Steroid Hormones and Brain Function [Reprint 2020 ed.] 9780520318625

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UCLA F O R U M IN M E D I C A L SCIENCES VICTOR E . HALL, JUDITH HELLER, M A R T H A AXELROD,

Editor

Assistant Editor Editorial Assistant

EDITORIAL BOARD

Forrest H. Adams Mary A. B. Brazier Carmine D. Clemente Louise M. Darling Morton I. Grossman

William P. Longmire H. W. Magoun Sidney Roberts Emil L. Smith Reidar F. Sognnaes

UNIVERSITY OF CALIFORNIA, LOS ANGELES

STEROID HORMONES AND BRAIN FUNCTION

UCLA FORUM IN MEDICAL SCIENCES NUMBER 15

STEROID HORMONES AND BRAIN FUNCTION Proceedings of a Conference held May 24-27, 1970 Sponsored by the School of Medicine and the Brain Research Institute, University of California, Los Angeles

CHARLES H. SAWYER and ROGER A. GORSKI EDITORS

UNIVERSITY OF CALIFORNIA PRESS BERKELEY LOS ANGELES L O N D O N 1971

EDITORIAL NOTE

The present volume contains the proceedings of a symposium on Steroid Hormones and Brain Function, organized by Charles H. Sawyer and Roger A. Gorski of the Department of Anatomy, UCLA School of Medicine. Acknowledgement for the support of this conference is owed to the following: Abbott Laboratories, CIBA Pharmaceutical Company, the Kroc Foundation, Eli Lilly & Company, Ortho Pharmaceutical Corporation, G. D. Searle & Company, Schering Corporation, Schering AG, Wallace Laboratories and The Squibb Institute for Medical Research. The conference was co-sponsored by the International Brain Research Organization (IBRO/UNESCO).

CITATION FORM Sawyer, C. H. and Gorski, R. A. (Eds.), Steroid Hormones and Brain Function. U C L A Forum Med. Sci. N o . 15, Univ. of California Press, L o s Angeles, 1971

University of California Press Berkeley and Los Angeles, California

University of California Press, Ltd. London, England

© 1971 by The Regents of the University of California I S B N : 0-520-01887-7 Library of Congress Catalog Card Number: 77-633321 Printed in the United States of America

PARTICIPANTS IN THE CONFERENCE ROGER A. GORSKI, Co-Chairman and Editor Department of Anatomy, UCLA School of Medicine Los Angeles, California 90024 CHARLES H. SAWYER, Co-Chairman and Editor Department of Anatomy, UCLA School of Medicine Los Angeles, California 90024

YASUMASA A R A I

Department of Anatomy, Juntendo University School of Medicine Hongo, Tokyo, Japan CHARLES A . BARRACLOUGH

Department of Physiology, University of Maryland School of Medicine Baltimore, Maryland 21201 F R A N K A . BEACH

Department of Psychology, University of California Berkeley, California 94720 CARLOS BEYER

Departamento de Investigación Científica Apartado Postal 73-032 México 73, D. F. México KEITH BROWN-GRANT

Department of Human Anatomy, Oxford University Oxford, England LYNWOOD CLEMENS

Department of Zoology, Michigan State University East Lansing, Michigan 48823 V A U G H N CRITCHLOW

Department of Anatomy, Baylor University College of Medicine Houston, Texas 77025

BARRY A . CROSS

Department of Anatomy, University of Bristol Medical School Bristol, England JULIAN DAVIDSON

Department of Physiology, Stanford University Stanford, California 94305 ELEMER E N D R Ó C Z I

Medical Research, Post Graduate Medical School Budapest, Hungary JOHN W . EVERETT

Department of Anatomy, Duke University School of Medicine Durham, North Carolina 27706 JACQUES FAURE

Chaire de Physiopathologie et Neurophysiologie Faculte de Medecine et de Pharmacie PI. de la Victoire, Poste 84 Bordeaux, France SHAUL FELDMAN

Department of Neurology Hadassah University Hospital Jerusalem, Israel BELA F L E R K Ó

Anatomical Institute, University Medical School Pécs, Hungary W . F . GANONG

Department of Physiology, University of California San Francisco, California 94122 ROBERT G O Y

Oregon Regional Primate Research Center Beaverton, Oregon 97006 JAMES N . H A Y W A R D

Department of Anatomy, UCLA School of Medicine Los Angeles, California 90024 ROBERT I . H E N K I N

National Institutes of Health Bethesda, Maryland 20014

G U N N A R HEUSER

UCLA School of Medicine Los Angeles, California 90024 JESSAMINE HILLIARD

Department of Anatomy, UCLA School of Medicine Los Angeles, California 90024 MASAZUMI KAWAKAMI

Department of Physiology, Yokohama University Urafune-Cho, Minami-Ku Yokohama, Japan BARRY KOMISARUK

Institute of Animal Behavior Rutgers, The State University Newark, New Jersey 07102 ROBERT D . LISK

Department of Biology, Princeton University Princeton, New Jersey 08540 LUCIANO M A R T I N I

Department of Pharmacology, Università Degli Studi 20129 Milano, Italy S. M . MCCANN

Department of Physiology, University of Texas Southwestern Medica] School Dallas, Texas 75235 BRUCE M C E W E N

The Rockefeller University New York, New York 10021 BENGT J . MEYERSON

Department of Pharmacology, University of Uppsala Uppsala, Sweden J O H N MONEY

Department of Psychiatry and Pediatrics Johns Hopkins University Baltimore, Maryland 21205 PRESTON L . PEARLMAN

Director of Biological Research Schering Corporation Bloomfield, New Jersey 07003

D O N A L D PFAFF

The Rockefeller University New York, New York 10021 V . DOMINGO RAMÍREZ

Department of Physiology, Austral University Valdivia, Chile FELIX A .

STEINER

Department of Experimental Medicine F. Hoffmann-LaRoche & Co. Ltd. 4002 Basle, Switzerland WALTER E . STUMPF

Laboratories for Reproductive Biology University of North Carolina Chapel Hill, North Carolina 27514 A N N A TAYLOR

Department of Anatomy, U C L A School of Medicine Los Angeles, California 90024 SAMUEL TALEISNIK

Instituto de Investigación Medica Mercedes y Martin Ferreyra Cordoba, Argentina ANTONIA VERNADAKIS

Department of Psychiatry University of Colorado School of Medicine Denver, Colorado 80220 RICHARD E . W H A L E N

Department of Psychobiology, University of California Irvine, California 92664

PREFACE The steroid hormones of the testes, ovaries and adrenal glands exert profound influences on the pituitary gland to control their own secretion via the release of gonadotropins and A C T H . The steroids also influence behavior. Both of these phenomena, the control of behavior and the stimulation and inhibition of pituitary secretion, involve the brain. The effects of hormones on the brain have been studied by a wide variety of methods including electrophysiology, psychology, autoradiography, labeled protein binding techniques, and steroid chemistry, with the hormone applied systemically or locally to cerebral neurons by stereotaxic implantation or ionotophoresis. Fresh applications of these methods were discussed authoritatively by more than 30 experts from 12 countries assembled in Los Angeles for a three-day workshop conference on "Steroid Hormones and Brain Function." Emphasis was placed on the differential effects of the steroids in the fetus, the newborn, the prepubertal and the mature subject. Sites and mechanism of the feedback actions of the steroids were given broad consideration in their facilitatory and inhibitory effects on both behavior and pituitary secretion. Special emphasis was placed on the sterilizing and behavioral effects of early androgen treatment. Finally, the clinical implications of steroid deficit and hypersecretion were discussed. The wealth of new data leave no doubt that certain regions of the brain are influenced directly by steroid hormones with references both to behavior and to control of pituitary secretion by the hypothalamic "releasing factors." The conference was co-sponsored by the U C L A F o r u m in Medical Sciences, the U C L A Brain Research Institute and the International Brain Research Organization ( I B R O / U N E S C O ) . Dr. Victor E. Hall, Editor-in-Chief of the U C L A F o r u m publications, played a very active role in editing the manuscripts. Mrs. Judith Heller, Assistant Editor, skillfully managed the major assignment of assembling the manuscripts, discussions, corrections, figures and proofs into a unified volume. Without their invaluable services the book could not have been published in a reasonable period of time. Financial support for the conference by U C L A Forum funds was supplemented by generous gifts from Abbott Laboratories, C I B A Pharmaceutical Company, the Kroc Foundation, Eli Lilly & Company, Ortho Pharmaceutical Corporation, G. D. Searle & Company, Schering Corporation, Schering A G , Wallace Laboratories, and The Squibb Institute for Medical Research. C. Sawyer and R. Gorski

CONTENTS 1. STEROID HORMONES AND BRAIN F U N C T I O N :

PROGRESS,

PRINCIPLES,

AND

1

PROBLEMS

Roger A. Gorski ELECTROPHYSIOLOGICAL AND BEHAVIORAL INFLUENCES OF ADRENAL STEROIDS 2 . EFFECTS OF CORTISOL ON SINGLE CELL ACTIVITY IN THE HYPOTHALAMUS

27

Shaul Feldman 3 . INFLUENCE OF CORTISOL ON BRAIN AND SPINAL C O R D EXCITABILITY IN D E -

35

VELOPING R A T S

Antonia

Vernadakis

4 . LOCAL EFFECTS OF ADRENAL STEROIDS ON CEREBRAL NEURONS

43

Felix A. Steiner 5 . SELECTIVITY OF A C U T E FEEDBACK EFFECTS OF CORTICOSTEROIDS ON A C T H

51

SECRETION

Vaughn Critchlow 6 . PITUITARY-ADRENOCORTICAL ACTIVITY, EXPLORATION AND AVOIDANCE BE-

59

HAVIOR IN THE R A T

Elmer Endroczi 7 . MODIFICATION OF THE RESPONSIVENESS OF COMPONENTS OF THE L I M B I C - M I D -

67

BRAIN CIRCUIT BY CORTICOSTEROIDS AND A C T H

Anna Newman Taylor, G. Keith Matheson and N. Dafny ELECTROPHYSIOLOGICAL CORRELATES OF SEX STEROID ACTIONS 8. EFFECTS OF SEX HORMONES AND OVULATION-BLOCKING STEROIDS AND D R U G S

79

ON ELECTRICAL ACTIVITY OF THE R A T BRAIN

Masazumi Kawakami, Ei Terasawa, Tomoko Ibuki and Manaka

Mikihiko

9 . CYCLIC CHANGES IN NEURONS OF THE ANTERIOR HYPOTHALAMUS D U R I N G

95

THE R A T ESTROUS CYCLE, AND THE EFFECT OF ANESTHESIA

Barry A. Cross and R. G. Dyer 10. STEROID SEX HORMONES IN THE R A T BRAIN: SPECIFICITY OF U P T A K E AND

103

PHYSIOLOGICAL EFFECTS

Donald W. Pfaff 11. EFFECTS OF ESTROGEN ON SINGLE U N I T ACTIVITY IN THE HYPOTHALAMUS OF THE BEHAVING RABBIT

Jacques Faure and J. D. Vincent

113

CONTENTS 12. EFFECT OF ESTROGEN ON BRAIN STEM NEURONAL RESPONSIVITY IN THE CAT

121

Carlos Beyer 13. INDUCTION OF LORDOSIS IN OVARIECTOMIZED RATS BY STIMULATION OF THE

127

VAGINAL CERVIX: HORMONAL AND NEURAL INTERRELATIONSHIPS

Barry A. Komisaruk 14. DISCUSSIONS OF ELECTROPHYSIOLOGICAL CORRELATES OF THE ACTION OF STE-

137

ROID HORMONES

PERINATAL INFLUENCES OF STEROIDS ON HYPOTHALAMOHYPOPHYSIAL FUNCTION AND SEXUAL BEHAVIOR 15. HORMONES AND THE ONTOGENESIS OF PITUITARY REGULATING MECHANISMS

149

Charles A. Barraclough 16. ANDROGENIZATION AND PITUITARY F S H REGULATION

161

Be la Flerko 17. SEX DIFFERENCE IN HYPOTHALAMO-HYPOPHYSIAL FUNCTION

171

Samuel Taleisnik, L. Caligaris and J. J. Astrada 18. SOME ASPECTS OF THE MECHANISMS INVOLVED IN STEROID-INDUCED STERILITY

185

Yasumasa Arai 19. THE EFFECTS OF TESTOSTERONE PROPIONATE ADMINISTERED BEFORE BIRTH ON

193

THE DEVELOPMENT OF BEHAVIOR IN GENETIC FEMALE RHESUS MONKEYS

Robert W. Goy and C. H. Phoenix 2 0 . PERINATAL HORMONES AND THE MODIFICATION OF A D U L T BEHAVIOR

203

Lynwood G. Clemens HORMONES AND CONTROL OF SEXUAL BEHAVIOR IN THE ADULT 2 1 . HYPOPHYSIOTROPIC NEURONS IN THE PERIVENTRICULAR BRAIN: TYPOGRAPHY

215

OF ESTRADIOL CONCENTRATING NEURONS

Walter E. Stumpf 2 2 . THE PHYSIOLOGY OF HORMONE RECEPTORS: PATTERNS OF UPTAKE AND R E -

227

TENTION OF ESTRADIOL AND PROGESTERONE IN RELATION TO REPRODUCTIVE CAPABILITY

Robert D. Lisk and Leonard A. Ciaccio 2 3 . OPTICAL ISOMERS OF ESTROGEN AND ESTROGEN-INHIBITORS AS TOOLS IN THE

237

INVESTIGATION OF ESTROGEN ACTION ON THE BRAIN

Bengt J. Meyerson 2 4 . BIOCHEMICAL STUDIES OF CORTICOSTERONE BINDING TO CELL NUCLEI AND CYTOPLASMIC MACROMOLECULES IN SPECIFIC REGIONS OF THE R A T BRAIN

Bruce S. McEwen, Carew Magnus and Gislaine Wallach

247

CONTENTS 2 5 . DISCUSSIONS OF THE ACTION OF STEROID HORMONES ON NEURONAL F U N C -

259

TION

STEROID FEEDBACK A N D BRAIN-PITUITARY

MECHANISMS

2 6 . THE ROLE OF STEROID HORMONES IN THE CONTROL OF GONADOTROPIN

269

SECRETION IN A D U L T FEMALE MAMMALS

Keith

Brown-Grant

2 7 . FEEDBACK MECHANISMS AND THE CONTROL OF THE HYPOTHALAMOHYPO-

289

PHYSIAL COMPLEX

Z. Kniewald, R. Massa, M. Motta, and L. Martini 2 8 . SEX AND BRAIN-PITUITARY FUNCTION AT PUBERTY

V. Domingo

301

Ramirez

2 9 . STUDIES ON THE FEEDBACK ACTIONS OF GONADAL STEROIDS ON GONADOTRO-

311

PIN AND PROLACTIN SECRETION: EFFECTS, SITES AND MECHANISM OF A C TION

S. M. McCann, P. S. Kalra, H. P. G. Schneider, J. T. Watson, K. Wakabayashi, C. P. Fawcett and L. Krulich CLINICAL ASPECTS 3 0 . CLINICAL ASPECTS OF PRENATAL STEROIDAL ACTION ON SEXUALLY DIMORPHIC

325

BEHAVIOR

John Money 3 1 . O N THE MECHANISM OF ACTION OF CARBOHYDRATE-ACTIVE STEROIDS ON

339

TASTANT DETECTION AND RECOGNITION

Robert I. Henkin and D. F. Bradley SUMMATION 3 2 . OVERVIEW AND SUMMARY OF CONFERENCE

355

JULIAN M. DAVIDSON LIST OF ABBREVIATIONS

373

NAME INDEX

375

SUBJECT INDEX

378

1. STEROID HORMONES AND BRAIN FUNCTION: PROGRESS, PRINCIPLES, AND PROBLEMS ROGER A. GORSKI* University of California Los Angeles, California

What are the fundamental principles of neuroendocrinology? Assume the neuron illustrated in Figure 1.1 to be a representative neuroendocrine cell. Essentially by definition it must terminate in the median eminence (ME) in association with portal vessels where it releases its neurosecretory product. This cell is greatly influenced by the environment, and may respond directly to neural input from the external environment, or indirectly through an induced change in the chemical internal environment. Neural input emanating from the internal environment can also influence this cell. In response to this varied input the neuroendocrine cell changes its secretion of releasing (or inhibiting) factors, thus changing pituitary output and ultimately target organ secretion. A fundamental principle of neuroendocrinology is that hormones in turn feed back upon this system and regulate its activity. Gonadal or adrenal steroids can feed back directly upon the pituitary or on to nerve cells. In addition, however, there is evidence to suggest that the pituitary hormones themselves feed back and alter neural function. This has been called internal or short-loop feedback. It is even possible that the releasing factors themselves alter th'eir own production by an "ultra-short" feedback loop. For recent reviews of feedback see references 16, 85, and Chapter 27 in this volume. Another fundamental principle is that hormone action on the brain is not limited to the feedback regulation of pituitary secretion. Hormones also exert a profound influence on the nonendocrine brain functions such as behavior. Since several excellent review texts of this complex field already are available (18, 33, 76, 114), I do not feel compelled to provide yet another comprehensive review. Instead I hope merely to prepare the stage upon which this conference will unfold by considering a few examples of recent progress in neuroendocrine research. Where should I begin a consideration of recent progress? Slightly over seven years ago a conference entitled "The Brain and Gonadal Function" (43) was held in this very room. I think it appropriate, therefore, to relate my remarks to that conference. I want to stress that this was an arbitrary decision rather than evidence that 1963 was a banner * Original research from the author's laboratory supported by USPHS grant No. HD-01182, and a grant from the Ford Foundation. The author would also like to acknowledge the generous gifts of CN-SS, 945-27from the Parke, Davis Research Laboratories, of Actinomycin-D from Merck, Sharpe & Dohme, and of cycloheximide from the Upjohn Company, and the bibliographic services of the UCLA Brain Information Service, which is a part of the National Information Network of the NINDB and supported under contract PH43-66-59.

1

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STEROID HORMONES AND BRAIN

FUNCTION

year for neuroendocrinology. Accepting this as a starting point, one should also be warned that my choice of advances in our field is frankly biased. In any case, where, roughly, was the field of neuroendocrinology at the close of that conference? What has happened since then? And, hopefully, what might we expect in our forthcoming discussions? The Neural Control of the Hypophysis Reichlin (98) opened the 1963 Conference by reviewing the anatomy and function of what he called the "median eminence gland." McCann (77) and Guillemin (46) described their attempts to isolate, purify, and identify separate hypothalamic factors for the various adenohypophyseal hormones. At the present time these hypothalamic factors have reached the stage where it may be justified to call them hormones (78, 101). One of these, t h y r o t r o p i c hormone releasing factor, has been purified from porcine and ovine sources and actually synthesized (10, 13). It can be concluded that the existence of hypothalamic "releasing" factors is firmly established. At the present time attention is turning to the possible mechanisms of release and action of these agents. McCann and his collaborators have conducted a series of studies on the possible role of the biogenic amines in the release of these agents. When pituitary and stalk-ME were incubated with dopamine, there was a significant increase in the release of FSH in vitro (59). Other amines were not effective at comparable doses, and dopamine was without any effect on pituitary tissue incubated in the absence of stalk-ME fragments, thus eliminating the possibility of the direct action of dopamine on the pituitary. Dopamine similarly induced the release of LH when pituitaries were incubated with ME tissue (104). Again, because there was no effect of dopamine directly on the pituitary, McCann and his collaborators have proposed that dopamine may be the neurotransmitter for the release of these two GTH-releasing factors. These investigators have suggested that releasing factor secretion follows transynaptic activation of the L R F cell by dopamine, or that dopamine is released within the ME at axoaxonic contacts and there causes the release of LRF. Finally, in what would amount to an intriguing blend of distinctly neurotransmitter and neurosecretory function, they have suggested that dopamine released from vesicles in nerve terminals may act on the same terminal to cause the release of LRF that might be contained in a second population of vesicles (see Figure 29.5). There is considerable anatomical evidence to support this general view. First of all, the fluorescence technique has been used to advantage to demonstrate that the ME is rich in dopamine-containing nerve endings (30). In addition, Hokfelt (53) has presented electron microscopic evidence that amines are located within vesicles of nerve endings in the ME. On the basis of a quite different type of study, that of isolation of vesicles by centrifugation, Ishii (56) has reported that L R F is associated with subcellular granules. Finally, electron microscope observation of the ME clearly demonstrates the existence within axons of two types of vesicles, electron-lucent synaptic and dense core vesicles (see Figure 1.2). I should emphasize that the precise correlation between vesicular size and content is still unsettled (see reference 25). With the existence of releasing factors firmly established and experiments underway which may elucidate their release mechanisms, it would seem logical to expect a crystallization of our understanding of the physiology of hypophyseal regulation. In

PROGRESS, PRINCIPLES AND

PROBLEMS

3

one sense, however, this does not seem to be the case; the role of direct feedback upon the pituitary has not been eliminated from consideration and probably should not be. Although numerous experiments have demonstrated that ME implants of crystalline hormones alter hypophyseal function, and similar implants into the pituitary are without effect, the conclusion that these experiments establish the neural feedback of steroids is clearly debatable. The principal antagonist of this view has been Bogdanove (9) who in 1963 proposed the "implantation paradox". This argument states that because of the concentration of the vascular supply of the pituitary in the ME, it is possible that a hormonal implant in the ME is a better method of applying hormone to a wide region of the pituitary gland than is direct pituitary implantation of that hormone. Where does this concept stand today? There is one experiment which definitely appears to support neural feedback. Smith and Davidson (108) studied the effect of ME implants of testosterone propionate (TP) in two male rat preparations: in intact males, and in hypophysectomized rats in which testicular weight was maintained above that of hypophysectomized controls by four pituitary grafts placed under the kidney capsule. Presumably such testicular maintenance is supported by gonadotropin secretion depending on circulating levels of releasing factors. In both preparations, implantation of TP suppressed testicular weight. The fact that an ME implant of TP suppressed testicular size and function in this animal without, however, reaching the general circulation (accessory tissues were not stimulated indicating that the implanted hormone did not reach the general circulation) is offered as evidence that androgen, at least, acts at the level of the ME and not necessarily upon the pituitary. Palka, Ramirez and Sawyer (88) reported data which offer a compromise picture. With the use of an ME implant of tritiated estradiol they observed that the label does reach the pituitary in general support of the implantation paradox. Moreover, they reported a significant increase in pituitary weight in response to either intrahypothalamic or intrahypophyseal estrogen. In contrast, only ME implants appeared to facilitate the release of LH as measured by circulating levels five days after implantation of estrogen. They interpreted their data as support for both hypothalamic and hypophyseal sites of estrogen action, but with the regulation of release occurring at the neural level. More recently several experiments have confirmed a direct effect of steroid hormones on the pituitary. In both the rabbit (109) and the rat (110) progesterone renders the pituitary less responsive to the direct infusion of ME extract. Weick et al (123) have observed that intrahypophyseal EB but, interestingly, not intrahypothalamic EB, will advance ovulation in cyclic female rats. It might be argued that the effectiveness of implants away from the ME supports direct neural feedback. This question has recently grown more complex. Docke and Dorner (19) reported that intrahypothalamic implants of EB were more effective in inducing ovulation in the immature female if they were in contact with the third ventricle. Kendall, Grimm and Shimshak (64) have studied the influence of intraventricular implants of Cortisol, which effectively blocked the response to ether stress when the Cortisol was applied to a portion of the ventricular system which will rather directly bathe the ME. They also reported that trypan blue injected into the lateral ventricle reaches the ME region within five to ten minutes. Knigge and Scott (66) have reported that labeled amino acids rapidly reach the pituitary from the ventricular system as well.

4

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AND BRAIN

FUNCTION

The mere transfer of substances in the ventricle beyond the ME is not proof of their function at the pituitary level. With elegant techniques for portal blood collection, and intraventricular injection by an inferior route after visualization of the base of the brain, Porter and his colleagues have demonstrated that dopamine causes the release of L R F (57), PIF (58), and F R F (personal communication). Infusion of dopamine into a cannulated portal vessel, however, is without effect in confirmation of the incubation studies (59, 104), and the results suggest that dopamine is acting at the level of the ME, not beyond. The possibility that substances in the ventricles may act on the ME, or may be transferred to the pituitary, suggests that the floor of the third ventricle may be a particularly important region. In fact, the attention of several laboratories is now focused on a specific anatomical component of this region — the ependymal cell. The ependyma of this region of the third ventricle is intimately in contact with both nerve endings and with the capillary epithelium because of the complex branching of ependymal processes (see 67). At their ventricular surface, these ependymal cells have microvilli which markedly increase their surface area. In scanning micrographs produced by Knigge and Scott (66), one can see the surface characteristics of the lining of the third ventricle at three levels (Figure 1.3). Knowles and Kumar (67) have reported changes in the ventricular surface with the menstrual cycle of the monkey, and Kobayashi and Matsui (71) have observed changes following castration and subsequent estrogen treatment in the ventricular surface of the rat ependyma and in the various inclusions in both the ependyma and the glial cells of this region. Are these cells adapted for absorption of materials from the ventricle, or perhaps for secretion into the ventricle? Thus, as has been suggested (66), it is conceivable that factors produced in various regions of the brain reach the ME via the ventricular circulation where they may act directly or where they may pass rapidly to the pituitary. In this regard, Endroczi and Hilliard (22) have reported the presence of LRF-like substances in extracts of various regions of the brain. This story of the ependyma is not complete, and in spite of our considerable progress in identifying the various releasing factors, rather fundamental aspects of the neurohumoral control of the pituitary appear similarly unclear. Extrahypothalamic Regulation of Pituitary Function In the early 1960's the attention of neuroendocrinologists was not restricted to the ME, but was broadly focused on the hypothalamus. In the female rat study, several techniques led to the development of the view that there was a dual level hypothalamic regulation of LH. The ventromedial-arcuate nuclear region was considered to be responsible for the tonic secretion of GTH, and more anteriorly the preoptic area (POA) was considered to be necessary for ovulation or the cyclic release of GTH (6). Flerko (27) also proposed the existence of estrogen sensitive cells active in the control of FSH in this general region. And in the 1963 conference, Lisk (75) reviewed his studies on the implantation of estrogen which supported an important role of the POA in sexual behavior as well. But in that same conference, Taleisnik (115) described the release of LH in response to spreading depression of the cerebral cortex, and Sawyer (100) asserted that the hypothalamus was in intimate relation with other regions of the brain by recounting his studies with Kawakami. Sawyer and Kawakami studied the effects of ovarian

P R O G R E S S , P R I N C I P L E S AND

PROBLEMS

5

hormones on specific brain thresholds, namely, the threshold for cortical arousal upon reticular formation stimulation and the threshold for what they termed the EEG after-reaction upon limbic stimulation. Changes in these thresholds were correlated with behavioral and internal feedback components, respectively, of the rabbit neuroendocrine axis. This work emphasized that the hypothalamic regulation of reproductive function was closely related to activity of extrahypothalamic circuits as well. Study of the role of extrahypothalamic influences in neuroendocrine processes has developed into an area of active research. In this general area, I consider the Halasz technique of deafferentation of the medial basal hypothalamus (MBH) to represent an important advance. As you remember, Halasz et al. (50) initially implanted pituitary tissue into the brain of hypophysectomized rats and found maintenance of both pituitary structure and function only if the pituitary tissue was localized in a rather circumscribed region they then called the hypophyseotrophic area (HTA). If, as they believed, this area represented the site of production and release of the various releasing factors, the next obvious question was how dependent is the activity of this region on afferent neural input? To answer this question Halasz designed his now famous knife which permits one to transect all afferents (and neural efferents as well) leading to the HTA without interrupting this system's efferent route of neuroendocrine interest, the portal vessels. At UCLA, Halasz extended this technique by performing what we called partial deafferentation of the MBH. We investigated the control of G T H (49) secretion in female, and the control of ACTH (51, 52), TSH (48) and STH (see 47) in male rats. The usefulness of these partial deafferentations can be illustrated briefly by reviewing our results on ovulation. In confirmation of the view that the MBH is incapable of supporting ovulation by itself, complete deafferentation prevented ovulation. Again in confirmation of the concept that the POA was especially important for ovulation, we observed that posteriorly incomplete deafferentation did not block ovulation, while by merely transecting neurons passing from the general area of the POA back to the MBH, that is the anterior deafferentation, ovulation was blocked. Figure 1.4 schematically summarizes our results with this knife. It appears that the dual level hypothesis proposed initially for LH (6) and FSH (27) can be more widely applied. After complete deafferentation, the only MBH function which appears to be depressed is TSH secretion, while in our hands, ACTH secretion was elevated although ether stress was still effective. Complete deafferentation also abolished the daily fluctuation in ACTH secretion as determined by changes both in pituitary content of ACTH and in plasma corticosterone. This block in daily change appeared to be due to an elevation of normally low morning samples possibly due to the transection of inhibitory afferents. Recently, Palka, Coyer, and Critchlow (87) have confirmed the absence of the diurnal rhythm. Reference to gonadal steroid and thyroxin feedback is purposely vague in this figure since hormone-sensitive elements active in the regulation of these hormones appear to lie at approximately the level of the anterior cut. Of course, as I have already indicated, ovulation is dependent upon anterior input. In general terms, one may consider the cells of the MBH as one level, in fact the final level of neural control of pituitary activity. Obviously this is a gross oversimplification; there may well be functional differences within this region. Above this level we still find considerable regulation of pituitary activity, in fact, probably most of the activity which correlates the external environment with pituitary secretion. As a broad

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AND BRAIN

FUNCTION

generalization, however, all the neural tissue outside of the MBH represents a potential release-regulating system as summarized in the final chapter of the Hungarian monograph (114). Is it possible to identify components of the brain which are specifically involved with any one aspect of MBH function? An obvious example would be the control of ovulation since the POA has been already specified as critical for the cyclic release of GTH. By varying the position and size of the partial deafferentations, Halász and I observed that the percentage of animals still able to ovulate also varied (49). One explanation of these results would be that the anatomical substrate for ovulation is more diffuse anteriorly but converges toward the ME. This concept agrees well with that of Everett (23) who proposed a similarly converging septopreopticoanterior hypothalamico-ME pathway for ovulation on the basis of the size of the electrolytic lesion needed to induce ovulation. Thus, in a contemporary concept of hypothalamic localization of LH release mechanisms, we see that the POA cyclic LH release system is under the influence of an open funnel extrahypothalamic system which might well be labeled Higher Neural Control (Figure 1.5). Several recent studies emphasize the diffuse nature of the tissues which can influence ovulation. Electrolytic stimulation of the arcuate nucleus (6, 15, 63, 116), medial POA (23, 54, 63, 116), amygdala (12, 63) and septum and bed nucleus of the stria terminalis (63) have all been shown to induce ovulation. Velasco and Taleisnik (119) demonstrated that chemical stimulation with carbachol, or electrochemical stimulation of the amygdala induced ovulation in the rat. On the other hand, stimulation of the hippocampus blocked both spontaneous ovulation and the release of LH in response to amygdaloid stimulation (120). In the deermouse, the amygdala also appears to influence GTH secretion (21). Thus, in the scheme of Figure 1.5, is the POA truly an integrative center for ovulation, or does it merely lie within an important pathway? This question is difficult to answer, but recently Halász (47) has reported that an anterior deafferentation placed rostral to the POA, is consistent with at least irregular but spontaneous ovulation in the few animals which survive the surgery. It is likely that the POA does perform an integrative function for ovulation, and although highly dependent upon afferent input for appropriate timing in relation to other events or to the environment, it may well be the location of the spontaneous trigger for ovulation. Finally, to return to the biogenic amines for a moment, in addition to the possible role of dopamine within the ME, numerous studies recently reviewed by Fuxe and Hokfelt (31) report changes in the biogenic amines with various neuroendocrine events. Suffice it to say that the complex anatomy of the biogenic amine system within the CNS supports the role of complex neural circuits in the control of pituitary function (1). The neuroendocrinologist of today cannot restrict his attention to the hypothalamus. Localization of Steroid Receptors Although the concept that neurons of the hypothalamus must be sensitive to changes in steroid levels was generally accepted, the conference in 1963 contained only one reference to the selective uptake of hormones by neurons (82). Since that time several investigators have measured the uptake of labeled estrogen by scintillation counting and have suggested that the hypothalamus, in contrast to other brain areas, behaves like a peripheral target organ in that the steroid is retained (20, 29,41, 62, 99). Recently

PROGRESS, PRINCIPLES AND

PROBLEMS

7

the technique of autoradiography has been used to localize possible steroid receptive neurons (2, 92, 111). Although much more on this subject appears in Dr. Stumpfs presentation (see Chapter 21), I would like to mention several points in this place. First is the apparent nuclear localization of the exposed silver grains, which indicate a tagged molecule. Second is the fact that the hypothalamic neurons which appear to concentrate estrogen most readily are located in precisely those areas which we have proposed to be important in the control of GTH secretion (Figure 1.5). Anderson and Greenwald (2) also observed that a specific region of the amygdala also retained estrogen. Stumpf (111) has reported that a larger area of the amygdala takes up labeled estrogen, and as his autoradiographs are allowed to develop for longer periods before processing, he observes some uptake in the hippocampus as well (see Chapter 21). Pfaff (92) has already reported a rather general distribution of label following estrogen administration. Thus, in any scheme for the control of GTH secretion, we now must consider the possibility that some or all of the extrahypothalamic input is steroidsensitive as well. A very basic question remains unanswered. What is the relation between steroid uptake and function? With uptake restricted to regions of the brain shown by other techniques to be functionally important in feedback regulation, one feels confident in assigning significance to this uptake. However, is this technique sufficiently specific to suggest that we must incorporate into our thinking all areas that pick up and retain labeled hormone? In this regard it is interesting to note the recent data of Kato and his colleagues on the change in the in vivo (61) and in vitro (60) uptake of tritiated estradiol with the estrous cycle. The observation that uptake in the anterior hypothalamus appears to be specifically decreased in proestrus implies to these authors that the anterior hypothalamus plays a role in the regulation of the estrous cycle by virtue of estrogen feedback upon receptors located there. Uptake in the pituitary, the cerebellum, or the mid and posterior hypothalamus did not change with the estrous cycle, indicating that these areas do not participate in this regulation, or that uptake mechanisms are different since endogenous estrogen does not compete for binding sites in these areas. Estimation of Plasma Hormone Levels In the conference in 1963, several references were made to the then relatively new and precise bioassay method for LH, the ovarian ascorbic acid depletion assay of Parlow (59). Although specific, this assay method still was unable to detect basal plasma levels in the intact laboratory rodent, an animal with perhaps the richest store of biological data. Nevertheless, with this assay a dramatic fall in pituitary LH was detected after ovulation (42, 69, 106) and LH was detected in the plasma in relation to the critical period for ovulation (69, 79, 107). Ramirez and Sawyer (97) also reported changes in L R F content within the stalk-ME during the estrous cycle. Recently the field of hormone assay has been stimulated by the development of sensitive radioimmunoassays for pituitary hormones, as well as by more precise assays for the steroids. Neuroendocrinologists, and of course endocrinologists, now appear to possess tools which will enable them to determine plasma levels of various hormones under physiological conditions and simultaneously in the same animal. It is now firmly established, for example, that ovulation is preceded by a monumental burst of LH secreted into the plasma (34, 35, 83, 84, 90; see also Chapter 26).

8

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HORMONES

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FUNCTION

As one might have predicted, however, the development of the radioimmunoassay has also given rise to new problems. Although FSH was classically assumed to be high during the preovulatory phase of the estrous cycle, or at least relatively constant, bioassay results suggested that FSH secretion changed dramatically with the cycle (37, 80). Recent radioimmunoassay data confirm that FSH is low throughout the cycle except at the critical period just prior to ovulation (34, 90). The increase in FSH is not as dramatic as that for LH, and is less sharp since plasma FSH remains elevated even on the day of estrus. Studies of Goldman and Mahesh (38) with antibodies to pituitary hormones, have suggested that FSH may well be an active component of an ovulating complex of hormones. Recent experiments have also detected a marked increase in the secretion of LTH at the time of ovulation in the virgin rat (34, 86). Is this hormone also part of an ovulatory complex? In one sense the accurate measurement of GTH levels in the plasma appears to challenge the concept of the fine neural regulation of pituitary function. After having painstakingly documented the existence of that neural control, are we now faced with the concept that the hypothalamus literally dumps its content into the portal blood and causes a vast overreaction of the pituitary? Stress, mating, and now the ovulatory critical period seem to result in the simultaneous secretion of several pituitary hormones. Is this almost chaotic picture consistent with the view of precise neural regulation? Obviously, whatever the consequences to contemporary hypotheses, the opportunity to measure accurately plasma levels of hormones is welcome. At the present time such accurate measurements are not restricted to pituitary hormones, and it is now possible to begin to consider the interrelations between plasma levels of steroids and pituitary activity. Focusing on ovulation again, we can say with confidence that estrogen levels in the plasma increase prior to the ovulatory discharge of GTH; thus, the concept that estrogen facilitates the release of ovulating hormones seems to be supported by current data (26, 55, 70, 105, 125; see also Chapter 26). On the other hand, the possible role of progesterone in ovulation is still controversial. Several reports have shown that progesterone in the plasma does not begin to rise until after the surge of LH secretion has begun, or at least, the increase in progesterone seems to be coincident with G T H activity (65, 118). Ferin et al. (26), using antibodies to steroid hormones also suggest that progesterone plays no critical role in the preovulatory state. On the other hand, Goldman et al. (36) have indicated that progesterone could be increasing just before the critical period in rats, and the ability of exogenous progesterone to facilitate ovulation is well known (24, 32). Although the existence of a preovulatory secretion of progesterone, and its possible role in spontaneous ovulation is controversial, work of more biochemically oriented endocrinologists suggests a further complication. It has been suggested that testosterone is converted within the nuclei of the prostate into dihydrotestosterone, which may be the physiologically active form of this steroid (11). In very recent experiments, Armstrong and King (4) have suggested that progesterone may be converted to its active 5-alpha (5a-pregnan-3, 20-dione) form within uterine nuclei. Of great potential significance, if it is found to apply to the brain as well as the uterus, is the further observation that estrogen appears to influence the enzyme which converts progesterone

PROGRESS, PRINCIPLES AND

PROBLEMS

9

to the 5-alpha form. Is it possible, therefore, that a rising preovulatory titer of estrogen might, through enzyme induction, increase the concentration of the intracellularly active form of progesterone in spite of a constant plasma level of progesterone? Thus, in this day when we can accurately measure circulating hormones in the blood of the normal animal, are we witnessing the birth of a demand to know the precise level of the active form of a hormone in its target tissue, perhaps even at the subcellular level? Sexual Differentiation of the Brain At the time of the 1963 conference, it was becoming well established that androgenization permanently changed the hypothalamic regulation of ovulation in the female, and, as well, her expression of female sexual behavior. In the intervening years numerous investigators have studied this problem, which has now developed into a consideration of a very fundamental principle, sexual differentiation of the brain (for review see references 5, 28, 40, 41, 95 and 124). Although this topic will be the subject of an entire session in this conference, I would like to summarize our own experience with this preparation in regard to the control of ovulation (see 40, 41). Although much emphasis has been placed on the female, it is clear that at least in the rat this process of androgenization is included in normal development of the male. Figure 1.6 summarizes the basic principle of sexual differentiation of the brain. That principle states that in the rat at birth the region represented by the POA is undifferentiated; moreover, it is at least bipotential if not inherently feminine. When the brain develops in an environment containing androgen, as in the physiological example of the intact male, the cyclic activity or capacity of the POA fails to develop or is suppressed. When adult, and given ovarian grafts both as a source of gonadal hormones and as an index of the pattern of GTH secretion, the male appears to secrete GTH more or less tonically. At the other end of this normal process we see that the absence of steroids (the intact female is the physiological example here) leads to the development of what we have earlier considered the cyclic GTH release system. Evidence that the absence of steroids is the critical factor is supplied by the observation that ovariectomy of the newborn female is without effect, whereas castration of the male leads to this feminine pattern of cyclic GTH release in spite of any genetic differences in the male and female brain. Another important observation documenting the influence of the postnatal steroid environment on development of neuroendocrine regulation is the fact that within the first five days or so of life in the female, as well as in the male which had been castrated earlier, androgen injections, testicular implants, and even estrogen injections reverse this inherent biological capacity and masculinize the pattern of GTH secretion in the adult, apparently by an action at the level of the POA. We have also indicated by circumstantial evidence that the anatomical substrate which regulates the cyclic discharge of G T H in the neonatally castrated genetic male with an ovarian graft is identical to that of the normal female. Ovulation in this animal just as in the female is prevented by anterior deafferentation, a POA lesion, and exposure to constant illumination. At least the first two provide strong evidence that the ability of the preoptic system to regulate the cyclic release of GTH is dependent almost exclusively on the nature of postnatal steroid secretion. Interestingly, Wagner

10

STEROID H O R M O N E S AND BRAIN

FUNCTION

(122) has shown that the timing of at least precocious ovulation in the genetic, but neonatally castrated male is identical to that of the female. Recently, Schindler and Wagner (103) have reported that this alteration in pituitary ovulating hormone secretion seems to be a specific hypophyseal consequence of neonatal castration. The differentiation of sexual behavior regulating mechanisms is also well documented, but is further complicated by the development of peripheral sexual tissues (124), and the reactivity of the animal to environmental influences (14). These subjects receive detailed discussion in this conference. Recently, attention has been directed toward the possible mechanisms of androgenization. Flerko and his collaborators (29), and Anderson and Greenwald (2) have reported that the uptake and retention of labeled estrogen is reduced in the adult ovariectomized androgenized rat. In a detailed study of ovarian compensatory hypertrophy, Petrusz and Nagy (91) have reported that androgenized females are less sensitive to estrogen in functional terms as well. Although these studies might be criticized on the basis that endogenous ovarian activity is markedly different in these animals, Barraclough and Haller (7) have published data which suggest that even after ovariectomy, the androgenized female is functionally less sensitive to estrogen. We have observed that androgenization produces a rather selective decrease in progesterone sensitivity of a behavioral system (14). In experiments still in progress we are studying the question of the possible mechanism of androgen action by the experimental use of antibiotics. Although the systemic injection of either actinomycin-D or puromycin is effective in attenuating the action of subcutaneously administered TP, these results were dependent upon the relative time of administration of these two agents, and moreover, were not readily interpretable (68). In an attempt to overcome some of the objections against using these powerful agents systemically, we began to implant antibiotics directly into the brain in the region of the POA which is a likely site of androgen action. If we consider for the moment that androgenization involves a permanent change in fundamental biosynthetic mechanisms within certain neural elements, antibiotics placed in the brain might have either of two consequences. If androgen acts by disrupting biosynthetic processes in the developing brain, local application of antibiotics or other potential inhibitors might be expected to mimic androgenization. Although still a possibility, we have not obtained evidence to support this view (Table 1.1). On the other hand, androgen's action might well depend on the initiation of new synthetic processes. Therefore, we have attempted to block the action of androgen with implants of these agents. As seen in Table 1.1, implantation of cycloheximide in the POA at the same time as subcutaneous androgen injection, significantly reduces the incidence of sterility at 45 days of age. No other agent was effective. The interpretation of this effect of cycloheximide is difficult. The lack of an effect of these other agents may argue against a nonspecific antibiotic-in-the-brain explanation, and the absence of any effect of antibiotics alone in the POA argues against a significant physical lesion. Although subcutaneous implants of rifampicin and sarkomycin, antibiotics which were ineffective intracerebrally, inhibited androgenization possibly at a peripheral level, cycloheximide implants were not effective subcutaneously. Thus, this may be evidence of a local involvement of androgen with biosynthetic processes within the developing hypothalamus. This speculative interpretation obviously requires further documentation.

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A s can be seen in T a b l e 1.3, t h e act of i m p l a n t i n g c o c o a b u t t e r ( G r o u p 3) or s h a m s u r g e r y ( G r o u p 2), m a r k e d l y depressed t h e L Q a l t h o u g h it t u r n e d o u t t h a t this s u p p r e s sion w a s not statistically significant. W h e n w e i m p l a n t e d b u t t e r pellets c o n t a i n i n g v a r y i n g a m o u n t s of A c t - D , w e o b t a i n e d a m a r k e d a n d significant suppression of b e h a v ior, d e p e n d i n g u p o n t h e d o s e e m p l o y e d . W h e n a g r e a t e r dose of estrogen (15 fig) w a s used to p r i m e similar animals, A c t - D did n o t inhibit sexual b e h a v i o r , a n observation t h a t a r g u e s against t h e p r o d u c t i o n of a n o n s p e c i f i c lesion by t h e antibiotic ( G r o u p 9). A n o t h e r observation in f a v o r of a r a t h e r specific i n t e r a c t i o n of t h e antibiotic with estrogen is also illustrated in T a b l e 1.3. In this case, i m p l a n t a t i o n of c o c o a b u t t e r o r b u t t e r with t h e effective dose of A c t - D (0.18 ;ug) was delayed until 33 h o u r s h a d elapsed. T h e r e w a s n o effect of A c t - D at t h i s t i m e ( G r o u p 11). T h u s , it is possible, if not likely, t h a t estrogen m a y p r o m o t e sexual b e h a v i o r by an A c t - D sensitive process. T h e use of these p o t e n t antibiotics is n o t t h e only a p p r a o c h to t h e intracellular level of h o r m o n e action. In o u r discussions in this c o n f e r e n c e w e deal with t h e latest results f r o m studies of t h e localization of h o r m o n e u p t a k e by t h e b r a i n (see C h a p t e r 21). C o n s i d e r a t i o n of h o r m o n e action in t h e b r a i n w o u l d be greatly a d v a n c e d by a clear d e m o n s t r a t i o n of t h e localization of n e u r o n s a p p a r e n t l y responsive t o h o r m o n e s . In a d d i t i o n , t h e question of intracellular localization is also a m e n a b l e t o s t u d y . T h e a u t o r a d i o g r a p h studies m e n t i o n e d a b o v e i n d i c a t e d specific n u c l e a r u p t a k e of estrogen. M c E w e n describes initial a t t e m p t s to c h a r a c t e r i z e a n u c l e a r r e c e p t o r f o r a d r e n a l a n d p r o b a b l y g o n a d a l steroids (see C h a p t e r 24). In their respective c h a p t e r s , Steiner s h o w s t h a t m i c r o e l e c t r o p h o r e s i s of a d r e n a l s t e r o i d s suggests t h a t h o r m o n e s m i g h t m o d i f y m e m b r a n e physiology, while M e y e r s o n describes studies utilizing optical isomers of steroid h o r m o n e s . H o w specific is t h e p o t e n t i a l n e u r a l steroid r e c e p t o r p a r t i c u l a r l y in c o m p a r i s o n with p e r i p h e r a l receptors? A n o t h e r a p p r o a c h to this level of s t u d y is typified by M c C a n n ' s w o r k , described in his c h a p t e r , i.e., w h a t a r e t h e c o n s e q u e n c e s of h o r m o n e action u p o n s y n a p t i c e v e n t s leading to t h e release of R F (see C h a p t e r 29). A t this c o n f e r e n c e , w e c o n s i d e r in great detail t h e i n f l u e n c e of h o r m o n e s o n the electrical activity of individual n e u r o n s , f r o m t h e level of local application, w h e t h e r it is t h e m i c r o a p p l i c a t i o n of Steiner, o r t h e m a c r o a p p l i c a t i o n of h o r m o n e s to t h e P O A t h a t P f a f f discusses, t o t h e level of systemic i n j e c t i o n in t h e n e o n a t a l a n i m a l . Steroid h o r m o n e s can i n f l u e n c e t h e s p o n t a n e o u s firing r a t e of single u n i t s in t h e h y p o t h a l a m u s . F o r e x a m p l e , L i n c o l n (74) h a s observed t h a t o v a r i e c t o m y increases, while estrogen t r e a t m e n t decreases unit activity in t h e general p o p u l a t i o n of p r e o p t i c o r septal n e u r o n s in r a t s u n d e r c o n s t a n t i l l u m i n a t i o n , while reciprocal c h a n g e s w e r e observed in t h e lateral h y p o t h a l a m u s . F e e d b a c k is not limited t o steroids. A s reviewed by Beyer a n d Sawyer (8), p i t u i t a r y h o r m o n e s also m o d i f y s p o n t a n e o u s unit activity. W i t h this t e c h n i q u e it m i g h t be possible to d e t e r m i n e w h e t h e r or not a p a r t i c u l a r cell is responsive t o t h e f e e d b a c k of m u l t i p l e h o r m o n e s . C a n integration of m u l t i p l e feedback signals o c c u r at t h e level of t h e single cell? P r e l i m i n a r y results of Steiner a p p e a r i n g in his c h a p t e r i n d i c a t e t h a t this is i n d e e d possible. T h e e x p e r i m e n t of P f a f f a n d P f a f f m a n (93) w h i c h d e m o n s t r a t e d an effect of a n d r o gen o n s p o n t a n e o u s unit activity in the m a l e rat e m p h a s i z e s an obvious fact. A l t h o u g h t e s t o s t e r o n e causes a n increase in s p o n t a n e o u s activity of p r e o p t i c units, t h e a n d r o g e n also reverses t h e n a t u r e of t h e response of t h e s a m e n e u r o n t o various p e r i p h e r a l stimuli (see also t h e c h a p t e r s of F e l d m a n , T a y l o r , a n d P f a f f ) . It is obvious t h a t a n y single unit

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is in intimate contact with other neurons and elements. With these electrical techniques can one actually study the single cell? Since we cannot yet study intracellular potentials of hypothalamic neurons, the answer to this question appears to be no. Two strategies at least approach this ideal, however. The first I have already mentioned, the microelectrophoretic application of hormones to the local environment of the single cell. Second is the technique of recording unit activity in hypothalamic islands which Cross and Feldman describe in their chapters. In this case, neural input to the cell under study is minimized although certainly not eliminated. B. Intercellular

Level

It is necessary to emphasize that any individual cell is in immediate contact with many nerve cells and glial processes. As Schneider and McCann (104) have indicated, it is conceivable that one cell releases dopamine in the ME, and the dopamine in turn, causes the release of LRF from an adjacent cell. Besides such neural connections, I refer again to the recent interest in ependymal cells and neuroendocrine function. When we consider the influence of hormones on neural function, must we assume that hormone levels are critical for all functions of a steroid sensitive neuron? Is it possible that certain elements, perhaps neuronal or perhaps glial, serve uniquely as steroid sensitive cells and influence several functional types of neurons under their sphere of influence? In this regard studies by de Vellis and Inglish (17) have demonstrated an action of hormones on glial enzyme activity. As described by Vernadakis, hormones can also alter glial potentials (see Chapter 3). Can we rule out the participation of nonneural elements when we consider the influence of hormones on brain function? When we consider neuronal electrical activity, almost by definition we are considering intercellular relations. Several of our participants will discuss the action of hormones in relation to afferent input. How do hormones modify the response to vaginal stimulation, or to olfactory stimuli? How does the sleep state of an animal alter responsiveness to hormones, or vice versa? One research strategy in particular utilizes the intercellular level of investigation. I am referring to the technique of multiunit activity. In this case populations of single cells are recorded with a single macromicroelectrode. An increase in firing rate, or in amplitude results in an upswing in the integrated output from neurons in the region of the electrode. A recent experiment of Terasawa and Sawyer (117) bridges the theoretical gap between the intercellular and the next highest level of study. They report an increase in multiunit activity in the arcuate nucleus upon electrochemical stimulation of the POA, which was correlated with ovulation. Electrochemical stimulation of the POA, which was not followed by this change in multiunit activity, did not result in ovulation. Utilization of ovulation as one end point in this study, takes us to the next higher level of study which I have called the systems level. C. Systems

Level

The results of the preceding experiment essentially reflect on the functional overview of hypothalamic regulation of GTH secretion illustrated in Figure 1.5. Teresawa and Sawyer showed that electrical activation of the POA resulted in an increase in electrical activity of the arcuate region only when that stimulus successfully induced ovulation.

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5. SELECTIVITY OF ACUTE FEEDBACK EFFECTS OF CORTICOSTEROIDS ON ACTH SECRETION* V. CRITCHLOW Baylor College of Medicine Houston, Texas

D u r i n g the last several years, in collaboration with Drs. Coyer, D u n n , Hamill, Mitchell, Palka, Smyrl, and Z i m m e r m a n n , we have been studying the control of pituitaryadrenal function in the rat. O u r experience is such that we have come to accept the views of Harris (8) and Slusher (11) that A C T H secretion is controlled by at least two separate neural systems. T h e first and most extensively studied is the stress system. It responds acutely and dramatically to a wide variety of noxious stimuli and, in our hands, appears relatively or totally refractory to physiological levels of corticosteroids. T h e second neural mechanism controlling A C T H secretion, because it is responsible for appreciable pituitary-adrenal function in the apparent absence of stress, is designated here as the nonstress component; our data suggest that this part of the A C T H control system is acutely and physiologically sensitive to corticosteroid feedback. The aim of this presentation is to summarize our experimental evidence for these views. T h e experiments to be described involved primarily female rats; plasma or adrenal concentrations of corticosterone, measured fluormetrically (5), were used as indices of A C T H secretion. If any single feature characterizes our approach to this system, it is our attempt to assess routinely the effects of experimental manipulations on both nonstress and stress-induced A C T H secretion. Nonstress pituitary-adrenal function is evaluated by determining corticosterone levels in plasma or adrenals or both obtained within 3 minutes of initiation of stress (cage opening). The stress-induced increment in steroid levels, i.e., the difference between a nonstress level and that existing at a specified time after initiation of stress (usually 15 minutes), is used as an index of the acute response to stress. In all cases, plasma and adrenal samples are obtained under controlled and defined experimental conditions (13). O u r first contact with this system impressed us with the considerable level of activity present under conditions designed to minimize sources of environmental stress (1). In the female rat, nonstress pituitary-adrenal function is characterized by a high amplitude circadian rhythm, nnd the diurnal peak in corticosterone levels is attained shortly before onset of the daily 10 hour dark period. Peak plasma concentrations of this steroid approximate 60 /ug/100 ml, a level that is often higher than that reached in males * The research discussed here was supported by grant A M 03885from the National Institutes of Health, United States Public Health Service.

51

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following stress. It is on this oscillating background that acute responses to stress are imposed (13). As shown in Figure 5.1, 3 minute immobilization or ether stress applied during the trough and peak periods of the rhythm produced increments in corticosterone levels, i.e., stress responses, that were completely superimposed upon significantly different backgrounds of pituitary-adrenal function. These results imply little or no interaction between processes subserving nonstress and stress-induced secretion of ACTH and support the concept that separate mechanisms are involved. These findings also suggest that maximal activation of the pituitary-adrenal system occurs only in association with stress applied during the diurnal peak. Because nonstress levels of corticosterone attained during the diurnal peak are approximately 50 per cent of those observed during maximal activation of both nonstress and stress systems, it is clear that under certain temporal conditions the nonstress component makes more than a minor contribution to circulating corticosterone levels. Our attempts to examine the role of corticosteroid feedback are in large part responsible for our view that separate mechanisms control nonstress and stress-induced ACTH secretion. A relatively small dose (100 ¡ig/kg) of dexamethasone-21 - P 0 4 (dex) given subcutaneously produced rapid (less than 30 minutes), complete, and long-lasting (approximately 12 hours) suppression of nonstress pituitary-adrenal function. Despite this clear-cut and highly reproducible effect on the nonstress system, this dose of dex failed to suppress, even partially, acute responses to several types of stress (14). Figure 5.2 shows complete suppression of the diurnal peak in nonstress corticosterone levels 4 hours after administration of dex and the unimpaired increments produced by 3 minute immobilization and ether stresses. Similar results were obtained with handling and cold stresses. The ability of dex to completely abolish nonstress pituitary-adrenal function without impairing acute responses to these several stimuli suggests functional dissociation of the nonstress and stress mechanisms on the basis of sensitivity to negative feedback. We have recently found that a heat stress, a 3 minute exposure to 56°C, produced a response which was blocked by this dose of dex. However, it is not clear, as indicated below, that this response utilizes pathways which are sensitive to physiological levels of corticosterone. Nevertheless, it may well be that some stress stimuli use corticosteroid-resistant pathways while others induce ACTH secretion via structures that are steroid-sensitive, as suggested by Dallman and Yates (2). To examine physiological implications of the preferential sensitivity of the nonstress system to dex, we have undertaken studies with corticosterone (16), the dominant glucocorticoid in the rat. As shown in Figure 5.3A, subcutaneous injection of 3 mg/kg of corticosterone produced plasma levels at 15 minutes of approximately 150 jug/100 ml; this level is considered high yet physiological because such concentrations are found in female rats subjected to stress during the diurnal peak in pituitary-adrenal function (13). Despite administration of corticosterone, levels of this steroid in plasma and adrenals were significantly lower 2 hours after injection than those in controls injected with vehicle. As shown in Figure 5.3B, this suppression was compatible with an intact acute response to 3 minute ether stress. These findings extend the results obtained with dex and suggest that physiological levels of circulating corticosterone acutely and preferentially affect mechanisms responsible for nonstress pituitary-adrenal function. Although the response to a 3 minute heat stress, as described above, was blocked by dex, such was not the case with this dose of exogenous corticosterone, despite significant suppression of nonstress plasma and adrenal steroid levels. We are still searching

F E E D B A C K C O N T R O L OF A C T H

53

for a stress response that is inhibited with physiological levels of corticosterone. W e have used several approaches in attempts to localize the site of feedback inhibition of nonstress A C T H secretion. Except for our inability to clearly implicate midbrain structures, the localization achieved corresponds to the distribution of dex-suppressible units described by Steiner and associates (10, 12). One of the approaches involved intracerebral implantation. Nonstress levels of corticosterone but not responses to ether stress were suppressed 8 hours after bilateral implantation of agar pellets, each containing 1 or 2.5 jug of dex, in ventral medial diencephalon and rostral midbrain. N o suppression was produced by dex placed in amygdala or pituitary (15). In efforts to obtain better localization of dex, a similar protocol was used recently to implant 0.03 jug of dex. Pellets containing this low dose of dex markedly suppressed nonstress corticosterone levels without compromising responses to immobilization stress when placed throughout medial hypothalamus (0.5 m m from midline); 2 of 6 pellets in lateral septum were similarly effective. In contrast, no suppression was observed with dex located in midbrain tegmentum or periventricular gray, hippocampus, lateral ventricle, or cerebral cortex. Thus, the several doses of dex placed in medial hypothalamus (0.03, 2, and 5 ng) all produced selective blockade of nonstress pituitary-adrenal function similar to that observed with larger amounts of dex given systemically. As another approach to localizing the site of feedback suppression of nonstress A C T H secretion, dex (100 jug/kg) was administered subcutaneously to rats following surgical isolation of the medial basal hypothalamus (MBH). Isolation of M B H was performed with a modification of the Halász-Pupp knife (6). As reported by Halász et al. (7), we found that the most consistent deficit in pituitary-adrenal function in such animals was disruption of the normal circadian r h y t h m in corticosterone levels (9). As shown in Figure 5.4, processes supporting nonstress A C T H secretion retained m a r k e d sensitivity to dex, suggesting the dex exerts its ACTH-suppressing effect on structures within the MBH-pituitary unit. Also, isolation of M B H was compatible with a significant though reduced response to immobilization. T o rule out the possibility of influences mediated by adjacent forebrain tissue or by residual or regenerated neural connections, we also studied effects of dex and stress 24 hours after complete or partial forebrain removal. T h e behavior of the pituitary-adrenal system of rats with basal hypothalamic islands, consisting chiefly of ventromedial and arcuate nuclei, median eminence, and pituitary, bears a striking resemblance to that of the intact rat. Such animals showed "nonstress" corticosterone levels comparable to those of intact rats and these levels were significantly depressed by dex (3). In addition, such preparations demonstrated significant steroid responses to ether or immobilization stress (4). Because dex also suppressed A C T H secretion from pituitary islands in rats subjected to complete forebrain removal, we consider it possible that dex acts at both pituitary and neural sites (3). T o summarize our views on the control of A C T H secretion, we conceive of two hypothalamic systems. One of these, the stress system, is activated by noxious stimuli, sometimes in the absence of fiber connections with other structures of the central nervous system, and it is relatively insensitive to feedback effects of glucorticoids. Although it is generally agreed that high or pharmacological levels of corticosteroids may inhibit responses to stress, it has yet to be demonstrated that such responses are acutely sensitive to physiological levels of circulating steroids. T h e second hypothalamic system supports nonstress pituitary-adrenal function and, because of connections with extrahypothalamic structures, demonstrates circadian rhythmicity. This

54

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FUNCTION

nonstress system is apparently not activated by stress input, but it is preferentially and acutely sensitive to suppression by physiological levels of circulating corticosterone. O u r results suggest that corticosteroids suppress nonstress A C T H secretion via structures residing in the MBH, the pituitary or both. If the units suppressed by the low doses of dex in the studies described by Steiner are involved in the physiological control of A C T H secretion, we suggest that consideration be given to the possiblity that such units are involved in the control of nonstress pituitary-adrenal function. The above schema is simplistic and not always in accord with interpretations of others, especially with respect to restricting the role of negative feedback to the regulation of nonstress secretion of ACTH. The literature is replete with differing views and findings concerning effects of corticosteroids on pituitary-adrenal function, and some of these differences appear to reflect variations in experimental conditions. In our experience, sex and even stage of the estrous cycle, dose of corticosteroids, duration of stress, time of sampling with reference to stress or time of day, and environmental conditions are variables that exert marked influences on feedback control of A C T H secretion. Although there is not time to present or describe effects of all of these variables, I would like to focus on a few points that seem particularly appropriate for this conference. First, we feel that considerable disagreement and confusion in the literature stem from failure to acknowledge the presence and substantial contribution of the nonstress system to overall pituitary-adrenal function. Effects of corticosteroids are often analyzed without differentiating between actions on nonstress and stress systems. Thus, depression of corticosterone levels following stress in steroid-treated animals is commonly used as evidence for interference with the stress response. Our findings suggest that such suppression always involves the nonstress component and only under certain conditions are responses to stress impaired. Another variable that may be important to the study of feedback in this system is the use of anesthetics. Figure 20.5A shows results of experiments designed to explore the possibility of interaction between dex and pentobarbital; the dose and timing of pentobarbital administration were as described in experiments which identified corticosteroid-resistant and corticosteroid-sensitive stress responses (2). Whereas either dex or pentobarbital given singly produced significant suppression of only nonstress corticosterone levels without affecting acute responses to immobilization, the combination of these agents clearly reduced responses to this stress. Because of these results, we were interested in the effects of the chloralose-urethane anesthesia used by Steiner and associates (10, 12). Figure 20.5B summarizes the results obtained. The combination of dex and chloralose-urethane suppressed the stress response. In contrast, only nonstress levels were affected when the agents were given individually. Similar results were obtained in males. These interactions suggest caution in interpreting dex-induced inhibition of stress responses in anesthetized animals as an effect of the steroid per se. In conclusion, I would like to raise several points regarding the findings of Steiner and associates (10, 12) for consideration. First, on the assumption that dex-suppressible units described are in fact concerned with regulation of A C T H secretion, we would suspect that they are concerned with the nonstress part of the system. That many of such units were located in the central gray of the hypothalamus is compatible with the crude localization we have achieved; however, two complications result from the use of chloralose-urethane anesthesia. The first derives from the finding that chloraloseurethane suppresses nonstress pituitary-adrenal function, presumably through effects

FEEDBACK

CONTROL

OF

ACTH

55

on neurons. This selective suppression may introduce a bias by eliminating those units most sensitive to physiological levels of corticosteroids. The second complication stems from the drug interactions observed; suppression of a neuron by dex in a chloraloseurethane-treated rat may not indicate its normal response to corticosteroids. REFERENCES 1. Critchlow, V., Liebelt, R. A., Bar-Sela, M„ Mountcastle, VV„ and Lipscomb, H. S„ Sex difference in resting pituitary-adrenal function in the rat. Amer. J. Physiol., 1963, 205: 807-815. 2. Dallman, M . F., and Yates, F. E., Anatomical and functional mapping of central neural input and feedback pathways of the adrenocortical system. Mem. Soc. Endocr., 1968, 17: 39-71. 3. Dunn, J. and Critchlow, V., Feedback suppression of non-stress pituitary-adrenal function in rats with forebrain removed. Neuroendocrinology, 1969, 4: 296-308. 4.

, Pituitary-adrenal response to stress in rats with hypothalamic islands. Brain Res., 1969, 16: 395-403.

5. Guillemin, R., Clayton, G. W., Lipscomb, H. S., and Smith, J. D., Fluorometric measurement of rat plasma and adrenal corticosterone concentration. J. Lab. Clin. Med., 1959, 53: 830-832. 6. Halasz, B., and Pupp, L., Hormone secretion of the anterior pituitary gland after partial or total interruption of all nervous pathways to the hypophysiotrophic area. Endocrinology, 1965, 77: 553-562. 7. Halasz, B., Slusher, M. A., and Gorski, R. A., Adrenocorticotrophic hormone secretion in rats after partial or total deafferentation of the medial basal hypothalamus. Neuroendocrinology, 1967, 2: 43-55. 8. Harris, G. W., Neural Control of the Pituitary Gland. Edward Arnold, London, 1955. 9. Palka, Y., Coyer, D., and Critchlow, V., Effects of isolation of medial basal hypothalamus on pituitaryadrenal and pituitary-ovarian functions. Neuroendocrinology, 1969, 5: 333-349. 10. Ruf, K. and Steiner, F. A., Steroid-sensitive single neurons in rat hypothalamus and midbrain: Identification by microelectrophoresis. Science, 1967, 156: 667-669. 11. Slusher, M. A., Effects of chronic hypothalamic lesions on diurnal and stress corticosteroid levels. Amer. J. Physiol., 1964, 206: 1161-1164. 12. Steiner, F. A., Ruf, K., and Akert, K., Steroid-sensitive neurons in rat brain: Anatomical localization and responses to neurohumours and A C T H . Brain Res., 1969, 12: 74-85. 13. Zimmermann, E., and Critchlow, V., Effects of diurnal variation in plasma corticosterone levels on adrenocortical response to stress. Proc. Soc. Exp. Biol. Med., 1967, 125: 658-663. 14.

, Negative feedback and pituitary-adrenal function in female rats. Amer. J. Physiol., 1969, 216: 148-155.

15.

, Effects of intracerebral dexamethasone on pituitary-adrenal function in female rats. Amer. J. Physiol, 1969, 217: 392-396.

16.

, Suppression of pituitary-adrenal function with physiological plasma levels of corticosterone. Neuroendocrinology, 1969, 5: 183-192.

DISCUSSION McCann: Your index of ACTH secretion really is the corticosterone levels in plasma, and with the marked diurnal variation in the corticosteroids, I wonder if there is a diurnal variation in ACTH sensitivity of the adrenal glands? Critchlow: Others have reported such variations in adrenal sensitivity to ACTH and in preliminary experiments we also observed significant differences. One hour after hypophysectomy, responses to ACTH were greater in the afternoon than in the morning. Therefore, we cannot be sure that the marked diurnal variations in plasma and adrenal corticosterone levels are due entirely to variations in ACTH secretion; however,

56

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FUNCTION

there is considerable evidence available from a number of species to indicate that variations in ACTH secretion are in large part responsible for rhythmic adrenal cortical secretion. McCann: How do you explain the response of your hypothalamic-island to the various stress stimuli? Critchlow: We are intrigued by the question you raise. We have repeatedly observed responses to ether and immobilization stresses in rats with chronic deafferentation and have seen responses to heat and cold stresses in preliminary experiments. We believe that the responsiveness of the isolated hypothalamopituitary unit to these several stimuli is real and not due to incomplete deafferentation because we have seen qualitatively similar responses from hypothalamic islands prepared by partial forebrain removal. We do not know what the signal is but are thinking in terms of something mediated by the systemic circulation. Ganong: The stresses to which these preparations will respond are ether and immobilization. Is there not general agreement on that? Critchlow: I do not believe that there is general agreement concerning persistence of the response to immobilization stress; unless I am mistaken, we are the only ones to date who have reported the use of this stimulus in deafferented rats. Several papers describe responses to ether after deafferentation, but as I remember, Voloschin (6) reported reduction of the response to this stress following complete deafferentation. I do not believe that Halasz, Slusher and Gorski (2) studied the effects of immobilization. Martini: As you probably know, we have data which are in complete disagreement with yours. We could block stress responses by giving 25 ¡J. of dexamethasone per 100 g of body weight (3). Critchlow: You can but we cannot. There are some basic disagreements between your results and ours which we feel are in part due to differences in time of sampling and other experimental conditions that we have discussed in our publications. As indicated in my presentation, the only stress response we have blocked with this dose of dexamethasone is that produced by a heat stimulus consisting of 3 minutes exposure to 56°C. However, this response is not blocked by a physiological level of corticosterone in blood which produces significant suppression of the nonstress part of the system. Ganong: I would like to clear up two fundamental points. Were you saying that in the hypothalamic island, the resting steroid level is elevated or normal? Critchlow: We see what appears to be a constant plasma level of steroid of approximately 40 jLtg per cent. This level is intermediate between normal peaks and troughs; this has been a common finding in several series of deafferented animals. This intermediate level is often significantly higher than that found in controls in the morning but lower than control levels in the afternoon. Ganong: I think you have answered my other question. There is no diurnal variation in the island animal. Critchlow: Yes, that is right and that is essentially what Halasz et al. reported (2). Feldman: Davidson and I have just published our results of a large number of animals, which were deafferented (1). We exposed those animals to various stresses. This included ether, immobilization, anoxia, light and acoustic stimulation, and we found normal, almost normal responses, to ether, to immobilization, to anoxia. We found considerably reduced responses to acoustic and photic stimulation. We found

FEEDBACK

CONTROL

OF

ACTH

57

that one could differentiate two kinds of stresses. Those which are mediated by neural input to the median eminence like light and acoustic stimulation; while the other nonspecific or nonneural stresses do not need this neural input in order to activate the island. We do all experiments between 8 and 10 am. It has been reported that in the island animals in the morning there was some rise in corticosterone. Our levels are exactly as those in the intact animal. Critchlow: I would like to make a comment on a few of these points. I think much depends on the morphology of the island. We have seen considerable difference depending on the type of cut we have. In response to your comments about photic and auditory stimuli, I would like to mention that we have tried similar experiments with these modalities and have been unable to elicit significant steroid responses. In the case of photic stimulation, we used strobe flashes under the conditions you described and did not see increments in steroid levels different from those obtained with sham stimulation. For auditory stimulation, we used an electric doorbell rather than the alarm clock described in your paper and again we failed to observe increments in corticosterone levels attributable to this modality. The animals showed clear behavioral responses to the sound but no changes in steroid levels. Unless you use sham stimulation procedures and look at stress-induced increments, I wonder how certain you can be that you have in fact triggered pituitary-adrenal responses with these specific stimuli. Davidson: We have not tried a doorbell, but there is no question that an alarm clock causes a response, which is rather similar to that of ether. This is controlled, of course. So this may be a factor of strain difference. Ganong: Since the question of diurnal fluctuation has come up, I would like to mention two results. Following up the observation that in the hypothalamic island preparation, the plasma B level is in an intermediate position throughout the 24 hours, Moberg and his associates in my laboratory sectioned the fornix selectively. This procedure produced the same type of curve; higher than normal in the morning and lower than normal in the evening (4). Scapagnini and associates in my laboratory noted that the diurnal fluctuation in the serotonin content of the hippocampus and amygdala paralleled the plasma corticosterone fluct^tion. They then administered p-chlorophenylalanine and showed that it produced the same plasma corticosterone curve as fornix section (5). It would seem to me that all these observations indicate there must be both an excitatory and inhibitory input into the hypothalamus from the limbic system. Critchlow: That certainly fits with our thinking; however, I might add to the confusion by mentioning that we have also transected the fornix and have seen no effect on the rhythm or on stress responses. We have also stimulated hippocampus with chronic electrodes and have seen no effect on the rhythm or on stress responses. In fact, we have been impressed with the lack of influence of the hippocampo-fornix system and find your observations most interesting. In a few animals with lesions in amygdala we have seen apparent disruption of the rhythm, similar to that observed following deafferentation; such lesions did not interfere with responses to several stresses. Stumpf: We have some preliminary data on the localization of Cortisol in the rat brain, and there is so far indications for concentration in the hippocampus. DISCUSSION REFERENCES 1. Feldman, S., Conforti, N., Chowers, I., and Davidson, J. M., Pituitary-adrenal activation in rats with

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medial basal hypothalamic islands. Acta Endocri,

BRAIN

FUNCTION

(Kobenhavn), 1970, 63: 405-414.

2. Halasz, B., Slusher, M. A., and Gorski, R. A., Adrenocorticotrophic hormone secretion in rats after partial or total deafferentation of the medial basal hypothalamus. Neuroendocrinology, 1967, 2: 43-55. 3. Mangili, G., Motta, M., Muciaccia, W., and Martini, L., Midbrain stress and ACTH secretion. Europ. Rev. Endocrin., 1965, 1: 247-253. 4. Moberg, G. P., Scapagnini, U., deGroot, J., and Ganong, W. F., The effect of sectioning the fornix on diurnal fluctuation on corticosterone levels in the rat. Neuroendocrinology, 1970, in press. 5. Scapagnini, U., Moberg, G. P., Van Loon, G. R., deGroot, J., and Ganong, W. F., Relation of the brain 5-hydroxytrptamine content to the diurnal variation in plasma corticosterone in the rat. Neuroendocrinology, 1970, in press. 6. Voloschin, L., Joseph, S. A., and Knigge, K. M., Endocrine function in male rats following complete and partial isolations of the hypothalamo-pituitary unit Neuroendocrinology, 1970, 3: 387-397.

A.

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8,

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Figure 5.1. Effect of diurnal variation in plasma corticosterone levels o n corticosterone responses t o 3 min immobilization (A) and ether (B) stress. Stippled areas indicate previously reported circadian patterns of corticosterone levels under similar conditions (1). T h e horizontal black bars m a r k the first portion of the daily d a r k period. In this and subsequent figures, numbers in the columns denote numbers of rats a n d vertical lines represent ± standard error. ( F r o m Z i m m e r m a n n and Critchlow, 13.)

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7. MODIFICATION OF THE RESPONSIVENESS OF COMPONENTS OF THE LIMBIC-MIDBRAIN CIRCUIT BY CORTICOSTEROIDS AND ACTH* A. NEWMAN TAYLOR, G. KEITH MATHESONt AND N. DAFNY University of California L o s A n g e l e s , California

The preceding discussion has focused on various aspects of the actions of corticosteroids on the central nervous system (CNS). These actions range from direct feedback effects in the regulation of pituitary-adrenal activity to more general effects on the CNS whereby steroids influence various aspects of behavior as indicated by the elegant studies of Dr. Endroczi. Our studies demonstrate actions of the corticosteroids at both hypothalamic and related extrahypothalamic portions of the limbic system-midbrain circuit. Two types of effects will be described: (a) modification of the central regulation of ACTH release by corticosteroids and (b) actions of corticosteroids and ACTH on the electrical activity of various regions in the brain. Steroid Levels and Reticular Formation Effects on ACTH Release We have previously reported (18) that the role of the reticular activating system (RAS) in ACTH release is directly influenced by circulating levels of corticosteroids. This conclusion was based on the types of ACTH responses which occurred upon stimulation of the RAS in encéphale isolé cats. The direction of the ACTH response, determined by adrenal venous steroid levels, was directly related to the prestimulation steroid titer. When initial steroid levels were low, stimulation of the RAS resulted in facilitation of ACTH release; however, when prestimulation steroid levels were significantly higher, ACTH release was either inhibited or not affected. These results indicated that the responsiveness of the RAS or connections between the RAS and the hypothalamohypophysial system is modified by the level of activity in the pituitaryadrenal axis, Thus, elevated steroid levels, or perhaps ACTH itself, can render a stimulus in the RAS ineffective or cause it to be inhibitory. * We should like to express our appreciation to Miss Berrilyn J. Branch and Mr. R. L. Casady for carrying out the steroid analyses, to Mr. David fVhitmoyer for his assistance with the electrical instrumentation, to Miss Arlene Koithan and Mrs. Beverly Bedard for the histological preparations and to Miss Jill Penkhus and Mrs. Keiko Akutagawa for preparing the figures. These studies were supported by NIH grants NB 07884 and AM 08468 and an NIH Postdoctoral Fellowship to G. K. Matheson. t Present address: Department of Anatomy, Loyola University Stritch School of Medicine, Maywood, Illinois.

67

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Steroid Levels and Amygdaloid Effects on ACTH Release Recently we have observed a similar modification of amygdaloid effects on ACTH release by circulating steroid levels. In these experiments we used seven freely behaving male cats chronically implanted with bipolar concentric stainless steel electrodes (27 ga) in the various amygdaloid nuclei (Figure 7.1) and with an indwelling catheter in the right atrium inserted via the external jugular vein. The cat was placed in the recording chamber where his behavior could be observed through a one-way window and his EEG recorded both from cortical and subcortical sites. Blood samples (1.5 ml) were collected from outside the chamber with.the aid of a catheter extension tubing connected to the cannula, thereby not disturbing the animal. The amygdaloid nuclei were stimulated for 30 minutes at stimulation parameters (10 or 100 cps, 0.1 or 0.75 ms/pulse, 0.05-0.5 mA, 15 sec. on-15 sec. off) which did not elicit any overt behavioral or EEG effects. Afterdischarges were observed only rarely and did not correlate with ACTH responses. Blood samples were drawn one-half hour before the stimulation, at the onset and termination of stimulation, and one-half hour later. Aliquots of plasma (0.2 ml) were analyzed in triplicate for Cortisol and corticosterone by extraction, separation of the steroids by thin-layer chromatography and quantitation by fluorescence (11). Stimulation was considered to have caused either facilitation or inhibition of ACTH release if changes in steroid levels during a period of stimulation exceeded normal prestimulatory fluctuations in steroid levels (±44 per cent) in that experiment. We systematically examined the corticomedial, basolateral (basal and lateral portions) and anterior amygdaloid nuclei (Figure 7.1). We found that the ACTH response to stimulation in all sites except the anterior amygdala was directly related to prestimulation steroid levels (Table 7.1). When steroid levels were low, facilitation of ACTH release was observed in response to stimulation of the corticomedial and basolateral (basal and lateral) nuclei. When prestimulation steroid levels were significantly higher,

TABLE

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Figure 8.6. Changes of integrated MUA in ARC (A) and MPO (B) after estrogen-progesterone treatment in the ovariectomized rats.

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Figure 8.7. A: Effects of electrical stimulation (duration, 0.1 msec. 100 Hz) of M P O on unit activity in the V M H and the A R C . Upper trace shows a case in which ovariectomized rats were treated with daily injection of 5 /ig estrogen for two successive days. Unit discharges in the V M H was suppressed during the stimulation (60 /iA), while those in the A R C increased markedly during the stimulation (40 ¿¿A). Lower trace shows a case that was treated by 10 mg progesterone only. Unit discharges both in the V M H and the A R C had no marked changes even with the higher current stimulation (200 /nA). Black bar indicates the repeated electrical stimulation of the M P O . Parenthesis indicates the threshold of electrical stimulation to induce the change of unit discharge. (From Kawakami, and K u b o , unpublished observations). Abbreviations: V M H , ventromedial hypothalamic nucleus; FC, frontal cortex; EST, estradiol benzoate; P R O G , progesterone propionate. B: Changes in evoked potential (EVP) of A R C elicited by stimulation* of the M P O (MPO-ARC-EVP) in the estrogen-progesterone treated ovariectomized rats. M P O - A R C - E V P consisted of a first negative (peak-latency 4-5 msec.) and a second positive (peak-latency about 20 msec.) deflection. Amplitude of both deflections increased slightly 6-8 hrs after (14:00-16:00) the first injection of 5 fig estrogen. Note especially that the amplitude of the first deflection was augmented increasingly without noticeable delay of peak-latency after the second injection of 5 fig estrogen after 1 mg progesterone injection, but that the peak latency of the second deflection was quite delayed.

* Parameter of electrical stimulation: duration 0.02 msec., 7 V, single square wave pulse. (From Kawakami, Ishida, and K u b o , unpublished observations).

co er Z)

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1 0 per cent) change in frequency occurred the time course of the response bore no obvious relationship to the duration of the anesthesia. These results forcibly demonstrate that the anesthetic potency of urethane is exerted elsewhere in the brain, for in these anesthetized rats not only is hypothalamic unit activity unaffected but the hypothalamus is disconnected from the brainstem and spinal cord. Our investigations with the barbiturate sodium methohexital produced diametrically opposite results. Here because of the short duration of action the method of choice was intravenous injection during the recording of a single neuron discharge, and the test could be repeated several times with different cells in the same island preparation. Forty-five units in 34 rats were tested in this way with 0.5-3.0 mg of methohexital. The response after a latency of about 5 sec. was a very consistent depression of spontaneous discharge rate. The response was highly reproducible and dose-dependent for any one cell but variable in intensity and duration for different neurons. Especially noteworthy was the fact that the depressant action could readily be observed with doses of the barbiturate too low to exert a significant anesthetic action (Figures 9.1 and 9.2). In these experiments we took polygraph recordings of arterial blood pressure and respiration, both of which were depressed by the methohexital injections (Figure 9.2). It was necessary to establish therefore that the depression of unit activity just described was not indirectly attributable to a reduced cerebral blood flow, hypoxia or hypercapnia. This was done by studying the effects of inhalation of amyl nitrite, nitrous oxide and carbon dioxide respectively. None of these procedures elicited a depression of unit firing rate comparable to that following methohexital injection although hypotensive and respiratory changes were induced (Figures 9.2 and 9.6). Evidently the barbiturate has a direct action on hypothalamic neurons which is not dependent on changes in blood flow or the partial pressures of carbon dioxide or oxygen. In summary, our results show that full anesthetic doses of urethane are without significant effect on hypothalamic neurons, but that even subanesthetic doses of sodium methohexital have a dramatic inhibitory action on hypothalamic cell discharge. Thus while exonerating urethane from the charge of directly influencing unit activity in the hypothalamus they cast serious doubts on the value of unit recording under barbiturate anesthesia. Indeed our results with methohexital would provide an adequate explanation of the efficacy of pentobarbital in blocking ovulation when administered during the critical period. Summary Diencephalic island preparations were made in 70 adult female rats in order to record single unit activity (700 units) that could be regarded as intrinsic to the hypothalamus. Firing rates of hypothalamic island units were found to be practically unaffected by urethane anesthesia, oxytocin injections, hypoxia, hypercapnia or hypotension. Subanesthetic doses of sodium methohexital consistently produced a reversible slowing or arrest of unit discharge, and chronic hypophysectomy resulted in a drastic reduction in unit activity. A significant increase (p < .01) was recorded in firing frequency of anterior hypothalamic units in proestrus over that in estrus, metestrus or diestrus. No significant change was seen in units from the lateral hypothalamic area in any day of the four day cycle.

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REFERENCES 1. Aub, J. C., Bright, E. M., and Forman, J., The metabolic effect of adrenalectomy upon the urethanized rat. Amer. J. Physiol., 1922, 61: 349-368. 2. Barraclough, C. A., and Cross, B. A., Unit activity in the cyclic female rat: effect of genital stimuli and progesterone. J. Endocr., 1963, 26: 339-359. 3. Cross, B. A., and Dyer, R. G., Does oxytocin influence the activity of hypothalamic neurones? J. Physiol., (London) 1969, 203: 70P-71P. 4.

, Characterisation of unit activity in hypothalamic islands with special reference to hormone effects. In: NATO Conference on Integration of Endocrine and Non-endocrine Systems in the Hypothalamus, (M. Motta, F. Fraschini, and L. Martini, Eds.). 1970, New York: Academic Press.

5. Cross, B. A., and Kitay, J. I., Unit activity in diencephalic islands. Exp. Neurol., 1967, 19: 316-330. 6. Cross, B. A., and Silver, I. A., Electrophysiological studies on the hypothalamus. Brit. Med. Bull., 1966, 22: 254-260. 7. Everett, J. W., Neuroendocrine aspects of mammalian reproduction. Ann. Rev. Physiol., 1969, 31: 383-416. 8. Everett, J. W., and Radford, H. M., Irritative deposits from stainless steel electrodes in the preoptic rat brain causing release of pituitary gonadotrophin. Proc. Soc. Exp. Biol. Med., 1961,108: 604-609. 9. Everett, J. W., and Sawyer, C. H., A 24-hour periodicity in the 'LH release apparatus' of female rats, disclosed by barbiturate sedation. Endocrinology, 1950, 47: 198-218. 10. Halasz, B., The endocrine effects of isolation of the hypothalamus from the rest of the brain. In: Frontiers in Neuroendocrinology, (W. F. Ganong and L. Martini, Eds.). Oxford University Press, 1969: 307-342. 11. Halasz, B., and Pupp, L., Hormone secretion of anterior pituitary gland after physical interruption of all nervous pathways to the hypophysiotrophic area. Endocrinology, 1965, 77: 553-562. 12. Kawakami, M., Terasawa, E., and Ibuki, T., Changes in multiple unit activity of the brain during the estrous cycle. Neuroendocrinology, 1970, 6: 30-48. 13. Komisaruk, B. R., McDonald, P. G., Whitmoyer, D. I., and Sawyer, C. H., Effects of progesterone and sensory stimulation on EEG and neuronal activity in the rat. Exp. Neurol, 1967, 19: 494-507. 14. Lincoln, D. W., Correlation of unit activity in the hypothalamus with EEG patterns associated with the sleep cycle. Exp. Neurol., 1969, 24: 1-18. 15. Lincoln, D. W., and Cross, B. A., Effect of oestrogen on the responsiveness of neurones in the hypothalamus, septum and preoptic area of rats with light induced persistent oestrus. J. Endocr., 1967, 37: 191-203. 16. Spriggs, T. L. B., and Stockham, M. A., Urethane anaesthesia and pituitary-adrenal function in the rat. J. Pharm. Pharmacol, 1964, 16: 603-610. 17. Taleisnik, S., Velasco, M. E., and Astrada, J. J., Effect of hypothalamic deafferentation on the control of luteinizing hormone secretion. J. Endocr., 1970, 46: 1-7. 18. Terasawa, E., and Sawyer, C. H., Changes in electrical activity in the rat hypothalamus related to electrochemical stimulation of adenohypophyseal function. Endocrinology, 1969, 85: 143-149.

DISCUSSION Everett: You mentioned in publication sometime in the past that it may be that one nerve cell somewhere would determine the timing of LRF release, or to the effect that the clock may be within one cell. On the other hand, your present data suggested that maybe the clock is not in any one cell, but widely distributed among many populations. Would that fit in? Cross: I do not think I ever held very seriously the idea of that one cell; however, I think it very likely each cell has its own clock, and what produces the diurnal clock

CYCLIC

CHANGES

IN H Y P O T H A L A M I C

UNITS

101

for the whole animal is an entrainment system, where the faster ones or the slower ones dominate the others and when they go into resonance, one gets the basis of a circadian rhythm. We suppose that whatever we have here every fourth day is an ovarian form of modulation that Everett and Sawyer (1) first described a long time ago on the basis of the pentobarbital block experiments. Everett: You determined whether or not ovulation has occurred, but did not indicate the histologic condition of the unruptured follicles, although commenting that some of them seemed to be enlarged. Was there any evidence of luteinization? Cross: We have not finished the histology yet. I would rather not commit myself. I would think it was minimal luteinization but cannot be dogmatic. Meyerson: You said firing rate is not affected by the urethane anesthesia. What is your impression about the number of neurons firing? Cross: The number of neurons firing is strictly comparable in the urethane and nonurethanized condition. There does not seem to be any reduction in the numbers of recordable units. Brown-Grant: Does urethane in the doses you have tested block ovulation in your colony of rats? Cross: There seems to be a certain amount of discrepancy. In our colony urethane given after the critical period reduces the amount of ovulation. This is the work of Dennis Lincoln (2). If I recall his results correctly, there is a fairly marked depression in the number of ovulations if the urethane is given intraperitoneally after the critical period and rather less depression if the urethane is given subcutaneously. I think he has some evidence that part of the effect is peripheral due to hyperemia in the peritoneal cavity with the intraperitoneal injection, giying a direct atresic effect on the ovary. However, there is some evidence too, that given before the critical period or during the critical period, there is some pentobarbital like action. I am not very clear at the moment about the precise stages of this; whether urethane has the classical pentobarbital effect or not. I think the evidence is conflicting. Taleisnik: Is the firing activity in the morning? Cross: Usually not before 11:30 to noon. Taleisnik: Is there any possibility to see changes in the sensitivity of the island? Cross: Do you mean diurnal changes? Taleisnik: No, changes in the sensitivity to drugs and hormones of the hypothalamus after the island was made. Cross: You mean comparing the island's response with an intact? Taleisnik: Yes. Cross: Well, I think it would be very difficult to get that sort of data, except on a population basis, since you would have to use many many units. You cannot have a situation where you measure a single cell response to some drug in the intact hypothalamus, then make an island and retest the same cell. Unfortunately, it is not possible. Ramirez: When you injected oxytocin, where were you recording? Cross: This was a mixed population of 65 units, and nine of them were in the paraventricular nucleus, which is the place one might expect to see a feedback effect. Others were in the anterior hypothalamus, ventromedial or lateral areas. In other words, they were a dispersed population. In fact, one cell in the anterior hypothalamus showed a brief 30 per cent increase,

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lasting a very short time with a large dose of oxytocin. This occurred fairly early in the series, and we were quite excited about it and looked again and again; but were never able to repeat it. Saline injections also were uniformly negative. Ramirez: Do you have any evidence that oxytocin arrived at the island? Cross: We do not know. We do not have any critical evidence that it is getting through the blood-brain barrier, but there is evidence that it does penetrate. Komisaruk: Do you think the increased excitability of these neurons might indicate an increased responsivity to input from either the environmental stimuli or the preoptic area? Cross: I do not think we can say we had an increased responsivity. All we get is an increased firing rate. Komisaruk: What do you think is the significance of the increased firing rate? Cross: We think we have removed more inhibitory inputs by deafferentation than we have excitatory inputs. There is a balance in favor of faster endogenous activity. This, of course, depends on the assumption that the accelerated firing is not due to incidental things, such as injury potentials from the edge of the tube or ischemia in the island. The evidence I showed you with the hypotension and the hypoxia rather suggested that ischemia cannot increase firing rate in the islands. We always coat our cutter with silicone to eliminate direct contact between metal and nerve tissue in an effort to cut down injury potentials, but, of course, we cannot eliminate this possibility. Feldman: I would just like to say that one would expect an increase in the rate of unit firing in such an island, because our other studies on the hypothalamus have shown that most of the hypothalamic inputs are inhibitory. I was surprised to see that the acceleration was so small. I would expect a larger acceleration. Our rate was 3.9 spikes/sec. What was the average rate in the island? Cross: About three. DISCUSSION REFERENCES 1. Everett, J. W., and Sawyer, C. H., A 24-hour periodicity in the "LH-released apparatus" of female rats, disclosed by barbituate sedation. Endocrinology, 1950, 47: 198-218. 2. Lincoln, D., and Kelly, W. A., Ovulation in the rat following urethane anaesthesia. J. Endocr., 1970, in press.

Figure 9.1. Comparison of the effects of intravenous methohexital and oxytocin on the firing rate of two hypothalamic island units (P. V. nucleus, above; lateral area, below). The ineffectual doses of oxytocin are in the supramaximal range while the doses of the barbiturate which depress unit discharge are below anesthetic level.

DIESTRUS

PROESTRUS

ESTRUS

METESTRUS

DIESTRUS

Figure 9.3. Histograms showing firing rates of 478 island units distributed between the anterior and lateral hypothalamic areas as a function of the stage of the estrous cycle. Because of the exponential distribution of firing frequencies the data were normalized by log transformation and plotted with the ordinate as geometric mean of firing frequency. The peak of firing rate seen in the anterior hypothalamic units in proestrus is significantly different (p .05).

N=83

N=78

N=75

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Urethane

Anaesthesia.

60-

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6

< 3

3-6

m etestrus

die st r u s

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SPIKES/SECOND. Figure 9.5. Histograms showing the similarity in the percentage distribution of firing rates of hypothalamic island units in cyclic rats under urethane and without anesthesia. These histograms show actual firing rates in contrast to Figures 9.3 and 9.4 where geometric means are used.

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10. STEROID SEX HORMONES IN THE RAT BRAIN: SPECIFICITY OF UPTAKE AND PHYSIOLOGICAL EFFECTS DONALD W. PFAFF* Rockefeller University New York, New York

Is radioactive estradiol or testosterone from the blood retained in one brain region and absent elsewhere in the brain? It is important to characterize brain uptake of labeled sex steroids accurately, as a guide for subsequent neurophysiological and neurochemical studies of hormone effects on brain function. Anatomical Specificity There is substantial agreement in the literature on certain important features of radioactive estradiol localization in the brain. Results from several laboratories using scintillation counting of dissected brain areas agree that estradiol is concentrated in the hypothalamus, the preoptic area and the septum to a much greater extent than in most other brain regions, such as in the cerebral cortex (6, 8, 12, 18, 21-23). Areas outside the septal-preoptic-hypothalamic axis showing quantitatively less uptake did not show a complete absence of radioactivity. Autoradiographic results with tritiated hexoestrol in cats (22) and estradiol-17B in rats (24) (Figure 10.1) showed this same regional distribution, and high estradiol-H 3 uptake in preoptic and ventromedial hypothalamic regions has also been shown with autoradiography in female mice (34). A similar regional pattern has been described for testosterone-H 3 using autoradiographic (25) and scintillation counting (19) methods, but testosterone was not concentrated from the blood to as great an extent as estradiol was. Results from different laboratories have also been consistent in further studies of more detailed aspects of estradiol uptake. Competition studies using unlabeled estrogens have demonstrated limited-capacity retention in the preoptic area and hypothalamus (6, 7, 13, 18, 21). Nuclear isolation procedures (35) and high resolution autoradiography (1, 33, 34) have shown estradiol concentration in the cell nuclei in these brain regions. In general, results with these procedures have magnified the difference between brain regions of relatively high and relatively low estradiol uptake levels. However, even in these studies, it has not been possible to dismiss possibly * Supported by NSF GB-4198X, NIH grant NS-08902-01 and the Biomedical Division of the Population Council. Much of this work was performed in collaboration with Drs. C. Pfaffmann, B. S. McEwen, E. Gregory and R. E. Zigmond, to whom the author is indebted.

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significant estradiol retention outside the septal-preoptic-hypothalamic axis. For example, some competition effects of unlabeled estradiol were observed in the amygdala, hippocampus and cerebellum (18). Autoradiographic results from counting reduced grains over cell bodies agree with this finding, in showing estradiol retention in limbic structures such as the amygdala and hippocampus, similar to that in the hypothalamus (24). Finally, isolated cell nuclei from amygdala and hippocampus contained four times or more the concentration of radioactive estradiol compared to the whole homogenates from these regions, and cerebral cortex cell nuclei concentrated estradiol to a still smaller extent (35). In light of these consistent findings from several laboratories, one question which remains is the meaning of relatively low estradiol uptake in many structures outside the septal-preoptic-hypothalamic axis. Although estradiol action in the medial preoptic area and ventromedial hypothalamus provides the most convenient model for biochemical study of estrogen action in brain tissue (especially with reference to pituitary control), it is also necessary, for neurophysiological study of brain function, to have an accurate picture of estradiol uptake outside this region (especially for the study of mating behavior control). The most misleading report of hormone uptake would be that of false negative evidence in a given brain region, which would powerfully imply an absence of physiological hormone action in that region. Differing degrees of positive results, that is relatively higher or lower retention levels, do not lead to such powerful implications because there is no proven correspondence between the amount of steroid retained and the presence or magnitude of physiological effects. One report of autoradiographic work (33) has been interpreted as showing 3 Hestradiol retention exclusively in the distribution area of the stria terminalis. It is important to note that a conclusion emphasizing such a restricted pattern of uptake relies on negative autoradiographic evidence, that is, the failure to see uptake in many preoptic-hypothalamic areas and the failure to see uptake outside the septal-preoptichypothalamic axis. Such autoradiograms seem to show uptake in structures where other investigators have reported high uptake, and the apparent absence of uptake where previous work has shown low uptake. The difference between Stumpf s conclusions and previous results could be explained by a possible loss of sensitivity incurred by his autoradiographic technique. Such a sensitivity loss could occur despite high spatial resolution and can even enhance the apparent spatial resolution. One reason for sensitivity loss could be the failure to expose autoradiograms long enough to show grain reduction over all the cells taking up the radiochemical (1). In addition, with Stumpf s technique the crucial contact between the surface of the tissue and the photographic emulsion is determined by finger pressure, and irregular or poor contact can lead to variable or low autoradiographic sensitivity. Positive results of previous autoradiographic work (24, 25) showing uptake outside the preoptic and hypothalamic locations listed by Stumpf cannot be ascribed to diffusion, because reduced grains were concentrated over cell bodies, with relatively low counts between cell bodies (Figure 10.1). Also, the positive results were not chemical artifacts because brain tissue from rats not injected with isotope was not associated with such grain reduction. Since quantitative autoradiographic results agree in major respects with scintillation counting experiments (see above), it can be concluded that there is not an absolute anatomical specificity of sex steroid hormone uptake for restricted cell groups in the

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105

medial preoptic area and hypothalamus, but rather quantitatively greater uptake there than elsewhere in the brain. The quantitative difference between relatively intense sex steroid uptake by some brain structures and relatively weak retention by other structures is not as great as, for instance, the difference in estradiol retention between uterus and skeletal muscle. Do steroid sex hormones affect electrical activity in one brain region and not in others? Direct application of testosterone to the preoptic region of castrated anesthetized male rats increased the responses of preoptic single units to olfactory bulb electrical stimulation (29) (see Figure 10.2). Systemic testosterone injections had more complex effects on preoptic units, including changes in spontaneous activity and changes in the magnitude and direction of responses to peripheral stimuli. Systemic injections also affected the activity of some units in the olfactory bulb and the mesencephalic reticular formation. Finally, testosterone effects were detected in the cortical EEG, olfactory bulb wave activity and nasal air flow, although blood pressure, heart rate and body temperature were rarely or never affected. Taken together, these results suggest that testosterone effects on the brain are not absolutely restricted to the preoptic area, unless the various effects recorded in other places after systemic testosterone injection were indirect effects mediated by testosterone action in the preoptic area. The presence of electrophysiological effects of testosterone in the olfactory bulb, reticular formation and EEG are consistent with the implications of Hart's demonstrations of testosterone (9, 11) and estradiol (10) effects on genital reflexes in spinally transected animals and with work on radioactive testosterone (19, 20, 25) and estradiol (18, 24) uptake. All of these results suggest possible physiological actions of steroid sex hormones outside the medial preoptic-hypothalamic region, even though that brain region has presented the best opportunity for initial study. Functional Specificity Can steroid sex hormones affect some functional aspects of neural mechanisms and not others? Regarding the single unit effects of sex hormone administration reported above, we may ask what role these effects play in the functioning of behaviorally relevant neural mechanisms. To begin working on this question, we have identified two functional aspects of preoptic single unit activity: (a) the relationship between unit activity and the EEG, a relationship which does appear to be androgen-sensitive; and (b) olfactory coding by differential responses to different odors by preoptic single units, which does not appear to be androgen-sensitive. Part A. Using urethane anesthesia for recording from male rats, we have seen the sudden spontaneous E E G transitions between desynchrony and synchrony reported by Ramirez, Komisaruk and coworkers (14, 31), Lincoln (16, 17) and others. As described in these reports, some units show activity changes correlated with EEG changes (Figure 10.3). In the mesencephalic reticular formation, the typical pattern for cells is to increase activity during E E G desynchronization (29, 32). We have found that a larger proportion of preoptic area single units in normal males than in castrates shows significant act'vity correlations with EEG state, primarily due to the larger proportion of cells showing the typical reticular-like pattern (see Figure 10.4 and reference 28). This effect was not brain-wide, and, therefore, not likely to be due to such a completely nonspecific factor as susceptibility to anesthesia, because no difference was seen in the olfactory bulb between normals and castrates. Further work on

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T A B L E

10.1

M E T H O D OF T A B U L A T I N G

DIFFERENTIAL

R E S P O N S E S BY E A C H U N I T TO T W O

Response to one and not the other? Excited by one and inhibited by other? Responses in same direction but of consistently different magnitude? None of the above. Totals

FUNCTION

ODORS

Differential Response Yes No V V V V A

Percent units responding differentially = —

B

A

— X100

the identification of EEG-related preoptic units, still in progress, has led to three tentative conclusions: (a) those units are not clearly located in a different preoptic subregion from units not related to the EEG, (b) units showing the typical reticular-like relation to the EEG tend to be those showing excitatory responses to odors, and (c) some preoptic units whose activity was related to the EEG showed a definite tendency to decrease activity seconds before the EEG changed from desynchrony to synchrony, suggesting that basal forebrain changes may be causally related to that EEG transition. Part B. The other aspect of preoptic unit activity examined for differences between normal males and castrates was olfactory coding (Figure 10.5). For this analysis, the presence, direction and magnitude of responses to different odors were tabulated for each preoptic unit. Two female rat urine odors (from estrous and ovariectomized females) and three nonurine odors (amyl acetate, cineole and benzaldehyde) were used. For each possible pair of odors, a check list of the type shown in Table 10.1 was constructed, and each unit tested for responses to both odors was placed in one of the four mutually exclusive categories shown. When all units were thus analyzed for that odor pair, the proportion of units responding differentially to the two odors of that pair was calculated (Table 10.1). Then the procedure was repeated for each of the other possible odor pairs. One pair compared the two urine odors (estrous versus ovariectomized female), other pairs compared two nonurine odors, and, finally, still other pairs compared urine odors with nonurine odors. With this analysis, preoptic area unit responses showed an interesting difference from olfactory bulb responses (30). A high proportion of preoptic area units responded differently to estrous female urine odor than to ovariectomized female urine, while only a low proportion of olfactory bulb units did so (Figure 10.6). The opposite was true for discriminative responses among nonurine odors: a higher proportion of olfactory bulb units than preoptic area units responded differently to the members of each pair of nonurine odors (Figure 10.6). A high proportion (70-80 per cent) of units in both brain regions responded differently to urine odors than to nonurine odors. The results of these differential response analyses were essentially the same in castrated as in normal male rats, suggesting that this olfactory coding function of sharpened discrimi-

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OF SEX S T E R O I D

ACTION

107

nation for female urine odors is not androgen-dependent (27). This is consistent with previous results of injecting testosterone acutely while recording from castrated males (29). In those experiments, even though spontaneous activity or absolute magnitudes of olfactory responses by preoptic area units were affected by testosterone injection, there was no consistent tendency for responses to the two female odors to be affected differently from each other. That is, responses to the two odors were not regularly made more or less discriminable from each other. These electrophysiological results agree with behavioral work on the reactions of male rats to female rat urine odors. The preference of male rats for female urine odors, measured by the amount of time they spontaneously investigate such odors is androgendependent (3, 4, 30) but their detection and discrimination of female urine odors is not (2, 5). Thus, it is possible that the androgen-independent differential responses by single units, reported above, are related to androgen-independent behavioral functions of detection and discrimination, while other electrophysiological measures, such as spontaneous activity, absolute response magnitudes, relation to EEG state and others, underly odor preferences. This is supported by the nature of the preference shown by normal male rats for estrous female odors, which has been interpreted as the "activiation" of exploratory sniffing response tendencies by the estrous female urine odor (30). The two sets of electrophysiological results reported above may reflect a difference between the "informational" (coding) and "motivational" (EEG relation) aspects of unit activity in the rat preoptic-hypothalamic area. Hormone Specificity Do estradiol and testosterone have mutually exclusive receptors and effects in brain tissue, or do estrogen-sensitive and androgen-sensitive systems overlap? Results from scintillation counting of dissected brain regions after injection of radioactive testosterone or estradiol have demonstrated several similarities between the two hormone systems (18-20) (Table 10.2A). Autoradiographic results, also, showed similarities between estradiol and testosterone regional distributions in rat brain, both being high in basal forebrain structures and lower in nonlimbic regions (24, 25). On the other hand, estradiol seemed to show a more specific form of uptake than testosterone due to its greater concentration in preoptic-hypothalamic tissue relative to blood or cerebral cortex, and to its larger, more specific competition effects (Table 10.2A) (18-20, 35). Parallel results have come from behavioral studies of gonadectomized male and female rats, in which each animal was tested both for male and female behavior under a variety of hormone conditions (26) (Table 10.2B). Both in male and in female rats, 10 microgram daily doses of estradiol benzoate stimulated masculine mating responses to a marked degree, although not as effectively as testosterone propionate (200 micrograms/day). In female behavior tests, testosterone alone did not raise receptivity above control levels, but daily testosterone propionate injections (200 jug/day) supplemented by 0.5 mg progesterone on the day of testing did induce receptivity in about 50 per cent of the female rats (ave. Lordoses/Mounts = 0.35). Finally, particularly for masculine behavior by either male or female rats, the same animals responding best to androgen also tended to respond best to estrogenic stimulation. Similarities between estradiol and testosterone effects were unlikely to be due to conversion of estradiol into androgen after injection, because estradiol doses were 20 times smaller than testoster-

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10.2

COMPARISONS B E T W E E N ESTRADIOL AND

A. Uptake (18-20, 35) Similarities between Radioactive Estradiol and Testosterone Uptake 1. Pattern of uptake across brain: Both hormones high in preoptic area and surrounding regions. 2. Distribution of limited-capacity sites across brain (competition experiments). 3. Significant cross-competition. 4. Both hormones taken up in cell body. 5. Both hormones taken up in both male and female brains. B. Behavior (26) Similarities between Estradiol and Testosterone Behavioral Effects 1. Male behavior: estradiol mimics testosterone effect. 2. Female behavior: to a small extent, testosterone can mimic estradiol effect.

FUNCTION

TESTOSTERONE

Estradiol Uptake More Specific than Testosterone Uptake Estradiol-H 3 IA. Higher concentration in brain relative to blood. IB. Better uptake in preoptic-hypothalamic regions relative to cortex. 2A. Larger competition effects. 3A. Uptake more specifically susceptible to cold estradiol competition compared to cold testosterone competition. 4A. Uptake in cell nucleus (35).

Estradiol Effect much better than Testosterone Effect

2A. Estradiol effect on female behavior much better than testosterone, even with smaller dose.

3. Male and female behavior: the same rats that respond best to one hormone also tend to respond best to the other.

one doses. Similarities were probably not due to testosterone conversion to estrogen, because in studies of radioactive estradiol uptake in rat brain, high doses of nonradioactive testosterone did not compete for estradiol-H 3 binding sites in the pituitary, even though low doses of nonradioactive estradiol did (18). Taken together, these behavioral results suggest some commonality between estradiol and testosterone actions on the rat brain, with the major exception that estradiol stimulates female behavior much more effectively than testosterone. REFERENCES 1. Anderson, C. H., and Greenwald, G. S., Autoradiographic analysis of estradiol uptake in the brain and pituitary of the female rat. Endocrinology, 1969, 85: 1160-1165. 2. Carr, W. J., and Caul, W. F., The effect of castration in rat upon the discrimination of sex odours. Anim. Behav., 1962, 10: 20-27. 3. Carr, W. J., Loeb, L. S., and Dissinger, M. L., Responses of rats to sex odors. J. Comp. Physiol. Psych., 1965, 59: 370-377. 4. Carr, W. J., Loeb, L. S., and Wylie, N. R., Responses to feminine odors in normal and castrated male rats. J. Comp. Physiol. Psych., 1966, 62: 336-338.

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5. Carr, W. J., Solberg, B., and Pfaffmann, C., The olfactory threshold for estrous female urine in normal and castrated male rats. J. Comp. Physiol. Psych., 1962, 55: 415-417. 6. Eisenfeld, A. J., and Axelrod, J., Selectivity of estrogen distribution in tissues. J. Pharm. Exp. Ther., 1965, 150: 469-475. 7.

, Effect of steroid hormones, ovariectomy, estrogen pretreatment, sex, and immaturity on the distribution of 3 H-estradiol. Endocrinology, 1966, 79: 38-42.

8. Green, R., Luttge, W. G., and Whalen, R. E., Uptake and retention of tritiated estradiol in brain and peripheral tissues of male, female and neonatally androgenized female rats. Endocrinology, 1969, 85: 373-378. 9. Hart, B. L., Testosterone regulation of sexual reflexes in spinal male rats. Science, 1967, 155: 1283-1284. 10.

, Mating behavior in the female dog and the effects of estrogen on sexual reflexes. Hormones and Behavior, 1970, 1: 93-104.

11. Hart, B. L„ and Haugen, C. M., Activation of sexual reflexes in male rats by spinal implantation of testosterone. Physiol. Behav., 1968, 3: 735-738. 12. Kato, J., and Villee, C. A., Preferential uptake of estradiol by the anterior hypothalamus of the rat. Endocrinology, 1967, 80: 567-575. 13.

, Factors affecting uptake of estradiol-6, 7- 3 H by the hypophysis and hypothalamus. Endocrinology, 1967, 80: 1133-1138.

14. Komisaruk, B. R., McDonald, P. G., Whitmoyer, D. I., and Sawyer, C. H., Effects of progesterone and sensory stimulation on EEG and neuronal activity in the rat. Exp. Neurol., 1967, 19: 494-507. 15. LeMagnen, J., Les phenomenes olfacto-sexuels chez le rat blanc. Arch. Sci. Physiol. (Paris), 1952, 6: 295-332. 16. Lincoln, D. W., Correlation of unit activity in the hypothalamus with EEG patterns associated with the sleep cycle. Exp. Neurol., 1969, 24: 1-18. 17.

, Effects of progesterone on the electrical activity of the forebrain.J.Endoc. 1969, 45: 585-596.

18. McEwen, B. S., and Pfaff, D. W. Factors influencing sex hormone uptake by rat brain regions. I. Effects of neonatal treatment, hypophysectomy and competing steroid on estradiol uptake. Brain Res. 1970, 21: 1-16. 19. McEwen, B. W., Pfaff, D. W., and Zigmond, R. E„ Factors influencing sex hormone uptake by rat brain regions. II. Effects of neonatal treatment and hypophysectomy on testosterone uptake. Brain Res. 1970, 21: 17-28. 20.

, Factors influencing sex hormone uptake by rat brain regions. III. Effects of competing steroids on testosterone uptake. Brain Res. 1970, 21: 29-38.

21. McGuire, J . L., and Lisk, R. D., Estrogen receptors in the intact rat. Proc. Nat. Acad. Sci., USA 1968, 61: 497-503. 22. Michael, R. P., Oestrogens in the central nervous system. Brit. Med. Bull., 1965, 21: 87-90. 23. Michael, R. P., and Glascock, R. F„ Cited by Michael, R. P. in Hormonal Steroids, Vol. / / ( L . Martini and A. Pecile, Eds ). Academic Press, New York, 1965: 474. 24. Pfaff, D. W., Uptake of 3 H-estradiol by the female rat brain. An autoradiographic study. Endocrinology, 1968, 82: 1149-1155. 25.

, Autoradiographic localization of radioactivity in rat brain after injection of tritiated sex hormones. Science, 1968, 161: 1355-1356.

26.

, Behavioral responses of rats to sex hormones: specificity of hormone effects and individual patterns of response. J. Comp. Physiol. Psych., 1969, in press.

27. Pfaff, D. W., and Gregory, E., Coding of olfactory input to the olfactory bulb and medial forebrain bundle in normal and castrated male rats. Submitted for publication. 28.

, Correlation between preoptic area unit activity and the EEG: difference between normal and castrated male rats. Submitted for publication.

29. Pfaff, D. W., and Pfaffmann, C., Olfactory and hormonal influences on the basal forebrain of the male rat. Brain Res. 1969, 15: 137-156. 30.

, Behavioral and electrophysiological responses of male rats to female rat urine odors. In: Olfac-

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lion and Taste, III (C. Pfaffmann, Ed.). Rockefeller University Press, New York, 1969: 258-267. 31 Ramirez, V. D., Komisaruk, B. R„ Whitmoyer, D. I., and Sawyer, C. H., Effects of hormones and vaginal stimulation on the EEG and hypothalamic units in rats. Amer. J. Physiol., 1967, 212: 1376-1384. 32. Schlag, J., and Balvin, R., Background activity in the cerebral cortex and reticular formation in relation with the electroencephalogram. Exp. Neurol., 1963, 8: 203-219. 33. Stumpf, W. E., Estradiol-concentrating neurons: topography in the hypothalamus by dry-mount autoradiography. Science, 1968, 162: 1001-1003. 34. Warembourg, M., Fixation de l'oestradiol 3H au niveau des noyaux amgdaliens, septaux et due systeme hypothamo-hypophysaire chez la souris femelle. C. R. Acad. Sei., Paris, 1970, 270: 152-154. 35. Zigmond, R. E., and McEwen, B. S., Selective retention of estradiol by cell nuclei in specific brain regions of the ovariectomized rat. J. Neurochem., 1970, 17: 889-899.

DISCUSSION

From the Floor: I take it you would have expected the results in the castrate since one can obtain this discrimination in a castrate with training. Pfaff: Yes, exactly. Behavioral results show that the detection and discrimination of female urine odors are not androgen sensitive (1,2). So if I had to speculate, I would suggest this androgen-independent neural coding function is related to detection and discrimination of female urine odors. This is to be contrasted to the preference for female urine odors, which is androgen-sensitive (5). In other words, if you ask the animal if he can distinguish estrous from ovariectomized female urine, the castrated male is the same as the normal. But if you ask him what he wants to do about it, then the castrate is much different from the normal. Feldman: What do you think is the meaning of the reversal in responsiveness with testosterone? Pfaff: That is one of two questions about the reversal of response direction after testosterone that I really do not know how to answer. The first question is how the reversal is produced. I agree with your suggestion that the inhibitory response may be secondary to the increase in spontaneous activity. It is just possible that once the spontaneous activity is raised, then the inhibitory response follows. The second question, which you asked, is about the meaning of it. Well, I really do not know. Feldman: Dr. Cross has reported a few years ago that the responsiveness would depend upon the spontaneous activity, but I do not think that in our case this is true because cells with different rates of firing showed facilitory or inhibitory responses. It had nothing to do with the basic activity. We have just recently studied a large series of units, above 1000 units in the hypothalamus of the cat, and examined just this point to see if the direction of responsiveness has anything to do with the spontaneous activity; we found no relation whatsoever. Pfaff: During recording from the preoptic area, we tend to see a relationship of this sort: increased spontaneous activity is associated with more inhibitory responses. Sawyer: There are certain species that depend more on olfaction for mating, for instance, than the rat. Are you working on any other species? Pfaff: Yes, Dr. John Scott and I did quite a bit of recording from mice that Dr. W. Whitten sent us, which were from a strain showing the Whitten effect (estrous acceleration effect) after exposure to normal male urine odor, but not castrated male urine odor

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(7). In fact, we replicated that effect in our own laboratory. Initially we though that maybe the lack of absolute specificity of responses to female urine odors was due to the unfortunate choice of the rat as a species to use, and if we used a mouse that showed one of the nice olfactory-hormonal relationships, olfactory coding would be more specific. However, the coding results in these mice were just about the same as in rats. We found some units that gave significantly different magnitude responses to normal male urine odors, compared to castrated male urine, but we found no units which would respond exclusively and specifically to one of these stimuli (6). Then Mr. Robert Johnston did some recording from hamsters, because there is some evidence, as you know, of hamsters having better olfactory control over sex behavior (4). But there again, at least in our preliminary sample of olfactory bulb units, we have not seen absolutely specific responses to odors from hamster flank gland or vaginal secretion. Feldman: Have you made any recording on a more long-time basis so you look at the difference after a couple of days? Pfaff: We have not yet applied the chronic recording method to the study of testosterone effects. Beach: We have administered a single injection containing 10 mg of TP to male rats two months after castration and found that three of eleven ejaculated when tested with receptive females 24 hours postinjection. At 72 hours, five of eleven ejaculated. Apparently, if it is sufficiently overloaded, the system can respond fairly rapidly. Pfaff: I also have some results that are something like that, using brain implants of testosterone. Three castrated male rats that had preoptic testosterone implants responded in 24 hours after implantation with male mating behavior. We are now following up these results. Barraclough: As you know, many units in the hypothalamus respond to a variety of sensory modalities. Several years ago with Dr. Cross we came across one cell in the lateral hypothalamus whose spontaneous activity could be accelerated by vaginal cervix stimulation or tail pinch. When we applied an olfactory stimulus, the cell's spontaneous activity ceased. Now if we applied both stimuli simultaneously, the olfactory stimulus dominated, and we could not accelerate the cell by either vaginal cervix stimulation or tail pinching. I might say we tested for these responses in many other cells, and this was the only cell which showed these characteristics. Pfaff: As far as the competition between modalities is concerned, I am thinking of the anatomical results of Milhouse (3) in which he shows a predominance of olfactory input to the anterior MFB and a predominance of reticular input to the posterior MFB. DISCUSSION REFERENCES 1. Carr, W. J., and Caul, W. F., The effect of castration in rat upon the discrimination of sex odours. Anim. Behav., 1962, 10: 20-27. 2. Carr, W. J., Solberg, B., and Pfaffmann, C., The olfactory threshold for estrous female urine in normal and castrated male rats. J. Comp. Physiol. Psychol., 1962, 55: 415-417. 3. Milhouse, O. E., A golgi study of the descending medial forebrain bundle. Brain Res., 1969, 15: 341-363. 4. Murphy, M. R., and Schneider, G. E., Olfactory bulb removal eliminates mating behavior in the male golden hamster. Science, 1970, 167: 302-304. 5. Pfaff, D. W., and Pfaffmann, C., Behavioral and electrophysiological responses of male rats to female rat urine odors. In: Olfaction and Taste, / / / ( C . Pfaffmann, Ed.). New York, Rockefeller University Press, 1969; 258-267.

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6. Scott, J. W., and Pfaff, D. W., Behavioral and electrophysiological responses of female mice to male urine odors. Physiol. Behav. 1970, 5: 407-411. 7. Whitten, W. K., Pheromones and mammalian reproduction. Adv. Reprod. Physiol., 1966, 1: 155-177.

Figure 10.1. Autoradiographs of neural tissue f r o m rats injected with tritiated steroid hormones ( i . v . ) . Combined osmium and formalin fixation of unembedded frozen sections. Following evaporation of excess fixative, slides were dipped in N T B - 3 , exposed f o r several months and developed in D19. Lightly stained with cresyl violet. A : Labeled cells in the preoptic area of an ovariectomized female rat injected with estradiol-H 3 2 hrs before sacrifice. Cells in the preoptic region and certain other hypothalamic and limbic areas regularly showed intense labeling. Brain regions outside the limbic-hypothalamic estrogen-concentrating zones showed fewer average grains per cell ( 2 4 ) . B : Examples of scattered labeled cells found occasionally in the cervical spinal cord of ovariectomized female rats, injected Vi hr before sacrifice with estradiol-H 3 . C : Labeled cells in the preoptic area of a castrated male rat injected with testosterone-H 3 2 hrs before sacrifice. T h e preoptic region and surrounding basal forebrain areas showed higher uptake than most other brain areas ( 2 5 ) . D : Example of a small number of labeled cells occasionally found in the cervical spinal cord of castrated male rats, injected Vi hr before sacrifice with testosterone-H 3 .

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i—110 seconds Figure 10.3. Record from a single unit in preoptic area which showed increased resting discharge during EEG desynchrony. Recording from urethane-anesthetized normal male rat. (From Pfaff and Gregory, 28.)

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