact alone reveals that regulations cannot be ignored. The study ofregulatory arrangements was a constant emphasis in James Stevenson's researches and teachings . He enjoyed constructing diagrams of regulatory relations in the mammalian nervous system and in the inter-

12 E.F. Adolph connections among its parts. Thus, in two of his feedback diagrams (Stevenson 1967, p. 331) he diagrammed four regions of the brain that are known to receive sensory information, to co-ordinate it , and to sort it into actions that bring food and water into the body. I here inquire how certain men came to realize (a) that organisms selfregulate their processes, and (b) that experiments can ascertain how organisms do it. I purposely omit many non-experimental approaches that by hindsight (Adolph, 1961) can be seen to have contributed concepts of regulation. Of the seven experimenters cited, the first three lived before my day: Lavoisier, Bernard, Ludwig. The remaining four were : Haldane, Henderson, Cannon, and myself. A man comprehends most realistically the minds that he sensed in action. All seven experimenters stumbled initially into regulatory studies independently of one another; ultimately their concepts converged . LAVOISIER

(1743-94)

How did Lavoisier come across phenomena of physiological regulation? He undertook studies of respiration in animals because air was proven, chiefly by Joseph Priestley (1775) and himself, to consist of two fractions; a fraction consumable by an animal· or a candle, and a non-consumable fraction. Combustion and respiration were therefore chemically similar. They proved, however, to be dissimilar in that the animal dictated how fast oxygen was taken up by the body . 'Whether animals respire in pure vital air [ = oxygen] or whether they respire in the same air mixed with a large or small proportion of azote [ = nitrogen] the quantity of vital air that they consume is the same' (Lavoisier, 1920, p.39). This experiment demonstrated for the first time a regulation by 'non-conformity,' a modem label expressing the fact that oxygen uptake was not proportional to oxygen pressure in the air breathed . Actually A. Seguin and A.L. Lavoisier (1789) were the first scientists to measure oxygen uptakes under controlled conditions . When a guinea-pig was the subject, it was placed in a belljar over water, together with a capsule containing alkali to absorb carbon dioxide; the diminution in volume of the enclosed gas equalled the oxygen uptake (Lavoisier, 1920, p.38). When Seguin was the subject, he breathed from a copper face-mask into which room air or oxygen was drawn and from which the expired air was collected, and later analysed by allowing phosphorus to bum in it. By their control of conditions they stumbled upon a second demonstration of regulation: 'in a cold atmosphere a man decomposes a greater quantity of

Seven discoveries of physiological regulations 13 air; more heat evolves and repairs the loss of heat occasioned by the cold environment' (p.46) . 'So it is that an almost constant temperature of32° (on Reaumur's thermometer) prevails, and that many animals and man in particular conserve it under any circumstance' (p.47). Lavoisier recognized three pathways through which heat was gained and lost. 'The animal machine is chiefly governed by three principal regulators: respiration, which consumes hydrogen and carbon and furnishes heat; transpiration, which increases or decreases, according as it is necessary to give off more or less heat; and digestion, which puts into the blood what the blood has lost by respiration and transpiration' (p.47) . The facts were plain ; body heat was self-managed . Lavoisier made a remarkable generalization. He pictured 'the equilibrium of the animal economy' as follows: 'Digestions, in different instances, introducing into the blood more substance than respiration can consume , establish in the blood an excess of carbon and hydrogen. Nature then resists this alteration of the components ; it hastens the circulation by fever, it compels itself to repair, by augmented respiration , the disorder that disturbs its progress; often it succeeds without any outside help, and the animal recovers its health. In the contrary instance, it succumbs unless nature finds other means of reestablishing equilibrium' (p.49). Thus Lavoisier postulated that 'Nature resists alteration.' One must avoid imputing to Lavoisier a general twentieth-century concept ofbio-regulation, but it seems fair to say that he drew three pertinent inferences from the above experiments: (a) the quantity of oxygen taken is regulated within the body ; (b) oxygen uptake results in body heat in amounts that maintain body temperature ; and (c) constancy of substances or of heat is restored after a disturbance of 'equilibrium.' These three concepts of Lavoisier were in advance of his time; no one else had devised experiments from which they could be drawn . Lavoisier's conclusions about physiological regulations acquired significance in the light of what later generations have done and said . Bernard would have gloried in Lavoisier's acute inference that oxygen intake is controlled internally, if Lavoisier's statements had struck him at the right time. Delays in appreciation were noted by Seguin and Lavoisier themselves; they spoke of the 'necessary concatenation in the sequence of ideas, an indispensable order in the progress of the human mind' (p.31) . A period of 15 years had elapsed from the time Lavoisier learned how to prepare oxygen before he realized that a man's oxygen uptake would respond to change of circumstance. Identical publications are also contained in the collected works of

14 E.F. Adolph Lavoisier (1862). Biographies of Lavoisier have been published, e.g., that of McKie (1952); none describe in depth his contributions to physiology. Lusk (1925) indicated some of the techniques used. BERNARD

(1813-78)

Early in his scientific career Claude Bernard experimented on the metabolic utilization of sugars. He found sugar (glucose) in the blood of dogs that ate only meat, and those that ate nothing. The supply of glucose to the blood may have led Bernard to the concept of constancy of blood composition. At the time (1855) he said: The liver is an organ containing sugar that it injects slowly into the blood. What we do with a syringe full of sugar solution, the liver can do in a natural manner. If in a given time this organ throws into the blood an amount of sugar greater than suits the physiological state, the excess sugar immediately appears in the urine (p.234) ... When an animal is starving for a certain period of time, sugar is produced in the liver ... Appearance of sugar in the urine is a matter of threshold, of more or less. In a physiological state, sugar exists in all blood but without being seen outside the blood. If its quantity increases a little, the individual becomes diabetic either continuously or intermittently' (p.235).

The entrance of sugar into the blood when sugar is deficient, and the exit of sugar from the blood when sugar is excessive, clearly demonstrate regulation of the sugar present in blood. Nothing could at that time be said about the degree of constancy of sugar concentration in the blood, for the reason that the methods for measurement ofreducing sugars in blood were in 1855 barely able to show the presence of any sugar at all. In the end Bernard declared that 'sugar is one of the constant components of blood' (1877, p.165). Even then this statement was not supported· by specific data, but rested upon general knowledge. He further said, 'The animal has reserves that assure the fixity of composition of its internal environment' (1878, p.121). The more general concept that blood furnishes a constant internal environment to tissues, incubated in the mind of Bernard during twenty years or more. His first thoughts were recorded in his lectures of 1857 (published 1859), and vaguely in 1854 (Holmes, 1967; Grmek, 1967). 'The blood is a fluid into which all tissues expel their decomposition products, and in which they find, for performance of their functions, invariable conditions of temperature, moisture, oxygen, nitrogenous compounds, carbohydrates and salts, without which the organs could not be nourished' (1859, p.43). In

Seven discoveries of physiological regulations 15 the present context the words 'invariable conditions' seem especially significant; the tissues live in a fluid that protects them from harsh disturbances. In 1876 (lectures published in 1878) Bernard described comprehensively the regulatory significance of four 'conditions for free life.' These were : (a) water requirement, (b) heat loss, (c) oxygen supply, and (d) nutritive reserves . Without these the life of mammals was impossible (p.114) . Two points were emphasized: the nervous system was indispensable for maintenance of these conditions; and each condition employed specific 'arrangements regulating the losses and the gains.' The result of regulation was a constancy of composition and performance, acquired by appropriate activities . From the original concept (1857) of an internal environment for living cells, Bernard had developed the greater concept (1876) that this internal environment was actively maintained in respect to each of its physical and chemical components . A biography of Bernard was published by Olmsted (1938). There were other biographies as well . LUDWIG

(1816-95)

AND CYON

(1843-1912)

The widely known paperofE. Cyon and C. Ludwig: 'Die Reflexe eines der sensiblen Nerven des Herzens' described, though with incomplete evidence, what has come to be known as a feedback regulator. What did Cyon and Ludwig do and say? When they gave multiple electrical stimuli to the caudal end of a certain cut nerve leading from the rabbit' s head to its chest , the arterial blood pressure and the pulse rate did not change. But when they imposed like stimuli on the head end of the same cut nerve, both pressure and pulse rate decreased by 30 to 55 per cent. The effect was the same even after both of these 'depressor nerves' had been cut. Further, when both vagi and both stellate ganglia were excised, the decrease in blood pressure still took place . Evidently motor nerve impulses elicited from the head, said Cyon and Ludwig, widened the arterial blood vessels and thus reduced their resistance to the flow of blood from the heart (p . 132). Here was a self-activating adjustment of blood pressure . Cyon and Ludwig were then led to cut the splanchnic nerve that innervated most of the viscera. Now excitation of the depressor nerve had little effect on the arterial blood pressure. 'The stimulation of the depressor nerve reflexly decreases the tonus in the vascular nerves,' they said (p.139). Through this novel activity the 'motor of the blood flow can control the resistances which it itself must overcome ... The heart, when it fills

16 E.F . Adolph excessively either from lack of propulsive force or from fast inflow, and as a result is stimulated therefrom, changes not merely its pulse rate but also reduces the resistance to its emptying' (p.139). The assumption that excitations of the depressor reflex ordinarily arise within the heart was unwarranted; the investigations of others eventually showed that the endings of pertinent afferent nerves were located in the walls of the aorta (aortic sinus). Strictly speaking, one may assert that Cyon and Ludwig did not prove that the depressor reflex is part of a regulatory system (Wagner, 1961). The concept was established, however, that information arising from the stretch of pressure-sensitive elements acts reflexly to diminish the pressure in large arteries. Automatic machinery was almost unknown in 1866. There was no category of feedback . A few automatons that had been invented remained largely as toys . Mayr (1970) suggests that the water clock of Ktesibios of Alexandria employed the earliest known regulator of water flow, and that the self-acting damper of Drebbel about 1620 constituted the first regulator of temperature. Only in 1868 was a generalized theory of mechanical governors developed by Clerk Maxwell . In the following decades the use of electrical sensing devices and electromagnets to control supplies became practical. Hence in 1866 physiologists were justly surprised when a damper of blood pressure was found . Bernard himself chaired a committee of the Paris Academy of Sciences that awarded the annual prize in experimental physiology to Cyon. In a special review of Cyon' s work , Bernard (1868) noted that 'the heart can, with the aid of its sensory nerves, regulate to some extent its amplitude of beat in accord with its needs .' At that time no one supposed that reflex controls had anything in common with rates of oxygen uptake or with constancy of glucose in the blood . Neither Cyon (1900) nor Ludwig contributed further researches that directly concerned self-regulations. Ludwig's life and work were reviewed by Schroer (1967). HALDANE

(1860-1936)

In 1905 , J .S. Haldane and J .G. Priestley described the effects of varying the composition of inspired air upon the breathing of man. By adding CO2 to inspired air or by absorbing CO2 from rebreathed air, they demonstrated that 'the hyperpnea produced by re-breathing expired air was due solely to excess of CO2 .' From this demonstration they inferred that inspired CO2 changed the CO2 content of arterial blood and of bodily tissues, and 'the

Seven discoveries of physiological regulations 17 CO2 pressure in the respiratory center is the factor which normally determines the lung ventilation.' They also studied the effects of high altitude, atmospheric compression, exercise, and other influences; the CO2 pressure in the alveolar air and the arterial blood and the respiratory centre was the most constant feature of breathing. Today one says that a set point for pressure of CO2 in arterial blood is maintained. Although one can uncover ways of modifying this set point, one can nevertheless state that ventilation varies in ways that restore the pressure of CO2 after any disturbance ofit. In 1920 I worked in Haldane's laboratory at Oxford. The laboratory was attached to Haldane's house, and daily discussions were usual. Often he talked about physiological regulations and how they operated. So far as I can ascertain, Haldane's intensive thinking about physiological regulations began from his breathing tests. In 1906 Haldane brought several other aspects of regulation into his thinking, and wrote: 'An organism displays unmistakeable autonomy in maintaining its normal structure and physiological activities and so asserting its identity as a living organism. In face of injury or disease or disturbance of any kind, it tends to return to the normal. It regulates the breathing and the ingestion of food; its own growth; its temperature; its secretions and excretions; and other bodily functions' (p.104). In later years Haldane actively experimented not only on responses to high altitudes with Douglas et al. (1913); but also on responses to excess body water with Priestley (see Haldane and Priestley, 1916). In 1917 (Haldane, 1917) he intimately related his own notions to those of Bernard. He insisted especially that a whole organism was essential for selfregulation: studies of its parts allowed only partial results. His viewpoint helps to correct the reductionist notion that a model can give the complete story. A biographical account of Haldane was written by his collaborator C.G. Douglas (1936). A history of Haldane's idea of controls was published by G.E. Allen (1967). HENDERSON

(1878-1942)

L.J . Henderson was long an observer of physiological regulations. In 1939 he recorded the fact that a paper of Maly (1876) on the phosphates in blood aroused his interest. Maly showed that acid phosphate diffuses faster than alkaline phosphate, and suggested that acidity of the urine might result therefrom. Henderson, however, studied the equilibrium between the two

18 E.F. Adolph phosphates; since these two compounds would maintain neutrality in solution , they were powerful buffers . Their buffering capacity, he discovered, resulted from the fact that the dissociation constant had a numerical value approximately equal to the hydrogen ion concentration (1908) . The buffering of hydrogen ion concentration in urine and in blood depended on bicarbonate as well as phosphate (Parascandola, 1971b). Henderson realized that regulation by buffering was part of a larger system in which organs and tissues joined for mutual maintenance. By the time he published his famous book, The Fitness ofthe Environment, he could write : 'automatic regulations of the environment and the possibility of regulation of conditions within the organism are essential to life'(l913, p.31). 'The milieu interieur for its cells , - like the blood and lymph, - serves the same purpose as stability of the external environment' (p.33) . He stated his own aim as a biologist when he said: 'The task of the investigator has been to make known the facts concerning the regulation of the ultimate physical and chemical constitution of the organism. In this undertaking he has always kept in mind the idea that the organism exists in a state of dynamic equilibrium ,' (1917, p.82) . The final achievement is represented in his book Blood (1928) . He had devised methods of describing the multiple interrelations of seven components of blood. During the cycle of respiration and circulation the coordinate changes represented regulatory activities; some were bufferings, some were corpuscular exchanges , some were chemical transformations. Mutual relationships represented biological organization at a high level of complexity . Each interaction minimized any single chemical shift by spreading the change through related chemical shifts . Hence arose the stability of the respiratory change in blood as a whole, and in tissues dependent upon it. But, the stability was reciprocal , each tissue depending upon others. Henderson, aware of the thoughts expressed by forerunners, combined the experimental approach with the historical approach to the study of regulations . Henderson sponsored my student researches in 1919, frequently imparting his concepts . For biographical material on Henderson , see the papers of Cannon (1945a) and Parascandola (1971a) . CANNON

(1871-1945)

W .B. Cannon stated that he pursued six areas of physiological research

before he realized that a common theme of regulation pervaded those areas (1932 , preface) . Some of the areas were even represented in book form, in which, of course , general statements are expected . The six areas were: (a)

Seven discoveries of physiological regulations 19 phenomena of swallowing, (b) motions of the stomach and intestines, (c) nervous factors in digestive processes, (d) influences of emotional excitement on adrenal secretion, (e) the traumatic shock syndrome, and (f) functions of the autonomic nervous system. 'Researches on the service of the autonomic in providing for stability of the organism had been completed and published,' wrote Cannon, 'before the connection of that system with regulatory arrangements was clearly understood.' What experiments of Cannon led to his comprehensive view? His investigations of traumatic shock deeply impressed him . During World War I he found how quickly an inadequate blood flow would permanently damage certain tissues. The autonomic nervous system governed blood distribution, and kept the 'fluid matrix' flowing to each tissue. For him the autonomic system became the prototype of a regulatory tool; and starting from it, the other studies from his past fell into place . But a decade was required for them to do so. Meanwhile he had explored sugar regulation and other types of automatic adjustment that made the co-ordinations for recovery from shock seem overwhelmingly remarkable. In the first paper (1926) in which stability of bodily states was reviewed, Cannon 'advanced a number of tentative propositions concerned with steady states in the body.' There were six of these propositions, such as: 'Constancy is in itself evidence that agencies are acting, or ready to act, to maintain this constancy.' Again, 'The regulating system which determines a homeostatic state may comprise a number of co-operating factors brought into action at the same time or successively.' Later in 1926 he coined the word 'homeostasis' for the steady states maintained in an organism. What influence did Cannon receive from Henderson's historical approach to the study of regulations and his researches on acid-base equilibria? No one can say. The two men were great friends. Henderson explicitly stated (1917, p.83) that 'the phenomena of emotional excitement investigated by Cannon are all regulatory .' Eventually the atmosphere of Henderson's thought may have helped to arouse Cannon's concepts. Cannon published an autobiography entitled The Way of an Investigator (1945b). ADOLPH

(1895-

)

I add the story of my own discovery of physiological regulations because I know precisely how certain experiments revealed them to me. Water exchanges were investigated by weighing frogs that had been sitting in water. Urine accumulated in the bladder when a ligature closed

20 E.F. Adolph the cloaca. Some frogs were injected with known amounts of water; others were made to lose water during a preliminary period of evaporation in air. Thus excesses and deficits of body water of various amounts were created . When the frogs were put back in water, the original body water content was restored, from an excess through faster renal excretion, and from a deficit through faster cutaneous absorption (Adolph, 1939). Further, the same relation of output (by urine formation) and of uptake (by drinking) to body water content were observed in dog and man (Adolph, 1943). A human subject force-drank a measured quantity of water, or a dog was given the water through a stomach tube. In a half hour the collected urine increased in volume and in three or four hours nearly all of the water taken was excreted through the kidneys. The water given was an increment added to the body's water content; the renal output of water was the means of adjustment of body water excess. Constancy of water content was achieved by a response keyed to content. In the inverse situation, a deficit of water content was established by subtraction of body water through evaporative loss (sweating in man, panting in dog). After a measured deficit was created, the man or dog drank water ad libitum. The amounts of water drunk on the average were proportional to the body deficits. The excess or deficit was a stimulus, the output or intake was a response. Only at one water content, the norm, did the average intake equal the average output. A graph in which water exchanges (intake, output) were plotted against previously established water contents represented the 'equilibration' of body water. Inevitably I realized that the gains and losses of other bodily components, such as total nitrogen, were the means by which the content of nitrogen was adjusted. Exchange and content were related in the manner of response and stimulus, a general means by which the amount of component could be restored to its norm after any disturbance (Adolph, 1943). Perhaps for each component there exists a specific detector of increment or decrement that sets in motion a specific correction of the content. The relevant views of Haldane and Henderson did not, at first, connect with the phenomena I saw in the laboratory. My own insight had to come in its own manner and its own time; then the concept became more than a quote. Finally I understood what previous investigators had written about regulation. LESSONS FROM THESE CASE HISTORIES

The discoveries described above show how experiments led to cardinal concepts of physiological regulations. There is clear evidence that each experimenter started without any conscious grasp of them. Each experi-

Seven discoveries of physiological regulations 21 enced the general observation of Whitehead (1954): 'There is always more chance of hitting on something valuable when you aren't too sure what you want to hit upon.' Today all the above paradigms of regulation can be reduced to a single rule, which I would state as follows: What an organism is and does is regulated through selected interrelations among its constituent properties and actions. Present-day appreciation of physiological regulations may be said to result from equal parts of physiological experiment and conceptual thinking. Pertinent concepts were available 2300 years ago, but by themselves gave no substance to the study. In the nineteenth century, experiments were done that contributed to the notion of regulations but some were not recognized as doing so . To be convincing to any physiologist, detailed descriptions of how regulations operated were needed, and had to be seen in the light of his own conceptual thinking. When each of the seven physiologists mentioned here stumbled into the experiments quoted, he aimed to study a special phenomenon. Long after he had completed that study he realized that the organism actively controlled the phenomenon. He noted that the animal body and certain of its parts maintained its own structures and activities. Finally, some realized that their predecessors had recorded similar thoughts. PROSPECT

There are many kinds of biological regulation: evolutionary, genetic, developmental, subcellular, enzymic, etc. Physiological regulation may be considered to be co-ordinate with these. But further, there are varieties of physiological regulation: homeostatic, non-homeostatic, substitutional, reparative, etc. The seven discoveries here recounted all concern homeostatic regulation. We now realize that Bernard spoke for everyone . Yet every regulation was observed differently, and it took a long time for similarities among them to become apparent. Today we can list specifications for homeostatic regulations: feedback, proportional detector, set point, closed loop, etc . Consequently, examples that belong to this category can be quickly recognized. Regulations are visible in the following diverse phenomena: growth, differentiation, repair, metabolism, turnovers, compositions , variabilities, sensory arrangements, communications, enzyme productions. Each regulation is known to manifest fixed norms and limiting factors, yet each is susceptible to shift in norm or rate in accord with specified conditions. Special characterizations of physiological regulations in current use are:

22 E.f. Adolph cybernetic diagrams, cycles of enzymic act1v1tles, blocking agents, maintenance of set points, tests of tolerance, interactions among properties, etc. Since some studies of regulations can be planned in the same degree as studies of other physiological phenomena, both old and new approaches are needed. How are the patterns created during ontogeny? What factors determine which of the available regulatory responses shall predominate at each moment, and how rapidly shall each action proceed? Quando et quanto? However, the investigation of physiological regulations, like that of other biological principles, rarely follows a programme . Bernard (1878, p.50) did not pre-empt this branch of biology when he wrote: 'There is an arrangement in the living being, a kind of regulated activity which ... is in truth the most striking characteristic of living beings' (transl. Henderson, 1927 p. vii) . He was pointing to an unknown territory for scientific exploration; he was asking his successors to devise methods for examination of it. For any physiologist the thrill may come not from a lecture on Bernard's concept of physiological regulation, but from a sudden realization that he has been researching for years on regulations without knowing it. The opinion is sometimes expressed that studies of physiological regulation are to be undertaken after certain 'mechanisms' have been analysed. But, it is unnecessary to wait. Every response of an organism or its parts is a constituent of a regulation. How does this response participate in the life of the body or cell? Unopened black boxes need not delay the study. In my estimation, regulatory actions of organisms permeate all physiological studies and do not occupy a separate compartment. This was James Stevenson's attitude; it is one that prevails in this memorial volume, and one that enlarges the outlook of the science of physiology. REFERENCES Adolph, E.F. 1939. Ann. physiol., 15: 353-67 - 1943. Physiological Regulations. Lancaster: Cattell - 1961.Physiol.Rev.,41:737-70 Allen , G.E. 1967. J. Hist. Med. , 22: 392-412 Bernard, C. 1855. Lerons de physiologie experimentale appliquee a la medecine. Paris: Bailliere. Tome 1 - 1859. Lerons sur /es proprietes physiologiques et /es alterations pathologiques des /iquides de /'organisme . Paris : Bailliere. Tome 1 - 1868. J. anal. physiol. , 5: 337-45 - 1877. Lerons sur le diabete et la glycogenese animale . Paris: Bailliere - 1878. Lerons sur /es phenomenes de la vie communs aux animaux et aux vegetaux. Paris : Bailliere. Tome 1

Seven discoveries of physiological regulations 23 Cannon, W.B. 1926Am. J. Med. Sci. , 171: 1-20 - 1932. The Wisdom of the Body. New York: Norton - 1945a. Biographical Memoirs Nat. Acad. Sci., 23: 31-58 - 1945b. The Way of an Investigator. New York: Norton Cyon, E. 1900. In C. Richel: Dictionnaire physiol ., 4: 88-160 et 774-94 Cyon, E. und Ludwig, C., 1866. Arb. physiol. Anstalt zu Leipzig, I: 128-49 Douglas, C.G. 1936. Obit. Notices Roy. Soc. London, 2: 115-39 Douglas, C.G., Haldane, J.S. , Henderson, Y., and Schneider, E.C. 1913. Phil. Trans. Roy. Soc.London,203e : 185-318 Drebbel, C. About 1620. See Mayr(1970) Grmek, M .D. 1967. In E. Wolff et al., Philosophie et Methodologie Scientifiques de Claude Bernard. Paris: Masson, 117-50 Haldane, J .S. 1906. Guy's Hosp . Repts . , 60: 89-123 - 1917. Organism and Environment. New Haven : Yale Haldane, J .S. and Priestley, J .G. 1905. J. Physiol., 32: 225-06 - 1916. J. Physiol., 50: 296-303 Henderson, L.J. 1908. Am. J. Physiol., 21 : 427-48 - 1913. The Fitness of the Environment. New York: Macmillan - 1917. The Order of Nature . Cambridge: Harvard University Press - 1927. Pages v-xii in C. Bernard (trans . H.D. Greene), An Introduction to the Study of Experimental Medicine. New York: Macmillan - 1928. Blood, A Study in General Physiology. New Haven: Yale - 1939. Memories. A Manuscript in Harvard Univ. Library. Cited by Parascandola (1971b) Holmes, F.L. 1967. Pages 179-91 in F. Grande and M.B. Visscher (eds .), Claude Bernard and Experimental Medicine. Cambridge: Schenkman Lavoisier, A.L. 1862. (Euvres. Paris: lmperiale. Tome 2: pp. 688-703 - 1920. Memoires sur la respiration et la transpiration des animaux. Paris: Gauthier-Villars Lusk, G. 1925. J. Am . Med. Assoc., 85: 1246-47 Maly, R. 1876. Ber. deut . chem . Ges. Berlin, 9: 164-72 Maxwell, J. Clerk. 1868. Proc. Roy. Soc. London, 16: 270-83 Mayr, 0. 1970. The Origins of Feedback Control. Cambridge: M.I.T. McKie, D. 1952. Antoine Lavoisier. London : Constable Olmsted, J.M.D. 1938. Claude Bernard, Physiologist . New York : Harper Parascandola, J . 1971a. J . Hist. Biol., 4: 63-118 - 1971b. Medizin-historisches J. 6: 297-309 Priestley, Joseph. 1775. Experiments and Observations on Different Kinds of Air. London : Johnson . Vol. 2 Schroer, H. 1%7. Carl Ludwig . Stuttgart: Wissensch . Verlagsges Seguin, A. et Lavoisier, A. 1789. Mem . Acad. Sci. Paris, Annee 1789, 185-96 Stevenson, J.A.F. 1%7. Chapter23 in M.R. Kare and 0 . Maller, (eds.). The Chemical Senses and Nutrition . Baltimore: Johns Hopkins Wagner, R. 1961. Naturw . Rundschau, 14: 65-8 Whitehead, A.N. 1954. Page 344 in L. Price: Dialogues of Alfred North Whitehead . Boston: Little Brown

Anatomical and neurobiochemical bases of the central nervous control of physiological regulations and behaviour*

P.J. Morgane

INTRODUCTION AND GENERAL ORIENTATION

Experimenters who entered the field of hypothalamic physiology in the 1950s and 1960s and who were fortunate enough to interact and conceptualize with Stevenson came away very much enriched . One of his main attributes was that he appreciated all types of approaches and methodologies and could see the value of each in unravelling the functions of the hypothalamus. Most especially, he espoused holistic views of the central nervous system operating at several integrative levels well beyond · the confines of the hypothalamus . He had an inherent interest in limbichypothalamic relations in a broad context, including neuroendocrine interactions. We should stress this concept of interrelations between brain areas since, after all, that is what nervous integration is all about. Obviously, such a holistic view cannot possibly be obtained simply by micro-minutia! analysis of the effects of lesions or any other manipulations restricted exclusively to the hypothalamus. If I could get one message across it would be to think beyond the confines of the hypothalamus and begin to *Supported by NIMH Grants MH-02211 and MH-10625

Control of physiological regulations and behaviour 25 deal with the brain based on the way it is really organized, i.e., at various integrative levels interlinked by recurrent loops of chemical circuitry, with interactions occurring because of changes in brain chemistry over wide areas. Thus, so called 'syndromes' or other constellations of events that might once have been thought to be relatable to the hypothalamus appear, rather, to be caused by changes in distant brain chemistry which is altered by manipulations of the fibres which merely pass through the area. In this sense the pathway is the message! Particularly so, if we know the chemical identity of the pathways and the nature of the influences operating on the cells of origin of these pathways . Unfortunately, not only has too much research been focused strictly at the hypothalamic level but many purely descriptive behavioural analyses have been derived purporting to 'explain' mechanism. Further, most of these behavioural speculations have been derived on the basis of the least resolving approach to the central nervous system functions, i.e., the electrolytic lesioning procedure. With respect to lesions, part of the results obtained with such approaches may be attributable to lost as well as stimulated (excited) function. As Hughlings Jackson (1958) astutely observed, we have two factors to consider: first, a negative factor - a loss of function which 'resided' in the damaged structllre - and, second, a positive factor - a release of function normally inhibited by the damaged structure. The former may be the less important of the two because of compensations and vicarious functions developed by other structures. In hypothalamic physiology we have had far too much emphasis on 'what's missing.' In reality, what we need is more work on release of function and neurophysiological and neurochemical analyses of changes in brain structures linked with the hypothalamus, particularly the limbic and reticular formations. Little information has been gathered about the identity of the nervous structures whose excitation leads to the observed effects, since this requires information on the thresholds for different nervous elements in the vicinity, their connections, and normal discharge characteristics. This type of approach has rarely been applied to the hypothalamus. Also, it has often been impossible to decide if the response is due to activation of cell bodies or of fibres of passage, or both. It is well known that accurate localization of electrolytic lesions to a prescribed nucleus or region is difficult, if not impossible. In chronic lesion experiments especially, there is always a rapidly evolving population of normal, abnormal (recovering and dying), and dead neurons on the periphery ofa lesion with passage of time. Remote effects are also present because of vascular damage, retrograde, and, perhaps most important,

26 P.J. Morgane transneuronal degeneration. The relative effects of these at different time periods are difficult to assess. Further, the distant chemical pathology of the brain has rarely been assessed until recently. In interpreting the effects of lesions, there is the additional problem of distinguishing those resulting from the loss of a discrete hypothalmic system and those arising incidentally from the disturbance of spatially overlapping systems. We might observe immediately that, if ever there was a nervous complex organized in terms of spatially overlapping systems, it is that composed of the two large longitudinal fibre systems of the hypothalamus, i.e., medial forebrain bundle and the periventricular pathways. This is discussed in some detail below but, in this regard, it should be emphasized that the presence of dendritic overlapping does not necessarily prove the morphological or functional equivalence of neighbouring regions. Rather it can be used to question the dogmatic value of many proposed functional parcellations. This means that when extensive dendritic overlapping exists between adjacent territories, such as along the entire medial forebrain bundle system, there is some reason to regard them as one single region until the contrary is indicated. The widespread practice of defining as the hypothalamic locus of a given function a small anatomical area common to a series of large lesions in different animals is of doubtful validity. It is regrettable that so great a proportion of hypothalamic research has been limited to the large lesion approach followed by purely descriptive behavioural analyses, without any regard to underlying processes. It has even been put forward almost as 'doctrine' that loss of feeding and drinking activity seen after lateral hypothalamic lesions is completely due to 'behavioural' deficits, and, to make matters worse, this has frequently been described as 'loss of motivation to eat or drink,' as though this categorization has any semblance of neurological meaning. Does not 'behaviour' have its essence in the underlying dendritic geometry, nerve impulse activity, and brain chemistry? What is the magic of the word 'motivation' in coming to terms with the brain itself? We should not continue attempts to perpetuate a false dichotomy between behaviour and underlying physiological mechanisms, where 'behaviour' becomes some sort of epiphenomenon divorced from underlying processes beyond the reach of physiological and biochemical approaches. Micro-behavioural descriptions of so-called evolving hypothalamic 'syndromes' have been mistakenly put forward as 'elucidating' the hypothalamic mechanism of feeding and such psychologizing has even been offered as 'differentiating the various components that make up

Control of physiological regulations and behaviour 27 normal feeding behaviour.' Obviously 'mechanism' cannot be approached from such a sideline view and describing stages and behavioural sequences hardly defines 'components' of a feeding or drinking mechanism. Even the most perceptive behavioural analyses remain speculative when underlying neural events are not considered! By indiscriminately scooping out large aliquots of hypothalamus willy-nilly some have thought to reveal something substantive about function . Pribam (1961) stated this case quite succinctly and decisively, 'ablation of the brain per se does not lead to a predictable change in behavior when that behavior is complex - prediction can be made only when one can detail the situation in which the behavior takes place as well as the structure that has been manipulated .' The reinterpretations of hypothalamic organization and function by Valenstein and co-workers (1970) also bear on the above observations. In these studies they have presented a variety of evidence to support the view that there is much less anatomical specificity within the hypothalamus than is commonly assumed . They have described significant differences between natural states of hunger and thirst and those associated with the artificial elicitation of eating and drinking. They suggest an alternative interpretation of hypothalamic function in the regulation of behaviour and question the view that relatively discrete hypothalamic regions are involved in the regulation of behaviour related to specific biological needs. They found an impressive amount of overlap of the anatomical sites yielding different behaviours in their own work and in that of others . Thus , it is difficult to justify the impression given by many investigators, who have restricted their observations to one behaviour, that there are discrete hypothalamic areas associated with a specific behaviour. It is obvious that the behaviour observed in many studies has been determined by the particular experimenters' interests and so has led to consequent limitations of the whole testing situation. One primary conclusion of the Valenstein group was that the behaviour elicited by hypothalamic stimulation may be maintained by some variable other than the satisfaction of a simulated need state such as hunger or thirst. It was further shown that the sites eliciting eating, drinking, and gnawing co-exist with the sites that elicit only nonspecific exploratory and generalized locomotor behaviour. Valenstein's group has also pointed out that, in the rat, where chemical stimulation is most effective , non-specific activation has an especially high probability of evoking eating and drinking. Taken together, the Valenstein, Cox, and Kakolewski reinterpretation of hypothalamic organization stresses the importance of viewing the behaviour elicited by hypothalamic

28 P .J. Morgane stimulation in terms of biologically essential behaviour, species-specific response patterns, and the environmental conditions that normally elicit them in an aroused animal. This new look at the hypothalamus has important general implications in terms of discarding 'centre' theory and concepts of functional localization and, most important, involves, in its broader sense, consideration of non-specific systems in the brain and the role of possible equipotential extrahypothalamic formations in the genesis of any complex, sequential behaviour. In general, it should not be implied that there have not been important results derived from judicious and cautious interpretation of small, overlapping lesions in the hypothalamus, provided these are considered in a broader context of hypothalamic interrelations with other brain areas. The 'full-sham' procedures, used effectively by Morrison and Mayer(1957) and Morgane (196la,b), certainly have borne out the fact that the most effective regions of the lateral hypothalamic area related to different aspects of feeding behaviour are much more closely circumscribed than the size of most electrolytic lesions reported in the literature might suggest. All functional interpretations drawn from large, 'defoliating' lesions in such a complex area as the lateral hypothalamus are questionable indeed . Needless to observe, bulldozer approaches such as large electrolytic lesions simply do not have adequate resolving power to define the relative roles of the maze of chemo-specific pathways criss-crossing in the lateral hypothalamic area or in any other reticular zone of the brain. In a restricted region such as the hypothalamus, even small incidental parts of the lesion, such as the electrode tracts, can cause substantial damage to fibre pathways which, in some cases, are individually compact. For the analysis of the effects of such lesions, it is therefore essential to establish the course and mode of termination of the hypothalamic afferent fibres of extrahypothalamic origin, the extent of functional impairment caused by destruction of the afferent pathways alone and, most importantly, determine the widespread alterations in brain chemistry resulting from damage to amine-specific fibres pervading this region. But, even with all the shortcomings of the lesioning method, it has given some crude insights into the workings of the hypothalamus and other brain areas. The significant fact to emphasize is that lesions, being indiscriminate, afford little idea of the nature of the working mechanisms. It is up to other approaches (reviewed below) to provide data upon which appropriate models of mechanisms can be constructed. Some of these studies have indicated that there is a high degree of afferent convergence on hypo-

Control of physiological regulations and behaviour 29 thalamic neurons, which emphasizes that interactions between sensory modalities are thus rendered possible. In this regard, the heterogeneous nature of the afferent supply to its constituent cells is one of the most typical of all attributes of hypothalamic cells and bears directly on the newer conceptualizations which consider the hypothalamus as an extension of the reticular formation (Morgane and Stem, 1974). Ramon-Moliner and Nauta (1966) have emphasized that a considerable degree of dendritic overlapping is characteristic of this type of neuronal assembly. As a result of the generally great length attained by the dendrites, and their rectilinear course, a continuum of overlapping dendritic fields is formed that appears to extend throughout the length of the brainstem, including the hypothalamic fields (Millhouse, 1969, 1973a,b). Further, free intermingling of dendrites with passing myelinated and unmyelinated fibre bundles characterizes this entire complex. Cross (1964), using unit analysis, has noted that there is spatial dispersion oflike-responding cells, which argues strongly against a rigid notion of any one nucleus/one activity-type organization. He and others have further stressed that activity and responsiveness to incoming stimuli can also be modulated by the hormonal environment of the brain. All of these considerations must, of course, be taken into account in constructing a model for any hypothalamic control system. Most hypothalamic systems are clearly not closed systems, for in every case there are other nervous or humoral influences that can be shown to operate and which can alter the output of the system. Perhaps the greatest simplifications in this field are those suggesting that glucoreceptors, osmoreceptors, and thermoreceptors, among others, are organized as separate functional entities. As is well known, some experimental evidence indicates that thermoregulatory and osmoregulatory mechanisms are linked with the mechanisms controlling food intake (Andersson et al., 1963), and many other examples of overlapping systems, both in the chemo-anatomical and functional sense, can be given. The very principle of overlapping systems implies that consideration of relations between hypothalamic and extrahypothalamic mechanisms are critical for any understanding of the role of the hypothalamus in any organized behaviour. Obviously, the hypothalamus regulates its outputs according to the memory stores available to it and to the signals continuously impinging on it from both nervous and humoral pathways and it is a step forward that we are at last beginning to look at these within the broad framework of the limbic-hypothalamic-reticular axis. Before examining hypothalamic functioning in terms of synaptology,

30 P.J. Morgane electrophysiological relations, and chemo-morphology, we should scrutinize briefly some of the concepts that the 'syndrome-makers' have advanced in the name of hypothalamic physiology. First of all, if one examines the hypothalamus as it is really organized, it becomes obvious there can be no such entities as 'hypothalamic' syndromes. Only for convenience sake would there be justification for naming them as such and only then if this served some specific heuristic purposes in terms of understanding hypothalamic organization or else revealed something definitive about underlying mechanisms and physiological processes - or, in the broader sense, if 'syndroming' revealed something of the nature of mechanisms of compensation and reorganization in the central nervous system following damage to foci of convergence of many neural pathways and interdigitated dentritic trees. The whole organization of the hypothalamus as a diencephalic extension of the lower brainstem reticular formation and as a bridge in overlapping circuitries of limbic and reticular fields bespeaks against defining functional loci and perturbations of these in circumscribed areas in the brain. Otherwise, to emphasize this point, in the field of energy homeostasis we would now have globus pallidus 'syndromes,' amygdalar 'syndromes,' substantia nigral 'syndromes,' mesencephalic 'syndromes,' and a host of others. It becomes a rather ludicrous exercise in 'syndroming' to regard the nervous system in such terms. Earlier (1961), I produced varieties of quantitative and qualitative disturbances in feeding behaviour by the use of discrete lesions and even full-sham electrode insertions directed to the far- and mid-lateral hypothalamic areas. There was no difficulty in distinguishing several varieties of disturbances in feeding and drinking behaviour across the continuum from the perifornical area, through the far-lateral hypothalamus, and into the globus pallidus and its conglomerate fibre systems . These latter were gathered in the upper far-lateral hypothalamic area, in the internal capsule, and in both the internal and external segments of the pallidi. Most important, the large lesions of the lateral hypothalamus had little resolving power in distinguishing the subtleties of these differences, which appeared to me at the time to be strictly related to damage to different fibre systems passing through the lateral hypothalamic area. Damage to the striatal fibres of passage were described by me in 1961 as resulting in a type of adipsia and aphagia qualitatively different from that seen with lesions in the lateral perifornical (mid-lateral) hypothalamic area. These differences might also be illustrated with just two further examples.

Control of physiological regulations and behaviour 31 Stevenson and Montemurro (1963) produced a type oflateral hypothalamic lesion following which administration of water and food by stomach tube did not prevent weight loss and death. Their finding of a dramatically increased metabolic rate points up the clear differences in the effects these lesions may produce . Also, Smith et al. (1972) stu~ied the chemical organization of the lateral hypothalamus by injecting 6-hydroxydopamine into the medial forebrain bundle at several antero-posterior levels. They noted that, if the rats were maintained by tube feeding, some ate and drank again and their behavioural 'recovery' differed from that often described as 'the' lateral hypothalamic 'syndrome' in three ways : 'recovered' rats (1) drank water in the absence of food ; (2) drank water in response to i.m. NaCl or isoproterenol ; and (3) ate dry food in the absence of water. Their major thesis was that catecholaminergic neurons form part of the neural network for feeding and drinking behaviour in rats, but the generalizing principle one can derive from such work is that we are not dealing with single syndromes and prescribed, invariant 'recovery' phases as defined originally by large electrolytic lesions. In over 200 rats I have produced every conceivable variation on the 'lateral hypothalamic theme' by select small lesions in the lateral hypothalamus itself, in the globus pallidus, subthalamus, pallidal efferent pathways , etc . Never did I see anything resembling an invariant constellation of events and recovery process - this varied clearly with the type, extent, and anatomical locus of the manipulation. Lately it has been shown that, when individual chemo-specific amines and other transmitter systems are separated out by means of chemical lesions, certain subtleties of the functional impairments are revealed which can never be elicited by any kind of 'molar' analyses of behaviour following indiscriminate simultaneous disruption of the many chemical pathways in the lateral hypothalamus . The over-all implication here is that microbehavioural fractionation techniques should not be applied to studying effects following large, disruptive lesions. At the stage of research sophistication of 15 years ago we were more confined and limited to cruder approaches, such as electrolytic lesioning procedures which had, as noted above, inherent limitations in their resolving power. However, our approach, even in that day , took full cognizance of the hypothalamic circuitry and fibres of passage in its various zones . Since chemical lesioning based on chemical anatomy has advanced us considerably past the ' syndroming' stage, we can look back on that type of analysis as serving rather limited purposes in descriking behaviour rather than in elucidating mechanisms

32 P.J. Morgane involved. Molar analysis of behaviour may serve some valuable, iflimited, purposes, but sooner or later the nuts and bolts of the brain have to be explored. EXTRINSIC CONNECTIVITIES OF THE HYPOTHALAMUS

With the above as a broad preamble, I wish first to point out some principles of hypothalamic-limbic functional interaction. One aspect of this view is that the hypothalamus is seen as part and parcel of a large and complex series of interacting, overlapping subcircuitries comprising, in large part, multiple components of the medial forebrain bundle and periventricular fibre systems. Thus, it becomes inextricably tied to limbic and reticular systems and forms, in essence, an anatomical continuum with both. The many details of hypothalamic and limbic connectivities, so amply reviewed as to basic anatomy by Nauta and Haymaker (1969) and as to chemical anatomy by Morgane and Stem (1974), would serve no useful purpose within the framework of this book. Rather I will concentrate on particular connectivities as these serve to illustrate special aspects and functional relations of components of the hypothalamus, amygdaloid complex, and striatal system related most closely with problems of energy homeostasis. For the present account I would point out the pronounced reciprocity in the connections between the hypothalamus and limbic forebrain and reticular formations (including the limbic midbrain zone). Also, there is now considerable evidence that neural pathways between the limbic components of the cerebral hemisphere and hypothalamus are predominantly organized in the form of neural circuits . As shown in Figure 1, the hypothalamus forms part of two neural circuits , one of which connects with the limbic forebrain structures, the other with the limbic midbrain fields . Nauta (1963) considers both circuits to be subdivisions of one and the same major circuit formed largely by multisynaptic neural connections linking the limbic forebrain structures with the limbic midbrain area and vice versa, both limbs of the circuit relaying, in large part, in the hypothalamus . Viewed in this manner the hypothalamus appears as a nodal point in a vast neural 'system-field' extending from the medial wall of the cerebral hemisphere caudalward to the lower boundary of the midbrain. Thus, even if based strictly on morphological evidence, it is clear that the functional state of the hypothalamus is influenced continuously by the prevailing activity patterns in the limbic forebrain-limbic midbrain circuit as a whole . As discussed

Control of physiological regulations and behaviour 33

Figure I Comprehensive schema of the limbic forebrain-limbic midbrain system. Upper drawing indicates the system in detail. Lower drawing reduces the system to its essential elements for description. Note the powerful reciprocities developed between the entire limbic forebrain zone with the paramedian area (limbic midbrain zone) of the midbrain. Abbreviations: 1c, inferior colliculus; sc, superior colliculus; PAG, periaqueductal grey; DG, dorsal nucleus ofGudden; VG, ventral nucleus ofGudden; NCS, Nucleus centralis superior of Bechterew; P, pons; IP, interpeduncular nucleus; oec, decussation of the brachium conjunctivum; PTA, pretectal area; HAB, habenular nuclei; M: mammillary nuclear complex; VTA, ventral tegmental area of Tsai; LHA, lateral hypothalamic area; VM, ventromedial nucleus of hypothalamus; ST, subthalamic area; PV, periventricular area of hypothalamus; oc, optic chiasm; LPO, lateral preoptic area; HIP hippocampus; cc, corpus callosum; F, fomix; AC, anterior commissure; s, septa! area; OT, olfactory tubercle; OFC, orbito-frontal cortex; oe, olfactory bulb; AM, amygdaloid complex; IL, intralaminar nuclei of thalamus; A, anterior nuclei of thalamus; TH, thalamus; CG, central grey; RF, reticular formation; LMA, limbic midbrain area; LFA, limbic forebrain area

below, more and more neurophysiological and neurochemical studies are bearing this out. One of the most important studies for consideration in this field is

34 P .J. Morgane analysis of the afferent neural pathways by which this limbic-midbrain circuit and its components can be activated. Unfortunately, these afferent connections are not always compact and often do not form bundles as is the case with the classical sensory afferent systems. However, chemical mapping of pathways has now revealed several distinct bundles within these major paths, especially the medial forebrain bundle. Nevertheless, many afferent connections to the limbic forebrain-limbic midbrain circuit form part of more diffusely organized pathways ascending from the spinal cord and brainstem reticular formation and likely convey a wide variety of sensory modalities, as well as impulses of visceral origin. Cowan et al. ( 1964) have pointed out that there are three ascending pathways which link the midbrain to the hypothalamus:·the mammillary peduncle, the medial forebrain bundle, and the periventricular fibre system. From the region of the dorsal and deep tegmental nuclei of Gudden (Figure 2) the fibres of the mammillary peduncle run ventro-laterally through the midbrain tegmentum, finally sending most of its fibres into the medial and lateral mammillary nuclei, and some into the medial forebrain bundle and lateral hypothalamus, with a few reaching as far as the septum . Caudally, the medial forebrain bundle itself is first recognized in the ventral tegmental area of Tsai from which it can be traced forwards, just dorsal to the mammillary peduncle, and thence through the lateral supramammillary, lateral hypothalamic, and preoptic regions to the septum and olfactory tubercle. The periventricular fibres enter the hypothalamus from the periaqueductal region of the midbrain. There is an extensive interchange between the medial forebrain bundle and the periventricular fibres at posterior hypothalamic levels (Nauta and Kuypers, 1958), with both systems having the same basic structure of cells mingled with ascending and descending fibres, the length oT the fibres being highly variable. Before chemical anatomy came to the forefront, not much could be said with certainty regarding functional relations of this ascending circuitry with the neocortex, but now, as summarized below, many of these connectivities are being revealed. The details of the synaptic organization of the descending pathways to the motor neurons are also not known, but it appears likely that numerous synaptic relays occur at successively more caudal levels of the brainstem reticular formation . Autoradiographic techniques are now finally revealing some of the details of some components of this descending circuitry (Lasek et al., 1968; Cowan et al., 1972; Edwards, 1972). A few additional overviews concerning hypothalamic organization within the framework of the limbic forebrain-limbic midbrain system are in order to set the stage adequately for discussing the functional organization

Control of physiological regulations and behaviour 35

Central

Grey

MFB - - - - -

IMedSop

N...•H'PPI

Figure 2 Representation of some of the midbrain fibre systems that contribute to the medial forebrain bundle. In particular, note that the dorsal and ventral tegmental nuclei ofGudden send ascending projections through the mammillary peduncle and tegmento-peduncular tract. Fibres from the nucleus raphe dorsalis are shown forming the ventromedial component of the medial forebrain bundle. The nuclei of Gudden are primary midbrain nuclei receiving descending limbic projections and form part of the 'limbic midbrain area.' Abbreviations: DLF, dorsal longitudinal fasciculus of Schiitz ; Dor Teg Nu Gud, dorsal tegmental nucleus of Gudden ; Ven Teg Nu Gud , ventral tegmental nucleus ofGudden; Nu Cent Sup, nucleus centralis superior of Bechterew; Mamm Ped, mammillary peduncle; Mamm Nu, mammillary nuclei; LHA, lateral hypothalamic area; Lat PreOp Ar, lateral preoptic area; MFB (Med Sep Nu+ Hipp, Medial Forebrain Bundle; medial septa( nucleus and hippocampus); MFB (Ventro-Med Paths), medial forebrain bundle (ventromedial paths); Ted Ped Tr, tegmento-peduncular tract; Hab IP Tr, habenulo-interpeduncular tract; Hab, habenularnuclei; Nu Raphe Dors (e7), nucleus raphe dorsalis (histoftuorescence code e7)

of this complex system. The medial forebrain bundle is a highly complex and chemically heterogeneous system and extends throughout the entire lateral hypothalamus and continues rostrally through the preoptic area to the olfactory regions, and caudally to the midbrain. It is composed of many short relaying fibres as well as longer projections. It is with the medial forebrain bundle, rather than the medial hypothalamic nuclei, that most of the extrinsic connections of the hypothalamus are made. Relationships between the medial forebrain bundle and the medial hypothalamus have,

36 P.J. Morgane however, been quite clearly shown in recent years by Golgi studies and electrophysiological analyses (see below). In general, the directions of conduction appear to be predominantly from the lateral hypothalamic areas to the more medial areas, including the periventricular zone. One of the major problems concerned with hypothalamic connections is the question of which pathways mediate the well-known effects of both exteroceptive and interoceptive stimuli upon central endocrine, behavioural, and autonomic mechanisms. Newer approaches such as chemical anatomy and autoradiographic techniques are revealing some of these relations, but it should be emphasized that there appears to be no well-established connections of the hypothalamus with the sensory lemniscal systems of the brainstem. Two further problems related to the effector aspects of the hypothalamus also merit consideration. First, as regards the concept of the hypothalamus as an autonomic effector structure, it is noteworthy that the descending hypothalamic connections do not appear to make direct contact with any known visceromotor areas. Second, from the point of view of endocrine control, such functional regions as have been mapped out in the medial (tuberal) zones of the hypothalamus do not correspond well with cell groups defined by conventional anatomical methods (Fuxe and Hokfelt, 1969; Bjorklund et al., 1970). Further, the fact that such areas as the hippocampus and amygdala, which are known to be so intimately connected with the hypothalamus, are themselves still relatively little understood functionally makes it easier to appreciate that we are presently only at the threshold of correlating hypothalamic anatomy and physiology. Some projections from the hippocampus and amygdala are reviewed by Dr Hall in this volume and will only be touched upon here. It is of special interest that the components of these projections differ in regard to their fields of origin within the amygdala and hippocampus and that strong reciprocities are established from the hypothalamus back upon these areas. Anatomically, the connections between the hypothalamus and the rest of the forebrain are dominated by the interconnections with limbic structures (Nauta, 1963), i.e., the hippocampus, septum, amygdala, and pyriform cortex . The amygdala and the hippocampus are connected with the hypothalamus by many direct and indirect fibre pathways. These pathways probably participate in any of the whole range of endocrine, autonomic, and behavioural responses that may be regulated by the hypothalamus. Broadly, we can summarize hypothalamic relations as follows: (1) its most massive associations are with the limbic forebrain structures and the paramedian region of the midbrain; (2) the hypothalamus receives few, if

Control of physiological regulations and behaviour 37 any , direct projections from the generally recognized sensory pamways; (3) its projections to known visceral motor nuclei are established via as yet unworked out pathways in the mesencephalic and bulbar reticular formation. The reciprocal connections of the hypothalamus with the limbic forebrain structures and the paramedian mesencephalon are of such magnitude that Nauta (1958), in particular, interprets the hypothalamus as a waystation in both the ascending and descending limbs of a multisynaptic neural circuit extending between the limbic forebrain, on the one hand, and the paramedian mesencephalic region (limbic midbrain area) on the other. From this it can be seen that the functional state of the hypothalamus is continuously determined by the neural events taking place in this limbic forebrain-midbrain circuit as a whole. The lateral hypothalamic area appears to be most directly involved in the circuit , particularly via several components of the medial forebrain bundle (Figure l) . However, more medial hypothalamic cell groups , presumably including the 'hypothalamo-hypophysial motor neurons,' receive direct connections from both the upper and lower poles of the circuit (e.g., via the stria terminalis and dorsal longitudinal fasciculus , respectively), and are furthermore connected with the lateral hypothalamic region by short transverse oriented fibres. It is stressed that virtually all of the hypothalamic mechanisms fall within the sphere of influence of this larger forebrainmidbrain organization. Of course, even within this holistic concept there is always the possibility and likelihood for further differentiation. Both the upper (limbic) and lower (mesencephalic) poles of the mechanism are highly heterogeneous, in structure as , no doubt, in functional significance of their component parts. Physiological studies, especially those teasing apart the chemical subsystems, are only now delineating the different functional elements within the over-all limbic forebrain-midbrain mechanism . It is well recognized that several hypothalamic connections exist besides components of the limbic forebrain-midbrain circuit, among these being thalamo-hypothalamic connections. However, the medial subdivision of the dorsomedial thalamic nucleus and, probably , the thalamic midline nuclei, receive afferents from the limbic forebrain structures as well as from the paramedian mesencephalic region . For this reason Nauta (1963) regards the known thalamo-hypothalamic pathways as representing, at least in part, a transthalamic-hypothalamic link with the limbic forebrain structures and the medial midbrain regions . Thus, there has developed the concept of the hypothalamus as a component of a widespread, but nonetheless highly definable, telo-di-mesencephalic organization (Figure 1). Of

38 P.J. Morgane course, the question as to how this complex mechanism is linked to the organism's outer and inner environments is of obvious immediate relevance within the context of this symposium. Discussion of this is beyond the scope of this overview but has been summarized recently by Nauta (1971). We have dealt mainly with hypothalamic afferents which could be interpreted as relatively primordial communication channels common to the brains of most, if not all, vertebrates . With the notable exception of the olfactory connections, such pathways reach the hypothalamus only via the relays in the non-specific apparatus of the brainstem, represented by the bulbar and mesencephalic reticular formation (including the limbic midbrain area), the subthalamus, and the intralaminar and paramedian cell groups of the thalamus. Nauta (1963) has pointed out that pathways have been traced from several neocortical regions to both the upper and lower poles of the limbic forebrain-midbrain circuit. Of added importance is the evidence of direct neocortical projections from the frontal lobe to the hypothalamus. Thus, there is now sound anatomical evidence suggesting that the sphere of neocortical influence extends not only to the somatic but also to visceral and endocrine mechanisms . Obviously, our viscera are as much a part of our behavioural armamentarium as our skeletal muscles. It now appears from a variety of evidence that functional interactions within the limbic forebrain-limbic midbrain circuits provide the integrations between visceral and somatic activities that comprise the total behavioural response of all animals. Finally, one of the more exciting recent extensions of knowledge of hypothalamic-reticular relations with neocortical formations has been derived from histofluorescence mapping (Morgane and Stern, 1974). With respect to an evaluation of the basic pattern of connections between the limbic system and hypothalamus, a few general principles of organization should be summarized. We will concentrate on the connectivities of two major components of the limbic fore brain and their relation with the hypothalamus, i.e. , the amygdaloid complex and hippocampal formation. From the amygdala two pathways project to the hypothalamus, the stria terminalis to the ventromedial nucleus and area, and the ventral amygdalofugal pathway mostly entering the medial forebrain bundle. Both pathways also convey hypothalamic efferent fibres to the amygdala. The presence of two amygdaloid efferent systems raises the problem of whether each system has a functional individuality and a variety of recent work indicates that they do. Connections between the amygdala and the dorsomedial thalamic nucleus (amygdalothalamic fibres) have also been de-

Control of physiological regulations and behaviour 39 scribed (Nauta, 1961; Valverde, 1965). The dorsomedial thalamic nucleus projects specifically upon the orbitofrontal cortex (Nauta, 1971) and fibres from the orbitofrontal cortex then converge upon the basal forebrain area and the preoptic-lateral hypothalamic region (Nauta, 1961). Thus, in this region, it can be seen how the stria terminalis, the ventral amygdalofugal pathway and the amygdalo-thalamo-orbitofrontal system converge, and here these three mechanisms articulate with the medial forebrain bundle . It is of special relevance to discuss, in terms of the limbic projection systems, some of the short intrahypothalamic connections. Conduction pathways between hypothalamic cell groups appear to consist mostly of relatively short and poorly myelinated axons. Such short intrinsic fibre connections are largely inaccessible to experimental anatomical techniques, and only studies by the Golgi method are now revealing their intricacy (Millhouse, 1969, 1973). Some of these connectivities have recently been demonstrated in degeneration studies by Chi (1970) who found two divisions of the hypothalamus which may be differentiated by their connections with the amygdala. The first division consists of the lateral hypothalamus and lateral preoptic area. These areas are dominated by an input from the ventral amygdalofugal pathway, but they also receive fibres ascending from the posterior hypothalamus . They project to the dorsomedial hypothalamic nucleus, but do not send any significant contribution of fibres to the ventromedial nucleus. The second division of the hypothalamus is dominated by amygdala projections through stria terminalis. It consists of (1) the medial preoptic and medial anterior hypothalamic areas which receive the projections of the preoptic component of the stria, and (2) the ventromedial nucleus, the outer shell of which receives fibres from the supracommissural component of the stria. The central part of the nucleus receives a relay of short fibres from the medial anterior hypothalamic area. Thus, the ventromedial nucleus appears largely under the influence of the stria terminalis, the peripheral parts directly, and the central part after a relay. As discussed further below, the interpretation of the distribution of the afferent fibre pathways to the hypothalamus is complicated by the fact that the dendrites of quite different cell groups commingle extensively. However, relations between the lateral and ventromedial hypothalamic areas have been described recently in a series ofGolgi studies by Millhouse (1969, 1973). The hippocampal complex is less directly related to the olfactory system than is the amygdala. However, from the pyriform cortex many short association fibres arise and project back into the entorhinal area, which is the main source ofafferents to the hippocampus. The pyriform cortex gives

40 P.J. Morgane rise to a massive projection (Nauta, 1961; Powell et al., 1965) which passes to the remainder of the amygdala (the baso-lateral nuclear group), to the entorhinal area, and , by the so-called ventral amygdalofugal pathway, to the medial forebrain bundle . Thus , the whole of the amygdala and the medial forebrain bundle are brought into very close synaptic relation with the primary olfactory pathways, and, in tum, the entorhinal area acts as the major source of extrinsic afferent fibres to the whole of the hippocampal complex. The hippocampus projects to the hypothalamus both directly and indirectly. The efferent fibres of the hippocampus divide into precommissural and postcommissural fomices . The precommissural fomix, which comprises about half of the hippocampal efferents, is principally directed to the nuclei of the septum, although direct connections with the hypothalamus are formed by fibres which traverse the diagonal band region and run caudally in the medial forebrain bundle . This projection to the hypothalamus is reinforced by a similar projection relayed in the septum (Nauta, 1958; Szentagothai et al . , 1968; Valverde, 1965). For all these pathways a reciprocal connection is established by fibres from the medial forebrain bundle to the diagonal barid and medial septa) nuclei (Guillery, 1957) since these nuclei themselves send fibres back to the hippocampus . The various possible routes for limbic control of the hypothalamus have been discussed by Heimer and Nauta (1969). Raisman (1970) showed that following lesions of the stria terminalis there is heavy degeneration in the outer part (i.e ., in the dendritic radiation) of the ventromedial nucleus. He observed that the normal neuropil of this region consists mostly of axodendritic synapses, axosomatic synapses forming only a small proportion of the total number of contacts . There is considerable evidence that the limbic system projects information from the olfactory system and from many other cortical regions, and hence other sensory systems, into the hypothalamus . In particular, the hippocampal complex is arranged as a series of circuits of increasing complexity, each playing upon the succeeding circuit until finally the prosubiculum is reached . Raisman (1970) feels that the projection of the prosubiculum through the medial cortico-hypothalamic tract to the arcuate nucleus of the hypothalamus can be considered as the final common pathway from the hippocampus. He notes that from the limbic system two major direct projections reach the medial hypothalamus : (l) the prosubiculum projects through the medial cortico-hypothalamic tract to the arcuate nucleus ; and (2) the amygdala projects through the stria terminalis to the ventromedial nucleus . As noted, the medial forebrain bundle is a complex tract made up of

Control of physiological regulations and behaviour 41 finely myelinated and unmyelinated fibres interconnecting the hypothalamus, the basal telencephalon, and the midbrain . The connections of the medial forebrain bundle with the limbic forebrain and midbrain, together with the rather close correspondence of the so-called 'reward' zone to the bundle's trajectory, suggest the medial fore brain bundle as a neuronal field important in what psychologists are fond of referring to as drive states and 'motivational' mechanisms. Moreover, with the realization of the hypothalamic role in endocrine function, the medial forebrain bundle has been considered as a pathway for funnelling diverse information into the neuroendocrine regulating areas of the hypothalamus. Suffice to note that earlier normal and degeneration studies of this bundle had defined its sources, components, and general extent, within the bound of limitations of the methods available before histochemical mapping and autoradiographic neuronal analysis re-opened the medial forebrain bundle for more systematic chemical reinterpretation. Some of the findings relating to hypothalamic connectivities and synaptology will now be reviewed, especially those that bear on the role of the hypothalamus in energy homeostatic mechanisms. The descending medial forebrain bundle consists of dorsal and ventral portions which merge in the lateral preoptic area and project caudally through the lateral hypothalamus before finally sweeping upwards into the midbrain (Figure 1) . As seen in sagittal sections, the medial forebrain bundle swings dorsolaterally to the mammillary body into the mesencephalon and, fanning out in the tegmentum, the fibres climb dorsally and synapse, via short collaterals, with tegmental reticular neurons. Rostrally the medial forebrain bundle is intersected by the stria terminalis bundle, while caudal to this it contributes fibres that enter the stria medullaris and inferior thalamic peduncle (Figs. 3 and 4). Along the course of the bundle, especially dorsally, are fibres of the ascending reticular formation (Fig. 3) which send numerous collaterals to neurons along the path of the medial forebrain bundle. The dorsal portion of the medial forebrain bundle is composed of fibres derived from the hippocampus, septum, nucleus of the diagonal band of Broca, frontal cortex, and nucleus accumbens. The precommissural fornix fibres course through the septum and they emit collaterals extending through the entire rostro-caudal depth of this structure . Practically all portions of the septum contribute to the rostral end of the medial forebrain bundle and septa! fibres can easily be traced into the lateral hypothalamus. Along the way they discharge collaterals not only to the lateral preoptic and lateral hypothalamic neurons but also to the medial preoptic and anterior hypothalamic nuclei. Rostrally and caudally project-

42 P.J. Morgane

Figure 3 Illustration showing the rostral projection of the reticular formation . Components in the ventral leaf of this system form the more dorsal components of the medial fore brain bundle. Note that these project through the subthalamus and zona incerta . Abbreviations : RF , reticular formation; su BTH, subthalamic area; 21, zona incerta; MTH, mammillo-thalamic tract; M, mammillary nuclear complex; CM-PF, centre median-parafascicular thalamic complex; TH IL+ DM FIELDS, intralaminar thalamus, anterior and dorsomedial fields; DM, dorsomedial thalamic nucleus; AD , anterodorsal thalamic nucleus; AM, anteromedial thalamic nucleus ; RE, reuniens nucleus of thalamus; NRT , reticular nucleus of thalamus; ITP , inferior thalamic peduncle ; AH, anterior hypothalamic area; MFB, medial forebrain bundle; HYPOTH, hypothalamus; BFA, basal forebrain area; OFC, orbito-frontal cortex; AC, anterior commissure; F, fomix

ing axonal patterns, which are quite characteristic of reticular formation arrangements, are also typical ofneurons along the medial fore brain bundle and have also been well described by Millhouse (1969) in the septum, where one part of an axonal system enters the medial forebrain bundle and another goes toward the hippocampus. The Golgi studies of Millhouse (1969) reveal clearly that a major portion of both the stria medullaris and inferior thalamic peduncle are composed of medial forebrain bundle collaterals and show unequivocally that both contain afferents to the lateral preoptic and lateral hypothalamic zone. INTRINSIC HYPOTHALAMIC ORGANIZATION

Because of their location in the pathway of the medial forebrain bundle,

Control of physiological regulations and behaviour 43

Figure 4 Illustration of frontal section of the hypothalamus at the level of the optic chiasm showing some of the relations of the medial forebrain bundle to the inferiorthalamic peduncle and ansa peduncularis . The ansa peduncularis is a complex fibre system extending from the amygdalo-pyriform area medially to the diencephalon. Passing through the region of the substantia innominata it divides into (a) a fibre contingent which continues its medial course and disperses in the lateral hypothalamic area, and (b) a fibre contingent which curves dorsalwards around the medial edge of the internal capsule and enters the medial thalamus as the inferior thalamic peduncle which distributes to paramedian thalamic nuclear groups , including the mediodorsal nucleus. The ansa peduncularis conducts in both directions and it contains fibres from the thalamus as well as from the amygdalo-pyriform area (the latter form the so-called ventral amygdalo-hypothalamic pathway) . Abbreviations: as in previous figure and : AP, ansa peduncularis; SM, stria medullaris; so, supraoptic nucleus; sc, suprachiasmatic nucleus; oc, optic chiasm

44 P.J. Morgane the neurons of the lateral preoptic and lateral hypothalamic area have been referred to by Millhouse (1969) as path neurons, i.e., the bed 'nucleus' of the medial forebrain bundle (Fig . 5). The dorso-ventral and medio-lateral spread of the dendritic system of a single path neuron usually cuts across much of the depth and width of the medial forebrain bundle. The long fibres of the medial forebrain bundle emit collaterals which usually parallel these dendritic systems and, ramifying among the dendrites, terminate as small boutons or thin fibres. Thus, a single fibre of the medial forebrain bundle contacts many path neurons along its trajectory and synapses profusely with dendrites from medial hypothalamic nuclei which extend laterally into the bundle (Fig. 5). From the functional point of view the extent of the dendrites allows convergence of activity in the multiple sources of the bundle upon a single path neuron. As has been emphasized by Millhouse (1969), a major fibre component of the medial forebrain bundle, which is largely ignored in the earlier literature, originates from the path neurons. These neurons have been identified in the lateral preoptic and lateral hypothalamic zones with their axons projecting caudally, rostrally, and sending still another collateral into the stria medullaris or inferior thalamic peduncle. Along their course these fibres emit short projections to other path neurons as well as to various medial hypothalamic nuclei. Beginning at the level of the ventromedial hypothalamic nucleus, this axonal pattern changes slightly in that a collateral arches medially to enter the periventricular fibre system. Of particular interest in the framework of this volume are the relations of lateral hypothalamic area neurons to those of the ventromedial hypothalamic nuclear area (Millhouse, 1969, 1973). At the level of the ventromedial hypothalamus, dendrites of path neurons extend medially into the ventromedial hypothalamic nucleus and neurons of this nucleus, in turn, have long spiny dendrites, many of which enter the lateral hypothalamus (Fig. 5). Most important, there is no separation of the dendritic fields of the lateral and ventromedial hypothalamus. This makes their relations and properties most complex and beyond simple vectorial analysis, either in terms of unilateral or reciprocal inhibition, which are usually applied to relations between the ventromedial nucleus and lateral hypothalamic area. When neurons interact strongly in such a complex manner, there emerge new collective properties that demand a level of conceptualization entirely different from that used heretofore. Also, the more ventrally located ventromedial neurons send axons laterally to terminate on dendrites of neighbouring path neurons. This projection is reciprocated by medial forebrain bundle collaterals, some originating from path

Control of physiological regulations and behaviour 45

Figure 5 Golgi analysis of cells of the medial fore brain bundle (path cells) and medial hypothalamic nuclei . Note the wide rangingdendritic tree spreading out in all directions from the path cells into the medial hypothalamic nuclear zone, into the internal capsule , as well as dorsally and ventrally . Dendrites of cells in the ventromedial nucleus oft he hypothalamus are shown extending far out into the lateral hypothalamic area. There is extensive co-mingling of the dendrites between the path neurons in the lateral hypothalamic area with those of the medial nuclei, especially with those of the ventromedial nucleus . Abbreviations: 3v , third ventricle ; VM , ventromedial nucleus of hypothalamus ; DM, dorsomedial nucleus of hypothalamus ; F, fornix; LH , lateral hypothalamic area; 1c, internal capsule ; OT, optic tract; AM , amygdaloid nuclear complex

neurons at this level and entering the lateral third of the ventromedial nucleus . Furthermore, neurons whose perikarya are adjacent to the fomix (perifornical cells) have axons with collaterals passing to both the ventromedial nuclear area and lateral hypothalamus. These neurons, in turn, receive a rich input from both the medial and lateral hypothalamus . Other collaterals of path neurons at this middle hypothalamic level cascade over the ventromedial nucleus into the periventricular fibre system. Along their course are additional collaterals which synapse with dendrites of cells of the ventromedial and dorsomedial nucleus of the hypothalamus and the periventricular neurons. Ramon y Cajal (191 I) first mentioned that many fibres in the stria medullaris are collaterals of fibres running caudally through the lateral hypothalamus. It is of special import that collaterals of medial forebrain bundle fibres, including those of path neurons , are distributed in both the

46 P.J. Morgane stria medullaris and inferior thalamic peduncle. This emphasizes that the medial forebrain bundle activity is not confined to just the lateral preoptic and lateral hypothalamic zones but is conveyed to other diencephalic fields, especially the entire midline thalamic nuclear group. Although the long medial forebrain bundle fibres and their various collaterals supply the path neurons with a common afferent pool, one or more of the components of the medial forebrain bundle appear to synaptically dominate the path neurons depending on the transverse level (Millhouse, 1969). Thus, path neurons ventral and caudal to the anterior commissure receive the brunt of hippocampal and amygdaloid input to the medial forebrain bundle. Millhouse's study indicates that medial hypothalamic nuclei project only to adjacent path neurons such that rostral path neurons receive a unique contribution from the medial preoptic and anterior hypothalamic nuclei, whereas progressively more caudal path neurons receive afferents from the ventromedial and premammillary nuclei. However much the foregoing might suggest the path neurons are segregated by input into several transverse segments, the long medial forebrain bundle fibres do constitute an afferent source common to the path neurons. Moreover, these path neurons are structurally related by their axonal systems, their axons usually bifurcating near the soma, projecting both rostrally and caudally, and giving off collaterals to other path neurons. By this means a series of neuronal loops is formed which synaptically link the path neurons throughout the lateral hypothalamic zones. Collaterals from path neurons, as well as other medial forebrain bundle components, relay information to medial hypothalamic nuclei. Thus, either directly by collateral fibres or by relay through a medial nucleus neuron, the medial forebrain bundle would appear to influence wide areas of the tuberal zone of the hypothalamus and, via this system, no doubt affects anterior pituitary function. The final terminations of the rostrally going axonal divisions of path neurons are still not fully worked out. It should be emphasized that along their trajectory in the midbrain, the medial forebrain bundle fibres have many collaterals . Consequently, before terminating somewhere in the caudal midbrain, a single bundle offibres has recruited a series ofreticular neurons which, in turn, have extensive collateral systems reaching throughout the brainstem and spinal cord . In Golgi sections Millhouse (1969) has traced path cell fibres that leave the medial fore brain bundle and pass medially at the level of the caudal hypothalamus to join the periventricular fibre systems. It is quite likely that these fibres join the dorsal longitudinal fasciculus of Schiltz and project caudally in the periaqueductal grey substance.

Control of physiological regulations and behaviour 47 Functional loci have been difficult to isolate and define precisely in the hypothalamus, notwithstanding our present fascination for ' centres' and 'syndromes.' The numerous collaterals of medial forebrain bundle fibres, their convergence upon path neurons, plus the rostrally and caudally projecting axonal divisions of individual path neurons, as well as the extensive overlap of their dendritic fields with those of medial hypothalamic nuclei, would certainly indicate why many have been unable to define circumscribed loci for different hypothalamic functions. This is a classical example , using strictly morphological analyses, for arguing against functional 'centre' theory. It is possible that the variation of the input to the rostral and caudal path neurons may serve as the structural bases for the polarization of the lateral hypothalamus into the two major functional domains defined by Hess (1954), although these, too, are under considerable question. Of course, the structural characteristics discussed above, which obviate dividing the lateral preoptic and lateral hypothalamic areas into discretely limited functional areas, may possibly be the very features which permit the co-ordination of the various autonomic activities accompanying complex behaviour patterns. Generally speaking, the structural features of the medial forebrain bundle appear to be superbly organized to furnish the anatomical and functional substrate for viscerosomatic integrations accompanying different behavioural sequences. ELECTROPHYSIOLOGICAL ANALYSES OF HYPOTHALAMIC AFFERENT SYSTEMS

It has become abundantly clear that hypothalamic physiology is inextricably tied to limbic and reticular processes and , therefore, a closer look at these processes may help in the understanding of their interactions in regulating energy homeostasis as well as other behaviours. It is, therefore , more and more obvious that the functional state of the hypothalamus is directly dependent upon the patterns of activity within the limbic system as a whole, both its forebrain and midbrain extensions. The fact that impulses arising in the hippocampus and in the midbrain impinge upon hypothalamic neurons which are shown to be connected to the septum and amygdala suggests that the hippocampus and the midbrain participate actively in the mechanisms which determine the functional state of the tuberal and lateral hypothalamic neurons. Dreifuss et al . (1968) have studied the electrophysiological characteristics of the two amygdaloid projection systems (stria terminalis and ventral amygdalofugal pathway) by use of extracellular microelectrode tech-

48 P.J. Morgane niques. They focused on the tuberal portion of the hypothalamus because they obtained the most conspicuous evoked responses to both corticomedial and basolateral amygdaloid stimulation in the region of the ventromedial nucleus of the hypothalamus. It is clear, therefore, that this nucleus and its immediate environs represent a focal point within the subcortical amygdaloid projection system. Significantly, they found that both the stria terminalis and ventral amygdalofugal efferents project to the same tuberal hypothalamic region, that this overlap extends to the single cell level (convergence onto the same ventromedial cell), and that there exists a direct relationship between the discharge patterns of single hypothalamic ventromedial neurons and the evoked slow potentials, both being distinctive for each one of the two projection systems. Discharge specific patterns for each projection system were observed and convergence upon single ventromedial neurons and interaction of the opposite effects mediated by the stria terminalis (inhibition) and ventral amygdalofugal pathways (activation followed by inhibition) were demonstrated in this important study. The functional state of the hypothalamus at any given time may, therefore, be, in part, dependent upon the algebraic summation of these effects. Murphy et al. (1968) also demonstrated that the functions of the hypothalamus are under the influence of the midbrain tegmentum and the limbic forebrain system, notably the amygdala and the septum. There has been a paucity of single-unit studies in this area because of the difficulty in obtaining satisfactory recordings from these neurons. The chiefreason for this is their small size (about 15µ. in diameter in the cat) and also the slow spontaneous firing rate of hypothalamic neurons. Nevertheless, these workers showed that the ventromedial hypothalamic region is a prime recipient for both the amygdaloid and the septa! projection systems, whereas fibres originating in the midbrain tegmentum were not shown to reach this region in significant numbers. One unexpected finding was that lateral hypothalamic neurons were consistently activated at longer latencies with amygdaloid stimulation than were ventromedial neurons, since it is believed that basal amygdaloid efferents synapse in the lateral hypothalamus and do not reach the medial hypothalamus directly. It would be expected that lateral hypothalamic neurons would be excited earlier than ventromedial neurons whereas clearly the reverse was found, thus suggesting that lateral hypothalamic excitation may have been more indirect. The functional implications of the input distribution from the septum and amygdala, of course, include the possibility that the ventromedial nucleus may be an important link in the descending control, usually attributable solely to the lateral hypothalamus,

Control of physiological regulations and behaviour 49 of certain behavioural, autonomic, and endocrine responses that have been reported with septa) or amygdaloid stimulation. In other studies, Murphy and Renaud (1968) demonstrated inhibitory interneurons in the ventromedial nucleus of the hypothalamus. They showed that the lateral edge of the ventromedial nucleus contains dendritic terminals of inhibitory interneurons which are excited by the amygdaloid efferent pathways . Their findings suggest the hypothesis that the bipolar neurons, at heaviest concentration at the lateral edge of the ventromedial nucleus, may be the morphological representation of these inhibitory interneurons. Most significantly, they failed to confirm the finding ofOomura et al . ( 1967) of a powerful inhibitory pathway from the lateral hypothalamic area to the ventromedial nucleus. Rather, they proposed that one of the mechanisms by which experimental lesions or stimulations in the lateral hypothalamus influence feeding behaviour may be by the interruption or activation of fibre systems from other sources, such as the amygdala, which terminate in converging fashion at the lateral edge of the ventromedial nucleus. Murphy and Renaud (1969) also studied the mechanisms of inhibition in the ventromedial nucleus of the hypothalamus . They emphasized that, since the stria . terminalis has a predominantly inhibitory effect on ventromedial neurons and the more diffuse ventral amygdalofugal system produces activation-inhibition sequences, both acting on the same cells, the question should be raised whether each produces its inhibitory effect by the same mechanism. They were not able to demonstrate powerful inhibitory connections from the lateral hypothalamus to the ventromedial nucleus and observed that the ability of lateral hypothalamic stimulation to inhibit the firing of ventromedial neurons would appear to be a function of the proximity of the stimulating electrode to the dendritic terminals of the inhibitory interneurons or the pathways synaptically exciting these terminals. The behavioural implications of these findings are important since the widely accepted view that the lateral hypothalamic area serves to control some behavioural functions by virtue of a negative feedback arrangement with the ventromedial nucleus is directly challenged. At the chemical level, Dreifuss and Matthews (1972) studied the effects of inhibitory agents and their blockers on ventromedial nucleus neurons. Their results provided evidence that strychnine reversibly blocks the action of glycine upon hypothalamic ventromedial neurons, whereas bicuculline antagonizes the reduction of cell firing observed during the application of GABA. Thus, in the hypothalamus, as elsewhere in the mammalian central nervous system, the action of these antagonists appears to permit a

50 P.J. Morgane distinction to be made, in electrophoretic experiments, between GABAreceptors and glycine-receptors. These convulsant alkaloids are, therefore, proving to be of considerable value in assessing whether a particular pathway impinging upon hypothalamic neurons could function by releasing GABA or glycine as an inhibitory transmitter. HYPOTHALAMIC UNIT STUDIES AND BEHAVIOUR

It is now important to discuss a few highly selected examples of studies illustrating how unit analysis of properties of hypothalamic neurons and their relations with limbic mechanisms may reveal information as to underlying processes involved in such complex behaviour as feeding. Olds et al. (1969) first showed that hypothalamic units were relatively uninfluenced during waiting periods prior to feeding, while midbrain and hippocampal units showed significant changes in firing rate. More recently Hamburg (1971) analysed firing rates of hypothalamic neurons in rats during eating periods, using chronic microelectrode techniques. Units were found in the lateral hypothalamus that decreased or completely stopped producing spike activity during eating behaviour. No medial hypothalamic unit activity was affected by eating and no increases in spike activity were observed in any area of the hypothalamus during eating. Moreover, upon food withdrawal, several units whose activity decreased during eating immediately resumed firing, even though chewing and swallowing continued. In some cases, units of the lateral hypothalamus whose activity decreased during eating were similarly affected by electrical stimulation oft he medial hypothalamus. Hamburg noted that, if lateral hypothalamic cells do function in the control of food intake, then their responsiveness to changes in food availability rather than simply food consumption suggests that they might be participating in the guidance of behaviour in the search of food reward. It is consistent with this hypothesis that the activity of these cells is inhibited by making food available or by terminating the drive for food, as with medial hypothalamic stimulation. Rolls (1972) carried out studies of activation of amygdaloid neurons in 'reward,' eating and drinking elicited by electrical stimulation of the brain. In order to analyse so-called 'rewarding' and ' motivating' effects, recordings were made from single neurons activated by hypothalamic or rhinencephalic stimulation in animals previously tested for self-stimulation. He found a population of brainstem neurons that is excited by 'rewarding' lateral hypothalamic stimulation and has provided evidence

Control of physiological regulations and behaviour 51 that these neurons synaptically excite other neurons in the brainstem, and, finally, activate brainstem and thalamic neurons which have firing rates that are closely correlated with arousal. Through this neural system the hypothalamic stimulation produces arousal as measured by desynchronization of the EEG, and locomotor activity, and it increases the rate of self-stimulation, perhaps by means of a 'priming' effect. Morgane (1969) previously pointed out a close relation of lateral hypothalamic activation with arousal mechanisms and in our studies of sleep have seen various alterations of the vigilance states following medial forebrain bundle damage. These effects on the arousal state appear to relate to what I (1961) originally termed 'motivational inertia' following some types of lateral hypothalamic lesions. Perseveritive and arousal states are clearly deranged in these medial forebrain bundle-damaged animals . In this regard, Wampler (1970) also observed somnolence and drowsiness following lateral hypothalamic damage and suggested that disruptions of sleep and arousal after lateral hypothalamic lesions contribute to aphagia and anorexia. After bilateral hypothalamic lesions he observed that rats were slower to arouse, did not respond to handling or other stimuli, and showed continuous fluctuations in the arousal state. As qualitatively more normal states of sleep and arousal returned the animals again began to eat. Wampler's findings suggest that regulation of ingestion and sleep and arousal depend on independent systems that overlap in the lateral hypothalamic area. Rolls ( 1971) has evidence that limbic system neurons drive other neurons in the brainstem and finally activate brainstem and thalamic structures having firing rates that are closely correlated with arousal. He indicates that, through this latter system, hypothalamic stimulation produces arousal. Rolls and Kelly (1972) observed that the most prevalent behaviour elicited by electrical stimulation of the lateral hypothalamus is locomotor activity. They noted that there are marked similarities between the locomotor activity and the arousal measured electrophysiologically, which are produced by stimulation of lateral hypothalamic sites. One additional study of note along similar lines is that of Ito (1972) who analysed the excitability of medial forebrain bundle neurons during self-stimulation behaviour. Quite surprisingly, during the condition of normal 'reward,' neurons in the supposed lateral hypothalamic 'feeding area' were not excited, but were suppressed . His data, in general, suggest that intrahypothalamic fibres mediate the inhibitory effects, and extrahypothalamic fibres mediate the excitatory effects, with both sets offibres forming components of the medial forebrain bundle.

52 P.J . Morgane CHEMICAL ANATOMY OF THE BRAIN

It is well known that there are now entirely new means of looking at brain organization in terms of chemical neuroanatomy (Morgane and Stem, 1974) . This is based primarily on histofluorescence mapping of transmitter-specific neuronal pathways, many of these forming subsystems of the medial forebrain bundle (Figs . 6 and 7). Before these direct means of visualizing neuronal systems were available, we had to rely solely on 'wiring charts' derived from studies of normal anatomy and degenerating fibres, since we knew nothing of the specific chemical identity of pathways. Many of these transmitter systems are composed of fibres too small to visualize by light microscopy (0.1-0.3µ, in diameter) and, in addition, their chemical properties appear to have made some of them refractory to silver staining techniques. Now, if we look at the medial fore brain bundle at the mid-hypothalamic level in terms of its chemical organization, we see it as shown in Figures 6 and 7. There is a large and rapidly developing literature on the functions of the component chemical pathways of the medial forebrain bundle. A great number of studies indicate that aspects offeeding and drinking behaviour, self-stimulation, among others, may involve noradrenergic, dopaminergic, and cholinergic pathways that comprise subsystems within the medial forebrain bundle at this level. From this type of circuit organization it is now easier to formulate redefinitions of the lateral hypothalamic area and the entire reticular continuum based on the topography of specific chemical pathways occupying its different sectors (Fig. 8). At the neurochemical level, we appear to be at the forefront of teasing out chemospecific pathways relating to many behaviours . This organization tells us again loud and clear that the time has long passed when we can consider the hypothalamus as a sort of separate, isolated entity operating as a series offunctional 'centres.' Nothing about its chemical, morphological, or physiological organization suggests any hint of restricted functional loci. In such a complexly organized region as the lateral hypothalamus, chemical mapping is for the first time revealing circuits we did not know existed just five years ago . With transmitter histochemical approaches we directly visualize neuronal systems in a functional way and thus, in large part, the pathway does become the message in terms of chemical reading of the transmitter itself. It must be remembered that ultimately the brain is a chemical machine and we now have some of the tools at hand to identify these chemical circuits and begin to tease them apart based on their

Control of physiological regulations and behaviour 53

SEP

Medial Forebrain

DA System Ventral NA

Bundle MES

( LHA)

Anterior Raphe' Complex ( B6 -B9 ) PONS

Posterior Raphe Complex

( BI -BS

MED

Figure 6 Illustration ofmonoaminergic chemical systems in the brain showing, in particular, that they form components of the medial forebrain bundle. The coding system for the Raphe nuclei (e series) and more lateral reticular nuclei (A series) is according to Dahlstrom and Fuxe ( 1964) . This figure shows that the medial fore brain bundle is composed of serotonergic fibres from the raphe, ventral, and dorsal noradrenergic components, and at least two dopamine systems .

54 P.J. Morgane • Dorsal Noradrenergic System ®

®

Ventral Noradrenergic System Medial Serotonergic System

+ Lateral Serotonergic System o N igro-striat al Dopamine System x M eso-1 im bic Dopamine System

STR

AM

Figure 7 Illustration of the topography of chemical systems comprising the medial forebrain bundle . The parcellation of the bundle is based on histoftuorescence tracing of the component fibre systems and, more particularly, on amine-specific effects of small lesions in different sectors of the lateral hypothalamic area . Abbreviations: OT, optic tract; AM, amygdaloid complex; STR, neostriatum; GP, globus pallid us; IC, internal capsule; VMH, ventromedial nucleus of hypothalamus; F, fornix; MT, mammillothalamic tract; DM, dorsomedial nucleus of hypothalamus; RE, reuniens nucleus of thalamus; 3v, third ventricle; ENTO, entropeduncular nucleus

chemical properties. Thus, psychopharmacological agents may act in some cases on particular amine and other systems in the brain, and chemical lesioning agents exert their effects by their ability to be picked up rather specifically in particular chemical neurons. Obviously, we are still a long way from understanding the role of many of these chemical pathways, but at least we now know that they are there and can be reasonably manipulated somewhat selectively. We should now look briefly at two major components of the medial forebrain bundle which indicate an entirely different chemical organization of the far-lateral and mid-lateral hypothalamic areas (Figs. 6, 7, and 8). Is there any wonder that the effects of manipulating these two broad sectors,

Control of physiological regulations and behaviour 55 i\ DORSIL NI

©NICRO·STRIUIL 01 eilNTRll NI

v SKT

~ ZI

\~

' ZI

IC

3V

oc

Figure 8 Schematic representation of dispersion of chemical systems at three different levels of the hypothalamus . In the first drawing at level of posterior hypothalamus, second drawing at level of middle hypothalamus, and third drawing at level of optic chiasm . This figure shows, in particular, the nigro-striatal fibres leaving the medial forebrain bundle, penetrating the internal capsule, and projecting into the striatum . Abbreviations : FIELD H, prerubral field of Fore I: SN. substantia nigra : PED, Cerebral peduncle: F, fornix: PMN . posteromedial hypothalamic area; 21, zona incerta ; VT, ventral thalamic nucleus; re, internal capsule; vM , ventromedial nucleus of hypothalamus; MT, mammillothalamic tract; 3v, third ventricle; oc, optic chiasm: OT. optic tract; EN , entopeduncular nucleus; GPI, globus pallid us (internal segment): GPE, globus pallidus (exterior segment) : P. putamen

for example, produce different physiological, biochemical, and behavioural changes? Chemical profiles over wide areas of the brain are altered, depending on where in these pathways we place a lesion or electrically or chemically stimulate. These changes are seen in distant brain areas to which these fibres of passage specifically project. Just a single millimetre away from one chemical system there are produced by these same manipulations entirely different chemical changes, either in the same regions related to the other system or, in many instances, in entirely different brain

56 P.J. Morgane loci . In the central nervous system the medial forebrain bundle is a focus of attention in this field since it appears to be essential for the mediation of afferent input necessary for the regulation of enzyme levels needed for the biosynthesis of neuroamines over wide areas of the brain, including the entire neocortex. Certainly, identification of the neurons responsible for the biosynthesis and storage of neuroamines is essential for understanding the functional role of these substances in the central nervous system. Thus, one aspect of this problem is the study of the roles of specific fibre systems in the regulation of monoamine biosynthesis across multisynaptic systems in the brain as pioneered by the Heller, Harvey, and Moore group (Heller et al., 1962; Moore et al., 1965; Heller and Moore, 1965, 1968; Heller, 1972). The technique of highly selective lesions of discrete groups of central nervous system neurons has provided a valuable tool for establishing the 'chemical identity' of neuronal systems in the brain whose integrity is essential for the regional maintenance of neuroamines. Along these lines Morgane and Stem (1972) explored the entire limbic forebrain-limbic midbrain circuit and separated out lateral and medial components of the medial forebrain bundle whose integrity was shown to be responsible for maintenance of forebrain levels of norepinephrine and serotonin, respectively (Table 1, Fig. 9). Figure 10 provides details of some possible chemical systems that may regulate aspects of energy homeostasis and water balance and serve as a model or neurological skeleton for placing 'motivation,' that 'phlogistan of psychology,' in some neurological terms that we may some day, if necessary, come to grips with . It is likely that distant neurochemical effects of a lesion may be of key importance in determining the physiological, behavioural, and clinical sequelae of brain lesions . The possibility that the neurochemical effects of central nervous lesions are mediated across polysynaptic systems and occur in otherwise 'intact' neurons is fascinating from a variety of viewpoints. This leads to the corollary, and generalizing principle, that manipulation of afferent input may be capable of reversing the neurochemical deficits in neuroamine biosynthesis induced by damage and may eventually help us better understand compensatory mechanisms in the brain. With respect to lesioning based on chemical properties, there is much more hope with chemical lesioning procedures which are induced by the uptake of the chemotoxins by particular types of chemical neurons . Thus, 6-hydroxydopamine, with a moderate degree of specificity, damages or destroys catecholaminergic neurons (Iversen and Uretsky, 1971) while 5,6and 5,7-dihydroxytryptamine (Baumgarten et al ., 1971; Baumgarten and Lachenmayer, 1972) are more selective in destroying serotonin-containing neurons. But even these agents may exert non-specific effects or, at the

Control of physiological regulations and behaviour 57 TABLE 1 Mean concentration of serotonin and norepinephrine in the hypothalamus 1 and/or basal forebrain area 2 of cats with brain lesions and of sham-operated controls Serotonin (µg/g)

Norepinephrine (µg/g)

Lesion

Number

Medial forebrain bundle at level of ventromedial hypothalamic nucleus (far-lateral hypothalamic area) Controls Dorsomedial tegmental area ('Limbic midbrain area') Controls Ventromedial tegmental area ('limbic midbrain area') Controls Central grey (ventral half) Controls Ventrolateral tegmental area Controls

3

1.33 ± 0.17

0. 78 ± 0 . 13

3 3

1.44 ± 0.20 0 . 51 ± 0 . 12

1.55 ± 0 . 23 1.11±0.13

3 3

1.46 ± 0.17 0.48 ± 0 . 11

1.29 ± 0 . 18 1.05 ± 0 . 15

3 3 3 3 3

1.42 0.56 1.49 1.65 1.48

0 . 19 0.12 0.17 0.12 0.13

1.39 ± 0.21 1.31±0, 11 1.36 ± 0. 18 0.89 ± 0 . 18 1. 58 ± 0 . 20

- · - · - - - - - - - -·---- ---- - - ·

-·

-- -- - --

± ± ± ± ±

- - --

-

---- - - - -·

When lesion is in hypothalamus, assay is in hypothalamus remaining rostra/ to lesion plus basal forebrain areas. 2 Basal forebrain area includes preoptic area, septal area, and basal olfactory areas beneath anterior perforated substance.

very least, produce a gradient-type lesion with all neurons destroyed near the cannula tip (or nearest the ventricle), and radiating outward from these sites lesser amounts of chemotoxin are taken up. Chemical lesioning is a complex story and most of the answers are not yet available but the method does at least offer an approach to the central nervous system based on chemical morphology and transmitter function. CHEMICAL PATHWAY MANIPULATION: RELATION TO FEEDING-DRINKING BEHAVIOUR

A few prime examples from the literature will indicate how the field of transmitter histochemical anatomy has evolved entirely new and dynamic approaches to the field of hypothalamic physiology. One study of Ungerstedt has provided a major impetus in correlating anatomical loci, brain chemistry, and altered behaviour.just as has been done so profitably in the sleep field by Jouvet and others. Noting that the nigro-striatal dopamine pathway ascends through the far-lateral hypothalamus, Ungerstedt (1970, 1971a, b) injected 6-hydroxydopamine along this pathway in rats. The animals that developed adipsia and aphagia all showed complete degeneration of the nigro-striatal dopamine system, and, ifnot tube fed, died within 4 to 6 days. Ungerstedt observed that the effects were similar to those seen

58 P.J. Morgane

Figure 9 Example of an amine-specific lesion which lowered norepinephrine levels in the basal forebrain area on the same side as the lesion without affecting norepinephrine levels on the contralateral side. There was no depletion of serotonin following this lesion. Cat brain, approximately 20 x

Control of physiological regulations and behaviour 59 after lesions in the lateral hypothalamus and postulated that the critical tract (nigro-striatal) for feeding behaviour was thus identified. In this regard, as noted by degeneration studies, Moore et al. (1971a, b) have shown that degenerating fibres following lesions in the substantia nigra come together into a compact bundle lying ventro-laterally in Field H of Foret dorsal and medial to the subthalamic nucleus . At the premammilliary level the bundle elongates in a dorso-ventral direction and most axons are located in the far-lateral hypothalamic area as originally defined in my work in 1961. Rostrally, as the subthalamic nucleus disappears, the bundle of degenerating axons is located in the ventro-medial part of the zona incerta, in the far-lateral hypothalamus, and in the medial edge of the internal capsule. On their course they intermingle extensively with other fibre systems identified by fluorescence mapping techniques. At rostral tuberal levels of the hypothalamus the fibres run entirely in the medial internal capsule and adjacent lateral hypothalamus (Figs. 4, I0). Degenerating axons leave the main bundle to run laterally as the entopeduncular nucleus develops and they traverse it and the globus pallidus to reach the putamen. Notably, scattered degenerating terminals are also seen in the entopeduncular nucleus and globus pallidus. There is little question that this nigro-striatal dopamine pathway overlaps crucial lateral hypothalamic areas related to feeding behaviour. Ungerstedt (1971b), in further studies, produced both electrolytic and 6-hydroxydopamine lesions along the pathway and observed long-lasting adipsia and aphagia, hypoactivity, difficulties in initiating activity, as well as loss of exploratory behaviour. To pin down the chemical aetiology he found that experiments with dopamine receptor stimulating and blocking drugs supported the lesioning result. He concluded that a number of symptoms earlier related to the hypothalamus may, in fact, be due to disruption of the nigro-striatal system . It is possible that lesions placed by Morgane (1961) in pathways earlier identified as 'pallidofugal' (because these were the primary pallidal efferent pathways known at that time) involved elements of this complex tract passing between the striatum and substantia nigra. Since recent cholinesterase mapping studies by Olivier et al. (1970) show powerful cholinergic contingents passing from striatum to the substantia nigra (strio-nigral pathways), these could also play a critical role in producing the disturbances resulting from lateral hypothalamic damage. When I first defined the pallidofugal fibre systems as responsible for the far-lateral hypothalamic effects on feeding behaviour most of the chemical pathways were not known. Obviously it could just as well be damage to these cholinergic strio-nigral pathways that is producing the effects described for nigro-striatal damage. So far we have been unable to

60 P.J. Morgane

CP

----- ---

Figure IO Schematic representation of projection routes of ascending cholinergic (ACH) reticular fibres and dopamine (DA) fibre systems in their diencephalic trajectory as viewed from the right side seen from behind. Cholinergic axons from the dorsal hypothalamic neurons run rostrally by a more medial route to the reticular nucleus and globus pallid us (GP) and thence on to the caudate-putamen (P-c). Some of these fibres may terminate in the entopeduncular nucleus (EN) . Other components of this system project forward in the medial fore brain bundle through the lateral hypothalamic (LHA) and lateral preoptic areas (LPO) to relay in the diagonal band area (DB), and some pass directly to the cerebral cortex (c). Fibres from cells of the subthalamic nucleus (ST) take a more lateral route across the cerebral peduncle (cP) to the entopeduncular nucleus and globus pallidus . The dopamine fibre systems, especially emanating from the substantia nigra (SN), pass rostrally in groups of fibres, one forming the nigrostriatal system that winds its way through the cerebral peduncle and projects to the globus pallid us and putamen-caudate . Another component of the dopamine system (meso-limbic component) passes rostrally in the medial forebrain bundle through the lateral hypothalamic area, and the lateral preoptic region towards limbic forebrain terminal sites. Other abbreviations: 1c, internal capsule

separate these elements but are presently testing the various alternatives. Ernst (1969) too has observed that cholinergic nerve fibres end synaptically on the dopaminergic nigral cells and feels that cholinergic stimulation causes dopamine production in the corpus striatum via the nigroneostriatal dopaminergic fibres. Parent et al. (1969) note that the nigrostriatal dopaminergic neurons could be under the control of the striatonigral pathways. Bedard et al. (1969) have described a striato-nigro-striatal

Control of physiological regulations and behaviour 61 loop which is involved in the synthesis of striatal dopamine. In this regard, Olivier et al. (1970) observe that the striatum is the richest cholinergic structure in the brain and that the striatal efferents to the pallidum and substantia nigra represent the richest cholinesterasic pathway within the entire central nervous system. This pathway may very well be essential in maintaining the proper level of dopamine in the striatum and should, accordingly, be considered a possible pathway involved in regulation of feeding behaviour. All of these studies clearly indicate that, at present, it is impossible to characterize the polarity of the chemical systems whose disruption results in disturbed feeding behaviour. Most certainly these three areas that have been implicated in the regulation of eating and drinking, the lateral hypothalamus , the medial portions of the internal capsule, and the globus pallid us are of especial neurochemical interest. A few additional chemical lesion studies relating to these pathways should be mentioned. One of particular importance is that ofOltmans and Harvey (1972) . They observed that electrolytic lesions of the nigro-striatal bundle produced a more severe aphagia, adipsia, and disturbance of water regulation than did lesions of the medial forebrain bundle itself. They concluded that the inability to regulate water intake in the absence of food, one of the often-described and long-lasting effects of lateral hypothalamic lesions, appeared to be the destruction of the nigro-striatal bundle and the consequent decline in telencephalic content of catecholamines. It is significant that nigro-striatal bundle lesions produced a more severe aphagia and adipsia, as well as a more severe effect on drinking in the absence of food, than did even larger lesions located in the medial fore brain bundle. Along similar lines, Baum et al. (1971) have found that lesions in the lateral part of the substantia nigra result in severe aphagia and adipsia . They feel this can be attributed either to a decrease in the number of dopaminergic receptors or to a decrease of concentration of dopamine or acetylcholine in the striate bodies. Using chemical lesioning techniques, Evetts et al. (1971) also found that repeated injections of 6-hydroxydopamine into the preoptic area of rats provoked a progressive diminution in the eating response. Zigmond and Stricker (1972) injected 6-hydroxydopamine intraventricularly in rats and found that they maintained body weight at subnormal levels and failed to increase food intake in response to a short-term decrease in glucose utilization. After treatment with the monoamine oxidase inhibitor pargyline, 6-hydroxydopamine produced no further norepinephrine depletion but increased the dopamine depletion to 95 per cent and produced complete aphagia. Of course, pargyline potentiates the effects of 6-hydroxydopamine on dopamine deptetion but not on norepinephrine depletion.

62 P.J. Morgane Thus, these findings pointed more to involvement of a dopamine system in the behavioural alterations. Smith et al. (1972) found that 6-hydroxydopamine produced deficits in thirst by selective damage of catecholaminergic neurons in the lateral hypothalamic area. They concluded that lateral hypothalamic catecholaminergic neurons are essential components in the central neural networks subserving feeding behaviour. Finally, Friedman et al. (1973) have presented findings indicating a role for both dopamine and norepinephrine in the regulation of food intake . They noted that brain dopamine synthesis rate appears to be accelerated in the hypothalamus as well as in extra-hypothalamic sites (nigro-striatal pathway) by food deprivation . Dependence of eating behaviour on catecholamine synthesis in the hypothalamus was evident from the ability of hypothalamically applied alpha-methyl-para-tyrosine to inhibit food intake in food deprived rats. They proposed that in sated animals norepinephrine acts in a disinhibitory capacity to elicit feeding while in the hungry rat dopamine has an excitatory influence which induces increased eating. REGENERATION IN CHEMICAL SYSTEMS IN THE BRAIN AND RELATION TO REORGANIZATION OF THE HYPOTHALAMUS FOLLOWING LESIONS

Finally, some newer chemical aspects of regeneration in the central nervous system are pointed out because it appears to occur in particular chemical systems and it also may relate to the evolution of behavioural processes and restitution offunction following brain damage. I do not think any workers in energy homeostasis will have difficulty in developing variations on the lateral hypothalamic recovery theme when it is remembered that sprouting of injured axons in the medial forebrain bundle is now a well-recognized occurrence . Raisman (1969) has strong evidence suggesting that medial forebrain bundle axons may grow to reinnervate medial septa) nucleus neurons that have been partially denervated by section of the fimbria. The validity of this conclusion is largely dependent on the reliability of the multiple versus single synaptic contact observations as an indicator of synaptic reorganization in the nucleus . His evidence suggests that collaterals from local axons can occupy deafferented synaptic sites which is, of course, a quite striking finding . Further, by means of histochemical fluorescence techniques, Katzman et al. (1971) have provided evidence for regenerative sprouting of axons from catecholamine neurons in the rat mid brain 1-7 weeks after electrolytic destruction of the substantia nigra and part of the ventromedial midbrain tegmentum . In other studies Moore et al. (l97la,b) used the Falck-Hillarp fluo-

Control of physiological regulations and behaviour 63 rescent histochemical method for the cellular localization of monoamines to demonstrate that catecholamine-containing axons of the medial forebrain bundle will reinnervate the denervated septum. They sought to establish the potential of this group of neurons for displaying plastic changes, and felt it would allow the designation of the probable neurotransmitter for the new terminals . Most important, the latter point raises the possibility ofpharmacologic manipulation both of the regenerative process itself and of the functional alteration which might be associated with reinnervation. They found that central catecholamine-containing neurons have a capacity for exhibiting regenerative changes greater than that described for any other group of central neurons. The source of the adrenergic innervation in both the normal and denervated septum are axons traversing the medial forebrain bundle since section of this tract produces a substantial reduction in the adrenergic innervation of the septa) nuclei. In other exciting approaches , Bjorklund and Stenevi (1972) demonstrated the growth of new axonal sprouts from transected , ascending noradrenergic axons into transplants of iris tissue in the caudal hypothalamus of rats. A single intraventricular injection of nerve growth factor, given at the time of axonal damage, resulted in an increased formation and growth of new noradrenaline sprouts seven days later. Relative to this, Berger, Wise, and Stein (1973) claimed that a single intraventricular injection ofnerve growth factor, given at the time of brain lesions, facilitated the progression to normal feeding behaviour in rats. They concluded that this factor may facilitate behavioural restitution by several means such as promoting the development of supersensitivity to norepinephrine and possibly also by stimulating the growth ofregenerating noradrenergic neurons in the brain. Glick et al. (1972) found that rats treated with alpha-methylpara-tyrosine for three days prior to lateral hypothalamic surgery eat , drink, and gain weight spontaneously after surgery. According to a denervation supersensitivity model, time-dependent supersensitivity of partially denervated neurons to other intact inputs might mediate recovery. They postulate that lateral hypothalamic lesions probably result in removal of various intra-hypothalamic connections to surrounding intact tissue . If the recovery process involves supersensitivity ofnoradrenergic neurons and if such neurons were functionally denervated and made supersensitive prior to lateral hypothalamic damage , then facilitation ofrecovery after the latter might be expected. Using alpha-methyl-para-tyrosine, a drug that selectively interferes with catecholamine synthesis, these workers provided data consistent with the denervation supersensitivity hypothesis. In further studies involving parallel and sequential ablations, Glick and Greenstein ( 1972) theorized that, if lateral hypothalamic neurons were

64 P.J. Morgane partially denervated by ablation of frontal cortex at some time before damage to the lateral hypothalamus itself, delayed supersensitivity from the cortical lesion should result in facilitated recovery. They have presented data in support of such a hypothesis. It is possible in these types of experiments that 'reorganization' following lateral hypothalamic lesions is contingent upon sprouting of intact inputs to remaining lateral hypothalamic tissue. As noted by Glick and Greenstein, the removal of one such input (frontal cortex) may induce enough sprouting of other intact inputs to facilitate compensations following subsequent lesions of the lateral hypothalamus. It is also possible that both supersensitivity of adrenergic hypothalamic synapses and sprouting of inputs to non-adrenergic synapses underlie this example of plasticity in the central nervous system . Obviously, these are areas at the forefront of neurobiology and their application to the field of energy balance is encouraging. Coupled with chemical anatomy, circuit analyses are providing clues as to brain organization and, perhaps even more important, are showing the hypothalamus in its true perspective, a subsidiary component in a major brain formation. Ultimately, for any treatment of the various obesities and other disturbances of energy balance, we shall have to rely largely on pharmacological agents that exert their effects on these chemical networks to alter brain chemistry in the particular areas to which they project. SUMMARIZING REMARKS AND OVERVIEWS

One of the main themes I have sought to develop is the view that one critical key to unlocking hypothalamic functioning lies in the limbic system - the other in the lower brainstem reticular formation. The view has been expressed that the hypothalamus bears relations with the limbic system organizationally analogous to that the thalamus bears to the neocortical formations. If so, can it be doubted that hypothalamic functional organization cannot be derived until we study the nature of its interactions with limbic forebrain and limbic midbrain areas and lateral reticular systems? Analysis of any one of these components in isolation is but a small part of the picture, and, ultimately, a dead end. Rather than attempting any unyielding pronouncements offunctional localization in the hypothalamus, it is best that we concentrate on defining its afferent and efferent relations and relate it physiologically and biochemically within a broader framework of systems in which it forms but one component. Most of the studies reviewed indicate clearly that we must move away from the view that ' behaviours emanate from the hypothalamus' or that this area 'orchestrates' behaviour. It is certainly a key element in one of the

Control of physiological regulations and behaviour 65 major subsystems in the brain but however quasi-independent any of these are with respect to specific behaviours is still not clear. The prime issues in this field, and in brain physiology in general, are not those concerned with the locus of behaviour but rather with the nature of its controlling conditions. Unfortunately, for too long we have been dealing with descriptions of outcome rather than with fundamental processes involved in bringing it about. Constellations of alterations seen after brain manipulation really need precise physiological and biochemical characterization. Energy homeostasis has been a field where too many things have been prematurely tagged with labels that tend to perpetuate themselves and grow like Topsy . The real danger here has been that this has focused too much attention and, accordingly, research itself, strictly on the hypothalamus. Thus, brain interrelations have been largely ignored. Giving something a name is of value only if it serves heuristic purposes for studies of brain processes that are responsible for an integrated 'output' we term 'behaviour.' In the field of energy balance we have, until recently, had almost no morphological, biochemical, or physiological approaches to the underlying mechanism and the field, almost by default, has gravitated to the realm of pure psychologizing about this entire domain of brain functioning. Behaviour emanates from intersegmental transmitter activity along hosts of chemical circuits. Von Gudden once said a truism that holds better today, perhaps, than it did when first written almost a century ago: 'Zuerst also Anatomie und dann Physiologie, wenn aber erst Physiologie dann nicht ohne Anatomie.' Now with chemical or transmitter anatomy we see even more the fundamental truth in this observation. Chemical anatomy is not static it is active, dynamic, 'forever constant yet forever changing'!! This latter characterizes homeostasis in the best possible manner. Permission has been kindly granted by Spectrum Publications, Flushing, NY, for the reproduction of Figures I, 2, 3, 4, 8, 9, and 10, Advances in Sleep Research, Vol. I; and by the New York Academy of Sciences for Figures 6and 7 and Table I. Ann. N . Y. A cad. Sci . , 193: 95-111, 1972.

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Control of physiological regulations and behaviour 67 - 1974. In E. Weitzman (ed.) , Advances in Sleep Research, Vol. I. Flushing, New York: Spectrum Publications pp. 1-131 Morrison, S.D. and Mayer, J. 1957. Am . J. Physiol . , 191 : 248-54 Murphy , J. T., Dreifuss , J .J. , and Gloor, P. 1968. Am . J. Physiol., 214: 1443-53 Murphy, J .T. and Renaud, LP., 1968. Brain Res. , 9: 385-9 - 1969. J. N europhysiol., 32: 85-102 Nauta, W.J.H . 1958. Brain, 81: 319-40 - 1961.J. Anat. ,95:515-31 - 1%3 . In A. V. Nalbandov (ed.) Advances in Endocrinology . Urbana: U. of Illinois Press pp. 5-28 - 1971. J. Psychiatr. Res . , 8: 167-87 Nauta , W.J.H. and Haymaker, W. , 1969. In W. Haymaker, E. Anderson, and W.J.H . Nauta (eds .), The Hypothalamus. Springfield: Charles C. Thomas pp. 136-209 Nauta , W.J.H . and Kuypers , H.G.J.M. , 1958. In H. Jasper, L. Proctor, R. Knighton , W. Noshay , and R. Costello (eds.), Reticular Formation of the Brain . Boston: Little, Brown Co. pp . 3-30 Olds , J., Mink, W., and Best, P., 1969. Electroenceph . clin. Neurophysiol., 26: 144-58 Olivier, A., Parent , A., Simard , H., and Poirier, L.J., 1970. Brain Res . , 18: 273-82 Oltmans, G.A . and Harvey J.A ., 1972. Physiol. Behav. , 8: 69-78 Oomura, Y., Ooyama, H., Yamamoto, T., and Naka , F . 1967. In: W.R. Adey and T. Tokizane (eds.), Progress in Brain Research : Structure and Function of the Limbic System . Amsterdam : Elsevier, vol. 27 pp. 1-33 Parent , A. , Saint-Jacques , C., and Poirier, L.J ., 1969. Exp . Neural . , 23: 67-75 Powell , T.P.S. , Cowan, W.M. , and Raiman , G. 1965. J . Anar. 99: 791-813 Pribram, K. 1%1. J. Neurosurg . , 18: 375-7 Raisman, G. 1969. Brain Res. , 14: 25-48 - 1970. Am.J.Anal. , 129: 197-202 Ramon y Cajal, S. 1911. Trabajos Lab. Rech. Biol. , 2: 31-{i9 Ramon-Moliner, E. and Nauta, W.J .H., 1966. J. comp . Neuro/. , 126: 311-36 Rolls, E.T. 1971. Brain Res . , 31 : 275-85 - 1972. Brain Res. , 45: 365-81 Rolls , E.T. and Kelly , P.H., 1972. J . comp. physiol. Psycho/ . , 81 : 173-82 Smith , G.P., Ervin, G.N. , and Reis , D.J. , 1972. Proc. Soc. Neurosci., 2nd Annual Meeting, Abstract 10.4 Smith , G.P., Strohmayer, A.J ., and Reis, D.J ., 1972. Nature (New Biology), 235: 27-9 Stevenson, J .A.F. and Montemurro, D.G., 1%3. Natu re , 198: 92 Szentagothai , J. , Flerk6, B., Mess , B., and Halasz, B., 1%8. Hypothalamic Control of the Anterior Pituitary , Akademiai Kiad6 Budapest, pp. 56-78 Ungerstedt, U. 1970. Acta physiol. scand . , 80: 35A-6A - 1971a. Acta physiol. scand. Suppl., 367: 1-48 - 1971b. Acta physiol. scand. Suppl., 367: 95-122 Valenstein, E.S., Cox, V.C., and Kakolewski, J.W., 1970. Psycholog . Rev. , 77 : 16-31 Valverde, F. 1%5. Studies on the piriform lobe . Cambridge, Mass.: Harvard University Press Wampler, R.S. 1970. Dissertation Abstracts, pp. 3741-2 Zigmond, M.J. and Stricker, E.M., 1972. Science, 177: 1211-13

The anatomy of the limbic system

Elizabeth Hall

The term limbic system, although appearing more and more frequently in the literature, continues to elude precise definition, especially with regard to its anatomical components. As this difficulty is likely to be enhanced by certain recent anatomical studies which will be mentioned later in the text, a few comments regarding the use of the term will be given to provide historical perspective. Although there is some uncertainty regarding the original use of the word limbic (White, 1965a), there is no doubt that its present meaning has been derived from an anatomical expression introduced by Broca in 1878. Noting that the cingulate and hippocampal gyri form a border or limbus about the diencephalon, and thinking that the olfactory bulb projected to both cortical areas, he grouped all these structures together as 'le grand lobe limbique .' He also noted the constancy of configuration of this lobe in comparison with the neocortical convolutions in a number of mammalian species. Numerous anatomical studies were done on the components of the limbic lobe during the ensuing years, but no particular function, other than that of olfaction, was ascribed to it until 1937. It was in fact the discovery of the hypothalamic influence on the autonomic and endocrine systems that eventually led to the suggestion that the cortex of the limbic lobe is concerned with emotion. Bringing together data about the function and connections of the

Anatomy of the limbic system 69 hypothalamus, and clinical and anatomical data about the cingulate cortex and hippocampal formation, Papez (1937) hypothesized that the hypothalamus-anterior thalamic nuclei-cingulate cortex-hippocampusfornix-hypothalamus circuit was concerned with emotion. More specifically, he considered that the cingulate cortex was important for emotional awareness and that through its intercortical connections might lend emotional colouring to psychic activity occurring elsewhere. Presumably the cingulate projection to the hippocampal region and the hippocampal projection to the hypothalamus could play a role in emotional expression. MacLean (1949, 1952, 1954) expanded Papez's concept and appears to have been the first to use the term limbic system, which he defined as follows (Mac Lean, 1952, p. 407): 'The limbic system is comprised of the cortex contained in the great limbic lobe of Broca together with its subcortical cell stations.' He considered that the limbic cortex included (p.407)' .. . cortex adjacent to the olfactory striae; pyriform area; hippocampal gyrus and hippocampus; parasplenial, cingulate and subcallosal gyri.' and that (p.407) 'Nuclear structures associated with the limbic system include: amygdala, septa) nuclei, hypothalamus, epithalamus, anterior thalamic nuclei, and parts of the basal ganglia.' On the basis of the phylogenetic age of these structures, the relative constancy of the limbic lobe noted by Broca (1878), the evidence from physiological studies, and certain clinical data regarding psychosomatic disease, MacLean came to think of the limbic system as man's primitive inheritance, the area of the brain concerned with the preservation of the individual and of the species (see MacLean, 1949, 1952, 1955, 1958; MacLean and Delgado, 1953). In general MacLean's concept has proven useful and has stimulated some of the increasing interest in limbic structures. However, the limbic system is not so easy to delimit as MacLean's (1952) definition might suggest. First, the anatomical characteristics of a 'subcortical cell station' have not been specified, so that any structure in direct connection with the limbic lobe may be considered a part of the limbic system. Secondly, there are no precise requirements which must be met regarding physiological and behavioural effects. It appears that in the absence of such anatomical and functional limitations the components included in the limbic system continue to increase in an unregulated manner (for further comments see Brodal, 1969, pp.537-9). Finally it should be noted that the term 'limbic' is still used occasionally in a strictly anatomical sense, with no implication regarding function. In the anatomical review which follows, examples of these problems will be encountered.

70 Elizabeth Hall THE CINGULATE CORTEX (Fig. I) STRUCTURE

It is generally accepted that the cingulate cortex can be subdivided into a posterior granular and an anterior agranular region according to the presence or absence of granular layer 1v. Further parcellations, which vary in position and extent from species to species, have been made according to additional cytoarchitectonic features (see Rose and Woolsey, 1948). According to Rose and Woolsey (1948) an anterior limbic region can be distinguished from a posterior limbic region which consists of a cingular area and a retrosplenial area in the rabbit and the cat. Their terminology is somewhat different from that of M. Rose which has been employed recently by Domesick (1969) in her study of the cingulate cortex in the rat. (Fig. I) On the basis of retrograde cell changes it has been established that the anterior thalamic nuclei project to the limbic cortex of the cingulate gyrus, although details concerning the origin and termination of this connection vary. Rose and Woolsey (1948) found that the anteromedial nucleus projected to the anterior limbic field, while the anteroventral and anterodorsal nuclei projected to the posterior limbic region in both the rabbit and the cat. However, in the monkey Yakovlev et al. (1960) observed that fibres from all three anterior nuclei reached the anterior limbic area (area 24 of Brodmann), while the granular (that is, posterior) limbic region received fibres from the nucleus lateralis dorsalis. The latter projection has also been observed by Locke et al . ( 1964) in both the cat and the monkey. More recently the studies ofDomesick (1969, 1973) have provided additional information regarding these connections. In a study of anterograde degeneration after lesions in the anterior thalamic nuclei of the rat she observed some fibres entering the cingulate gyrus. However, the majority continued in the cingulum bundle beyond the cingulate cortex and terminated in the presubiculum, parasubiculum, and entorhinal cortex . Thus some of the discrepancies regarding the retrograde degeneration seen in certain thalamic nuclei may be due to a varying degree of damage to thalamic-subicular and thalamic-entorhinal fibres in the cingulum rather than to ablation of the cingulate cortex itself, especially where these reports present different results in the same species (cat: Rose and Woolsey, 1948;Lockeeta/., 1964). Experimental studies have demonstrated that the cingulate cortex projects back to several thalamic nuclei . These connections were first seen THALAMIC CONNECTIONS

Anatomy of the limbic system 71 with the Marchi method (e .g., Showers, 1959) and have been analysed recently in detail by Domesick (1969) with the Fink and Heimer method. Domesick noted that all of the cingulate cortex appears to project to the anteromedial nucleus; in addition, she showed that the anterior cingulate region projects to the mediodorsal and medioventral thalamic nuclei and the posterior region to both the anteroventral and lateral dorsal nuclei, but she did not observe any fibres extending to the anterodorsal nucleus. OTHER SUBCORTICAL PROJECTIONS

Some investigators have described a projection from the cingulate gyms to the hypothalamus (Nauta, 1953; Showers, 1959). These fibres were not observed by Domesick (1969), nor could she identify any degeneration extending to the septum or limbic midbrain tegmentum. However, she was able to confirm that the cingulate cortex projects to the caudate nucleus, zona incerta, pretectal area, and certain other brain stem regions (see Nauta, 1953; Showers, 1959). CORTICAL PROJECTIONS(Fig . I)

Several investigators have confirmed the observation of Ramon y Cajal (1911, 1955) that fibres in the cingulum bundle extend to the presubicular and entorhinal areas. As these studies were done with different staining methods and in different species, and as the lesions varied in size and position, there are some variations in the results. There has, however, been general agreement that these fibres arise in the cingulate gyms and extend through the cingulum to terminate in the entorhinal area (monkey: Showers, 1959; rat: Raisman et al., 1965; Cragg, 1965), the presubiculum (rabbit: Adey, 1951; monkey: Adey and Meyer 1952a; rat: Raisman et al., 1965; Cragg, 1965), and subiculum (rabbit: Adey, 195 ]). It has also been suggested that some fibres reach the hippocampus (monkey: Showers, 1959) and dentate gyms (rabbit: Adey, 1951). Employing the Fink and Heimer method to trace degeneration from relatively small lesions, Domesick (1969) was unable to identify a projection from the anterior cingulate cortex to any of these regions . Further, she observed that the more posterior lesions gave rise to only a small amount of degeneration which was confined to the presubiculum. On this basis she suggested that the more extensive distribution described by earlier investigators may have been due to involvement of the cingulum bundle which, in the rat at least, is predominantly thalamic in origin. In contrast, the cingulate cortex is strongly interconnected with a number of neocortical areas (see Showers, 1959; Jones and Powell, 1970).

72 Elizabeth Hall

CINGULATE

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Figure I Schematic summary of the main limbic connections of the cingulate cortex. In this and the other figures each solid dot, whether at the beginning or along the shaft of the arrow, indicates a site of origin of the fibre bundle. Each arrowhead, whether along or at the end of the shaft, indicates a site of termination. An interrupted arrow shaft indicates there is some uncertainty regarding that segment of the projection which has been mentioned in the text. The list of abbreviations applies to all figures. ACC AH AMY AO AOB AT BL CEN COR CTX ER FD

H HYP LO LH LHYP LRPO M

nucleus accumbens anterior hypothalamus amygdala anterior olfactory nucleus accessory olfactory bulb anterior thalamic nuclei basal and lateral amygdaloid nuclei central amygdaloid nucleus cortical amygdaloid nucleus anterior cortical regions entorhinal cortex fascia dentata habenula hypothalamus lateral dorsal nucleus of the thalamus lateral habenular nucleus lateral hypothalamus lateral preoptic region mammillary nucleus

mediodorsal nucleus of the thalamus medial amygdaloid nucleus medial preoptic region medioventral nucleus of the thalamus nucleus of the diagonal band of Broca olfactory bulb olfactory tubercle periamygdaloid cortex premammillary region pp prepyriform cortex regio inferior of the hippocampus RI preoptic region RPO regio superior of the hippocampus RS subiculum (including all its s subdivisions) septum SEP substantia innominata SI TEMP CTX temporal cortex ventromedial hypothalamic nucleus VM I, 2, 3, 4 hippocampal fields CA I, CA2, CA3, and CA4

MD MED MRPO MY NDB OB OT PA PM

Anatomy of the limbic system 73 On this basis the importance of the gyrus as a link in Papez' s circuit and its role in the limbic system must be seriously re-examined. THE PYRIFORM CORTEX (Figs . 1, 2, and 3) TERMINOLOGY AND STRUCTURE

Before embarking upon a morphological description of the pyriform cortex it may be useful to mention some of the terms presently applied to this region of the brain. Ramon y Cajal (1911, 1955), who was among the first to describe the pyriform cortex, divided it into two main parts according to its structure and afferent connections . He subdivided the more anterior part , which displays fewer cortical layers and receives an olfactory projection through the lateral olfactory tract, into 'ecorce du lobe frontal sous-jacente a la racine externe' and 'region olfactive principale ou centrale de l'hippocampe.' In the more posterior part (his ' noyau angulaire ') he observed that the cortical layers were more numerous , structurally more elaborate , and apparently did not receive olfactory fibres. For some years , these three areas have been referred to as prepyriform, periamygdaloid, and entorhinal cortex respectively, although Gray's (1924) terminology (areae piriformis anterior, medialis, and posterior) has also enjoyed considerable popularity. It is common practice to include all three regions in the pyriform lobe, but occasionally the entorhinal area is omitted. Conversely, those who base their terminology on that of Brodmann (for details see Vaz Ferreira, 1951) consider that only the entorhinal cortex belongs in the pyriform lobe and that the two more anterior regions form the prepyriform lobe. (See also the excellent review by Pigache, 1970.) Each of the three pyriform regions has been further subdivided according to cytoarchitectural differences, and for information and references on this subject the reader is referred to Ramon y Cajal (1911, 1955) , Gray (1924), Rose (1929), Lorente de No (1933), Valverde (1965) , and Scalia (1966). (Fig. 2) The olfactory bulb sends a large contingent offibres to the prepyriform and periamygdaloid regions through the lateral olfactory tract which courses posteriorly on the inferior surface of the brain and fans out in the molecular layer of these cortices. The first detailed descriptions of this connection were based on normal material (Ramon y Cajal 1911, 1955), and have since

OLFACTORY CONNECTIONS

74 Elizabeth Hall AO

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Figure 3 Schematic summary of the main subcortical connections of the pyriform cortex. See caption for Fig. I for explanation of symbols and abbreviations .

been confirmed experimentally with the Glees method (Le Gros Clark and Meyer, 1947, and others) and more recently with the Nauta and modified Nauta methods (White, 1962, 1965b, Powell et al., 1965, Scalia, 1966; Mascitti and Ortega, 1966; Heimer 1968; Lohman and Mentink, 1969). These authors agree that the fibres and their terminals are confined primar-

Anatomy of the limbic system 75 ily to the molecular (that is, the most superficial) layer, a point which has been demonstrated recently with the electron microscope (Westrum, 1969). In addition, it appears that the projection is dense anteriorly, but becomes sparser as it continues posteriorly through the periamygdaloid cortex. There is some evidence that the more anterior part of this olfactory cortex projects back to the olfactory bulb (Cragg, 1962; Heimer, 1968). However, it has also been suggested that these fibres arise from the ventral limb of the diagonal band of Broca (Price and Powell, 1970a) and that in previous studies they were damaged either at their origin or as they coursed towards the bulb through the lateral olfactory tract. Evidence is accumulating (Scalia, 1966; Heimer, 1968; Price and Powell 1971) in support of White's (1965b) contention that some olfactory fibres continue through the periamygdaloid cortex to enter the ventrolateral part of the entorhinal area, an area which until recently had been considered to receive no olfactory projection. Following a re-evaluation of the cytoarchitecture of this region, Scalia (1966) has concluded that it is incorrect to consider the terms noyau angulaire of Ramon y Cajal (1911, 1955) and entorhinal cortex synonymous . He considers that the entorhinal cortex includes a larger area, and that the part of the entorhinal area which receives olfactory fibres was not included by Ramon y Cajal in the noyau angulaire. It is probable that the accessory olfactory bulb (vomeronasal organ) does not project directly to any part of the pyriform cortex (Winans and Scalia, 1970). A projection from the pyriform cortex to the anterior olfactory nucleus has also been described (Price and Powell, 1970a). (Figs. 1 and 2) The pyriform cortex appears to have relatively few cortical connections. Adey and Meyer(1952b) were the first to describe fibres extending from the temporal to the entorhinal cortex in experimental material, a projection that Whitlock and Nauta (1956) described as coursing from the inferior temporal cortex into the immediately adjacent part of the entorhinal and periamygdaloid areas in the monkey. Jones and Powell (1970) have suggested that these fibres terminate in a transitional zone, the perirhinal area 35 of Brodmann. In the cat, Cragg (1965) demonstrated that the most ventral part of the temporal cortex projects to the entorhinal cortex, and that after entorhinal lesions degenerated fibres can be seen curving around the rhinal fissure and extending for a very short distance (two mm) back CORTICAL CONNECTIONS

76 Elizabeth Hall into this area ofneocortex . In the rat, however, Powell et al. (1965) did not observe a neocortico-pyriform projection even though the lesion bordered on the rhinal fissure . Afferent projections from the frontal cortex (Showers , 1958) and suprasylvian gyrus (Cragg, 1965) have also been suggested but have not yet been confirmed. The extent of the cingulate projection was discussed above . Connections between the entorhinal cortex and hippocampus are discussed below in the section on the hippocampal region . Degeneration has been observed in the entorhinal cortex following lesions in the prepyriform and periamygdaloid regions (Cragg, 1961b, rabbit , cat , rat; Powell et al., 1965 , rat; Price and Powell , 1971 , rat) . However, as lesions in these areas necessarily involve fibres of the lateral olfactory tract, and as the olfactory tract projects to the entorhinal cortex , there is some uncertainty regarding the origin of this projection . The pyriform cortices have been shown to be interconnected through the anterior commissure by the Golgi (Ramon y Cajal, 1955 ; Valverde, 1965) retrograde degeneration (Broda!, 1948) and Marchi methods (Fox and Schmitz, 1943). (Fig. 3) Most of the studies dealing with the subcortical projections of the prepyriform and periamygdaloid cortex present similar results . After lesions in the anterior pyriform cortex of the rat (Powell et al., 1965) heavy degeneration was traced caudally and medially, the subcortical component of the caudal projection terminating in the lateral and basal amygdaloid nuclei , and the medially directed fibres ending in the nucleus of the diagonal band, the olfactory tubercle, the cortex lying anterior and inferior to the corpus callosum, and the preoptic region . From the last area, degenerated fibres were observed entering the medial forebrain bundle and continuing to about the mid-lateral hypothalamic level. A second projection continued into the inferior thalamic radiation and terminated in the medioventral and mediodorsal thalamic nuclei and the lateral habenular nucleus. The degeneration was distributed bilaterally in the last two nuclei . After lesions in the more posterior part of the pyriform cortex (interpreted here as mainly periamygdaloid cortex on the basis of Powell et al. 's diagrams, 1965), degenerated fibres were observed coursing into the lateral and basal amygdaloid nuclei where some of them terminated. However, the majority of the fibres continued anteriorly and medially to the preoptic region and from here followed the same course and had the same terminal distribution as was described for the prepyriform region.

SUBCORTICAL CONNECTIONS

Anatomy of the limbic system 77 Lundberg (1962) described degeneration extending more posteriorly in the lateral hypothalamus to terminate precisely in two small nuclear groups he named the nuclei gemini. Price and Powell ( 1970a,b) observed degeneration extending as far anteriorly as the anterior olfactory nucleus but found none in the nucleus of the diagonal band. Recently Heimer (1973) has cast doubt on the existence of the direct projection from pyriform cortex to rostral hypothalamus. Employing both light and electron microscopic techniques, he found extensive axonal but only occasional bouton degeneration in the lateral preoptic region and anterior part of the lateral hypothalamus . It would appear that the degeneration described by earlier authors probably consisted of fibres of passage coursing towards the mediodorsal thalamic nucleus or continuing posteriorly in the lateral hypothalamus to the nuclei gemini. In addition, Heimer's (1973) findings would indicate that the electron microscope should be employed to verify other sites of termination that have been described for fibres of the pyriform cortex with the silver impregnation methods. To date, no direct projection from the hypothalamus to the pyriform cortex has been described. However, there is evidence that the amygdaloid complex sends fibres to the periamygdaloid cortex (De Olmos, 1972). THE HIPPOCAMPAL REGION (Figs. 4 and 5) STRUCTURE

The hippocampal region includes hippocampus, fascia dentata, subiculum and entorhinal cortex. Although some anatomical features of the entorhinal area have already been discussed in the section on pyriform cortex, it was considered more appropriate to deal with the entorhinal-hippocampal formation connections here. The hippocampus (Ammon's horn) consists of a stratum lacunosummoleculare, a stratum radiatum containing the ascending apical dendrites of the pyramidal layer, a stratum pyramidale consisting of pyramidal cells, and an underlying stratum oriens beyond which lies the alveus (see Ramon y Cajal, 1911, 1968; Blackstad, 1956). It can be subdivided into two main parts on the basis of the density and the size of cells in the pyramidal layer. That part which lies adjacent to the subiculum and contains more densely packed smaller cells is the regio superior (CAI of Lorente de N6, 1934), while that part which meets the fascia dentata and contains the large pyramidal cells is the regio inferior (cA2, CA3, and CA4 of Lorente de N6, 1934). The fascia dentata is composed of a superficial molecular layer and a

78 Elizabeth Hall

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Figure 4 Schematic summary of the intrinsic connections of the hippocampal region . See caption for Fig. I for explanation of symbols and abbreviations .

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Figure 5 Schematic summary of the main subcortical afferent and efferent connections of the hippocampal region . See caption for Fig. 1 for explanation of symbols and abbreviations .

granular layer. These two, together with the hilar region which corresponds approximately to CA4 of Lorente de No (1934), are referred to as the area dentata (Blackstad, 1956). The term subiculum may be used in a more general sense to refer to the cortex between the entorhinal region and the hippocampus, but more specifically a distinction can be drawn between parasubiculum, presubiculum, subiculum, and prosubiculum (see Blackstad, 1956). Finally, the entorhinal cortex may be divided into medial and lateral parts (see Blackstad, 1956).

Anatomy of the limbic system 79 This parcellation of the hippocampal region is characterized not only by differences in cyto- and fibrearchitectonic features but also by differences in the distribution of acetylcholinesterase (Mathisen and Blackstad, 1964; Geneser-Jensen and Blackstad, 1971; Geneser-Jensen 1972a, 1972b), zinc (see Haug, 1967), and other substances (Mellgren and Blackstad, 1967; Blackstad et al., 1967; Geneser-Jensen, 1971) stained by histochemical methods. INTRINSIC CONNECTIONS (Fig. 4) Of the intrinsic connections of the hippocampal region the mossy fibre system has been studied in the greatest detail. The fibres arise from the granule cells of the area dentata (see Ramon y Cajal, 191 I, 1968) and course through the deepest part of the stratum radiatum of regio inferior. Here they display large boutons 'de passage' which synapse with elaborate branching spines or excrescences given off from the proximal part of the pyramidal apical dendrites (see Blackstad and Kjaerheim, 1961; Hamlyn, 1961, 1962). It has been shown that this fibre system is rich in zinc (Maske, 1955, and others) and further that the zinc is located exclusively within the giant boutons of the mossy fibres (Haug, 1967). This has been verified in a study employing both the Fink and Heimer technique and Timm's method for zinc. After lesions of the fascia dentata, it was observed that the areas of terminal degeneration and blanching of the zinc stain in regio inferior were spatially coincident (Haug et al., 1971). Blackstad et al. (1970) have observed that the mossy fibre projection from the fascia dentata follows a very precise topographic distribution in the regio inferior which, as the authors noted, suggests a comparable precision offunction. Fibres from at least part of the regio inferior project back to the fascia dentata (Zimmer, 1971) . Additional intrinsic connections have been described both within the hippocampus and from the hippocampus to the subiculum by experimental methods. Raisman et al. (1965) observed that CA4 and CA3 projected to CA2 and CA I but found no reciprocal pathway. Similarly, Hjorth-Simonsen (1973) has reported a non-reciprocal projection from regio inferior to regio superior and a projection from regio superior to the subiculum. The projection from the entorhinal cortex to the hippocampus and area dentata has been known for many years from normal preparations (Ramon y Cajal, 1911) and was demonstrated first by Blackstad (1958) in experimental material. The site of termination in the stratum lacunosummoleculare and molecular layers was confirmed at the electron microscopic level by Nafstad (1967) . In a more detailed experimental study,

80 Elizabeth Hall Hjorth-Simonsen and Jeune (1972) and Hjorth-Simonsen (1972) have observed that the entorhinal fibres project topographically, the more medial region terminating in the deep half of stratum lacunosum-moleculare of CA3 and the middle of the molecular layer of fascia dentata, while the lateral part ends more superficially in these two layers. Finally, it has been shown that the regio inferior projects back to the medial entorhinal area (HjorthSimonsen, 1971). AFFERENT PROJECTIONS (Fig. 5) The entorhinal cortex receives fibres from cingulate, temporal, prepyriform, and periamygdaloid cortical areas and from the olfactory bulb, while the hippocampal formation (which consists of hippocampus, fascia dentata, and subiculum) receives fibres from the anterior thalamic nuclei, the cingulate cortex, entorhinal cortex, and septum. All but the septal projection have been considered in the preceding text. Septal projections to the hippocampal formation have been described in experimental studies by several investigators. Daitz and Powell (1954) observed retrograde degeneration in the medial septal nucleus and the nucleus of the diagonal band following section of the fimbria of the fornix . Cragg (1961b) and Votaw and Lauer (1963) confirmed that the septum projects via the fornix to the hippocampal formation in the rabbit and the monkey. In the rat it has been observed that fibres arising in the medial septa! nucleus and the nucleus of the diagonal band terminate in the hippocampus and fascia dentata and that within the hippocampus they are restricted primarily to fields CA3 and CA4, although some were also seen in CA2 (Raisman et al., 1965; Raisman, 1966). No degeneration was seen in any of these regions following lesions of the lateral septal nucleus . More recently Siegel and Tassoni (1971b) have described a different distribution in the cat. They observed degeneration in all of the hippocampal fields and in the dentate gyrus, but noted that fibres from the medial septa) nucleus were restricted to the ventral, and fibres from the lateral nucleus to the dorsal part of the hippocampal formation. However, Ibata et al. (1971) found that the medial septa! nucleus of the cat projected fibres to all fields of the hippocampus throughout its entire dorsoventral extent and that the septa) projection to the hippocampal formation was bilateral. It has also been reported that the hippocampal formation receives a small projection through the fornix from the midbrain (Guillery, 1957; Cragg, 1961b) and from the hypothalamus (Cragg, 1961b).

Anatomy of the limbic system 81 (Fig. 5) The massive projection of the hippocampus through the fornix to the septal region and diencephalon has been described in normal material by many investigators (for references , see Sprague and Meyer, 1950; Nauta, 1956) and only the more recent experimental investigations will be considered here. Nauta ( 1956) in his study of the hippocampus and fornix in the rat traced degeneration to all the septal nuclei including the bed nucleus of the hippocampal commissure and the nucleus of the diagonal band, the lateral preoptic region, the dorsal and periventricular zones of the hypothalamus, the arcuate nucleus, the ipsilateral and contralateral anterior thalamic nuclei, and the mammillary nuclei. In addition , a few fibres continued into the most rostral part of midbrain . Guillery (1956) reported very similar findings in the same species . In a later study Valenstein and Nauta (1959) noted that the sites of termination of the fornix fibres differed slightly in the rat, guinea pig, cat, and monkey . The observations of Raisman et al. (1966) in the rat agree well with the earlier work of Nauta (1956) and Guillery (1956). However, they provided greater details regarding both the origin and termination of the hippocampal fibres , and at least three of these should be mentioned. First, they observed an additional projection extending to the nucleus accumbens. Secondly, they found that the projection to the periventricular region of the hypothalamus originated in the subiculum. Thirdly, they found that the posterior part of CA I projected to the medial septum , diagonal band nucleus , septofimbrial nucleus, and ventromedial quadrant of the lateral nucleus , while CA3 and CA4 projected to the septofimbrial and medial septal nucleus, and bilaterally to the dorsolateral quadrant of the lateral septal and to the diagonal band nuclei . The importance of the hippocampal projection to the septum has been emphasized recently by Raisman (1969a) who noted that it accounted for thirty-five per cent of the total bouton population in the medial nucleus and forty-three per cent of the ipsilateral and thirteen per cent of the contralateral boutons in the lateral nucleus . Siegel and Tassoni (1971a) found a different topographic projection in the cat, reporting that the dorsal part of all hippocampal fields projected to the medial septum and the ventral part of all fields projected massively and bilaterally to the lateral septum. Otherwise their observations concerning the distribution of fornix fibres are in agreement with those of the authors mentioned above.

EFFERENT PROJECTIONS

82 Elizabeth Hall A very detailed literature exists regarding the commissural connections of the hippocampal formation which, because of their complex nature, have been considered beyond the scope of this review. The interested reader is referred to Blackstad (1956), Alksneet al. (1966), and Laatsch and Cowan ( 1967) for discussion and references on this topic . AMYGDALA (Figs. 6and 7) STRUCTURE

The amygdaloid complex consists of several nuclei which can be distinguished according to the size and degree of staining of their neurons in N issl preparations . These cell groups, although displaying certain variations in their relative size and position (Crosby and Humphrey, 1944), present a relatively constant appearance in all the mammals in which they have been studied (for references, see Koikegami, 1963). However, there is a difference of opinion among investigators regarding the number of subdivisions which can be distinguished in the individual nuclei of a given species. In general, it can be said that the Japanese anatomists favour a greater number of subdivisions and base their terminology on that of the early German scientists , while most European and North American investigators describe fewer subdivisions and employ the simpler terminology of Johnston (1923). Johnston (1923) divided the amygdaloid nuclei into basolateral and corticomedial groups , the former consisting of the lateral and basal nuclei, the latter of the cortical, medial , and central nuclei and the nucleus of the lateral olfactory tract. The only change which has occurred in this terminology was introduced by Gurdjian (1928) who separated the central nucleus of Johnston into a more clearly defined part which retained the name central nucleus and a less clearly defined anterior part which he called the anterior amygdaloid area. Although the Niss! stains have provided the basic plan of the amygdala, other methods have revealed different patterns of grouping of the nuclei and subnuclei . It has been observed in Golgi preparations that the lateral, basal, and cortical nuclei of the cat are relatively homogeneous and the boundaries between them difficult to distinguish (Hall, 1972b). However, when the same nuclei are stained by histochemical methods patterns are observed which suggest not only differences in the structural organization within a given nucleus (Hall et al. , 1969, Hall and Geneser-Jensen, 1971; Hall, 1972a), but also similarities between parts of different nuclei . The significance of these findings in terms of the overall cyto- and

Anatomy of the limbic system 83

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Figure 7 Schematic summary of the dorsal and ventral amygdalofugal pathways (stria terminalis and the logitudinal association bundle) . Note reservations regarding the ventral pathway in the text. See caption for Fig. I for explanation of symbols and abbreviations.

fibrearchitectural organization of the amygdala has not yet been determined (see Hall, 1972a, for further discussion).

84 Elizabeth Hall (Figs . 6and 7) The first experimental studies on the terminal distribution of bulbofugal fibres indicated that the amygdaloid component of the projection extended into the central, cortical, and medial nucleus and the nucleus of the lateral olfactory tract (Le Gros Clark and Meyer, 1947). Further investigations provided conflicting data regarding this pathway which could not be explained entirely on the basis of either species variations or differences in the staining technique (Scalia, 1968). However, it was generally agreed that no fibres reached the central nucleus, and that fibres terminated in the nucleus of the lateral olfactory tract and at least part of the cortical nucleus. Findings regarding a projection to the medial nucleus and other areas of the amygdala were more variable (see the review by Scalia, 1968). The recent observations of Winans and Scalia (1970) appear to resolve these issues. These authors observed that after lesions of the olfactory bulb degeneration in the amygdala was confined to an anterolateral strip of the cortical nucleus, while after lesions of the immediately adjacent accessory olfactory bulb (vomeronasal organ) degeneration was limited to the medial nucleus and the medial and posterior parts of the cortical nucleus. These findings receive indirect support from the observation of De Olmos (1972) and De Olmos and Ingram ( 1972) that the posterior parts of the cortical and medial nuclei project via the stria terminal is to the granule cell layer of the accessory olfactory bulb, a finding that has been confirmed at the fine structural level by Raisman (1972). OLFACTORY CONNECTIONS

(Fig. 6) Neocortical projections to the amygdaloid nucleus have been observed by several investigators in both the monkey and the cat. These were first reported by Whitlock and Nauta ( 1956) who noted that fibres arising in the inferior temporal gyrus of the monkey coursed to the lateral, basal, and central nuclei. In the cat these nuclei receive fibres from the anterior and posterior sylvian gyri (see Druga, 1969; Lescault, 1969, 1971; Siegel et al., 1971) and from the posterior ectosylvian gyrus (Lescault, 1969, 1971; Siegel et al., I 971). Both Druga (I 969) and Lescault (I 969, I 971) observed that the terminal degeneration in the lateral nucleus was heaviest in its dorsolateral segment. Projections from part of the anterior ectosylvian gyrus to the lateral and central nuclei and from the secondary auditory area to the lateral nucleus have also been described (Lescault, 1969, 1971). Fibres arising in the amygdala course to the rostral parts of the inferior, middle, and superior temporal gyri and to the ventral insular and caudal CORTICAL CONNECTIONS

Anatomy of the limbic system 85 orbitofrontal cortex in the monkey (Nauta, 1961). In the cat, amygdaloid fibres reach the posterior sylvian gyrus (Valverde, 1965). Projections from the orbital gyrus to the amygdala have been described in the cat (Valverde, 1965; Hirata, 1965; Mizuno et al ., 1969; Lescault, 1971), but have not been observed in the monkey (Nauta, 1962). Whether these findings reflect a true species difference is uncertain, as the cortical regions referred to as orbital cortex in the cat and the monkey may not be strictly homologous. No neocortico-amygdaloid fibres have been observed in the rat (Powell et al . , 1965). Connections between the amygdala and pyriform cortex were described earlier in the text. (Figs . 6 and 7) The amygdala has two main subcortical efferent pathways, the stria terminalis and the longitudinal association bundle of Johnston, each of which shows species variations in its origin and termination. Numerous authors have described these fibre tracts in normal material, including Johnston (1923), Berkelbach van der Sprenkel (1926), Fox (1940), and Valverde (1962, 1965). Omukai (1958), however, was among the first to provide a detailed description of their origin and field of distribution based on experimental material. Using the Marchi method, this investigator observed that the medial , cortical , and small-celled part of the basal nucleus of the rabbit projected via the stria terminalis to the bed nuclei of the stria terminalis and anterior commissure, the septa! area, and the rostral part of the ventromedial nucleus of the hypothalamus, while the magnocellular part of the basal nucleus sent fibres to the lateral preoptic region. No contribution from the lateral nucleus to the dorsal trajectory was observed. Both Nauta and Valenstein (1958) and Nauta (1961) in their investigation of the monkey, and Cowan et al. ( 1965) in their study of the rat noted that amygdaloid fibres coursing via the stria terminalis terminated in the medial preoptic region and anterior hypothalamus, but none were observed extending into the lateral preoptic region or the ventromedial hypothalamic nucleus . However, more recently, in a study employing the Fink and Heimer method, Heimer and Nauta (1969) described dense terminal degeneration in the peripheral cell poor zone and sparser degeneration in the central core of the ventromedial nucleus following lesions of the stria terminalis in the rat. The presence of degenerating terminals in the nucleus has been confirmed by electron microscopical methods (Heimer and Nauta, 1969; Field, 1972) . Heimer and Nauta (1969) also reported that additional fibres continue posteriorly to the premammillary nucleus. Although his observations agree in general with those of previous work-

SUBCORTICAL CONNECTIONS

86 Elizabeth Hall ers, De Olmos (1972) has obtained a more complete impregnation of the stria terminalis with his cupric silver method, and has described additional regions of distribution, including the basolateral part of the septum, the nucleus accumbens, the olfactory tubercle, the medial division of the anterior olfactory nucleus, the granular layer of the accessory olfactory bulb and the lateral tuberal area. In addition, he has observed a commissural projection to the contralateral cortical and medial nuclei . The contribution from the amygdala to the longitudinal association bundle is difficult to assess. According to the description based on normal material, pyriform fibres course superiorly through the lateral and basal amygdaloid nuclei and are joined by fibres arising from both of these cell groups. Together these axons form the longitudinal association bundle which continues anteriorly through the amygdala and then turns medially into the preoptic region (see Johnston, 1923; Fox, 1940; Valverde, 1965). Using experimental methods Cowan et al. (1965) observed that the terminal distribution of this ventral pathway was the same following lesions of either the pyriform cortex or the amygdala (see previous section on pyriform cortex). However, they pointed out that the pyriform fibres were necessarily destroyed in passage when lesions were placed in the amygdala and concluded that the contribution of the lateral and basal nuclei could not be determined . More recently, Leonard and Scott (1971) have observed that only when amygdaloid lesions in the rat could have involved periamygdaloid fibres were there any long fibres observed projecting medially into the hypothalamus. Comparable control studies of the amygdaloid versus the pyriform contribution to the longitudinal association bundle have not been carried out in other species. After lesions in the amygdala of the monkey, Nauta and Valenstein (1958) and Nauta (1961) traced degeneration through this ventral pathway to the substantia innominata, lateral preoptic and hypothalamic regions, nucleus of the diagonal band, subcallosal gyrus, rostral cingulate cortex, olfactory tubercle, and magnocellular division of the dorsomedial thalamic nucleus. Hall (1963) reported a less extensive projection in the cat (see also Valverde, 1965). Using experimental methods, a few investigators have observed that the hypothalamus sends projections back to the amygdala. Nauta (1958) observed that, after a lesion in the preoptic region of the cat, degeneration coursed to all but the cortical and lateral nuclei over both the dorsal and ventral 'amygdalofugal' pathways. Similar observations were made in the rat by Cowan et al . ( 1965) who observed terminal degeneration in all but the

Anatomy of the limbic system 87 central nucleus. In a detailed study of the cat employing both light and electron microscopy, Wakefield (1972) noted that fibres arising in the lateral preoptic region terminated in both parts of the central nucleus and to a lesser extent in the medial, basal, and lateral nuclei. She also observed that the great majority of degenerating terminals contained flat vesicles and participated in the symmetrical type of synapse defined by Colonnier (1968). In addition, projections to the amygdala from the dorsomedial, medial geniculate, and lateral posterior thalamic nuclei have been mentioned by Nauta (1962), Ebner (1967), and Graybiel (1972) respectively. For a more detailed description of the connections of the amygdala, the reader is referred to the excellent papers by Lammers (1972) and De Olmos (1972). SEPTUM (Figs. 3, 5, 7, and 8) STRUCTURE

In describing this region it has been common practice to divide the septa) nuclei along a parasagittal plane into medial and lateral groups ; the medial group consisting of the anterior continuation of the hippocampus, the nucleus of the diagonal band ofBroca, and the septo-hippocampal, medial, and triangular nuclei; the lateral consisting of the lateral and fimbrial nuclei and additionally the bed nuclei of the anterior commissure and stria terminalis (see Fox, 1940). However, in 1959 Andy and Stephan broke with established practice and classified the nuclei into dorsal, ventral, medial , and caudal units in order to provide a more precise delineation of the

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Figure 8 Schematic summary of the subcortical connections of the septum. See also Figs. 5 and 7. See caption of Fig. I for explanation of symbols and abbreviations .

88 Elizabeth Hall region. These new categories proved to be useful guidelines in their detailed study of the comparative anatomy of the septum; a study which has led them to conclude that, contrary to general opinion, the septum increases progressively in size to reach its highest degree of development in man (Andy and Stephan, 1968). (Figs . 3 and 5) The interconnections of the septal nuclei with the hippocampal formation, and the projections from the pyriform cortex to the bed nucleus of the stria terminal is and nucleus of the diagonal band have been described in previous sections. Projections from the cingulate cortex have been suggested in studies of normal material but have not been confirmed experimentally (see Domesick, 1969). CORTICAL CONNECTIONS

(Figs. 7 and 8) A direct pathway from the olfactory tubercle to the septum has been noted in the experimental studies of both Brodal (1948) and Raisman (1966) . However, it is less certain that there is a direct reciprocal connection (Raisman, 1966). In his investigation of the distribution of the medial forebrain bundle, Guillery ( 1957) described both a medial and lateral component projecting to the septum. He observed that the medial component, probably arising in the midbrain or pons, coursed to the medial nucleus and ventral limb of the nucleus of the diagonal band, while the lateral component originating in the premammillary hypothalamus ended in the lateral septal nucleus. The more recent observations of Raisman (1966) are essentially in agreement with these findings. Further, the septum not only receives fibres from but also sends fibres into the medial forebrain bundle which are distributed to the lateral preoptic region and throughout the lateral hypothalamus (Nauta, 1956; Raisman , 1966). In addition, the septum sends a large projection to the medial habenular nucleus through the stria medullaris (Nauta, 1956; Cragg, 1961a) . Raisman (1966) observed that these fibres arise in the fimbrial nucleus and Mitchell (1963) suggested a reciprocal pathway from the habenula back to the septum. Some investigators have also reported a projection from the septa! region to the mediodorsal thalamic nucleus (Guillery, 1959; Cragg, 1961a), but Raisman (1966) suggested that these fibres may have a different origin. Fibres arising in the mediodorsal thalamic nucleus terminate in the nucleus oft he diagonal band of Broca (Nauta, 1962).

SUBCORTICAL CONNECTIONS

Anatomy of the limbic system 89 Finally, attention should be drawn to the recent observations ofRaisman (1969b) and Raisman and Field (1973) regarding the plasticity of septa) afferents coursing via the medial fore brain bundle and fimbria of the fornix . Following interruption of the fimbrial fibres, the normal afferent endings of the medial forebrain bundle undergo collateral sprouting and appear to occupy those synaptic sites which have lost their fimbrial terminals . After destruction of the medial forebrain bundle, normal fimbrial fibres undergo a similar growth phenomenon . The elucidation of the functional significance of these structural changes in connectivity will be a difficult and complex task. DISCUSSION

There are a number of problems associated with the investigation of the limbic telencephalic areas reviewed above which not only make it difficult to establish species variations, but also give rise to differences in the interpretation of a given set of experimental findings . Each of the regions presents problems of surgical approach because of its relative inaccessibility , its general configuration , the shape of its internal subdivision , or its relationship to an adjacent limbic structure. Fibres of passage constitute additional hazards which have been noted above in regard to fibres of the olfactory bulb coursing in the superficial layer of the pyriform cortex , fibres of the anterior thalamic nuclei coursing through the cingulum, and fibres of the pyriform cortex traversing the amygdala. In addition, the reduced silver staining techniques have not always displayed and may still not display the complete terminal field of a given efferent pathway (e.g., the stria terminalis) . In spite of these problems, a relatively large amount of reliable data has accumulated gradually on the structure and connections of the limbic areas reviewed above . The increasingly widespread use of a combination of techniques to demonstrate connections, as in the study of the mossy fibre system in the hippocampus , should prove extremely fruitful. Further cause for optimism is offered by the recent introduction of two additional methods for mapping pathways, one for determining their origin (the horseradish peroxidase technique, see La Vail and La Vail, 1972), the other for analysing their fields of termination (the radioautographic method, see Cowan et al ., 1972). Some of the observations emerging from the literature exemplify the difficulty of defining the limbic system in precise anatomical terms. The role of the cingulate cortex in the Papez (1937) circuit appears to be less

90 Elizabeth Hall significant than was previously assumed (see Domesick, 1969). On the other hand, strong interconnections between this gyrus and the surrounding neocortex have been confirmed, and these may, as suggested by Papez (1937), play an important role in producing emotional awareness and adding emotional colour to psychic activity occurring in other regions of the brain . The demonstration of a direct projection from the anterior thalamic nuclei to the entorhinal area (Domesick, 1969) reconfirms the importance of this cortex in the circuit of Papez (1937) . On the other hand, the more anterior part oft he pyriform cortex appears to have less direct influence on the rostral hypothalamus (see Heimer, 1973) than was indicated by earlier experimental investigations . The hippocampus, amygdala, and septum send heavy projections to the hypothalamus and preoptic region which are undoubtedly essential to the production of a variety of patterns of emotional expression. Their inclusion in the limbic system is unquestioned. However, the basis for including the mediodorsal, lateral dorsal, and lateral posterior nuclei of the thalamus is much more uncertain , even though all three are connected with limbic regions. The same dilemma exists for some anatomists regarding the nucleus accumbens, olfactory tubercle, and substantia innominata. Regarding the last three structures, Heimer and Wilson (1973) have presented a new concept. These authors point out that the hippocampal and pyriform cortex send a heavy projection to the nucleus accumbens and the deep part of the olfactory tubercle, both of which can be considered as part of the ventral striatum. Further, the nucleus accumbens and the deep part of the olfactory tubercle project to a part of the substantia innominata which can be interpreted as a ventral extension of the globus pallidus. On this basis the authors suggest that these cortical-subcortical relations are very similar to those found in the extrapyramidal system and that the dichotomy between the limbic and extrapyramidal systems should not be overemphasized. It is possible that the pathway described by Heimer and Wilson (1973) may play a role in producing the somatomotor component of emotional behaviour. One must agree that 'limbic system' is an imprecise anatomical term and that the accumulation of additional data tends to increase rather than decrease the difficulty of establishing a more specific definition . This situation is not unique. The same criticism could be extended to the terms 'pyramidal system' and 'extrapyramidal system.' The expression, however, is still too useful (possibly because of its very imprecision and thus its flexibility) to be abandoned. Perhaps the difficulties are not so great if one

Anatomy of the limbic system 91 accepts that inclusion of a structure in one system of the central nervous system does not preclude inclusion in a second system. The limbic system could then be considered as being formed of those parts of the brain which under varying conditions initiate different emotional patterns of behaviour through the autonomic, endocrine, and somatomotor systems in response to changes in both the external and internal environments.

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92 Elizabeth Hall Geneser-Jensen, F.A. 1971 . Z . Zellforsch . . 117: 46-64 - 1972a. z. Zel/jiJrsch., 124: 546~0 - 1972b. Z . Zellforsch., 131 : 481-95 Geneser-Jensen, F.A. and Blackstad, T.W . 1971. z. Zel/jiJrsch .. 114: 460-81 Gray , P.A. Jr. 1924. J . comp . Neural., 37: 221-63 Graybiel , A.M. 1972. Brain Res., 44 : 99-125 Guillery, R.W . 1956. J. Anal . (Lond .). 90: 565 - 1957. J.Anat . (Lond .),91 : 91-115 - 1959. J . Anal. (Lond.), 93 : 574 Gurdjian, E.S. 1928. J. comp. Neurol . • 45 : 249-81 Hall, E.A. 1963. Am . J . Anal., 113: 139-52 - 1972a. B.E. Eleftheriou (ed .), In The NeurohioloRY of the AmyRda/a. New York : Plenum Press, pp . 95-121. - 1972b. Z . Zellforsch . , 134: 439-58 Hall, E.A. and Geneser-Jensen , F.A . 1971. Z . Zel/jin·sch . . 120: 204-21 Hall, E., Haug, F.M.S., and Ursin, H. 1969. z. Zel(forsch .. 102 : 40-8 Hamlyn, L.H . 1961. Na/lire (Lond .). 190: 645-6 - 1962. J . Anal. (Lond.), 96: 112-20 Haug, F.M.S. 1967. Histochemie (Berlin), 8: 355-68 Haug, F.M.S ., Blackstad , T.W . , Simonsen. A.H., and Zimmer, J . 1971. J . comp . Neurol .. 142:23-32 Heimer, L. 1968. J. Anal . (Lond.). 103 : 413-32 - 1972. Brain, Behl/\'. Ei-ol . , 6: 484-523 Heimer, L. and Nauta, W.J .H. 1969. Brain Res . . 13: 284-97 Heimer, L. and Wilson . R.D. 1973. In press Hirata, Y. 1965. Acta Med. Biol. (Niigata) , 13 : 123-42 Hjorth-Simonsen, A. 1971. J. comp . Neurol., 142: 417-38 - 1972. J. comp. Neuro/., 146: 219-31 - 1973. J. comp. Neuro/ . , 147: 145-61 Hjorth-Simonsen, A. and Jeune, B. 1972. J. comp . Neuro/., 144: 215-32 lbata , Y., Desiraju, T., and Pappas, G.D. 1971. Exp . Neuro/ ., 33: 103-22 Johnston, J.B. 1923. J . comp . Neuro/ . , 35: 337-481 Jones, E.G. and Powell , T .P.S. 1970. Brain, 93 : 793-820 Koikegami, H. 1963. Acta Med. Biol. (Niigata) , 10: 161-277 Laatsch , R.H. and Cowan, W.M. 1967. J. comp. Neuro/., 130: 241-61 Lammers, H.J . 1972. In B.E. Eleftheriou (ed .), The Neurobiology of the Amygdala . New York: Plenum Press, pp . 123-144 La Vail,J.H . and La Vail, M .M. 1972. Science, 176: 1416-17 Le Gros Clark, W.E . and Meyer, M . 1947. Brain, 70: 304-28 Leonard , C.M. and Scott, J .W. 1971. J . comp. Neurol., 141 : 313-29 Lescault, H. 1969. Proc . Can . Fed. Biol. Soc., 12: 24 - 1971. Some neocortico-amygdaloid connections in the cat. Thesis, University of Ottawa Locke, S., Angevine, J.B. , and Yakovlev, P.I. 1964. Arch . Neurol . (Chicago), 11 : 1-12 Lohman, A.H .M. and Mentink, G.M. 1969. Brain Res., 12: 396-413 Lorente de No, R. 1933. J. Psycho/. Neurol . (Leipzig), 45: 381-438 - 1934. J. Psycho/. Neuro/ . (Leipzig), 46: 113-77 Lundberg, P.O. 1962. J. comp . Neurol . , I 19: 311-16 MacLean , P.D. 1949. Psychosom . Med . , 11 : 338-53

Anatomy of the limbic system 93 - 1952. Electroenceph . clin. Neurophysio/ . , 4: 407-18 - 1954. J. Neurosurg ., 11: 29-44 - 1955 . Psychosom. Med. , 17: 355-66 - 1958. A,n . J.Med. ,25:611-26 MacLean , P.O. a nd Delgado, J.M .R. 1953. Electroenceph . din . Neurophysiol., 5: 91-100 Mascitti, T .A. and Ortega , S.N . 1966. J . comp . Neural. , 127: 121-35 Maske, H. 1955. Naturwissenschajien , 42 : 424 Mathisen , J .S. and Blackstad, T.W . 1964. Acta Anal. (Basel), 56: 216-53 Mellgren, S.I. and Blackstad, T.W. 1967. Z . Zellforsch . , 78: 167-207 Mitchell , R. 1963 . J . comp . Neurol., 121 : 441-57 Mizuno, N. , Clemente, C.D., and Sauerland , E. K. 1969. J. comp . Neuro/. , 136: 127-41 Nafstad, P.H.J. 1967. z. Zel/forsch . . 76: 532-42 Nauta, W.J .H . 1953 . Anal. Record . 115 : 352 - 1956. J. comp . Neurol . , 104: 247-71 - l958. Brain , 81 : 319-40 - 1961.J.Anal.(Lond.),95:515-31 - 1962. Brain . 85: 505-20 Nauta, W.J.H. and Valenstein, E.S. 1958. Anal. Record , 130: 346 Omukai , F. 1958. Acta Anal . Nippon . , 33 : 499-522 Papez, J. W . 1937. Arch. Neuro/. Psychiatry (Chicago) , 38: 725-43 Pigache, R.M . 1970. Ergeb. Anal . Entwicklungsgeschichte (Berlin) , 43 : 7--02 Powell , T .P.S .. Cowan, W.M ., and Raisman, G. 1965. J. Anal. (Lond.), 99: 791-813 Price, J .L. and Powell, T.P.S. 1970a. J. Anal . (Lond .), 107: 215-37 - 1970b. J. Anal. (Lond .), 107: 239-56 - 1971. J . Anal. (Lond .), 110: 105-26 Raisman , G. 1%6. Brain, 89: 317-48 - 1%9a . Exp. Brain Res . , 7: 317-43 - 1969b. Brain Res . , 14: 25-48 - 1972. Exp. Brain Res . , 14: 395-408 Raisman, G. , Cowan , W.M .. and Powell , T.P.S . 1965 . Brain, 88: 963-% - l966.Brain , 89:83-108 Raisman, G. and Field, P.M. 1973. Brain Res ., 50: 241-64 Ramon y Cajal, S. 1911. Histologie du Systeme Nen·euxde /'Homme et des Vertebres. Paris : Maloine, Reprinted Madrid: lnstituto Cajal, 1952 - 1955. Studies on the Cerebral Cortex (limbic structures). Translated by L.M . Kraft. London : Lloyd-Luke (Medical Books) - 1968. The Structure of Amman 's Horn. Translated by L.M . Kraft, Springfield, Ill.: Charles C. Thomas Rose , J.E. and Woolsey , C.N. 1948. J . comp. Neural . , 89: 279-347 Rose, M . 1929. J. Psycho/. Neurol . (Leipzig), 40: 1-51 Scalia, F. 1966. J . comp . Neuro/ . , 126: 285-310 - 1%8. Brain, Behm·. faol., I: 101-23 Showers, M.J.C. 1958. J. comp. Neurol ., 109: 261-315 - 1959. J . comp. Neural. , 112 : 231-301 Siegel , A., Sasso, L.. and Tassoni, J.P. 1971. Exp. Neural . , 33: 130-46 Siegel, A. and Tassoni, J .P. 1971a. Brain, Behav. Eva/. , 4: 185-200 - 1971b. Brain , Behav. Eva/. , 4: 201-19 Sprague , J.M . and Meyer, M. 1950. J. Anat. (Lond .), 84: 354-68

94 Elizabeth Hall Valenstein, E.S. and Nauta, W.J.H. 1959. J . comp. Ne11ro/ . , 113: 337-63 Valverde , F. 1962. TrahajoJ inJI. Cajal im·eJt . hiol. (Madrid), 54: 291-314 - 1965. St11dies on the Pirij,mn Lohe . Cambridge , Mass .: Harvard University Press Vaz Ferreira, A. 1951. J. comp . Ne11ro/., 95 : 177-243 Votaw, C.L. and Lauer, E.W . 1963. J. comp . Ne11rol ., 121: 195-206 Wakefield, C. 1972. Hypothalamic projections to the amygdala in the cat : a light and electron microscopic study. Thesis, University of Ottawa Westrum, L.E . 1969. Z. Zel/forsch . , 98: 157-87 White , L.E. Jr. 1962. Anal. Record, 142: 333 - 1965a. /nt. Rei·. Neurohiol. , 8: 1-34 - 1965b. Anal. Record, 152: 465-79 Whitlock, D.G. and Nauta, W.J.H. 1956. J. comp . Ne11rol . , 106: 183-212 Winans, S.S. and Scalia, F. 1970. Science, 170: 330-2 Yakovlev, P. I., Locke, S., Kiskoff, D.Y., and Patton, R.A. 1960. Arch . Neural . (Chicago) , 3: 620-41 Zimmer, J . 1971. J. comp . Neuro/. , 142: 393-416

Current concepts in energy balance

J. LeMagnen

Concepts about body energy balance are current concepts, not because they are revisions of older ones, but rather because the concept of an overall regulation of body exchanges is a new one . For a long time, the three terms of energy balance: inflow of energy, outflow of energy, and body mass as potential energy, have been considered and studied separately . As pointed out by Brobeck ( 1960), physiologists confronted with the extreme complexity of the overall process found it simpler to treat the problem with one of the terms arbitrarily taken at zero or as a constant. By suppressing the balanced energy input, that is free food intake , classical bioenergetics prevented a possible study of the concept of a body energy balance or even the existence of the concept. Similarly, a majority of studies on food intake overlooked the fact that feeding is not regulated separately in a perfect and ideal steady state of energy expenditures . Thus , before analysing current concepts which permit the experimental study of mechanisms involved in the balance of energy exchanges, it seems useful to clear up the old mistakes and fallacious concepts, which until now have prevented a true understanding of the problem of energy balance . A CRITICAL SURVEY OF MISLEADING CLASSICAL CONCEPTS

The first serious source of misunderstanding was introduced by the old and extraordinary concept of 'basal metabolism' and its measurement. The

96 J. Le Magnen inclination of the human mind towards constant levels is so imperative that it leads to consideration of an unrealistic constancy by subtracting all sources of variations, even when the aim is to account precisely for an intrinsically variable condition. The condition of assessment of the metabolic rate at rest, fixed ambient temperature and after a 12-hour fast, the claimed 'basal metabolism,' is not a physiological condition. The subtraction of activity in a fasting animal, which in fact is hyperactive, is essentially an artificial condition. The sum of this minimum metabolism plus current activity taken as 'total metabolism' is erroneous since exercise in turn modifies various components included in the basal metabolism such as heart rate and respiratory expenditures. Activity and claimed minimal expenditures are not simply additive. The radical suppression of the 'embarrassing' free food intake in the food deprived condition is still more confusing. After 12 hours of food deprivation, the body is already engaged in one of the specific conditions of the regulation which must also be considered. Furthermore, the addition to the claimed 'basal' heat production in the fasting animal of the so-called 'heat increment of food' is misleading. The decreased metabolic rate in underfed or unfed subjects has been presented as hypometabolism; alternatively, the metabolic rate of fed subjects has been presented as a hypermetabolism due to a special heat increment offeeding or specific dynamic action which appears mysteriously different from a 'basal' heat production. The fixed ambient temperature may similarly be considered as a theoretical and artificial condition. Down to the so-called critical temperature of the onset of a specific thermogenesis, the overall heat production of active and fed subjects is used to warm the body but is not produced in order to warm the body, as a specific heat requirement. In the physiological condition, the critical temperature is not at all a constant point since it is then widely dependent and variable with the heat production due to voluntary muscle activity and to feeding. Finally, by considering and measuring a 'basal' metabolic rate over three hours, this rate is taken artificially as constant, when in fact in a free-moving and fed animal the continuous outflow of energy is widely fluctuating over time with the sleep-wakefulness and feeding patterns. BODY ENERGY CAPACITY, INFLOW, AND OUTFLOW OF ENERGY

Prior to the study of various control systems and of their relationships ensuring the regulation of energy exchanges, outflow and inflow of energy

Current concepts in energy balance 97 have to be separated from increment or decrement of body mass as energy capacity. As a whole, in the balance of body energy, the inflow plus the decrement or minus the increment of body energy content (BEC) matches the outflow . Thus, three series of control systems, controlling respectively the outflow, the inflow, and variations of the BEC, will have to be considered. As substituted for the measurement of basal metabolism, the study of the three terms of the energy balance must be carried out by a measurement of the metabolic rate in free-moving and fed animals. A standardized measure could be a 24-hour continuous recording of respiratory exchanges in animals given free access to food and water at all times, kept at a fixed ambient temperature and free moving in a given living space . In order to separate the various components and to determine the time relationships of outflow and inflow, activity and feeding patterns must also be measured . Such measurement of the daily total metabolic pattern has been performed rarely on laboratory animals or human subjects (Durnin, 1957, 1961; Lackey et al., 1970; Morrison , 1955, 1968) . Except for the quantitative measure of activity and of food eaten these requirements were met in Morrison' s work (1968) on rats . The relations between 02 consumption and bursts of feeding and non-feeding activity were studied . The notion of a basal metabolic rate is so strong that the author, despite the measurement of the metabolic rate in a condition of balanced outflow and inflow , looked for a reference basal level. He assumed that this level was represented by a straight line obtained by the joining of the lowest points oft he recorded 02 consumption throughout the fluctuating daily pattern . Referred to this baseline, the extra 02 consumption was ascribed exclusively to activity expenditures. According to this assertion , the part of activity expenditures in the total metabolism appeared to be a constant value of some 24 per cent of the basal metabolism . In addition, feeding and non-feeding activities were complementary in this constant range. However, it is difficult to accept such a low cost of free activity and that the increment from a supposed basal metabolism in fed rats is compsed only of activity expenditures . Nevertheless, this work gave evidence for a very important aspect of energy balance . Variations of the total metabolic rate of an active and fed animal are neither concomitant with, nor proportional to, variations in food intake. In comparable recordings of the daily metabolic pattern of rats, it has been shown (Fig. 1) (Le Magnen and Devos, 1969a,b; 1970) that fluctuations of 02 consumption throughout the 24 hours are not generally synchronous and quantitatively related to intermittent food intake. At night, rats ate up to double the amount of calories expended. In daytime,

98 J. Le Magnen

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damaged. In some dogs the syndrome was obtained with damage to the postero-ventral part only (Fig . 2B). Experiments performed on dogs with lesions of the ventromedial hypothalamus (VMH) have furnished evidence of a definite similarity between the syndrome produced by these lesions (Rozkowska and Fonberg, 1971, 1973) and that produced by lateral amygdalar (LA) lesions (Fonberg,

The amygdala and ingestive behaviour 179 1971). The only difference was in the general behaviour of the dogs (Table I). Dogs with lesions of the VMH were somewhat quieter than before the operation and only became extremely excited before meals; dogs with lesions of the LA were lively and playful like puppies, jumping up and inviting attention and petting. This excitement was less dependent on hunger and seemed to be more related to social intercourse with humans. These results supported my previous hypothesis that the role of the lateral amygdala is inhibitory. Further confirmatory evidence was provided by the following work . THE EFFECT OF LATERAL AMYGDALAR LESIONS ON THE OMA AND LH SYNDROMES

Recent experiments performed on dogs with OMA lesions have shown that subsequent lesions of the LA cancelled the effects of the first lesions (Fonberg, 1970, 1972a,b, 1973). After LA damage the dogs started to eat voraciously and instead of being apathetic and indifferent were lively and friendly. Their instrumental responses, which were either abolished or unstable after OMA lesions, were again restored to 100 per cent performance (Fonberg, 1972a). The change immediately after the LA lesions was remarkable. These results indicate that the neural mechanisms important to alimentary responses were not irreversibly abolished by OMA lesions but were likely caused by an 'overflow' of inhibition derived to a great extent from the LA . Further proof that the lateral amygdala is involved in inhibition has been furnished by our recent experiments in which dogs were subjected to OMA and LA lesions after being trained to differentiate between two different tones by instrumental response. One sound was positively reinforced and the other was not reinforced. After OMA lesions the response to both sounds was abolished or severely impaired, but with subsequent LA lesions instrumental response to the positive tone was restored and the response to the negative sound appeared (disinhibition) . This rebound of disinhibition caused by lesioning the LA (as the result ofremoval inhibition) was transitory and normal balance was restored within a few weeks. The 'cancelling' effect of lateral amygdalar lesions on the LH aphagic syndrome has also been demonstrated in our laboratory (Fonberg, 1969b; Fonberg, unpublished observations). Dogs which were hypophagic, apathetic, and underweight after LH lesions became voracious, lively, and gained weight after subsequent LA lesions. One dog, which had been consuming no more than 1/2 kg of food per day for several weeks following

180 Elzbieta Fonberg the LH lesions, immediately began to devour 3 1/2 kg of food per day after the LA was lesioned . The most striking effect was observed in dog A 151 which was completely aphagic for 75 days as a result of the two-stage combined lesions of the dorsomedial amygdala and lateral hypothalamus . When the lateral amygdala was then damaged , he immediately ran to the food bowl and consumed 1/2 kg of food . Thereafter, his daily food intake was maintained at the level of I kg (half of that before any lesioning) and his instrumental responses were also partly restored (Fig. 4). DISCUSSION

The results of our experiments indicate that in the amygdala of the dog there are two opposing systems which regulate alimentary function - one facilitatory in the dorsomedial amygdala and the other inhibitory in the lateral amygdala. Although it is true that one is not justified in taking the results obtained in one species and applying them in general, analysis of the behavioural results obtained by other investigators working with other species support the idea of the functional dichotomy of the amygdaloid complex. The electrophysiological studies ofKreindler and Steriade (1963) confirm this as well. Recently , Dreifuss et al. ( 1968) have found in cats that stimulation of the corticomedial nuclei of the amygdala (via the stria terminalis) elicited a different pattern of evoked potentials in the ventromedial hypothalamus compared to the stimulation of the basolateral nuclei (via the ventro-amygdalofugal pathways) . Moreover, the responses produced by stimulation of these two systems were negatively correlated and inhibited each other. Recent electrophysiological evidence obtained by other investigators has also indicated a similar dichotomy of amygdalopetal influences (Krachun, 1970- rats ; Happel and Bach, 1970- cats). The distribution of acetylcholinesterase in the basolateral nuclei of the guinea pig and monoamine-oxidase in the corticomedial nuclei , as demonstrated by Hall (Hall and Geneser-Jensen, 1971), may be relevant to the differentiation of the physiological functions of these divisions as well. Another interesting result is the similarities observed between hypothalamic and amygdalar 'feeding centres.' Not only food intake but also many different functions involved in the overall alimentary response were impaired in the same manner and to the same degree by lesioning either the dorsomedial amygdala or the lateral hypothalamus on the one hand, and by lesioning either the lateral amygdala or the ventromedial hypothalamus on the other. Whether the similarities between the amygda-

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lar and hypothalamic syndromes are superficial and produced by different mechanisms is open to speculation. The dog may refuse to eat because he is not hungry or because the food has lost or changed its reinforcing values. It may smell, taste , or appear different than before the operation. The anorexia may also be the result of general malaise or feeling of sickness; the consumption of food may produce nausea (this is very probable as the dogs vomited). The dog may not eat because he is generally depressed and indifferent. The general signs of decreased arousal and other depressive

182 Elzbieta Fonberg symptoms support this. All of these factors may summate and intermix in various proportions and may be different after lesions of the hypothalamus than after lesions of the amygala although the overt signs are the same. It should be noted that we did find some minor differences, connected with hypothalamic and amygdalar hyperphagia (see Table l). In 1951, Bruce and Kennedy proposed that the hypothalamus deals with hunger whereas appetite and other conditioned aspects of feeding are dependent on cortical influences. Similarly, Anand (1961) assumed that the hypothalamus is involved in hunger and the amygdala in appetite . Konorski (I 967) has proposed that the hypothalamus mediates unconditioned and the amygdala conditioned alimentary responses. According to Stevenson (1969), the amygdala is a secondary modulator of alimentary functions whereas the hypothalamus is the main locus of convergence of many sensory, metabolic, autonomic, and hormonal factors . On the other hand, although some authors feel that the hypothalamus is more involved with the metabolic mechanisms of hunger and satiety and that the amygdala influences and modulates sensory stimuli, most investigators have stressed the role of the hypothalamus itself in sensory input and the importance of sensory mechanisms in the act of eating as such (Pfaffman, 1960; Teitelbaum, 1961; Teitelbaum and Epstein, 1962; Mogenson and Stevenson, 1966; Stevenson, 1967; Wyrwicka, 1969, 1972; Marshall et al., 1971; Mogenson, 1971) . Our experiments demonstrate that, in the dog at least, the alimentary functions of the amygdala are important and comparable to hypothalamic functions; they are organized in a similar way and contain both excitatory and inhibitory areas. These similarities may exist because of their mutual interaction . Oomura et al . (1970) have observed that stimulation of the lateral amygdala in the rat produces an increase of spontaneous activity in the VMH and a decrease in the LH. Thus the amygdalar inhibition offeeding may be mediated either by excitation of the VMH or by inhibition of the LH. The latter explanation was suggested by Oniani and N aneisvili ( 1968) . The dorsomedial amygdala may also influence the LH and VMH directly. Murphy and Renaud (1969) have shown that the VMH contains two types of neurons and suggest that the smaller ones are involved in inhibition . Both the stria terminalis pathway and the ventral amygdalofugal pathway have fibres leading to these neurons; the first system may inhibit them and the other system excite them. If we assume that the DMA (via the stria terminalis) inhibits the ventromedial satiation centre, lesions of the DMA may produce activation of the VMH and consequently decrease the activity of the LH, thus producing aphagia. On the other hand, the DMA may through

The amygdala and ingestive behaviour 183 some other components of the stria terminalis directly exert positive influences on the LH. Besides the amygdala and the hypothalamus, midbrain structures also exert both positive and negative influences on ingestive behaviour (Skultety and Gary, 1962; Wyrwicka and Doty, 1966; Parker and Feldman, 1967; Skultety, 1966, 1968). Pathways from the amygdala to the mesencephalon may or may not pass through the hypothalamus (Gloor, 1967). Neocortical influences on feeding, although beyond the scope of this paper, should be taken into account when considering the overall regulation of ingestion. It is obvious that all these levels of the alimentary system interact with each other and an adequate balance of inhibition and excitation is essential in maintaining normal ingestive responses. The role of this paper has been to show that the alimentary functions of the amygdala, which often have been overlooked, are important and comparable to the functions of the hypothalamus, and are organized in a similar way, involving both facilitatory and inhibitory parts. ACKNOWLEDGMENTS

The author wishes to express her appreciation to Blanche M. Box for valuable suggestions and editorial corrections of the paper and to Mrs A. Kurzaj and Miss U. Miaczynska for technical assistance. This investigation was supported by Project 09.4.1 of the Polish Academy of Sciences and by Foreign Research Agreement No. 05 .275.2 of the u.s. Department of Health, Education and Welfare under PL 480.

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184 Elzbieta Fonberg 1968b.BrainRes. , 22 : 273-81 1969a. Physiol. Behav., 4: 739-43 1969b. Acta Biol. Exp., 29: 335-58 1970. In D.A.A . Primrose (ed .), Symposium 13: The Limbic System. Warsaw, Poland, Proc. 2nd Congr. IASSMD , pp. 578-80 - 1971.ActaNeurobiol.Exp. , 31: 19-32 - 1972a. Proc. XII Congr. Pol. Physiol. Soc. (Olsztyn, in Polish), pp . 65-6 - 1972b. In Porter and Knight (eds.), Ciba Symposium Physiology Emotion and Psychosomatic Illness . Amsterdam: Elsevier Excerpta Med . pp. 131-61 - 1973. Acta Neurobiol . Exp., 33: 449-66 - 1974. Acta Neurobiol. Exp . , 34: 435-66 Fonberg, E. and Delgado, J.M.R. 1%1. J. Neurophysiol. , 24: 651-64 Fonberg, E. and Rozkowska, E. 1968. Proc. III Int . Conj. Regulation of Food and Water Intake, Haverford, Pa. Fonberg, E. and Sychowa, B. 1968. Acta Biol. Exp . , 28: 35-46 Fuller, J .L., Rosvold, H.E., and Pribram, K.H . 1957. J . comp. physiol . Psycho/. , 50: 89-96 Gloor, P. 1955. Electroenceph. clin. Neurophysiol., 7: 222-43 - 1967. AMA Arch Neural . Psychiat . , 77: 247-58 Green , J.P., Clemente, C.E., and DeGroot, J . 1957. J . comp. Neuro/., 108: 505-36 Grossman , S.P. and Grossman , L. 1963. Am. J . Physiol., 206: 761-5 Hall, E. and Geneser-Jensen, F.A. 1971. Z . Zel/forsch , mikroskop. Anal., 120: 204-21 Happel, L.T. and Bach, L.M .N. 1970. Fed. Proc . , 29: 392, Abstr. #829 Kling, A. 1965. J . Psychiat . Res., 3: 263-73 - 1966. Psychosom. Med., 28: 155-61 Kling, A. , Orbach , J., Schwartz, N.B ., and Towne, J .C. 1960. Arch. Gen . Psychiat ., 3: 391-420 Kling, A. and Schwartz, N.B. 1961. Fed. Proc., 20: 335 Koikegami, H. 1964. Acta Med . Biol. , 12: 73-266 Konorski, J. 1967. Integrative Activity of the Brain . Chicago: University of Chicago Press Krachun, G.P. 1970. Zhurn . Vysshei Nervno, Dejat. Akad. Nauk USSR , 20: 130--8 Kreindler, A. and Steriade, M. 1963. Electroenceph . clin. Neurophysiol ., 15: 811-26 Lagowska, J. and Fonberg, E. 1972. Proc . XII Congr. Pol . Physiol. Soc . (Olsztyn , in Polish), p. 150 Lewinska, M .K . 1968. Acta Biol. Exp . , 28: 23-34 Machne, X. and Segundo, J.P. 1956. J. Neurophysiol., 19: 232-40 MacLean, P.O. 1958. J. nerv. men/ . Dis . , 127: 1-11 Marshall , J .F. , Turner, H.B., and Teitelbaum, P. 1971. Proc . IV Int . Conj. Regulation of Food and Water Intake , Cambridge, England Mogensen, G.J . 1971. Physiol . Behav., 6: 255-60 Mogenson, G.J . and Stevenson, J.A .F. 1966. Physiol. Behav. , I: 251-4 Morgane, P.J. 1961. Am . J. Physiol., 201: 420-8 - 1%2. Proc . xx11 /nt . Congr. Physiol. Sci., Vol. 1, Part 11 , pp. 670-6 Morgane, P.J . and Jacobs, H.L. 1969. In G.H. Bourne (ed.), World Review of Nutrition and Dietetics. Basel , Switzerland: Karger, pp . 100-213 Morgane , P.J. and Kosman , A.J. 1957. Nature, 180: 598-600 - 1959. Am . J . Physiol. , 197: 158-62 Murphy, J.T. and Renaud, L.P. 1969. J . Neurophysiol., 32: 85-101 Nauta, W.J .H. 1961. J . Anal., 95 : 515-31

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The amygdala and ingestive behaviour 185 Oomura, Y., Ono, T. , and Ooyama, H. 1970. Nature. 228: 1108-10 Oniani, T.H . and Naneisvili, T .L. 1968. In Problems of the Physiology oft he Hypothalamus (in Russian). Kiev : University of Kiev, 2, pp. 89-99 Papez, J .W. 1937. Arch. Neurol. Psychiat . , 38: 725-44 Parker, S.W. and Feldman , S.M . 1967. Exp . Neurol . , 17: 313-26 Pfaffman, C. 1%0. Psycho/ . Re,·. , 67: 253-68 Pribram , K.H . and Bagshaw, M . 1953. J. comp . Neuro/ . , 99: 347-75 Robinson, B.W. and Mishkin, M. 1962. Science, 136: 260-2 Rozkowska, E. and Fonberg, E. 1970. Acta Neurobiol . Exp., 30: 59-68 - 1971. Ac/a Neurobiol . Exp., 31 : 351-64 - 1972. Acta Neurobio/ . Exp . , 32: 711-20 - 1973. Ac/a Neurobiol . Exp., 33: 553-62 Sawa, M. and Delgado, J.M .R. 1963. EEG Clin . Neurophysiol., 15: 637-50 Schreiner, L. and Kling, A. 1953. J . Neurophysio/. , 16: 643-58 Schwartz , N.B. and Kling, A. 1%4. Acta NeunJl'eget . , I: 12-34 Schwartzbaum , ,J .S. 1961. Am . J . Psycho/., 74 : 252-8 Shealy, C.N . and Peele , T.L. 1957. J. Neurophysiol., 20: 125-39 Skultety, F.M . 1966. Arch. Neuro/., 14: 670-80 - 1968. Proc . 111 Int. Conf. Regulation of Food and Water Intake , Haverford, Pa. Skultety , F.M. and Gary, T.M . 1962. Neurology , 12: 394-401 Stevenson. J.A.F. 1%7. In C.F. Code (ed.) , Handbook of Physiology , Alimentary Canal, Vol. 11, Washington, o.c .: American Physiological Society, pp. 173-90 - 1969. In W. Haymaker, E. Anderson and W.J.H . Nauta (eds .), The Hypothalamus . Springfield, Ill.: C.C. Thomas, pp . 524--621 Stoller, W. L. 1972. Physio/. Behm· . , 8: 823-8 Teitelbaum, P. 1961. In J.R. Jones (ed .), Nebraska Symposium on Motivation . Lincoln : University of Nebraska Press, pp. 39-69 Teitelbaum, P. and Epstein, A.N. 1%2. Psycho/ . Rev . , 69: 74-90 Weiskrantz, L. 1956. J . rnmp. physiol. Psycho/., 49 : 381-91 Wilkinson, H.A. and Peele, T.L. 1962. Am. J . Physio/. , 203 : 537-40 Wood . C.D. 1958. Neuroloi:y , 8: 215-20 Wyrwicka, W. 1969. Physiol . Beh(II'., 4: 853-8 _ - 1972. The Mechanisms of Conditioned Behavior. Springfield: C.C. Thomas Wyrwicka, W. and Doty, R.W . 1966. Exp. Brain Research , I: 152-60 Yamada, T . and Greer, M.A. 1%0. Endocrinology, 66 : 565-74

Feeding and temperature*

C.L. Hamilton

Over the past decade a fruitful co-operation between physiologists and behavioural scientists has evolved in the area of the study of the regulation of body temperature. It was Richter (1927) who first introduced us to the concept that overt behaviour was involved in physiological regulations. Several decades later, Weiss (1957) and Weiss and Laties ( 1961) began their work, using operant techniques in the study of behavioural thermoregulation. Since then, it is a rare occasion for a conference on temperature regulation to be held without a complement of behavioural scientists present. The laboratory rat, when placed in a cold environment, readily learns to press a lever activating a heat lamp, thereby reducing heat loss. Under such circumstances the animal's behaviour is in effect part of the control loop of temperature regulation and no less important than the autonomic responses of vasoconstriction, piloerection, et cetera. Whether other behavioural adaptations, such as changes in food intake in response totemperature stress, can be considered behavioural thermoregulation is still a matter of controversy. •

Research from the author's laboratory was supported in part by Grant No.

NIMH.

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10179.

Feeding and temperature 187 According to Brobeck ( 1948) the thermostatic theory of food intake is related to 'conditions' of temperature regulation . At the time of his proposal , Brobeck's hypothesis was based primarily on studies of the effects of environmental heat and cold stress on feeding. We need not review all these data since this hypothesis has already been adequately covered (Brobeck, 1960; Hamilton , 1967) . Suffice it to say that in the laboratory rat, food intake may range from 18 kcal/day at an ambient temperature of35° c to 126 kcal/day at ambient 7° c (Hamilton , 1967). Further, if the rat is force-fed more calories than it usually eats at 35° c, it develops lethal hyperthermia (Hamilton and Brobeck, 1962). Such observations are the basis upon which speculation has developed as to whether feeding, at times , is controlled only indirectly as a consequence of other bodily adjustments made to thermal stress . For example, is the increase in food intake in the cold the consequence of increased energy expenditure and, therefore , merely a replacement of energy stores? To test this hypothesis , thermal stress of much less than 24-hours' duration was needed to separate the effects of the ambient temperature stimulus from the effects of energy depletion . It has been shown that normal and hypothalamic hyperphagic rats, maintained at room temperature (25° c) , increase bar press rates for food pellets when tested for only two hours/day at an ambient temperature of l0°c (Hamilton, 1963) . An example of an even shorter term effect of temperature on feeding is demonstrated in the following study, carried out in my laboratory . Three rats were trained to obtain their daily food rations by bar pressing for pellets on a continuous reinforcement schedule one hour/day at room temperature (25° c). On test days the room temperature was altered for the one-hour period only. Food intake was automatically recorded every 9 minutes and the cumulative records are shown in Figure l . Performances at ambient temperatures of 18° and 32° c were compared with those at 25°c. The suppression at 32° c is obvious and needs no further comment. At 18° c food intake during the first half hour exceeded the intake at 25° c and by the end of the test period , before energy depletion could have occurred, the intakes differed markedly. These data from short-term experiments provide a clear answer to the thermal stimulus-energy stores argument. Perhaps the most interesting finding in the above example was the absence of a plateau during feeding in the l 8° c record . This suggests that a study of the patterns of food intake in the cold may be more informative than the absolute amount of food consumed . The lack of suppression of feeding after 27 minutes at 18° c indicates that under conditions of mitd cold stress, rate of stomach emptying may be increased, enabling the animal to

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200 Sharma, Dua-Sharma, and Jacobs Also, the status of the control system profoundly influenced the pattern of the receptor discharge. For example, topical NaCl producing an enhanced effect on tactually evoked discharge, was further increased if applied after gastric vagotomy, but was reduced if repeated after cervical sympathectomy. If, on the other hand, gastric vagal stimulation preceded NaCl application, the tactually evoked activity was decreased, but was enhanced following sympathetic stimulation. The resultant effect thus depends on the level of vago-sympathetic activity and the interacting influence of chemoceptive and mechanoceptive stimuli on gastric receptors. 3 OSMORECEPTORS Jacobs (1964) has investigated the problem of osmometric mechanisms existing in oral and gastric cavities of rats and has suggested that the satiety signals evoked by intragastric glucose and NaCl act through different receptor mechanisms. The decrease in brain impedance observed after intragastric loads of NaCl or intraperitoneal sucrose were not observed after intragastric glucose or sucrose loads . Using a preference technique in a free-choice situation, Chemigovsky (1960) concluded that at least in dogs a salt preference depends on peripheral neural information coming from gastric vagal afferents. Both 3 and 5 per cent intragastric NaCl loads significantly inhibited NaCl-milk preference, the inhibition being practically abolished after subdiaphragmatic vagotomy. INTESTINAL RECEPTORS Herrin and Meek (I 933) demonstrated that loss of appetite produced by distension of innervated Thiry and Thiry-Vella loops was no longer seen after denervation, and the dogs showed normal ingestive behaviour. Similarly, rhythmic distension of innervated Thiry-Vella loops in cats produced a significant drop in both milk and meat intake on the days of distension; however such a drop in food intake was not observed after denervating the loops (Sharma, 1967b) . These and similar studies have suggested the existence of detector mechanisms in the intestine, excitation of which produces satiety signals . It has been possible to demonstrate that mesenteric and splanchnic afferents, particularly from the jejunum, project to hypothalamic areas implicated in feeding. The characteristics of evoked potentials obtained from ventromedial nucleus (VMN) and lateral hypothalamus (LH) substantiate the notion of a reciprocal relationship between hypothalamic 'satiety' and 'feeding' centres (Oomura et al., 1967) and indicate that intraluminal perfusion of the gut with glucose modulates the excitability ofvMN and LH neurons (Kumar, 1971). A chemoreceptor mechanism sensitive to nutrients like glucose and

Multilevel signals related to food intake 20 I amino acids has also been demonstrated in the intestine (Sharma and Nasset, 1962). Analysis of receptor responses reveal relatively specific features related to a particular class of substances used. Studies of electrical activity in mesenteric nerves after perfusion with glucose or various amino acids have shown the jejunum to be the most excitable region of the digestive tract (Zamiatina, 1957). Most excitable chemoreceptors responding to ammonia are also present in the jejunum and to a lesser degree in the duodenum, ileum, and large intestine in that order (Poliakova, 1959). If these receptors are sensitive to various nutrients and can selectively detect small changes in the composition of the diet, it is possible that such information could bring about appropriate changes in alimentary behaviour. Development of differential conditioned reflexes was achieved by selective intraluminal introduction of acids or glucose in innervated Thiry-Vella loops of dogs (Vasilevskaya, I 957); and may point to the nature of excitation and function of these receptors. Thus response characteristics and functional significance of oral and gastric detector systems are shared by intestinal mechanisms to a large extent. Intestinal mechanoceptive information interacts with local and systemic factors in a pattern similar to that observed in gastric receptors. In general, however, the intestinal mechanisms show prepotent chemoceptive information transfer, while the stomach seems to use predominantly mechanoceptive cues in influencing food intake. HEPATIC RECEPTORS

The postulation of hepatic glucoreceptors (Russek, 1963) has been recently confirmed by the electrophysiological studies of Niijima (1969). More recent evidence about hepatic glucoreception is reviewed by Russek (this volume). OTHER ABDOMINAL RECEPTORS

The hunger for sodium chloride after deprivation is well known and widely studied. Adrenalectomized animals show a preference for weak salt solutions to which they are normally indifferent. Though electrophysiological analysis in the adrenalectomized preparation does not indicate a change in gustatory receptor threshold, salt preference in these animals is claimed to be abolished after sectioning of gustatory afferents. Perhaps the gustatory afferent activity is handled against the 'background information' brought about by adrenalectomy. Though direct evidence of 'salt receptors' in the adrenals is lacking, chemoreceptors responding to catecholamines have been demonstrated (Niijima and Winter, 1968). Perhaps the information

202 Sharma, Dua-Sharma, and Jacobs from the adrenals interacts with gustatory information at some as yet unknown site in the central nervous system, thus bringing about changes in salt appetite . CENTRAL MONITORING HYPOTHALAMUS

I GLUCOSTATIC MECHANISMS Mayer (1955) put forward the glucostatic theory which postulated that somewhere , possibly in the hypothalamus, there are 'glucoreceptors' sensitive to blood glucose utilization and that these receptors bring about changes in feeding behaviour. Electrophysiological analysis of hypothalamic regions (Anand et al . , 1961) revealed that the activity of the VMH was significantly and selectively affected by changes in blood sugar level. Sharma et al. (1961) showed that intravenous or intracarotid glucagon injections also produced a selective increase in the activity of ventromedial hypothalamic regions coincident with increased arterio-venous difference in glucose concentration , but there was no significant change in the activity of the feeding centres . Simultaneously , the gastric 'hunger' contractions were inhibited markedly . Repeating glucagon injections after bilateral lesions of the VMN neither produced changes in VMH electrical activity nor showed any inhibition of the gastric contractions . The rise in blood glucose level was, however, similar to that seen in animals without such lesions. It was, therefore, surmised that increased arterio-venous difference in glucose concentration selectively activated some specific cells in the region of the VMH and their activation produced inhibition of gastric 'hunger' contractions. Thus, it could be shown that changes in blood glucose utilization influence central hypothalamic mechanisms, and through centrifugal pathways, influence peripheral mechanisms of hunger and satiety. The frequency of discharge of VMH neurons of starved animals was found lower than that from LH neurons (Anand et al., 1964). After intravenous glucose, the frequency of spikes recorded from VMH neurons increased and that of LH neurons decreased significantly; a reverse pattern of response was obtained from these units after intravenous insulin. Whether the unit activity changes in the neurons of these hypothalamic regions is the direct effect of glucose utilization by these neurons , or is secondary to glucose utilization at the periphery, especially in the gastrointestinal tract and liver, or both, has been investigated in recent years. It has been shown that intravenous glucose and insulin selectively affect hypothalamic LH and VMH neurons in cats transected at the midcollicular

Multilevel signals related to food intake 203 level (Chhina et al. , 1971); this has been interpreted by the authors as the activation of these hypothalamic neurons directly by changes in the level of glucose utilization. 2 THERMOSENSITIVE MECHANISMS Brobeck (1948) proposed the concept of'thermostatic ' regulation of food intake, and suggested that animals eat to keep warm and stop eating to prevent hyperthermia. Andersson and Larsson ( 1961) showed that direct local cooling of the preoptic area and the rostral hypothalamus induces eating, whereas warming the same area inhibits eating. The frequency of firing of single units in the anterior hypothalamus and preoptic area is known to be influenced by local heating (Nakayama et al., 1963), but raising of temperature in YMN and LH does not change the firing frequency of these neurons (Anand et al ., 1966). It seems that the anorexic effect of warming the preoptic area is not due to a direct thermal effect on the hypothalamic appetite centre; any changes in food intake produced in response to changes in temperature may be due to integration between the thermo-sensitive rostral hypothalamic region and the 'satiety' and 'feeding' centres in the middle hypothalamus . Perhaps , in those circumstances in which food intake or energy output may lead to an upset of temperature homeostasis, the heat regulating centres also influence the nervous mechanisms for feeding . 3 LIPOSTATIC MECHANISMS Lipostatic control has been postulated as a long-term modulating influence that corrects the errors of the short-term glucostatic control (Mayer, 1955). The electrical activity of lateral and ventromedial hypothalamic regions on intravenous injections of neutral fat particles, however, do not show any change (Anand et al. , 196 I). There is some suggestion that differential information from cutaneous and muscular thermoreceptors which would depend on the thickness of the fat layer located between them can influence feeding . Electrophysiological evidence about cold receptors in muscle (Banet and Seguin , 1967) gives some support to this hypothesis . 4 AMINO ACID-SENSITIVE MECHANISMS A reciprocal relationship has been suggested between the serum amino acid concentrations and appetite (Mellinkoff et al . , 1956), but no change in the electrical activity of hypothalamic 'satiety' and ' feeding' centres has been observed on intravenous infusion of protein hydrolysate which significantly increases the amino acid content of circulating blood (Anand et al., 1961, 1965). 5 OSMORECEPTORS Similarly, osmoreceptor discharges from supraoptic and anterior hypothalamus are well known to be influenced by changes in osmolarity of the circulating fluid, but they do not seem to influence hypothalamic feeding mechanisms directly . The anorexic effect of high

204 Sharma, Dua-Sharma, and Jacobs doses of NaCl may be regarded as an emergency mechanism showing priority of osmotic regulation over feeding control. 6 OTHER CHEMOSENSITIVE MECHANISMS Grossman (I 962) showed that cholinergic stimulation ofLH produces drinking , whereas adrenergic stimulation through the same implanted cannula induces eating. Unit recordings from LH neurons (Oomura et al ., 1969a,b), on the other hand, show that many cells are sensitive to both acetylcholine and noradrenaline and there is considerable overlap among populations of cells. With regard to VMH units, though firing rate decreased on acetylcholine application and noradrenaline increased as often as it decreased the discharge, the population of cells also showed considerable overlap in distribution. In light of these electrophysiological results obtained from single neurons in VMH and LH, it is difficult to interpret the behavioural observations of Grossman, suggesting specific chemical sensitivities for drinking and feeding responses . LIMBIC AND OTHER CNS STRUCTURES A number oflimbic forebrain structures as well as the cerebral cortex have also been implicated in feeding and drinking behaviour by electrophysiological studies . These studies have been discussed in the article by Mogenson (this volume). It is not possible to assign unitary functions to these structures , but they certainly seem to be forming the components of feedback loops in which the hypothalamus is the critical focus. Food intake is continued as long as hypothalamic feeding mechanisms are intact. However, the complete behavioural act of ingestion, its start and stoppage, obviously requires an integrated activity involving 'multifactors,' 'multihomeostasis,' and 'multilevels.' ORAL-GASTRIC INTERACTIONS AS A FUNCTION OF NUTRITIONAL STATE

The sections above have outlined the current knowledge of chemo- and mechanoreceptor activity at all levels of the nervous system as they might operate in providing signals for the control of food intake . Although overnight fasted animals are commonly used in these experiments, nutritional state has not been assumed to be an important variable in the results of these experiments. However, behavioural results on food intake described previously (Jacobs and Sharma, 1969) suggest that the nutritional state might be an important modulator of electrical activity in the detector system related to food intake. Thus we have studied the role of chronic

Multilevel signals related to food intake 205 hunger in oral-gastric receptor systems (Sharma et al . , 1967, 1968; Sharma, 1970; Jayaraj et al., 1972; Sharma et al., 1972). As will be seen in the discussion below, nutritional state is indeed a critical variable in intake related to sensory systems . GUSTATORY RECEPTORS

Our studies on unit analysis of gustatory receptors in rats and frogs show that topical application of glucose, NaCl, and amino acids, limited to one or a few gustatory papillae, produce differential responses related to the nutritional background of the animal. In well-fed animals (Type 1), fewer taste buds responded to glucose and amino acids than when the animal was chronically food deprived (Type 11). This difference was accentuated with increasing degree and duration of food deprivation. Also, the threshold of excitation in Type I animals was higher with longer latency, lower magnitude, and shorter duration ofresponse. Table l gives details ofresponses to various substances obtained from glossopharyngeal afferents in Type 1 and Type II frogs. In the response column, the numbers in parentheses indicate the number of frogs . The numbers with plus indicate intensity of response (frequency of unit discharges) based on a 6-point scale; l + indicates slight but definite activity, 5 + indicates intense activity, and zero indicates no response. It is clear that the number of positively responding papillae and the intensity of response to glucose is significantly higher in Type II animals. There is also some increase in amino acid response of Type II frogs, but no appreciable difference in Type I and Type II frogs is seen for NaCl response . GASTRIC RECEPTORS

In recent years, properties of gastric mechanoreceptors have also been analysed in our laboratory under varying conditions of nutrition, electrical, chemical, and distension stimuli, applied locally or in distant parts of the gastrointestinal tract (Sharma, 1972; Sharma et al . , 1972). In well-fed animals (Type 1), mucosa! or serosal applications of gastric tactile stimulus, evoke afferent discharges showing peak frequency response almost immediately . The increased activity remains elevated without decrement for an average of200 to 400 msec, and then ends abruptly. This is in contrast to the slow and graded adaptation seen in distension receptors, some of which may continue to fire for several seconds. When a gastric tactile stimulus is applied during maintained gastric distension, the tactually evoked activity is inhibited. The amount of inhibition depends on the rate and degree of distension, and on the temporal relation of the tactile stimulus to gastric

206 Sharma, Dua-Sharma, and Jacobs TABLE I Responses from glossopharyngeal afferents in well-fed (Type 1) frogs and in chronically food-deprived (Type 11) frogs Response (Type 1)

Test solution

n

Glucose

17

Response (Type 11)

n

1st trial

2nd trial

3rd trial

1st trial

(8)0 (5)1 + (2)1 + (2)2+

(8)0 (5)0 (2)1 + (2)2+

(8)0 (5)0 (2)0 (2)2+

19

NaCl

17

(2)0 (11)3 + (4)4+

(2)0 (11)3 + (4)4+

(2)0 (I 1)3+ (4)4+

19

Acetic acid

II

(2)0 (7)2+ (2)3+ (1)0 (3)1 + (4)2+ (3)3+ (3)0 (4)1 + (2)2+ (3)3 +

(2)0 (7)1 + (2)1 + (1)0 (3)0 (4)2+ (3)3+ (3)0 (4)0 (2)2+ (3)3+

(2)0 (7)0 (2)0 (1)0 (3)0 (4)2+ (3)3+ (3)0 (4)0 (2)2+ (3)3 +

8

Quinine II sulphate Amino acids

12

IO

12

(5)0 (5)1 + (7)3+ (2)4+ (2)0 (10)3+ (5)4+ (2)5+ (2)0 (6)2+ (2)0 (2)1 + (2)2+ (4)3 + (2)0 (3)1 + (2)2+ (4)3+ (1)4+

2nd trial

3rd trial

(5)0 (3)2+ (2)1 + (7)3+ (2)4+ (2)0 (10)3+ (5)4+ (2)4+ (2)0 (4)2+ (2)1 + (2)0 (2)2+ (2)2+ (4)3+ (2)0 (3)2+ (2)2+ (4)3 + (1)4+

(5)0 (4)2+ (1)3+ (7)3+ (2)4+ (2)0 (10)3 + (5)4+ (2)4+ (2)0 (3)1 + (3)0 (2)0 (2)1 + (2)2+ (4)3+ (2)0 (3)2+ (2)2+ (4)3+ (1)4+

distension. These inhibitory effects may appear at distension volumes, not sufficient to produce evoked distension discharge proper and demonstrate how sensitive the control system is. Release of gastric distension restores the evoked tactile response following a slow recovery which may be complete only after 3 to 5 minutes. Gastric vagotomy blocks the inhibitory effect of gastric distension upon the tactually evoked response . Removal of cervical ganglia has no such effect and fails to affect the inhibition of tactually evoked responses by gastric distension. In chronically food deprived animals (Type 11), contrary to the findings in Type I animals, maintained gastric distension facilitates rather than inhibits tactile-evoked afferent discharge. Gastric vagotomy fails to diminish the facilitatory effect of gastric distension upon the tactile response. Removal of cervical ganglia, by contrast, severely impairs the effect. Thus the facilitatory effects on gastric receptor discharge is mediated via sympathetic fibres while inhibition is routed via vagal fibres. In these studies it was also shown that topical application of glucose to the mechanoreceptor field produced inhibition in Type II animals, as though the hungry animal was

Multilevel signals related to food intake 207 'satiated' by the mucosa! application of glucose for approximately 15 minutes . This indicates that the analysis of events at the receptor site depends not only on the type and amount of particular change brought about by a substance locally, but is linked with the nutritional status and the activity level of the neuro-humoral control system . ORAL-GASTRIC INTERACTIONS

An extension of the above approach was attempted by studying the effects of gastric distension - a classic satiety signal, on gustatory responses - a classic hunger system , under varying conditions of nutritional state. Figure 3 depicts unit responses from the glossopharyngeal nerve of frogs . One ml of stomach distension inhibits the evoked activity of glossopharyngeal afferents obtained by topical application of glucose and NaCl in Type 1 animals (left half of the figure). The magnitude of inhibition of gustatory impulses depends on the degree and mode of gastric distension, and the previous state of the stomach. Distension of 4-6 ml may produce complete inhibition of the gustatory response. Similar inhibition may be obtained by a smaller amount of distension if repeated within 5 minutes of the previous distension. By contrast, an identical stimulus of gastric distension produces facilitation of the gustatory response obtained by glucose and NaCl application in Type II animals (right half of the figure). Bilateral gastric vagotomy considerably reduces the inhibitory effects of gastric distension in Type 1, but no appreciable change is seen in the inhibition of the gustatory response after cervical sympathectomy. In Type II animals, on the other hand, cervical sympathectomy potentially decreases the facilitation of gustatory response obtained by coupling gastric distension with topical application of glucose and NaCl to the tongue. Similar differential effects of gastric distension have been obtained in rats, but the nature and pathways involved in these interactions have not been worked out in detail. Working on toads, Brush and Halpern (1970) reported that gastric distension enhanced glossopharyngeal multi-unit neural activity elicited by lingual application of NaCl, but decreased quinine hydrochloride response and showed no change for dextrose. Gustatory neural responses in peripheral nerves have also been reported to be modulated by mechanical and chemical stimulation of the stomach in the frog (Esakov, 1961; Halpern, 1967), rat (Sharma et al., 1967; Hellekant, 1971), and man (Zaiko and Lokshina, 1962). Cabanac (1971) found that gastric loads of glucose and NaCl produced a differential modulating influence on the palatability of sweet solutions in obese human subjects as compared with well-fed individuals. The obese failed to show the increased dislike of sweet stimuli

208 Sharma, Dua-Sharma, and Jacobs TYPE II

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Figure 3 Glossopharyngeal afferent activity in Type 1 (/eji ha(f' offigure) and Type 11 (right haif offigure) frogs . (A) spontaneous activity ; (B) after 5.4 percent glucose application to the tongue; (C) response to coupling of lingual glucose application with I ml gastric distension . Strips (D) and (E) show respectively the effects after applying NaCl to tongue alone and coupled with distension ( I ml) stimulus .

after drinking to satiety shown by subjects of normal weight. It is hypothesized that the effects of gastric distension and chemoceptive stimulation may modify gustatory selection of food intake, the allesthesic changes being brought about by the 'internal signal' generated by the difference between the set point and the internal state (energy pool). MODEL

It could be suggested that the change in the nutritional background of the animal from well fed to chronically food-deprived is capable of biasing the signal pattern formalized in Figure 4, in a manner comparable to the capacity of the 'energy pool' to influence the behavioural response by the sensory and the metabolic qualities of the diet (Jacobs and Sharma, 1969). The fact that the gustatory and gastric receptor activities were different in the two cases (depending upon whether the frogs were well fed or chronically food deprived) provides strong evidence that the modulation of oral gastric receptor discharge is linked with some factors related to the metabolic state of the animal . The mechanism here proposed is not visualized as a double-throw switching system, in which either detector system can be completely shut off: it appears certain that oral-gastric receptors are in either case subject to both vagal and sympathetic

Multilevel signals related to food intake 209

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PHYSICO CHEMICAL CHANG[

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Figure 4 Model showing the dual detector system and the role of energy pool as a biasing system, in the over-all control of food intake . Each bar represents tactually evoked responses. The 'dark carpet' area shows the tactile response during maintained gastric distension under intact conditions (facing rear wall of box) and after gastric vagotomy (left side-wall of box) and cervical sympathectomy (right side-wall of box) . Bold Sat the top of the left box, indicates predominantly sympathetic activity, while Bold Vat the bottom indicates that vagal activity is low . In the right box, Bold Vis at the top indicating predominantly vagal activity with relatively low sympathetic activity (Bold Sat bottom) . Yagi cut - left gastric vagotomy; CG cut - left cervical sympathectomy (From Sharma et al., 1972). This same model can serve for oral and intestinal detector systems by substituting each bar for oral chemoreceptor or intestinal mechanoreceptor/chemoreceptor response.

influence. A significant change in nutritional background could merely change the relative importance of the vagus and sympathetic systems in registering, and responding to, visceral events. It could be that the information from oral-gastric receptors is handled by the central nervous system against certain 'background' changes brought about by the animal's nutritional (internal) state, and may account for the differential responses. Though the above model is discussed in the light of electrophysiological results obtained in frogs, and one is handicapped by knowing so little about food intake in this species, it affords an excellent experimental situation for

210 Sharma, Dua-Sharma, and Jacobs delineating the locus of receptor sites, and the nature and pathways of the control systems involved in alimentary responses . The next logical step would be to apply these techniques and analytical approach to mammalian systems. Already some work in this direction has been reported. Pager et al. (1972) hypothesized that olfactory sensitivity might be modified by nutritional state . They made clear that hunger, induced by food deprivation or insulin administration elicited a selective facilitation of electrical responses from the olfactory bulbs to food odour stimulation , but not in response to citral or amyl acetate. The selective modulation ofbulbar electrical activity disappeared with the induction of satiety . These results show a striking similarity to our behavioural model (Jacobs and Sharma, 1969). Norgren (1970) recorded hypothalamic unit activity in rats following gustatory stimulation with various substances, and showed that only units which responded solely to water or solely to sucrose were affected by the food-deprivation state of the animal. The unit responded to water alone when the animal was satiated, and to sucrose alone when the animal was food-deprived. These positively responding units were likely to be recorded from electrodes which support self-stimulation, while units which responded only to quinine were not likely to be recorded from positively reinforcing electrodes. Thus unit discharges could be shown to respond selectively, depending upon the nutritional state and appear to have behavioural implications of influencing motivational and reinforcing systems. Elsewhere (Jacobs and Sharma, 1969) behavioural correlates of gustatory and alimentary responses have been discussed as a function of the nutritional state of the animal. It will become apparent that, though the bridge between electrophysiological events and behavioural responses needs elucidation in many aspects, electrophysiological analysis may not only reinforce behavioural results but may, in fact , complement them . REFERENCES Adolph,E.F. 1947.Am . J . Physiol . , 151 : 110-25 Anand, B.K. 1961. Physiol. Rel' . , 41: 677-708 - 1963. In M.A.B. Brazier (ed .), Brain and Behal'ior . Washington : Amer. Inst. Biol. Sci ., Vol. 11, pp. 43-116 Anand, B.K ., Banerjee, M .G., and Chhina, G.S. 1965. Indian J. Med. Res., 53 : 1172-9 - 1966. Brain Res., l: 269-78 Anand, B.K. , Dua, S. , and Singh, B. 1961. Electroenceph. din. Neurophysiol ., 13 : 54-9 Anand , B.K., Chhina, G.S., Sharma, K .N., Dua, S. , and Singh, B. 1964. Am . I . Physio/., 207: 1146-54 Anand, B.K . and Pillai, R.V. 1967. J . Physiol . (Lond.), 192: 63-77 Andersson, B. and Larsson, B. 1961. Acta physio/. scand. , 52: 75-419

Multilevel signals related to food intake 2 I I Banet, M. and Seguin, J .J. 1967. Canad. J. Physiol. Pharmacol . , 45 : 319-27 Brobeck, J.R. 1948. Yale J . Biol. Med., 20: 545-52 - 1%0. In J. Field , H.W. Magoun , and V.E . Hall (eds .), Handbook (~{" Physiology, 'Neurophysiology .' Washington: Physiol. Soc ., Vol. 11, pp . 1197-1206 Brush, A.D. and Halpern , B.P. 1970. Physiol . Behav., 5: 743-6 Cabanac, M. 1971. Proc 1v Intern. Conf. Regulation of Food and Water Intake, Cambridge, England Chernigovsky, V .N. 1960. Pal'!o1· J . Higher Nervous Activity, English transl. , 10: 329-41 Chhina, G.S ., Anand, B.K., Singh, B.. and Rao, P.S. 1971. Am. J. Physiol . , 221: 662-7 Cohen , M.J., Hagiwara, S., and Zotterman. Y. 1955. Acta physio/. scand., 33 : 316-32 Dunlop, C.W. 1958. Electroenceph. din . Neurophysio/., 10: 297-304 Esakov, A.I. 1961. Bull. Exp . Biol. Med. USSR . (English transl.), 51 : 283-9 Ganchrow, J .R . and Erickson , R.P. 1970. J. Neurophysio/., 33: 768-83 Grossman, M.1. 1955. Ann . N. Y. Acad. Sci ., 63: 76-91 Grossman, S.P. 1962. Am. J. Physiol . , 202: 872-82 Halpern, B.P. 1967. In M.R. Kare and 0 . Maller(eds .), The Chemical Senses and Nutrition . Baltimore: Johns Hopkins Press, pp . 213-41 Hellekant, G. 1971. Aclll physiol. scand ., 83 : 527-31 Herrin, R.C . and Meek, W.J. 1933. Arch. Intern . Med . , 51: 152-68 Jacobs , H.L. 1964. In M.J. Wayner(ed.), Thirst . New York: Pergamon Press, pp. 117-37 Jacobs, H.L. and Sharma , K .N. 1969. Ann . N . Y. Acad. Sci., 157: 1084-1125 Janowitz, H.D. 1958. Am . J . Med., 25: 327-32 Jayaraj, A.P., Savithramma, M ., Gopal, V. , Dua-Sharma, S. , and Sharma, K .N . 1972. Exp . Neurol . , 36: 507-11 Jewell, B.A. and Verney, E.B . 1957. Phil . Trans . R. Soc . (Lond .), Ser. B., 240: 197-324 Kennedy, G.C. 1953. Proc . Roy . Soc . (Lond.), Ser. B., 140: 578-92 Kennedy , G.C. and Mitra, J. 1963. J. Physiol . (Lond .), 166: 395-407 Kumar Mohan, V. 1971. Role of intestinal afferents in the regulation of the activity of brain regions concerned in food intake . PH D thesis, All India Institute of Medical Sciences, New Delhi Landgren, S. 1957. Acta physiol. scand., 40: 210-21 Mayer, J. 1953. Physiol. Rei·. , 33: 472-508 - 1955. Ann. N . Y. Acad. Sci., 63 : 15-43 McCleary, R.A . 1953. J. comp . physio/. Psycho/ ., 46: 411-21 Mellinkoff, S.M., Frankland, M., Boyle, D. , and Greipel, M . 1956. J . app/. Physiol .. 8: 535-8 Nakayama, T. , Hammel , H.T. , Hardy, J.D. , and Eisenman, J .S. 1963 . Am. J. Physiol., 204: 1122-6 Niijima, A. 1967. Physiol. Behm·., 2: 1-4 - 1969. Ann. N. Y. A cad. Sci . , 157: 690-700 Niijima, A. and Winter D.L. 1968. J. Physio/ . (Lond.), 195: 647-56 Norgren, R. 1970. Brain Res . , 21 : 63-77 Oomura, Y., Ooyama, H. , Yamamoto, T. , and Naka, F. 1%7. Physiol . Behav . , 2: 97-115 Oomura, Y., Ooyama, H., Naka, F., Yamamoto, T .,Ono, T., and Kobayashi, N. 1%9a.Ann. N. Y. Acad. Sci ., 157: 666-89 Oomura, Y., Ooyama , H., Yamamoto, T. , Ono, T. and Kobayashi, N. 1969b. Ann . N . Y. A cad. Sci. , 157: 642-665 Pager, J. , Giachelli, I., Holley, A., and LeMagnen, J. 1972. Physio/. Behm•. , 9: 573-9 Paintal, A.S. 1954. J. Physio/. (Lond.), 126: 255-70

212 Sharma, Dua-Sharma, and Jacobs Pfaffman, C. 1957. In Nutritional Symposium SerieJ. New York : Nutritional Vitamin Foundation Inc., 14: pp. 40-5 - 1959. In J. Field. H.W. Magoun. and V.E. Hall (eds.). Handbook of Physiology. Neurophysiology . Washington: Am . Physiol Soc ., Vol. 1, pp. 507-33 Pfaffman, C .• Erickson. R.P .. Frommer, G.P .• and Halpern, B.P. 1961. In W.A. Rosenblith (ed.). Sensory Communication New York: M.I.T. Press and John Wiley and Sons, pp . 455-73 Poliakova, N.N . 1959. Sechenm · Physiol. J . USSR (English transl.) , 45: 36--45 Ramakrishna, T. and Sharma, K .N. 1971. Indian J. Physiol. Pharmacol .. 15: 38 Russek, M . 1963. Nature (Lond .), 197: 79-4l0 Sharma, K.N . 1967a . In C.F . Code (ed .), Handbook of Physiology, Alimentary Canal. Washington : Am. Physiol. Soc .. Vol. 1, pp . 225-37 Sharma, K.N. 1967b. In M .R. Kare and 0. Maller (eds .). Chemical Senses and Nutrition . Baltimore : Johns Hopkins Press, pp . 281-91 - 1970. Proc . Reg . Congr. IUPS, Romania , p. 67 - 1972. Proc. Indian Sci. Congr., 59: 1-16 Sharma, K .N . and Nasset, E. 1962. Am. J. Physiol .. 202 : 725-30 Sharma, K.N., Dua-Sharma, S., and Gopal, V. 1967. Indian J. Physiol. Pharmacol., 11 : 22 Sharma, K.N ., Dua-Sharma, S., and Jacobs, H.L. 1968. Proc. xxivth Intern . Congr. Physiol. Sci ., vu: 398, Abstr. 1192 Sharma, K.N ., Anand, B.K .. Dua, S .. and Singh, B. 1961. Am . J. Physiol .. 201 : 593-4l Sharma, K.N ., Dua-Sharma, S .. Gopal, V., and Jacobs, H.L. 1971. Proc. xxvth Intern . Congr. Physiol. Sci., 1x: 510, Abstr. 1517 Sharma, K.N ., Jacobs, H.L., Gopal, V .• and Dua-Sharma, S. 1972. J. Neural Trans .. 33: I 13-54 Sirotin, B.Z. 1961. Bull. Exp. Biol. Med. USSR (English trans .). 50: 873-7 Sudakov, K .V . and Rogacheva, S.K . 1963. Fed. Proc. (Trans. Suppl.), 22: 306-10 Vasilevskaya, N .E . 1957. Secheno1· Physiol. J. USSR. (English trans .), 43: 795-9 Zaiko, N.S . and Lokshina, E.S. 1962. Bull Exp. Biol. Med . USSR (English trans .), 53: 9-11 Zamiatina, O.N. 1957. Secheno1· Physiol. J. USSR (English Trans.), 43: 412-20

The central control of water and salt balance

Bengt Andersson

Professor J.A.F. Stevenson 's fundamental contributions to present knowledge of the hypothalamic control of fluid and energy balance are obvious to anyone interested in homeostatic mechanisms . I have, therefore, appreciate d the invitation to treat as a chapter of this book a subject which was of particular interest to Jim Stevenson . As always, however, it is the conceptio n that provides the delight, not the travail. Research on the importance of the limbic system in the regulation of water and salt turnover is continuously accelerating and much of the evidence appears to contain many contradict ions which make an attempt to condense this information into an intelligible story arduous and hazardous. The reading of such a story is bound to leave behind the paradoxical feeling that what has been said may well be coherent but not necessarily true. To evade these problems to some extent I have taken the liberty to limit this paper to a discussion of the ways in which limbic receptor systems may perceive the changes in the internal environme nt which arise as a consequen ce of various deviations from fluid balance. DEVIATI ONS FROM FLUID BALANC E AND COMPEN SATORY MECHAN ISMS

As a rough generalization it can be stated that the volume and the distribution of the fluids of the body are determined by its content of water and

214 Bengt Andersson sodium. Shortage or excess of either upsets the balance and compensatory mechanisms are brought into play. The pure loss of water results in absolute dehydration. Both the volume of the intracellular fluid (ICF) and of the extracellular fluid (ECF) are reduced, and the tonicity of the body fluids is increased. Absolute dehydration elicits thirst and accelerated release of antidiuretic hormone (ADH) from the neurohypophysis; both mechanisms are attempting to restore water balance. An excessive intake of water has the reverse effects. During hydration ICF and ECF volumes are expanded with reduced solute concentration . Thirst is no longer experienced, and the release of ADH is inhibited more or less completely -a condition leading to rapid elimination of surplus water via the kidneys. An excessive intake of salt raises the Na+ concentration of the ECF, which in turn causes a shift of water from the ICF to the ECF (relative, or cellular dehydration). Relative dehydration is compensated for in two ways: (I) thirst-induced drinking and accelerated release of ADH work to return the extracellular Na+ concentration to normal; (2) the renal Na+ excretion increases and excess salt is eliminated with the urine. Haemodynamic and hormonal events seem to be involved in this natriuresis (e .g., increased glomerular filtration rate, reduced aldosterone secretion, and possibly, release of humoral natriuretic factor(s)) (Orloff and Burg, 1971). During sodium depletion, on the other hand, the Na+ concentration of the ECF becomes subnormal. Consequently water is shifted from the ECF to the ICF. The ECF volume, including the blood plasma, is reduced (hypovolaemia), whereas the ICF volume may expand above normal (cellular hydration). Among the compensatory mechanisms induced by hypovolaemia and hyponatraemia are increased aldosterone secretion, salt appetite, and activation of the renin-angiotensin system (Denton, 1965). This last mechanism provides an explanation for the fact that sodium depleted animals continue to drink water (Cizek et al., 1951; Holmes and Cizek, 1951) and to release ADH, although their extracellular Na+ concentration is subnormal and their intracellular fluid volume may be expanded (Fitzsimons, 1972). HYPOTHALAMUS AND BODY FLUID HOMEOSTASIS

It has gradually become evident that the limbic system, particularly the hypothalamus, is involved in most of the homeostatic mechanisms mentioned above. This has been clearly demonstrated by clinical observations

Central control of water and salt balance 215 and by numerous experimental studies involving various kinds of stimulation, ablations, and electrophysiological recordings. Since this work has been reviewed excellently and comprehensively by Dr Stevenson (1967, 1969), two milestones only may be mentioned: (I) the demonstration in I 938 by Ranson' s laboratory (Fischer et al., I 938) that the release of ADH from the neurohypophysis is mediated by nerve impulses from the hypothalamus, and (2) Stevenson's own important discovery ten years later that medially placed hypothalamic lesions cause hypodipsia and a significant increase in serum Na+ concentration (Stevenson, 1949; Stevenson et al., 1950). The hypothalamus and other parts of the limbic system may receive information about deviations from fluid balance in two ways: (a) via afferent impulses transmitted from peripheral (e.g., vascular, hepatic and oropharyngeal) receptors of various kinds, and (b) by a direct influence of humoral factors on central sensors. Only the latter possibility will be considered here. However, the evidence for hypothalamic receptor systems of importance in the control of water and salt balance is by no means conclusive. The nature of such receptors cannot be described adequately until answers have been given to at least some of the following questions. Where exactly in the hypothalamus are these receptors located? Are they neurons, glial cells, modified ependymal cells, or cells of still another kind? Are they stimulated from the outside or from the inside of the blood-brain barrier? What are their physiological stimuli? As precise information on any of these points is lacking, much of the following has to assume the character of theoretical speculation. BRAIN-BARRIER SYSTEMS AND CENTRAL CONTROL OF FLUID BALANCE

The most prevalent view is that hypothalamic receptor systems which participate in the control of fluid balance are influenced directly by the composition of the blood. However, as long as it is not known whether the sensing part of such receptors is in direct contact with the blood, any discussion of their function must consider the possible influence of the blood-brain barrier and other brain barriers. The original and classical evidence for osmoreceptors in the hypothalamic region and for a central osmosensitive control of water balance was obtained by studying the effects of alterations in the solute composition of blood plasma (Verney, 1947). However, the presence of a blood-brain barrier may have received too little attention in these studies (see below).

216 Bengt Andersson The alternative route by which humoral factors might affect cerebral receptor systems is via the cerebrospinal fluid (csF). The CSF is formed by a secretory-reabsorptive process in the epithelium of the choroid plexuses so that the composition of the CSF in many respects differs from an ultrafiltrate of blood plasma and from the ECF in most other organs and tissues . The question whether an extracellular space really exists in the brain tissue has been debated extensively, but today it is established that the neurons and the glial cells of the brain, like most other cells in the body, live in an environment of ECF (van Herreveld, 1966). The brain ECF, however, is protected by the blood-brain and the blood-liquor barriers which tend to repress and modify the effects of rapid variations in the composition of blood plasma. Since smaller molecules and ions pass through the ependymal lining of the ventricular walls relatively freely, the ECF of the brain tissue has a composition similar to that of the CSF, and, consequently, differs from the ECF composition in most other tissues (van Herreveld, 1966). Therefore, it is evident that factors which interfere with the secretory and reabsorptive processes in the choroid plexuses may induce alterations in the ionic composition of the cerebral CSF. Under such circumstances the brain cells ,(especially those located near the ventricles) may be exposed to changes in their ionic environment which do not necessarily reflect corresponding changes in the composition of the blood plasma. Certain parts of the hypothalamus are heavily vascularized and this may facilitate the exposure of hypothalamic receptor systems to stimuli carried via the blood . However, the hypothalamus surrounds the ventral part of the third ventricle which makes its ECF easily accessible to changes in the composition of the CSF. At sites in the hypothalamus, and more caudally in the central nervous system, there are neurons which even make direct contact with the CSF by protruding through the ependymal cell layer (Vigh et al., 1967). Nothing seems to be known about the physiological significance of these protrusions. However, it was suggested 50 years ago that they may function as central sensors (Agduhr, 1922), and more recently the idea has been put forward that the hypothalamic part of this 'Liquorkontakt-Neuronensystem' may participate in the regulation of the ADH secretion (Vigh, 1971). Whatever may be the importance of these particular structures, it is evident that suitable physiological and anatomical conditions exist for humoral stimuli to reach hypothalamic neurons via the CSF. THE OSMORECEPTOR THEORY

In the 1940s Verney (1947) performed a series of ingenious experiments in

Central control of water and salt balance 217 the non-anaesthetized dog which form the basis for the conception of an osmotic regulation of ADH release. Verney found that a rise in the tonicity of the blood which flows through the hypothalamus and adjacent parts of the brain could cause a release of ADH in the hydrated animal. This was demonstrated when hypertonic solutions of sodium salts or sucrose were used to increase carotid blood tonicity, but when a hypertonic urea solution was employed for the same purpose no ADH release was obtained. The lack of response to urea was explained as due to the high diffusibility of the substance. On the basis of these experiments Verney postulated that there are osmoreceptors in the brain, probably in the anterior hypothalamus, which regulate the release of ADH . It was assumed that these receptors are not stimulated by a rise in blood tonicity as such, but rather by changes in the composition of the ECF which cause cellular dehydration, and thereby a reduction in the volume of the osmoreceptors. This assumption would explain how urea, which easily diffuses into the cells, was not found to stimulate the receptors. When later it was found that injections of small amounts ofhypertonic NaCl solution into the anteriomedial hypothalamus elicit drinking in the goat, this finding was taken as an indication that osmoreceptors in the hypothalamus also participate in the regulation of water intake (Andersson, 1953). Since both absolute and relative dehydration are characterized by cellular dehydration, the osmoreceptor theory provides an explanation for thirst and accelerated ADH release evolving from these disturbances of fluid balance. Yet, several aspects of the current idea of an osmotic control of water balance may be criticized when the presence of the blood-brain barrier is taken into consideration. The effects of the application of various hypertonic solutions to the central nervous system of the goat, both from the outside and from the inside of the blood-brain barrier, seem to contradict the osmoreceptor theory. An effective blood-brain barrier exists for many substances and ions, among them fructose (Crone, 1965a), urea, glycerol, and Na+ (Yudilevich and de Rose, 1971). There is also a barrier for the free exchange of glucose and galactose between the blood and the brain tissue (Crone, 1965b). It would be expected, therefore, that intracarotid infusions of these substances in equiosmolal, hypertonic solutions would cause approximately the same degree of cerebral dehydration, and would act as equally effective stimuli to hypothalamic osmoreceptors, provided such receptors were located inside the blood-brain barrier. However, a rise in the carotid blood osmolality obtained by infusions ofhypertonic NaCl and fructose acts as a much more potent stimulus to ADH release (Eriksson et al., 197 l) and thirst (Olsson, 1972a) than the equivalent rise elicited by all the other substances mentioned (Fig. I). That osmoreceptors

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Figure I A comparison between dipsogenic responses to intracarotid (left) and intravenous infusions of various hypertonic. equi-osmolal solutions in the goat. The intracarotid infusions ofhypertonic NaCl (11 = 18) and fructose (11 = 8) cause conspicuous cumulative drinking , whereas the corresponding infusions of hypertonic urea (11 = 8) and glycerol (11 = 7) induce considerably less drinking . lntracarotid infusions of equi-osmolal hypertonic solutions of galactose and glucose (not indicated in the figure) have very weak and inconsistent dipsogenic effects . (From : Olsson , 1972a, reproduced by permission of A cta Physiol. Srnnd. )

of importance for the control of water balance are located inside the blood-brain barrier in the hypothalamus also seems unlikely for another reason. Infusions of hypertonic sucrose (Olsson, 1969) and fructose (Olsson, 1972a) solutions into the third ventricle do not elicit ADH release and thirst, in contrast with corresponding infusions ofhypertonic NaCl. An alternative explanation would be that cerebral osmoreceptors are located outside the blood-brain barrier, or in a region of the brain which lacks an effective barrier of this kind. Recent observations in the goat seem to rule out this possibility. It was found that slow infusions of iso- or hypertonic solutions of sucrose or monosaccharides into the lateral cerebral ventricle repress the dipsogenic , antidiuretic , and natriuretic responses to intracarotid infusions of hypertonic Na Cl (Olsson, 1972b, 1973) (Fig . 2). This would hardly be the case if the intracarotid infusions of hypertonic NaCl acted via receptors located outside the blood-brain barrier. Verney's own later work (Jewell and Verney, 1957) also confuses somewhat the picture of osmoreceptors in the hypothalamus which are stimulated directly by an elevated blood tonicity. The ADH release in response to unilateral carotid infusions ofhypertonic NaCl was studied in a

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large number of dogs in which different branches of the internal carotid artery had been tied off. By elimination the conclusion was reached that osmoreceptors were located in the anterior hypothalamus and/or the preoptic region . However, Jewell and Verney were successful in supplying exclusively the anterior hypothalamus and the preoptic region with hypertonic blood in two of the dogs only. It is to be noticed that these two dogs did not respond to the infusions with ADH release. The hypertonic blood apparently did not reach the choroid plexuses in the two dogs, and for this reason probably did not have much effect on the composition of the csF. Therefore, Verney's experiments do not exclude the possibility that the cerebral mechanism that regulates the release of ADH is sensitive to changes in the composition of the csF rather than to osmotic stimuli acting directly over the blood-brain barrier.

220 Bengt Andersson Recently, the effects of lesions in the lateral preoptic region of the rabbit (Peck and Novin, 1971) and the rat (Blass and Epstein, 1971) have been considered convincing evidence that an osmoreceptor mechanism, which regulates water intake (but not the release of ADH) is located in the lateral preoptic region. A minority of animals with bilateral lesions in the lateral preoptic region were found to have lost the immediate thirst response to an extracellular load of hypertonic saline. However, drinking occurred at about one hour after the intravenous injection of hypertonic NaCl, and the lesions left the thirst response to 24-hr water deprivation completely unaltered. The interpretation was that these lesions had destroyed the sensors that mobilize drinking in response to cellular dehydration, but had left the volumetric regulation of thirst intact (Peck and Novin, 1971). However, if this interpretation is correct, it follows that thirst elicited by 24-hr water deprivation in the rabbit lacks the stimulatory component of cellular dehydration. In view of the possibility that blood-borne stimuli may affect central control offluid balance via alterations in the composition of the CSF, it would be interesting to know whether the effective preoptic lesions in the rabbits and rats had impaired the vascular supply to the choroid plexuses of the lateral and third ventricles. If so, it ought to have delayed considerably the transfer of Na+ from the systemic ECF to the csF . This may explain the much delayed thirst response to the intravenous injection ofhypertonic NaCl observed in some of the lesioned animals. In conclusion, this discussion infers that the presence of osmoreceptors in the hypothalamic region of the brain is an accepted truth which does not seem to be in accordance with reality. INDICATIONS OF SPECIFIC SODIUM SENSITIVITY

A series of studies of central control of fluid balance performed over the past several years in the goat indicate that a possible alternative to hypothalamic osmoreceptors might be a sodium-sensitive receptor system located close to the third cerebral ventricle (Andersson, 1971, 1972). The main evidence follows. Slow infusions ofhypertonic NaCl solution into the third ventricle elicit cumulative drinking in goats in normal water balance, and an inhibition of the water diuresis (due to release of ADH) in hydrated animals. Such infusions also induce natriuresis which is most pronounced in the saltsupplemented, pre-hydrated goat. The natriuresis appears concomitantly with an increase in glomerular filtration rate (Andersson, Dallman, and

Central control of water and salt balance 221 Olsson, 1969) and a moderate but sustained elevation of the arterial blood pressure. Similar infusions into the csF of the lateral ventricles have identical , but somewhat weaker and more delayed effects. However, no dipsogenic or antidiuretic responses are obtained as a result of equivalent infusions ofhypertonic saline into the fourth ventricle. As distinguished from the effects of hypertonic NaCl, no drinking or inhibition of water diuresis is obtained by infusions into the third ventricle of hypertonic solutions of non-electrolytes, like sucrose and fructose. Rather, such infusions appear to reduce the normal release of ADH, since they often induce positive renal clearance of free water in the non-hydrated goat (Eriksson , 1972). The results of combined intracarotid/intraventricular infusions also support the idea that a sodium-sensitive receptor system participates in the central control of water and salt balances. It was mentioned above that the dipsogenic, antidiuretic, and natriuretic effects of intracarotid infusions of hypertonic NaCl can be inhibited by infusions of iso- or hypertonic sucrose solutions into the CSF of the lateral ventricle (Fig. 2) . However, corresponding intraventricular infusions of sucrose dissolved in isotonic saline do not have any repressive effect on the responses to intracarotid infusions of hypertonic NaCl (Olsson, 1973). This makes it unlikely that the repressive effect of the intraventricular infusions of pure sucrose solutions is due to reduced concentration of some other csF ion or solute other than Na+ . This casuistry for a periventricular sodium-sensitive system may be full of flaws but it is strengthened by the well-demonstrated central sodiumangiotensin interaction described below. CENTRAL SODIUM-ANGIOTENSIN INTERACTION

The presence of regulated ADH release and water intake in hypovolaemic and sodium-depleted subjects makes it evident that deviations from normal ECF volume affect cerebral mechanisms of importance in the control of fluid balance . Reflex influences from capacitance vessels and humoral factors are apparently among the components of this complex volumetric regulation (Fitzsimons, 1972). The importance of the renin-angiotensin system in the regulation of water intake is treated in this volume in a separate chapter by Dr Fitzsimons. For this reason I shall mention only some evidence obtained from the goat that Na+ and angiotensin interact in the central control offluid balance (Andersson et al., 1972) . In this species the infusion into the third ventricle of angiotensin 11, dissolved in iso- or

222 Bengt Andersson RENAL FREE WATER

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slightly hypotonic NaCl affects the fluid balance in the same manner as the corresponding infusions of hypertonic NaCl solution (see above) . The intra ventricular administration of angiotensin 11, together with hypertonic NaCl, results in a conspicuous potentiation of the dipsogenic, antidiuretic, natriuretic, and hypertensive effects . In contrast, no or only weak responses are obtained when angiotensin is infused into the third ventricle in solutions of non-electrolytes, which reduce the CSF Na+ concentration . This central sodium-angiotensin interaction is illustrated in Figure 3 (antidiuretic responses) and in Figure 4 (hypertensive and natriuretic responses). CONCLUSIONS AND SUGGESTIONS

The studies in the goat which have been reviewed above seem to warrant the following conclusions: 1 An experimentally induced rise in the Na+ concentration in the csF of the third ventricle influences the water balance in a positive, and the salt balance in a negative direction, and elevates arterial pressure to some degree . 2 The local Na+ concentration determines to what extent angiotensin II

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rectly is particularly interesting in view first of the suggestive evidence that catecholaminergic mechanisms are also involved in angiotensin-induced drinking (Fitzsimons and Setler, 1971) and secondly that intraventricular noradrenaline causes increased salt appetite (Chiaraviglio and Taleisnik , 1969); this is discussed later. We still need better evidence, however, that circulating angiotensin produces at least some of its effects on drinking by central action , and, of course, the subsidiary hypothesis of a peripheral action of angiotensin remains untested . PEPTIDE SPECIFICITY OF THE ANGIOTENSIN-SENSITIVE CELLS

We were surprised in our original experiments to find that intracranial renin is a highly potent dipsogenic substance, indeed astonishingly potent compared with angiotensin 11 . Like angiotensin it induces drinking after a latency of about ½ to 1 minute, but unlike angiotensin the response continues for many hours so that very large quantities of water may be

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drunk - more than 100 ml in 6 hrs to 5mU renin is not uncommon. Renin substrate and angiotensin I are also effective intracranial dipsogens and, in fact, molecule for molecule are even more potent than angiotensin 11 (Fitzsimons, 1971b) (Fig . 5) . While it is largely true that other biological actions of renin, renin substrate, and angiotensin I are mediated through angiotensin 11 , both renin substrate and angiotensin I have been reported to have some actions which do not depend on the formation of angiotensin 11, and it may be that thirst is included among these. The peptide specificity of the angiotensin-sensitive neural systems remains to be elucidated. At the moment we know that the dipsogenic action of angiotensin II can be prevented by prior administration of anti-angiotensin II and that drinking to renin substrate is attenuated by the same antibody (Epstein, Fitzsimons, and Johnson , 1973) . In addition , we have recently learned that the Sar', Ala8 analogue of angiotensin 11 (Norwich Pharmacal, P-113) antagonizes the dipsogenic effect of angiotensin with which it is mixed both intravenously and in the brain, where it is effective against angiotensin 11, angiotensin 1 (Epstein, Hsiao , and Johnson, unpublished), renin substrate, and renin (Epstein et al., 1974) in molar ratios of hormone to analogue at l :50 or greater. An alternative explanation, however, has been provided by the discovery of a subsidiary renin-angiotensin system in the brain itself (Fischer-

238 J.T. Fitzsimons Ferraro, Nahmod, Goldstein, and Finkielman, 1971 ; Ganten, MarquezJulio, Granger, Hayduk , Karsunky, Boucher, and Genest, 1971). It seems possible that injected renin acts upon the locally available renin substrate and that injected renin substrate or injected angiotensin I causes drinking through angiotensin II which is formed locally as a result of the action of brain renin and/or brain converting enzyme. Though it provides a possible explanation of why renin, renin substrate, and angiotensin I are dipsogenic when injected into the brain, the function of the cerebral renin-angiotensin system, its relation to the renal renin-angiotensin system and its role if any in thirst mechanisms remain unknown . There is, however, the obvious possibility that the cerebral system functions as a neurotransmitter in limbic circuits for drinking. ANGIOTENSIN AND CHOLINERGIC THIRST

The relationship of angiotensin-induced drinking to the better established central neurotransmitters, acetylcholine and the monoamines, is extremely interesting because these substances are known to have marked effects on ingestive behaviour. Angiotensin causes release ofacetylcholine from brain tissue (Elie and Panisset, 1970), but it is improbable that intracranial angiotensin causes drinking by releasing acetylcholine because intracranial doses of atropine which completely block cholinergic drinking have no effect on angiotensin-induced drinking (Fitzsimons and Setler, 1971) (Table 4). Only intracranial doses of atropine high enough to cause systemic atropinization attenuate angiotensin-induced drinking. Atropine and methylatropine nitrate given subcutaneously are also relatively ineffective at preventing drinking in response to intracranial angiotensin though fully effective at preventing drinking to intracranial carbachol. Activation of cholinergic and angiotensin sensitive mechanisms simultaneously by giving mixtures of carbachol and angiotensin through the same intracranial cannula results in perfect additivity of effect on drinking (Fitzsimons, unpublished). There is no mutual interference and it would seem that the mechanisms are completely independent. MONOAMINERGIC INVOLVEMENT IN ANGIOTENSIN-INDUCED DRINKING

The question of possible monoaminergic involvement in angiotensininduced drinking needs to be considered , though noradrenaline itself is rightly regarded as being more important in feeding than in drinking . Indeed Grossman (1962a ,b), who first described the stimulatory effects of catecholamines on feeding, also pointed out that these drugs are antidip-

Endocrine mechanisms 239 TABLE 4 The amounts of water drunk by rats one hour after angiotensin or carbachol given 10-15 min after a receptor blocker. The blocker and dipsogen were given through the same pre-optic cannula Receptor blocker

N

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sogenic when given to thirsty rats . However, though intracranial noradrenaline inhibits drinking by water-deprived rats and also attenuates drinking induced by cellular thirst stimuli and by intracranial carbachol, it does not inhibit drinking elicited by extracellular thirst stimuli or by intracranial angiotensin (Setler, 1973) . The inhibition, when it occurs, is through specific block of adrenergic mechanisms since the inhibition can be prevented by adrenergic antagonists . The failure of noradrenaline to prevent angiotensin-induced drinking does, therefore, suggest a separate mechanism for the angiotensin response different from the cellular thirst mechanism. It is also worth noting that the lack of effect of noradrenaline on the angiotensin response is reciprocated by the failure of moderate doses of angiotensin (100 p-mole) to affect either noradrenaline or deprivationinduced eating, though larger doses of angiotensin do have some inhibitory effect on eating (Fitzsimons and Setler, unpublished). The effects of angiotensin and noradrenaline on ingestive behaviour are, therefore, not mutually exclusive, and noradrenaline does not inhibit all types of thirst. In fact, ever since Grossman's discovery that noradrenaline causes eating, there have been persistent reports that it may actually cause drinking (e.g., Slangen and Miller, 1969). It is common experience that this drinking

240 J. T . Fitzsimons precedes eating and consequently there can be no question of its being secondary to the ingestion of dry food. The observation that isoprenaline given subcutaneously (Lehr, Mallow, and Krukowski, 1967) or intracranially (Leibowitz, 1971) causes drinking led to the suggestion that drinking may be mediated by /3 adrenergic neurones centrally. Against this view is the finding that the response both to subcutaneous isoprenaline (Houpt and Epstein, 1971) and to intracranial isoprenaline (Fisher, 1973) is abolished by nephrectomy but not by ureteric ligation. This suggests that drinking to isoprenaline, no matter how administered , requires the intervention of some renal factor, most likely renin, since isoprenaline is known to cause a highly significant rise in plasma renin activity (Peskar, Meyer, Tauchmann, and Hertting, 1970). In the case of intracranial isoprenaline, a leakage hypothesis is supported by the finding of radioactivity in the blood stream following injection of radioactive isoprenaline into various brain structures, especially into the hippocampus (Fisher, 1973). However, the response of some intracranial sites to isoprenaline may also be abolished by interrupting the sympathetic outflow to the kidney by section of the spinal cord so that the leakage hypothesis is not the only explanation of the action of intracranial isoprenaline . At present it seems not unlikely that the increased drinking that follows intracranial isoprenaline is attributable to activation of the renal renin-angiotensin system, and that this activation is brought about partly by a direct action of isoprenaline that has leaked out of the brain and partly by increased sympathetic discharge to the kidney. The increased sympathetic discharge is interesting because it is reminiscent of central sympathetic effects produced by blood-borne angiotensin already mentioned (Buckley, 1972). ls it brought about by isoprenaline acting directly on brain structures or is it secondary to leakage of isoprenaline from the brain into the circulation, with consequent release of renal renin and stimulation of brain structures by circulating angiotensin? Interesting and dramatic though the dipsogenic action of isoprenaline may be , it casts little light on possible central catecholaminergic mechanisms of drinking ; isoprenaline is important because it is a way of activating the renal renin-angiotensin system. There is more direct evidence of participation of central catecholaminergic neurones, particularly dopaminergic neurones, in the angiotensin response . This evidence may be summarized as follows: (I) angiotensin causes release or interferes with the re-uptake ofnoradrenaline in the brain (Palaic and Khairallah , 1968); (2) there is a close correlation between the amounts of angiotensin and noradrenaline found in different parts of the brain (Fisher-Ferraro, Nahmod,

Endocrine mechanisms 241 Goldstein, and Finkielman, 1971); (3) angiotensin-induced drinking is markedly reduced by pre-treatment with intracranial 6-hydroxydopamine, which destroys catecholaminergic nerve terminals, whereas carbachol-induced drinking is unaffected (Evetts, Fitzsimons & Setler, unpublished) (Table 5); (4) neither angiotensin-induced nor carbachol-induced drinking is significantly reduced by centrally administered a or f3 adrenergic antagonists, but the dopamine antagonist haloperidol abolishes angiotensin-induced drinking without affecting carbachol-induced drinking (Fitzsimons and Setler, 1971) (Table 4); (5) dopamine, injected into the ventricle in very high doses (260 and 520 n moles) causes significantly higher intakes of water than in control animals , though curiously, injections into brain tissue up to now have been ineffective . Drinking to ventricular injections occurs despite marked weakness and ataxia. This evidence is by no means conclusive and the failure of catecholamines to enhance angiotensin drinking is disappointing, though the fact that catecholamines inhibit some forms of thirst but not angiotensin-induced drinking is more hopeful. 'Yet the awkward paradox remains that while the anti-dipsogenic effects of diminished catecholaminergic activity are profound, the dipsogenic effects of intracranially injected catecholamines are weak, much weaker than the effects of the extracellular thirst stimuli which seem to depend on adequate catecholaminergic function' (Setler, 1973) . THE RENIN-ANGIOTENSIN SYSTEM AND SALT APPETITE

In view of the effect of angiotensin in stimulating aldosterone production, it was and still is attractive to postulate a role for the renin-angiotensin system in Na appetite. Mineralocorticoids, including aldosterone, have been reported to augment consumption of Na solutions by normal rats (e.g., Weisinger and Woods, 1971), and bilateral nephrectomy abolishes formalin-induced Na appetite (Fitzsimons and Stricker, 1971). However, induction of anuria by other means, but leaving the kidneys in situ perfused by the animal's own circulation, also abolishes Na appetite and all efforts to restore the appetite by injecting renal extracts or renin itself, or by stimulating endogenous renin with isoprenaline, have failed (Fitzsimons and Stricker, 1971) . Single-bottle preference tests with water, 0.9, 1.8, or 2.7 per cent NaCl, carried out on rats given intracranial renin or intracranial carbachol revealed no differences in Na preference induced by the two dipsogens; the preference-aversion functions were similar to the curve after a 24-hour period of water deprivation (Fitzsimons 1971b). Fisher (1973) has claimed recently that undeprived animals with continuous access

TABLE 5 Effects of depletion of catecholamines by 6-hydroxydopamine (6-0HDA) on drinking induced in rats by angiotensin or carbachol Mean water intake in one hour After 6-0HDA

Before 6-0HDA Treatment 8 µg 6-0HDA

(preoptic area) 2 X 250 µg 6-0HDA (intraventricular) NA = Noradrenali ne DA = Dopamine * p < 0.05 tN=8

N

Angiotensin 100 ng

Carbachol 300 ng

Angiotensin 100 ng

Carbachol 300 ng

15

6.2

±

0 .8

7.2 ± 0.9

*1.9±0 .5

9.6 ± 0.9

8.2 ± 1.2

6.2±1.1

*2.2 ± 1.5

6.4 ± 1.3

4

- - -- - - - - - ·· ·- - -

% Depletion -NA

DA

Preoptict *30 ± 5 *46 ± 4 Whole brain *75 ± 3 *79 ± 4

Endocrine mechanisms 243 to both isotonic saline and water do show a shift in preference from water to saline in a one-hour test after intracranial angiotensin ( l 0, l 00, and l 000 ng) and a shift from saline to water after intracranial carbachol (0.25, 1, and 2.5 µ,g). In two-bottle preference tests lasting one hour on animals experienced in making a choice between two solutions but not allowed saline between tests, no significant differences were found in the preference-aversion functions of those stimulated with intracranial angiotensin (I, IO, 100, and 1000 ng), intracranial carbachol (3, 30, 300, and 600 ng), subcutaneous isoprenaline (50 and 100 µ,g/kg), or deprivation of water overnight (Fitzsimons, Fig. 6 and unpublished). These results and Fisher's differ partly because Fisher compares the percentage of isotonic saline drunk relative to water after intracranial stimulation with the baseline response of the unstimulated animal, and secondly because his present data refer to a situation in which the animal has to choose between water and isotonic saline, a concentration at which it is difficult to separate the effects of palatability from those of need. The first objection is major in that thirsty animals practically always drink substantial quantities of isotonic saline compared with unstimulated animals. It also seems extremely unlikely that angiotensin by itself would cause an immediate switch to saline, because experience with a variety of extracellular stimuli to thirst shows that water is preferred to saline initially and that the increased salt appetite becomes evident only some 6-12 hours after the stimulation (Fitzsimons, 1969; Stricker, 1973) . An observation of considerable interest because of the suspected relationship between angiotensin and catecholamines is that crystalline noradrenaline introduced into the third ventricle induces rats to choose saline when both water and 1% saline are offered, whereas water is chosen after crystalline acetylcholine (Chiaraviglio and Taleisnik, 1969). Bilateral lesions in the mesencephalon, where there are many ascending noradrenergic fibres, abolishes the increased saline intake to noradrenaline in the third ventricle or to sodium deprivation, whereas stimulation of the mesencephalon by deposition of crystalline FeCb lateral and ventral to the periaqueductal grey induced intake of 1% saline in preference to water (Chiaraviglio, 1972). Acetylcholine, noradrenaline, and dopamine were without effect on saline intake when placed in this part of the mesencephalon . It may be then that any effect of intracranial angiotensin on salt appetite is mediated through some sort of interaction with catecholaminergic circuits in the brain stem, though the evidence for this is weak at the moment. The position regarding the renin-angiotensin system in salt appetite may be summarized as follows. Angiotensin may ultimately be found to stimu-

244 J.T. Fitzsimons 30

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Figure 6 Fluid intake in one hour by rats given intracranial carbachol or angiotensin and offered water and saline to drink .

late salt appetite. The present evidence is that it does not do so by direct action on the brain but that it may do so indirectly through stimulating production of aldosterone by the adrenal cortex. Of course, we still do not know how mineralocorticoids stimulate salt appetite, but here as with angiotensin it seems unlikely that it is by direct action on the brain centres. A unifying hypothesis to explain the paradox that salt appetite may be increased both in the absence of the adrenal cortex and when mineralocorticoids are given in excess would be that increased salt preference depends on diminished salivary Na which is detected by the Na taste receptors on the tongue. Na depletion in adrenal insufficiency results in a reduction in

Endocrine mechanisms 245 Extracellular hypovolia - - - - - - - - - - - - - - - ,

Thirst --------~----Renin-angiotensin--ADH release

!

Na aTetite - - - - - - Aldosrrone V Water intake

Na intake

Na retention

Water retention

Figure 7 An outline of some mechanisms concerned in regulating the extracellular fluid volume . Note how the kidney may influence intake mechanisms through the renin-angiotensin system and aldosterone (from Fitzsimons, 1971a - slightly modified).

salivary Na at source, while excessive amounts of mineralocorticoids reduce salivary Na by increasing Na reabsorption in the salivary duct. The Na taste receptors are definitely important in Na appetite because lesions in the thalamic gustatory relay cause a significant reduction in saline intake by Na-depleted rats (Wolf, 1968), but the neurology of salt appetite is complicated ( see Striker, 1973) and needs much further investigation. CONCLUSIONS

It is becoming evident that the increased intake of water and sodium which follows the loss of body fluids is partly under the control of two endocrine systems, vasopressin and renin-angiotensin-aldosterone, the same two systems that are involved in the renal conservation of water and sodium. The link between the control of intake and excretion seems to centre on the renin-angiotensin system (Fig. 7) and it is appropriate that the kidney, the principal homeostatic organ for fluid balance, should influence both the i'ltake and excretion of water and salt. REFERENCES Adolph, E.F., Barker, J.P., and Hoy, P.A. 1954. Am. J. Physiol., 178: 538-62 Bailey, P. and Bremer, F. 1921. Arch. Inter. Med., 28: 773-803 Barker, J .P. , Adolph, E.F., and Keller, A.D. 1953. Am. J . Physiol., 173: 233-45

246 J. T. Fitzsimons Barlow, E.D. and de Wardener, H.E. 1959. Quart . J. Med., 28: 235-58 Bellows, R.T. 1939. Am. J. Physiol., 125: 87-97 Bellows, R. T. and Van Wagenen, W.P. 1938. J . nerv . ment. Dis., 88: 417-73 Buckley,J.P. 1972. Fed.Proc ., 31: 1332-7 Chiaraviglio, E. 1972. Brain Res., 44: 73-8 Chiaraviglio, E. and Taleisnik, S. 1969. Am. J. Physiol., 216: 1418-22 De Wied, D. 1966. Physiol. Behav., 1: 193-7 Elie, R. and Panis set, J.C. 1970. Brain Res., 17: 297-305 Epstein , A.M., Fitzsimons, J . T., and Johnson, A.K. 1973. J. Physiol. (Lond.), 230: 42-3P - 1974. J . Physiol. (Lond.), 238: 34-5P Epstein, A.N., Fitzsimons, J.T ., and Rolls, B.J. 1970. J. Physiol. (Lond .), 210: 457-74 Fischer-Ferraro, C., Nahmod, V.E., Goldstein, D.J., and Finkielman, S. 1971. J . exp. Med . , 133:353-61 Fisher, A.E . 1973. In A.N. Epstein , H. Kissileff, and E. Stellar(eds.), The Neuropsychology ofThirst . Washington : Winston, pp. 243-78 Fisher, C., Ingram, W.R., and Ranson, S.W. 1938. Diabetes lnsipidus and the Neurohormonal Control of Water Balance: A Contribution to the Structure and Function of the Hypothalamico-Hypophyseal System. Ann Arbor: Edwards Fitzsimons, J .T. 1961. J. Physiol. (Lond.), 159: 297-309 - 1969. J. Physio/ . (Lond .), 201: 349-68 - 1970. J . Physiol. (Lond.), 210: 152-3 - 1971a. In L. Martini and W. F. Ganong (eds.), Frontiers in Neuroendocrinology . New York : Oxford University Press, pp. 103-28 - 1971b. J. Physiol. (Lond.), 214: 295-303 - 1972. Physiol . Rev., 52: 468-561 Fitzsimons, J. T. and Setler, P.E . 1971. J. Physiol. (Lond.), 218: 43-4P Fitzsimons, J. T. and Stricker, E.M . 1971. Nature ( New Biol.), 231: 58-60 Ganten, D., Marquez-Julio, A., Granger, P., Hayduk, K. , Karsunky, K .P., Boucher, R., and Genest, J. 1971. Am. J. Physiol., 221 : 1733-7 Garrigues, M . and Montastruc, P. 1969. Compt. rend. Soc . Biol., 163 : 1432-5 Gilman, A.E. and Goodman, L. 1937. J. Physiol ., (Lond .). 90: 113-24 Grossman, S.P. 1962a. Am. J . Physiol . , 202: 872-82 - 1962b. Am . J . Physiol., 202 : 1230-6 Holmes, J.H. and Gregersen, M .I. 1950. Am . J. Physiol., 162: 326-37 Houpt, K.G. and Epstein, A.N . 1971. Physiol. Behav ., 7: 897-902 Kourilsky, R. 1950. Proc . Roy. Soc . Med., 43: 842-4 Kozlowski, S. and Szczepanska, E. 1969. xnh Congr. Polish Physiol. Soc., Warsaw Kozlowski, S. and Szczepanska-Sadowska, E. 1971. Acta Physiologica Polonica, 22: 799-818 Lehr, D.J ., Mallow, J., and Krukowski, M . 1967. J . Pharmacol. exp. Therap . , 158: 150-63 Leibowitz, S.F. 1971. Proc . Natl . A cad. Sci., 68: 332-4 Mohring, J., Dauda, G., Haack, D., Homsy, E., Kohrs, G., and Mohring, B. 1972. Life Sci., Part I, II : 679-83 Mohring, J., Schomig, A., Brekner, H., and Mohring , B. 1972. Life Sci . , Part 1, II: 65-72 Palaic, D. and Khairallah, P.A. 1968. J. Neurochem . , 15 : I 195-1202 Pasqualini, R.Q. and Codevilla, A. 1959. Acta Endocrino/., 30: 37-41 Peart, W.S . 1969. Proc. Roy . Soc. (Lond .), 173: 317-25 Peskar, B., Meyer, D.K., Tauchmann, U., and Hertting, G. 1970. European J. Pharmacol., 9: 394-6

Endocrine mechanisms 247 Pickford, M. 1939. J . Physiol. (Lond.), 95: 226-38 Radford , E.P. 1959. Am. J. Physiol., 196: 1098-1108 Rolls, B.J. 1971.J. Physio/ . (Lond.), 219 : 331-9 Setler, P.E. 1973. In A.N . Epstein, H . Kissileff, and E. Stellar (eds.) , The Neuropsychology of Thirst. Washington : Winston, pp. 279-91 Severs, W.B. , Summy-Long, J., Taylor, J.S., and Connor, J .D. 1970. J . Pharmacol. exp . Therap., 174:27-34 Share, L. 1969. In W .F . Ganong and L. Martini (eds.), Frontiers in Neuroendocrino/ogy. New York : Oxford University Press, pp. 183-210 Simons, B.J. 1968. Nature (Lond.), 219 : 1061-2 Slangen , J .L. and Miller, N .E. 1969. Physio/. Behav . , 4: 543-52 Smith, R.W. and McCann, S.M . 1964. In M.J . Wayner (ed.), Thirst. Oxford: Pergamon, pp. 381-92 Stricker, E.M . 1973. In A. N. Epstein, H. Kissileff, and E. Stellar(eds .), The Neuropsychology of Thirst . Washington : Winston, pp. 73-98 Szczepanska-Sadowska, E . 1973. Pjliigers Archiv . , 338: 313-22 Verney, E .B. 1947. Proc. Roy. Soc . London, Ser. , B, 135: 25-106 Volicer, L. and Loew, C.G. 1971. Neuropharmacol . , 10: 631--o Weisinger, R.S . and Woods, S.C . 1971. Endocrinology, 89: 538-44 Wolf, G . 1968. Physiol . Beha,·., 3: 997-1002

Electrophysiological studies of the mechanisms that initiate ingestive behaviours with special emphasis on water intake G .J. Mogenson

Much of what is known about the central control of ingestive behaviours has come from lesion and stimulation stud'ies. However, during the last few years electrophysiological techniques have been used increasingly to investigate the neural mechanisms that initiate drinking and feeding. The main contribution of electrophysiological studies has been to implicate several neural structures in the initiation of drinking and feeding behaviour, in most cases confirming previous results of lesion and stimulation studies. These investigations are considered in the first section. Another goal of electrophysiological studies, which so far has been accomplished only rarely, is the identification of receptors involved in providing the relevant information to the central nervous system. Although specific receptors are difficult to identify, recordings have been made from osmosensitive neurons and from neurons that respond to other thirst signals, and these studies are discussed in the second section. Another aspect of the central mechanisms for thirst and hunger to which electrophysiological techniques are likely to make important contributions is the investigation of the functional interrelationships of the various levels of the brain (midbrain, hypothalamus, limbic forebrain) that are involved in the initiation of drinking and feeding . Attempts have been made using electrophysiological techniques to determine the pathways and the manner in which one struc-

Electrophysiological studies 249 ture influences another for the initiation of drinking and feeding. Examples of such studies are considered in the final section. NEURAL STRUCTURES IMPLICATED IN THE CONTROL OF DRINKING

The first observations of the electrophysiological correlates of thirst and hunger were mass recordings from ventromedial (Brobeck et al., 1956; Brooks, 1959; Hockman, 1964) and lateral (Anand et al., 1962; Hockman, 1964; Steiner, 1962) hypothalamus, from the reticular formation (Hockman, 1964), the septum (Steiner, 1962), the hippocampus (Sadowski and Longo, 1962), and prepyriform cortex (Freeman, 1962; Sutton, 1967). Steiner (1962) observed that the electrical activity recorded from the hypothalamus, septum, and cerebral cortex in chronically prepared animals was a low-voltage, high-frequency pattern when rats were thirsty and a high-voltage, low-frequency pattern when they were satiated (Fig. la). Hockman (1964) recorded similar changes from the hypothalamus and reticular formation in food deprived rats. Steiner and Hockman suggest that the deprivation state, thirst or hunger, enhances neural arousal mediated by the reticular activating system, consistent with an activation theory of motivation (Hebb, 1955; Lindsley, 1951), whereas Sutton maintained that the increased amplitude of electrical activity recorded from prepyriform cortex was not merely a reflection of arousal but specific to food deprivation. Another electrophysiological correlate of ingestive behaviour has been designated post-reinforcement synchronization of the EEG. After cats had made an operant response to obtain food and while consuming the food, 'a high voltage slow burst of EEG synchronization (100-150 µv, 4-8 cycles/sec)' was recorded from the occipital and parietal cortices (Buchwald et al., 1964; Clemente et al., 1964), but not from the sensory motor cortex, suggesting 'that they are not directly related to the muscular activity instant to the act of drinking' (Buchwald et al., 1964, p. 830). The synchronized waves usually appeared 10-30 sec after the cat began drinking (Buchwald et al., 1964) and it has been suggested that they might be related to satiety processes (Clemente et al., 1964); 'Satiety may be the expression of a type of internal inhibition which, after consummation is achieved, plays an important role in bringing innate behavior to an end' (Magoun, 1962). This cortical synchronization is apparently not specific to feeding, however, since the relief or 'reward' of withholding painful shock is also accompanied by a similar EEG synchronization. In some cases EEG

250 G.J. Mogenson

A

DEPRIVATION

SATIATION

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Figure 1 (A) EEG recorded from the lateral hypothalamus and cerebral cortex of the rat when deprived and when satiated (from Steiner, 1962). (B) A synchronized EEG associated with food reinforcement recorded from the cat. Note that the synchronized potentials occur when a light that signals food is presented (from Roth, Sterman, and Clemente). Reprinted with permission from Electroenceph . clin . Neurophysiol .. 23 : 509-20, 1967.

synchronization, although appearing after the lever press response, preceded the food (see Fig. I b). This suggests that the EEG synchronization might also reflect the anticipation of food, an effect studied more recently in investigations by Olds and co-workers (see below). Recordings have been made of the activity of single neurons in areas believed to contain osmoreceptors or to play some role in the control of water balance. Standard microelectrode (Cross and Green, 1959) as well as microiontophoretic (Oomura et al., 1969) techniques have been used to study osmosensitive cells in the hypothalamus. Osmosensitive cells have been identified in the region of the supraoptic nucleus (Vincent et al., 1972a; Vincent and Hayward, 1970) and in regions of the brain implicated in the control of water intake, notably in the lateral hypothalamus (Oomura et al . , 1969; Vincent et al., 1972a), a site from which drinking is elicited by electrical stimulation (Mogenson, 1969), in the anterior hypothalamuspreoptic region, a site where osmotic stimulation elicits drinking (Blass and Epstein, 1971; Peck and Navin, 1971), and in the septum (Bridge and Hatton, 1973).

Electrophysiological studies 251 Changes in firing rates during thirst and hunger have also been observed for neurons that are not osmosensitive, in the reticular formation , thalamus, hippocampus, cingulate gyrus, and basal ganglia (Olds et al., 1969; Olds and Hirano, 1969; Phillips and Olds , 1969 ; Travis and Sparks, 1967). Presumably these neurons are not concerned with detecting primary thirst and hunger signals, but it is difficult to determine whether the changes are related to 'associative, motivational, emotional or motor mechanisms ' (Travis and Sparks, 1967, p. 176). One criterion that has been suggested in helping to decide whether a neuron has a specific role in a particular motivational state (e.g. , thirst) is the differential response to water, food, and other motivational stimuli. Travis and Sparks (1967) observed some neurons in the globus pallidus and in the region of the anterior commissure that responded differentially to tones which preceded food or electric shock and concluded that these 'reflect "specific" rather than "generalized" influences' (p . 171). Olds et al. ( 1969) reported an increase of firing rates of neurons in the reticular formation and, since the effects were not related to whether water or food was presented, they suggested that these were non-specific neural changes, perhaps related to arousal or a neuromuscular preparatory response . For some neurons in the hippocampus, however, they did observe differential changes in firing rates prior to the presentation of water and food (see Fig. 2a,b,c). Since this effect could be reversed or appeared one day and disappeared the next, they suggested that the hippocampus, which has been implicated in learning and memory, provides a 'temporary representation' of the anticipated water or food . This interpretation is supported by the observation that an auditory stimulus conditioned to the presentation of food produces large increments in the discharge rate of hippocampal neurons, with only small changes in frequency of firing of neurons of the reticular formation (Olds and Hirano, 1969). Since hippocampal neurons showed large differences during 'food-waiting' and 'water-waiting' periods , these authors favour the view that 'the neurons appeared to be involved in specific anticipatory representations rather than in general activation, inhibition, or arousal' (Olds and Hirano, 1969, p. 164) . Differential responses following drinking and feeding have also been demonstrated with DC potential recordings obtained between the occipital region of the cerebral cortex and the skull (Kawamura et al., 1967) of rabbits. Ten or 15 minutes after drinking there was a positive shift of the DC potential , whereas after feeding there were negative DC shifts . Kawamura and co-workers suggest that drinking and feeding alter osmolarity of the blood and influence blood-brain barrier potentials.

252 G.J . Mogenson

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Figure 2 Changes in the discharge rates of neurons of the hippocampus prior to food or water reinforcement are shown in Band C. In each case there is differential responding depending on whether food or water is anticipated. (A) The unit discharges faster when food is anticipated. (B) The unit discharges faster when water is anticipated. (C) The unit discharges faster when food is anticipated and slower when water is anticipated (after Olds, Mink , and Best. 1969). (D) Effect of electrical stimulation oft he LH on the discharge rate of a neuron in the vM (top) and effect of electrical stimulation of the VM on the discharge rate of a neuron in the LH (after Oomura et al . , 1967). (E) Correlation plot for discharge rates of neurons in the VM and LH. The activity of neurons in the two regions is reciprocally related. When discharge rates increase in the VM they decrease in the LH and vice versa (after Oomura :::,