EFFECTS OF ACETYLCHOLINE, GABA, AND PROLACTIN ON ANTERIOR PITUITARY HORMONE SECRETION A Disserfoflon Ior II‘Ie Degree OI pk. D. MICHIGAN STATE UNIVERSITY Lindsey James Grandison I976 '- "alfi‘lafll‘ilt '\ LIB R14 R I" -'.: Micfigm Smea- ‘ University fig; & X % // ll’llllllllllll Ill/I I #53:???“ ‘ "r.- n33 4;} This is to certify that the thesis entitled Effects of Acetylcholine, GABA, and Prolactin on Anterior Pituitary Hormone Secretion presented by Lindsey James Grandison has been accepted towards fulfillment of the requirements for Ph.D. Physiology degreein November 12, 1976 I)ate 0-7639 ABSTRACT EFFECTS OF ACETYLCHOLINE, GABA, AND PROLACTIN ON ANTERIOR PITUITARY HORMONE SECRETION BY Lindsey James Grandison l. The cholinergic agonist, pilocarpine, significantly reduced the elevated serum prolactin concentration of estrogen-primed male rats and of female rats during the proestrous surge. By contrast, pilocarpine failed to reduce serum prolactin concentration when adrenergic receptors were blocked by chlorpromazine, haloperidol, or pimozide, or when catecholamine stores were depleted by reserpine. The cholinergic (muscarinic) receptor blocker, atropine, did not affect serum prolactin concentration. However atrOpine, which penetrates the blood brain barrier, prevented pilo- carpine from reducing serum prolactin concentration. Methyl- atropine, which does not readily penetrate the blood brain barrier, did not prevent pilocarpine from reducing serum prolactin concentration. These observations indicate that cholinergic agonists act centrally to reduce serum prolactin concentration, and that they act through dopaminergic neurons. The observed effects of atropine suggest that Lindsey James Grandison cholinergic neurons are not acting tonically and thus are not involved in tonic inhibition of prolactin release. 2. Pilocarpine given before cervical stimulation of female rats on estrous morning, prevented pseudopregnancy induction and lordosis. Pilocarpine prevented pseudo- pregnancy induction in rats given methyl-atropine but not in rats given atropine. These results suggest that cholin- ergic neurons in the brain can inhibit pseudopregnancy induction and lordosis. 3. Pilocarpine given to male rats before ether- or restraint—stress prevented the rise in serum prolactin. Pilocarpine also prevented the rise of serum prolactin in stressed rats given methyl-atrOpine but not rats given atropine. In lactating rats separated from pups for 6 hours, pilocarpine given before pup replacement inhibited suckling. When lactating rats were given methyl-atropine, pilocarpine prevented the rise in serum prolactin during 20 minutes of suckling. These observations show that cholin- ergic agonists can act centrally to inhibit the rise in serum prolactin concentration following stress or suckling. These and the previous results suggest a role for the cholinergic system in the physiological control of prolactin secretion. Lindsey James Grandison 4. Two anterior pituitaries grafted beneath the kidney capsule at the time of ovariectomy reduced the rise in serum LH during the 5th to 11th days postcastration. The pitui- tary grafts increased serum prolactin concentration up to 5-fold in these castrated rats. In female rats bearing the anterior pituitary tumor MtTwlS' serum prolactin concentra- tion was greatly elevated (up to 3850 ng/ml) and castration was not followed by an increase in serum LH 20 days after castration. In male rats, 2 anterior pituitaries grafted 4 days prior to orchidectomy reduced the rise in serum LH for two weeks after castration. Median eminence implants of prolactin, or pituitary grafts beneath the kidney capsule in male rats reduced the rise in serum LH to 50% of that in control rats 24 hours after castration. Systemic administra— tion of prolactin, or pituitary grafts did not decrease the amount of LH released by the in situ_pituitary following injection of 50 ng LRH/100 gm body weight. In addition pituitary grafts prevented the decrease in hypothalamic LRH content observed in male rats 24 hours after castration. These results indicate that prolactin, acting on the hypo— thalamus, probably to increase dopamine turnover, can in- hibit the release of LH. They also suggest that prolactin may have a physiological role in reducing LH release during such states as lactation. Lindsey James Grandison 5. In male rats, the putative neurotransmitter, GABA, significantly reduced serum TSH and increased serum prolactin concentration. Amino-oxyacetic acid, which increases endo- genous brain GABA concentration, reduced serum TSH and pre- vented the rise in TSH following cold exposure. Although amino-oxyacetic acid did not affect basal prolactin concen- tration, it prevented the fall in prolactin during cold exposure. The GABA antagonist, bicuculline, at non-convul- sant doses, reduced serum prolactin concentration but did not affect TSH release. These results suggest that GABA neurons participate in regulation of TSH and prolactin release. EFFECTS OF ACETYLCHOLINE, GABA, AND PROLACTIN ON ANTERIOR PITUITARY HORMONE SECRETION BY Lindsey James Grandison A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1976 Dedication This dissertation is dedicated to my wife Peggy and my son Timothy. They have given endlessly to me during the completion of this_work. ii ACKNOWLEDGMENTS I wish to acknowledge thanks to Dr. Joseph Meites for providing me with the support and the opportunity to under- take these studies. Dr. Meites has created a challenging, stimulating atmosphere where extensive experience and scientific judgment are gained. I have developed a deep respect for him because of his genuine excitement with research and vast store of knowledge. I also wish to thank my fellow graduate students, past and present, for their help and encouragement during my graduate studies. iii TABLE OF CONTENTS Page LIST OF TABLESOOOOOOOOOOOOI.OOOOOOOOOOOOOOOOOOOOOOOO Vii LIST OF FIGURESOOOOOCOCOOOOOOOOOOOOOOO...0.00.0.0... Viii INTRODUCTION........................................ 1 REVIEW OF THE LITERATURE............................ 5 I. The Hypothalamus.............................. 5 A. Anatomy.................................... 5 B. Hypophyseal Portal System.................. 9 C. Connections to the Hypothalamus and CNS Structures Influencing Hypothalamic Activity................................... 10 II. Putative Transmitters of the Hypothalamus..... 17 A. Catecholamines............................. 17 B. Serotonin.................................. 24 C. Acetylcholine.............................. 27 D. Gamma Amino Butyric Acid (GABA)............ 29 III. Hypothalamic Hormones......................... 30 A. Distribution of Hypothalamic Hormones...... 31 B. Action of Hypothalamic Hormones............ 36 IV. Hypothalamic Regulaton of Pituitary Prolactin, Luteinizing Hormone (LH), and Thyroid Stimu- lating Hormone (TSH) Release.................. 41 A. Prolactin.................................. 41 B. Luteinizing Hormone........................ 50 C. Thyroid Stimulating Hormone................ 58 V. Effects of Prolactin on the Hypothalamus...... 60 A. Effects of Prolactin on Prolactin Secretion 62 B. Effects of Prolactin on Gonadotropin Secretion.................................. 65 iv TABLE OF C. D. E. CONTENTS—~continued Effects of Prolactin on ACTH Secretion...... Effects of Prolactin on Hypothalamic Activity.................................... Physiological Role for the Central Effects of Prolactin................................ MATERIALS ANDMETHODSCOOOOOOOOIOOOOOOOOOOOCOOOOOOCOOO I. II. Cannulation of the Lateral Ventricle of the Rat Arlima1800¢oooooooooooooococoooooooooooooooooooo III. Radioimmunoassay............................... EXPERIMENTAL DATAOOOOOOOOOOOOOOOOOOOOOOIOOOOOOCOOOOOO I. II. III. IV. Evidence for Adrenergic Mediation of Cholin- ergic Inhibition of Prolactin Release.......... A. B. C. D. Introduction................................ Materials and Methods....................... Results..................................... Discussion.................................. Effects of Pilocarpine on Pseudopregnancy Induction.0.0.0.000...0.00000000COOOOOOOOOOOOOO A. B. C. D. Introduction................................ Materials and Methods....................... Results..................................... Discussion.................................. Effects of Pilocarpine on the Rise in Serum Prolactin Concentration After Suckling and StreSSOOOOOOCOOOCOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO A. B. C. D. Inhibition by Prolactin of Post-Castration Rise in A. B. C. D. Introduction................................ Materials and Methods....................... Results..................................... Discussion.................................. LHOOOCCOOOCCOOOOOOOOOOOO0.000000000000000... Introduction................................ Materials and Methods....................... Results..................................... Discussion.................................. Page 68 69 70 72 72 73 74 76 76 76 77 81 91 93 94 95 101 104 104 105 106 111 113 113 114 116 127 TABLE OF CONTENTS--continued Page V. Effects of GABA on TSH and Prolactin Release... 131 A. IntrOduCtion. O O O I I O O O O O I O O I O O O O O I O O O O O O O I O O O 131 B. Materials and Methods....................... 132 C. ResultSOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.. I 134 D. DiscuSSj-on. I O O O O O C O I O O O O O O O C O O O O O O O I O O O O O O O O 140 GENEML DISCUSSION. 0 O O O I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 142 BIBLIOGRAPHYOOOOOOOCC O. O... O. O .0. 0.. 0.. .0. 00.... 0.. O. 148 APPENDIX : GLOSSARY. C O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 196 CURRICULUMVITAEOOOOOOOOOOCOOO0.000000000000000000000 200 vi TABLE 10. 11. 12. LIST OF TABLES Page Effects of Pilocarpine Nitrate (5 mg/kg) on Pseudopregnancy Induced by Cervical Stimulation. 97 Dose Response Relationship of Pilocarpine Nitrate on Cervical Stimulation Induced Pseudo- . pregnanCYOOOOOOOOOOOOOO0.0.0....OOOOOOOOOOOOOIOO 97 Effects of Atropine Sulfate and Methyl-atropine Bromide on Inhibition of Pseudopregnancy by Pilocarpine NitrateOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 99 Effects of Pilocarpine on the Rise in Serum Prolactin Concentration (ng/ml) after Ether Exposure.00.0.0.0...0.00.00.00.00...00.0.0000... 107 Effects of Pilocarpine on the Rise in Serum Prolactin Concentration after Restraint......... 107 Effects of Pilocarpine on the Rise in Serum Prolactin Concentration After Suckling.......... 110 Effects of GABA on Serum TSH and PRL in Male Rats.....OOOOOOOO000......0..OOOOOOOOOOOOOOOO... 135 Effects of AOAA on Serum TSH and PRL............ 136 Effects of Bicuculline Methyliodide on Serum TSH and PRLOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO000...... 136 Effects of Picrotoxin on Serum TSH and PRL...... 137 Effects of GABA and Bicuculline Methyliodide on Serum TSH and PRL in Estrogen Primed Male Rats.. 137 Effects of AOAA and Cold Exposure on Serum TSH and PRLOO00....OOOOOIOOOOOOOOOOOOOOO0.00.0000... 139 vii LIST OF FIGURES FIGURE 10. 11. 12. Anatomy of the hypothalamus..................... Biosynthesis pathway of catecholamines.......... Catabolism pathway of catecholamines............ Effects of pilocarpine on prolactin release in proestrous female and estrogen-primed male rats. Effects of pilocarpine on prolactin release in male rats given reserpine or chlorpromazine..... Effects of pilocarpine on prolactin release in male rats given haloperidol or pimozide......... Effects of atropine on prolactin release in.male rats...OOCOOOOOOOOOOOOOOOIOCOOOOOOIOOOOOOOOOOOOI Effects of pilocarpine on prolactin release in male rats given 0.85% NaCl, atropine or methyl- atropineOOOOOOOOOOOO...OOOOOOIOOOOOOOOOOO...O... Effects of pilocarpine on the serum concentra- tion of prolactin l and 4 hours after cervical SthIationOIOOOOOOOOOCOOOOOIOOOOOOOOOOOIOOOO... Effects of pilocarpine on the serum concentra- tion of prolactin 2-5 days after cervical stimu- lationOOOO0.00.0.0000...OOOOOOOOOOCOOOOOOOOOOOOO Effects of pilocarpine on serum prolactin con- centration of restrained rats given atrOpine or methyl-atropine.............................. Effects of AP grafts on serum LH and prolactin from 5 to 44 days after ovariectomy............. viii Page 23 25 83 86 88 89 90 98 100 109 117 LIST OF FIGURES--continued FIGURE Page 13. Effects of AP grafts on serum LH and prolactin from 5 to 17 days after ovariectomy............. 118 14. Effects of anterior pituitary tumor on serum LH after ovariectomy............................... 119 15. Effects of LRH on serum LH concentration in ovariectomized AP grafted rats.................. 121 16. Effects of LRH on serum LH concentration in ovariectomized rats given prolactin............. 123 17. Effects of AP grafts on serum LH during the 22 hours after orchidectomy........................ 124 18. Effects of AP grafts on serum LH concentration of rats 18 hours to 20 days after orchidectomy.. 125 19. Effects of median eminence prolactin implants or AP grafts on serum LH, hypothalamic LRH and serum prolactin 24 hours after orchidectomy..... 126 ix INTRODUCTION The importance of the anterior pituitary gland in physiological function is now well established. After hypo- physectomy, mammals are unable to reproduce and incapable of satisfactory adaptation to their changing environment. The anterior pituitary synthesizes polypeptides or proteins and releases them into the circulation. These polypeptide hormones are transported through the vascular system and act on peripheral end organs. The release of anterior pituitary hormones is a dynamic process, responsive to external stimu- li and endogenous rhythms. Yet changing rates of hormone release are not characteristic of the isolated anterior pituitary. In the rat translocation of the anterior pitui- tary from the sella turcica to the renal capsule or to the eye is associated with a steady, reduced rate of release for all anterior pituitary hormones except prolactin (Meites gt_al., 1963), and with atrophy of the target organs of the anterior pituitary hormones. Similarly, during incubation or organ culture of anterior pituitary tissue in_yitrg_ hormone release occurs at a steady, slow rate except for the release of prolactin. Removal of the anterior pituitary from the sella turcica stimulates release of prolactin. Relocation of a transplanted pituitary to the sella turcica restores normal anterior pituitary function (Nikitovitch— Winer and Everett, 1958). Such observations implicated the hypothalamus, located directly above the pituitary, in the regulation of anterior pituitary hormone release. In agree- ment with this concept, lesions of the median eminence of the hypothalamus produced effects similar to pituitary trans- location, while electrochemical stimulation of the hypo- thalamus induced hormone release. The hypothalamus is thought to act as a transducer, monitoring environmental stimuli and endogenous function, integrating input, and in- ducing hormone release to maintain homeostasis and to adapt the organism to its environment. Early investigation into the anatomical relationship between the hypothalamus and the anterior pituitary demon- strated no neural connections, but revealed a vascular portal connection. In the rat the entire arterial blood supply of the anterior pituitary courses from branch arter- ies of the circle of Willis through a capillary bed in the median eminence, down the hypophyseal portal vessels and into a second capillary bed in the anterior pituitary. For normal pituitary function the hypophyseal portal system must be intact (Harris, 1955). It is now well recognized that the hypothalamus synthesizes and releases hypothalamic hormones into the hypophyseal portal system, these hormones induce anterior pituitary hormone release. In turn hypo- thalamic hormone release appears to be regulated by hypo- thalamic neurons. Sawyer et a1. (1949) provided the first evidence for neural involvement in anterior hormone release when they blocked c0pulation-induced ovulation in the rabbit with the adrenergic receptor blocker dibenamine. Since then much investigation has been directed toward establishing the identity of neurotransmitters in the hypo- thalamus which induce release of each anterior hormone, and factors effecting neurotransmitter release. The hypothala- mus is rich in several neurotransmitters: dopamine (Carlsson 33 31., 1962), norepinephrine (Vogt, 1954), serotonin (Amin st 21., 1954), histamine (Lipinski et_al., 1970) and gamma amino butyric acid (Berl and Wealsh, 1958). The work of Sawyer in the 1940's and the histochemical mapping of cate- cholaminergic and serotonergic neurons into and within the hypothalamus during the 1960's (Ungerstedt, 1971) provided a great stimulus to investigate the role of catecholamines and serotonin in hypothalamic regulation of hormone release. Such efforts indicated that catecholamines or serotonin are involved in controlling the release of each anterior pitui- tary hormone. However, only 5% of the nerve terminals in the hypothalamus contain catecholamines (Hokfelt gt 31., 1970). Little is known about the involvement of the neuro- transmitters in hormone regulation in the other 95% of hypothalamic nerve terminals. The present studies examine the role of the putative transmitters acetylcholine and gamma amino butyric acid (GABA) in regulation of anterior pituitary secretion of prolactin and thyroid stimulating hormone (TSH). In addi— tion, the effects of increased dopamine turnover induced by prolactin on release of luteinizing hormone (LH) were investigated. A glossary is provided in the Appendix to aid the reader in interpreting abbreviations and effects of drugs. REVIEW OF THE LITERATURE I. The Hypothalamus A. Anatomy Wherever possible the function of the hypothalamus is related to its anatomy. With a recently developed procedure for isolating individual nuclei (Palkovits, 1973), the role of each hypothalamic nucleus in regulation of hormone re- lease is currently being explored (Kizer EE,El-r 1974, 1976a,b). Consequently the anatomy of the hypothalamus is taking on additional significance for future neuroendocrine research. The hypothalamus is a bilateral structure forming the floor and lower walls of the third ventricle. It extends caudally from the rostral edge of the optic chiasm to a plane immediately behind the mammillary bodies. The hypo- thalamic sulcus is the dorsal boundary separating the hypo- thalamus from the overlying thalamus, while the ventral surface of the hypothalamus forms the base of the brain. Located within the hypothalamus are dense masses of nerve cell bodies referred to as nuclei (Figure l). The nuclei are paired bilaterally on either side of the third ventricle. Figure 1. Anatomy of the hypothalamus. POA Preoptic Area AHA Anterior Hypothalamic Area SC Suprachiasmatic Nucleus PVH Paraventricular Nucleus DMH Dorsal Medial Nucleus VMH Ventral Medial Nucleus PH Posterior Hypothalamic Area PMD Dorsal Premammillary Nucleus ARH Archate Nucleus MM Medial Mammillary Nucleus MP Posterior Mammillary Nucleus (DeGroot, 1959) At the anterior hypothalamus of the rat is the unpaired median preoptic nucleus located just dorsal to the third ventricle. The preoptic area composed of a medial and lateral preoptic nucleus is at the rostral end of the third ventricle. The suprachiasmatic nucleus lies directly above the optic chiasm and the supraoptic nucleus is formed from two components, one dorsolateral and the other ventromedial to the Optic chiasm. Caudal to the preoptic area and dorsal to the suprachiasmatic nucleus is the anterior hypothalamic nucleus. The paraventricular nucleus is at the dorsal part of the hypothalamus caudal to both preoptic and anterior hypothalamic nuclei. The third ventricle is lined by ependymal cells; lateral to them is the periventricular area. At the rostal part of the hypothalamus the cell bodies in this region are scattered while in the middle portion of the hypothalamus the cell bodies of the periventricular region are grouped together forming the periventricular nucleus. Other nuclei in the middle section of the hypothalamus are the arcuate nucleus located at the ventral wall of the third ventricle. Dorsal lateral to the arcuate is the ventral medial nucleus, and dorsal to it, the dorsal medial nucleus. Lateral to this column of nuclei is the lateral hypothalamic area, a diffuse region of cell bodies. The dorsal hypothal— amic area is another region of scattered cell bodies extend- ing along the dorsal part of the hypothalamus from the anterior hypothalamic nucleus to the caudal limit of the hypothalamus. In the caudal region of the hypothalamus, the mammillary complex is the major anatomical structure con- sisting of a medial- and lateral-mammillary, and supramammil- lary nuclei. Surrounding the mammillary complex are the pre-mammillary nucleus anteriorly and the intercalate and mammillary cinereus nuclei basolaterally. The fornix, a nerve tract, terminates in the mammillary complex and is surrounded by the perifornical nucleus. The posterior hypothalamus contains two regions of diffuse cell bodies, the periventricular area and, dorsal to the mamillary com- plex, the posterior hypothalamic area. The remaining major hypothalamic structure is the medi- an eminence, the floor of the third ventricle surrounding the tubular infundibular stem. It is organized into three layers: an ependymal layer lining the third ventricle, a fibrous layer, and an outer palisade layer, site of the primary capillary bed. Within the ependymal layer of the median eminence are specialized ependymal cells of unusual shape and variable size, i.e., tanocytes. Processes from tanocytes project down to the palisade layer in the vicinity of the primary capillary plexus. Knigge et_§l, (1972) pro— posed that tanocytes participate in regulating anterior pituitary hormone release by transporting releasing factors from the cerebral spinal fluid (CSF) to the primary capillary bed of the median eminence. Releasing factors have been found in the CSF (Joseph 33 31., 1974; Knigge and Joseph, 1974; Morris and Knigge, 1975) and in tanocytes (Zimmerman gt_§l., 1974). The tanocytes are able to transport releas- ing factors, polypeptide hormones and steroids (Ondo 35 al., 1967; Kumen and Knowles, 1967; Silverman 35 $1., 1973). Intraventricular catecholamine administration induced morphological changes in tanocytes (Schecter and Weiner, 1972) and altered anterior pituitary hormone release. In the monkey changes in tanocyte morphology correlate with stages of the menstrual cycle and in gonadectomized monkeys can be induced with steroid treatment (Knowles and Kumen, 1974). However, the physiological significance of releasing factor tranSport by tanocytes has not been established. B. Hypophyseal Portal System There are no neural connections between the hypothala- mus and the anterior pituitary. However, Popa and Fielding (1930) described a portal system between the hypothalamus and the pituitary in which blood flowed from the pituitary to the hypothalamus. Later Wislocki and King (1936) sug- gested blood flow was in the Opposite direction. Harris (1955) conclusively demonstrated that blood flow was from the hypothalamus to the pituitary and proposed factors enter the portal vessels in the hypothalamus, travel to the anterior pituitary and induce hormone release. 10 In the rat, anterior hypophyseal arteries branch from the internal carotid arteries and form a coiled, looped capillary network in the median eminence and infundibular stem: the primary capillary plexus. The long portal vessels on the surface and inside of the stalk connect the primary capillary bed with a second capillary bed in the anterior pituitary. The inferior hypophyseal arteries form a primary capillary bed in the posterior pituitary. This bed is drained by short portal vessels into a secondary plexus in the anterior pituitary. As a result of these vascular connections, the arterial blood supply of the anterior pituitary first passes through the hypothalamus where it is enriched with releasing factors. C. Connections to the Hypothalamus and CNS Structures Influencing Hypothal— amic Activity The hypothalamus has the major role in regulating anterior pituitary hormone secretion. The basomedial hypo- thalamus, separated from the rest of the brain, maintained basal secretion of anterior pituitary hormones and prevented atrophy of the end organs (Halasz, 1969). Removal of end organs still induced hypersecretion of the respective trophic hormones though diurnal ACTH rhythms and ovulation were absent. The preoptic area is responsible for trigger- ing the ovulatory surge Of LH. Deafferentation of rostral, dorsal, and lateral connections to the preOptic area did not l 11 inhibit ovulation as long as the connections between the preoptic and basomedial hypothalamus were intact (Halasz and Gorski, 1967). Other central nervous system structures connected to the hypothalamus do modify its activity. Since drug treat- ment has been a major approach in examination of hypothala- mic regulation of anterior pituitary hormone secretion, these extrahypothalamic structures must also be considered sites of drug action. The amygdala is connected to the hypothalamus by the stria terminalis and the ventral amygdalofugal pathway. In the rat the stria terminalis connects the corticomedial component of the amygdala with the anterior hypothalamic nucleus, the zone surrounding the ventromedial nucleus, and sparingly with the ventral premammillary nucleus. The ventral amygdalo-hypothalamic pathway extends from the basolateral amygdala to the lateral hypothalamic area. The influence of the amygdala has been most clearly established in hypothalamic regulation of gonadotroPin secretion. Stimulation of the amygdala electrochemically (Bunn and Everett, 1957; Van der Schoot, 1974) or by carbachol injec- tion (Velasco and Taleisnak, 1969) caused ovulation in female rats induced into persistent estrus by continuous exposure to light. Similarly electrochemical stimulation of the amygdala induced ovulation in reserpine— or 12 atropine—treated rats, increased LH in estrogen-primed ovariectomized rats (Velasco and Taleisnak, 1969), and advanced the LH surge on proestrus (Taleisnak, 1976). Cutting the stria terminalis or coagulating the medial amygdala temporarily blocked ovulation for two or three estrous cycles (Velasco and Taleisnak, 1971; Kawakami and Terasawa, 1972; Brown-Grant and Raisman, 1972; Velasco, 1972) while lesion of the cortical amygdala inhibited ovarian compensatory hypertrophy (Smith and Lawson, 1972). In addition to its facilitatory role the amygdala has been found to inhibit gonadotropin release also. Thus, electro- chemical stimulation of the basolateral amygdala in proes— trous rats inhibited the preovulatory LH surge (Taleisnak, 1976; Kawakami et_al., 1976) and in rats on diestrous III blocked ovulation (Van der Schoot, 1974). Lesions of the cortical amygdala in ovariectomized female rats increased serum LH (Lawton and Sawyer, 1970) and electrocoagulation of the basolateral amygdala in male rats increased LH activ- ity in the blood (Eleftheriou gt_a1., 1969). In immature female rats lesions of the amygdala advanced puberty (Elwers and Critchlow, 1960) whereas stimulation delayed it (Bar Sela and Critchlow, 1966). Sawyer (1972), in review- ing the effects of the amygdala on gonadotropin secretion postulated the area of the amygdala stimulatory to gonado- trOpin release may be estrogen sensitive and dominant to 13 the amygdala inhibitory area under such conditions. The influence of the amygdala on the secretion of other pitui- tary hormones is not clearly defined. The amygdala may have a stimulatory role in prolactin secretion since estro- gen implants in the amygdala induced lactogenesis (Tindal §E_al., 1966, 1967) while amygdaloid lesions inhibited mammary tumor growth (Welsch gt_al., 1968), both prolactin- dependent phenomena. Recently Martin (1974) reported that stimulation of the basolateral amygdala increased growth hormone (GH) release while stimulation of the corticomedial portion decreased GH release. The hippocampus is another forebrain structure with a major efferent connection to the hypothalamus. The post- commissural fornix column from the hippocampus has its main termination in the lateral subdivision of the medial mammillary nucleus. Offshoots of this column terminate in the rostral hypothalamus. One distinguishable offshoot is the medial corticohypothalamic tract which terminates in the rostral half of the periventricular zone and the arcuate nucleus. Hippocampal influences on hormone release have been reported mainly for gonadotropin secretion. Hippo- campal stimulation prolonged vaginal estrous cycles from 4 days to 5 in rats (Kimura and Kawakami, 1972). Electro- chemical stimulation of the subiculum in the ventral hippo— campus blocked spontaneous ovulation and the ovulation 14 induced in rats manifesting persistent estrus by stimulation of the amygdala or preoptic area (Velasco and Taleisnak, 1969). The proestrus LH surge and the LH surge after ster- oid treatment were likewise blocked by electrochemical stimulation of the hippocampus (Taleisnak, 1976; Velasco and Taleisnak, 1969). Release of FSH is also inhibited by the hippocampus (Kawakami, 1972). The effects of hippo- campal stimulation on gonadotropin release are reversed in immature rats. Stimulation of the hippocampus advanced vaginal opening and increased FSH release (Kawakami and Terasawa, 1971) and hippocampal lesion delayed puberty (Riss et_§l., 1963). Sectioning the medial corticohypothalamic tract prevented hippocampal stimulation from effecting gona- dotropin release (Velasco and Taleisnak, 1969b; Gallo e: 31., 1971; Taleisnak, 1976). Prolactin release may be inhibited by the hippocampus (Kawakami et_al,, 1972) while growth hormone release was stimulated (Martin, 1972, 1974; Martin gt 31., 1973). Other forebrain structures project fibers to the hypo— thalamus in the medial forebrain bundle with terminations in the lateral preopticohypothalamic zone and premammillary nucleus. Stimulation of the bed nucleus of the stria terminalis, the septal nucleus and the nucleus accumbens increased LH release (Kawakami gt_al., 1970, 1972, 1973; Kawakami and Kumura, 1973). Although the medial forebrain 15 fibers from the anterior olfactory nucleus, the olfactory tubercle and the periform cortex have not yet been reported to change hormone release, this olfactory input might medi- ate the effects of pheromones in the Bruce and Whitten phenomena. In both circumstances it has been suggested that prolactin release is inhibited. Thalamicohypothalamic connections have been reported but not well defined and their significance on pituitary function remains unknown. From the brain stem reticular formation two well described pathways to the hypothalamus exist. The mammillary peduncle in the medial forebrain bundle (MFB) arises in the mediocaudal midbrain and projects to the mammillary body, the lateral hypothalamic area, and the preoptic nucleus. The dorsal longitudinal fasciculus connects the central grey of the mesencephalon mainly with the posterior hypothalamus. In the mesencephalon stimula- tion of the medial raphe nucleus, the periaqueductal grey of the rostral midbrain or the ventral tegmental area in- hibited spontaneous ovulation, progesterone facilitated LH release in proestrous rats and LH release in estrogen primed ovariectomized rats (Carrer and Taleisnak, 1970, 1972). The inhibitory signals from these areas reach the hypothalamus through the dorsal longitudinal fasciculus, the medial fore- brain bundle, and the medial corticohypothalamic tract via the MFB and the hippocampus (Carrer and Taleisnak, 1972). 16 In contrast to the ventral tegmental effects, stimulation of the dorsal tegmentum induced ovulation in persistent-estrous rats, and increased LH release in ovariectomized estrogen- primed rats (Carrer and Taleisnak, 1970). Other minor tracts from extrahypothalamic structures have been reported. A pallidohypothalamic tract projects from the globus pallidus to the ventral medial nucleus of the hypothalamus where it loses its myelination. However, its actual termination is not established. Direct connec- tions between the cerebral cortex and the hypothalamus via a frontohypothalamic tract were suggested and may convey the effects of cortical spreading depression onto LH and prolactin release (Columbo et_al., 1975). Retino-hypothala- mic connections were observed by some investigators but remain unconfirmed. Other indirect pathways may convey the effects of light on hypothalamic function. For example, the light-responsive pineal gland, although it lacks a direct neural connection to the hypothalamus, influenced hypothalamic activity. The pineal product melatonin in- hibited LH and FSH release and stimulated prolactin release when injected intraventricularly (Kamberi et al,, 1970a,b). Since melatonin alters hypothalamic serotonin metabolism, it may effect hormone release by this mechanism as well (Anton Tay et 31., 1968). Other pineal compounds also have antigonadotrOphic effects (Reiter, 1974). However, pinealec- tomy in the rat does not produce dramatic effects on 17 reproductive function (Blake, 1976), so that the physio- logical significance of the above observations is unresolved. Reiter (1974) suggested that the conventional 12 to 14 hours of light exposure of rats in domestic colonies may produce physiological pinealectomy. Further manipulation of the light-responsive pineal gland would therefore produce negligible effects. II. Putative Transmitters of the Hypothalamus Recent advances in biochemical pharmacology have per- mitted the mapping of biogenicvamine-containing neurons, and the measurement of neurotransmitters within individual hypothalamic nuclei. As a result, changes in hormone secre- tion can now be related to altered neuronal activity in discrete hypothalamic nuclei. A. Catecholamines 1. Distribution of Catecholamines in the Hypothalamus a. Norepinephrine Using the histochemical technique of Hillarp and Falck (1962), Dahlstrom and Fuxe (1964) described groups of nor- adrenergic cell bodies.. Each group is labeled using the letter A followed by a number. The axons from noradrenergic neurons of the medulla oblongata and pons (designated Al, 2, 4-7) enter the medial forebrain bundle as the ventral 18 noradrenergic system and terminate in the hypothalamus, basal forebrain and limbic structures. The noradrenergic neurons in the subcaeruleus (ventral part of group A6 and A7) terminate in a periventricular plexus along the third ventricle of the hypothalamus and preoptic area (Olsen and Fuxe, 1972). Lesion of the locus caeruleus reduced norepinephrine concentration in the para— ventricular and periventricular nuclei (Kobayashi gt_§1., 1974). The noradrenergic fibers from cell bodies A1, A2 and A5 terminate in the basal and lateral hypothalamus, and the preOptic area (Olson and Fuxe, 1972). DOpamine B-hydroxy- lase, an enzyme marker for noradrenergic neurons, is lost from the median eminence, and the arcuate, ventromedial and dorsal medial nuclei after hypothalamic deafferentation and in the ventral medial hypothalamus after transection of the ventral noradrenergic bundle (Brownstein et_al., 1976; Kizer 33 21., 1976). Ungerstedt (1971) described in more detail the termination of the noradrenergic neurons of the medulla oblongata and pons. Noradrenergic terminals were found in preoptic, supraoptic, retrochiasmatic, and paraventricular nuclei, in the periventricular, dorsal medial, and arcuate nuclei, the internal layer of the median eminence and the area ventral to the fornix. Measurement of the norepineph- rine concentration of hypothalamic nuclei (Palkovits et 21., 1974) showed the highest concentration in the l9 retrochiasmatic nucleus, the periventricular and dorsal medial nuclei and the median eminence agreeing with the histochemical localization of noradrenergic terminals. The distribution of dopamine B-hydroxylase is similar to that of norepinephrine (Saavedra et_al,, 1974a). b. Dopamine Unlike noradrenergic neurons, dopaminergic cell bodies are located in the hypothalamus. The dopamine cell bodies in the arcuate nucleus (cell group A 12) innervate the external layer of the median eminence (Carlsson et_al., 1962; Fuxe, 1963). This dopamine system has been the focus of much neuroendocrine research. However, 80 percent of the dopamine in the hypothalamus is found outside the arcuate-median eminence area (Bjorklund et_al., 1970; Brown et_al., 1972; Kavanagh and Weiss, 1973). Recently Bjorklund et_al. (1975) described an incerto—hypothalamic dopamine neurone system. There are two cemponents, not directly con- nected to each other: a caudal part, and a rostral peri— ventricular-preoptic part. The cell bodies of the caudal component are located in the periaquaductal grey of the rostral mesencephalon, the periventricular grey, and the parafascincular nucleus of the caudal thalamus (the A 11 cell bodies) and in the posterior hypothalamic area and zona incerta (A 13 cell bodies). The dopaminergic terminals from these cells are found in the dorsal part of the dorsomedial 20 nucleus and the dorsal and anterior hypothalamic areas. The rostral component arises in the anterior periventricular nucleus of the hypothalamus (A 14 cell bodies). It extends laterally to the medial preoptic area, rostrally to the periventricular and suprachiasmatic-preoptic nucleus and into the septal nucleus. These newly described incerto- hypothalamic dopamine neurons are regarded as a system of short intradiencephalic neurons. Substantiating the distri— bution of dopamine neurons, biochemical measurement of dopamine indicated very high concentrations in the median eminence and arcuate nucleus, with high concentration in the suprachiasmatic, paraventricular, and medial portion of the ventromedial and dorsal medial nuclei (Palkovits gt_al., 1974). Deafferentation of the medial basal hypothalamus did not significantly reduce its dopamine content (Weiner §t_al., 1972). Histochemical fluorescence has been applied at the ultrastructural level in the median eminence to determine the percent of monoamine boutons of total and the number of monoamine boutons per square unit (Ajika and Hokfelt, 1973). In the lateral part of the median eminence about one-third of the nerve endings were monoamine neurons, probably dopamine. In this area only glial cells and nerve endings were found. There was no evidence for axo-axonic synapses in this area but synaptic-like structures were reported 21 between boutons and tanocytes (Guldner and Wolff, 1973; Kobayashi and Matsui, 1967). Dopamine does effect the tanocytes (Schecter and Weiner, 1972; Hokfelt, 1973, 1974) and may influence releasing factor secretion in this way (Hokfelt, 1973, 1974). c. Epinephrine It has been only recently that the distribution of epinephrine containing neurons has been described. Immuno- histochemical localization of phenylethanolamine-N-methyl- transferase (PNMT), indicated a central tract projecting from the medulla oblongata to the arcuate nucleus, especial- ly the ventral lateral part, the internal and subependymal layers of the median eminence, and the magnocellular portion of paraventricular nucleus (Hokfelt et 31., 1973, 1974). Biochemical measurements of PNMT activity and epinephrine substantiate the immunochemical data (Saavedra et_al., 1974; Koslow and Schlumpf, 1974; Van der Gugten 25 31., 1976), but in addition suggest an equally high concentration of epi- nephrine is located in the periventricular, medial preOptic, supraoptic and dorsal medial nuclei (Van der Gugten §t_al., 1976). 2. Metabolism of Catecholamines The catecholaminergic neurons of the hypothalamus regu- late anterior pituitary hormone release by depolarizing and releasing catecholamines. There is no method presently 22 available to monitor the neuronal firing of hypothalamic catecholaminergic neurons selectively. However, catechol- amine synthesis is reflective of bioelectrical activity. Changes in catecholamine synthesis and turnover do correlate with changes in rate of anterior pituitary hormone release, and catecholamine metabolism can be altered by hormones. Thus, regulatibn of catecholamine metabolism is a central concern in neuroendocrinology. The synthetic pathway of catecholamines is depicted in Figure 2. Tyrosine from the blood enters the brain and is taken up into catecholaminergic neurons by an active trans- port mechanism (Chirigos et 31., 1960). The rate—limiting step in catecholamine synthesis is the hydroxylation of tyrosine to dopa by tyrosine hydroxylase. This enzyme is saturated under physiological conditions and inhibited by its end products. DOpa, dOpamine and norepinephrine compete with a pterin cofactor for binding to the enzyme. The remaining steps in catecholamine synthesis occur rapidly. Dopa is decarboxylated to dopamine by z—aromatic acid decarboxylase, a ubiquitous, non-specific enzyme. Noradre— nergic neurons contain an additional enzyme, dopamine-B- hydroxylase, which hydroxylates dopamine to norepinephrine. Phenylethanolamine-N-methyltransferase (PNMT) in epinephrine— containing neurons converts norepinephrine to epinephrine by transfer of a methyl group from S-adenylmethionine. EPINEPHRINE PHENYLETHANOL- AMINE-N -METHYL TRANSFERASE NOREPINEPHRINE DOPAMINE —8-OX|DASE DOPAMINE DOPA DECARBOXYLASE D O P A TYROSINE' HYDROXYLASE 'I’YRO SIN E PHENYLALANINEI HYDROXYLASE PH ENYLALAN IN E Figure 2. Biosynthesis pathway of catecholamines. 24 The catecholamines are stored in granules within the nerve terminals and released during cell depolarization. The storage vesicles protect these amines from catabolism by monoamine oxidase (MAO) present in the nerve terminal mitochondria. Catecholamines are converted to their reSpec— tive aldehydes by MAO and then reduced (Figure 3). MAO also exists extraneuronally along with catechol-o-methyl trans- ferase (COMT). The methyltransfer of catecholamines by COMT is depicted in Figure 3. Catabolism is not the mechanism terminating the action of catecholamines at the postsynaptic membrane. Re-uptake removes the transmitter from the synap- tic cleft, thus ending its effect. B. Serotonin 1. Distribution of Serotonin in the Hypothalamus Histochemical fluorescent studies located serotonergic cell bodies in the raphe of the mesencephalon that send fibers via the medial forebrain bundle to the hypothalamus (Dahlstrom and Fuxe, 1964; Fuxe, 1965). Serotonergic nerve terminals were observed in the suprachiasmatic nucleus, in the middle of the retrochiasmatic area and in the anterior median eminence (Fuxe, 1965). Biochemical measurement of serotonin has suggested a wider distribution (Saavedra §E_gl., 1974c). A high concentration of serotonin was found in the suprachiasmatic nucleus, the medial forebrain bundle 5 2 3 A- . u .. u .. .. u .. o_o< osmozxomo>:.v>xo:mz.m £551,154” 1 O! o 0 oo 5. m “9132 ”6:32.42 All 1225252 All! I. |>xomo>¥$>x015¥m O<¢< 1.02 .2209 mzaramnfimZA. d O p m E I mzaramzxu mZEIamZKEE LEOu Catabolism pathway of catecholamines. Figure 3. 26 and the arcuate nucleus. Moderate amounts were detected in the preoptic area, premammillary nucleus, the posterior hypothalamic area and the median eminence. The activity of tryptophan hydroxylase, the enzyme converting tryptophan to 5-hydroxytryptophan, was located in all hypothalamic nuclei so far examined, but high activity correlated with areas having high serotonin concentration (Brownstein gt_al., 1976). 2. Metabolism of Serotonin The serotonin precursor, tryptophan, is present in the blood and is actively taken up by brain serotonergic neurons. It is converted to 5-hydroxytrypt0phan by tryptophan hydroxylase and then rapidly to serotonin by l-amino acid decarboxylase. Tryptophan hydroxylase is not inhibited by 5-hydroxytryptophan or serotonin. Serotonin synthesis is thought to be regulated by precursor availability since under physiological conditions tryptophan hydroxylase is not saturated with substrate. Thus, the daily variation in plasma tryptophan or dietary tryptophan intake can alter serotonin biosynthesis. Catabolism of serotonin involves deamination by mono- amine oxidase to 5-hydroxyindolacetaldehyde and oxidation to 5-hydroxyindolacetic acid (S-HIAA). Termination of post- synaptic nerve stimulation by serotonin results from active reuptake of serotonin by the presynaptic neuron. 27 C. Acetylcholine 1. Distribution of Acetylcholine in the Hypothalamus The distribution of cholinergic neurons in the central nervous system has not been well described. Cholinergic tracts have been mapped using histochemical localization of acetylcholinesterase; however, the presence of acetylcholin— esterase is not restricted exclusively to cholinergic neurons. Shute and Lewis (1967, 1969) found many acetyl- cholinesterase containing fibers in the perifornical nucleus and the dorsal posterior region of the hypothalamus. Cholinergic tracts extend from the lateral preoptic area to the supraoptic nucleus and amygdala, from the supramammil- lary region of the medial and lateral mammillary nuclei and from the reticular system through the hypothalamus. Recently Jacobowitz and Palkovits (1974) described acetyl- cholinesterase-containing cell bodies in the paraventricular and supraoptic nuclei and in the dorsal medial, ventromedial and arcuate nuclei. Another enzyme, choline acetyltransferase is closely associated with acetylcholine in the brain. In the hypo- thalamus the highest choline acetyltransferase activity was observed in the median eminence, with high amounts in the medial forebrain bundle and the premammillary nucleus (Brownstein gt 31., 1975). Since deafferentation of the basomedial hypothalamus decreased choline transferase 28 activity in the dorsal medial, ventromedial and arcuate nucleus but not median eminence, Brownstein EE.§£° (1976) suggested cholinergic nerves may project from the area of the dorsomedial, ventromedial and arcuate nuclei to the median eminence. The distribution of acetylcholine in the hypothalamus is unknown owing to the comparatively low concentration found there and the difficulty of measuring it. However, Cheney et a1, (1975) have reported moderate amounts of acetylcholine in the lateral preoptic nucleus and the anteri- or hypothalamic nucleus. Receptors for acetylcholine (muscarinic) were found in moderate density in the dorso- medial and arcuate nuclei (Snyder gt_al., 1975). Other hypothalamic regions were not extensively examined. 2. Metabolism of Acetylcholine In cholinergic neurons the enzyme choline acetyltrans— ferase catalyzes the synthesis of acetylcholine from acetyl coenzyme A and choline. Both choline availability (Cohen and Nurtman, 1976) and uptake (Barker and Mittag, 1975; Mulder et_al., 1974; Simon and Kuhar, 1975) have been sug- gested to regulate acetylcholine synthesis. Other control mechanisms were proposed as well: end-product inhibition (Kaita et_al., 1969; Morris et_al., 1971), and mass action effects (Glover and Potter, 1971). After release into the synaptic cleft, acetylcholine is hydrolyzed by acetylcholin- esterase, thus terminating its postsynaptic effects. 29 D. Gamma Amino Butyric Acid (GABA) The hypothalamus contains high concentrations of GABA compared with other brain areas. Kuriyana and Kimura (1976) found that the hypothalamic areas of highest GABA concentra- tion did not coincide with nuclei. GABA content was high- est in the lateral hypothalamic area and intermediate in the anterior hypothalamic area and the ventromedial nucleus. The activity of 1 glutamic acid decarboxylase (GAD) was highest in the anterior hypothalamic, suprachiasmatic, para- ventricular and dorsomedial nucleus and the medial forebrain bundle (Brownstein et 11., 1976). A synaptic transmitter role for GABA in the hypothalamus is supported by the observ— ation of GABA receptors there (Young 35 a1., 1976). GABA is formed from glutamate, an intermediate metabol— ite of the tricarboxylic acid cycle, by the enzyme glutamic acid decarboxylase (GAD). There is some evidence to support end-product inhibition of GAD (Sze and Lovell, 1970). GABA is transaminated with alpha-oxoglutarate by GABA transferase to form succinic semialdehyde (SSA) and glutamate. SSA is then converted to succinic acid by succinic semialdehyde dehydrogenase. This metabolic pathway from glutamate to GABA and to succinic acid forms a shunt (GABA shunt) in the tricarboxylic acid cycle and may account for 10 to 40 per- cent of total brain metabolism. 30 III. Hypothalamic Hormones The effects on anterior pituitary hormone release of pituitary transplantation and of hypothalamic stimulation and lesion demonstrate hypothalamic regulation of anterior pituitary function. Absence of neural connections between the hypothalamus and anterior pituitary along with a hypo- physeal portal system suggested that hypothalamic neurohumors affect hormone release. Later, extracts of the hypothalamus were shown to increase release of adrenocorticotropic hormone (ACTH) (Saffran gt g1., 1955), luteinizing hormone (LH) (McCann gt g1., 1960), follicle stimulating hormone (FSH) Mittler and Meites, 1964; Igarashi and McCann, 1964; Igarashi gt g1., 1964), growth hormone (GH) Frantz gt_g1., 1962; Deuben and Meites, 1964), thyroid stimulating hormone (TSH) (Guillerman gt g1., 1962; Bowers gt g1., 1964) and inhibit release of prolactin (Pasteels, 1963; Talwalker gt g1,, 1963) and GH (Krulich gt_g1., 1972). Recently three factors from hypothalamic extract effecting pituitary hormone release have been isolated and identified: Thyrotropin releasing factor (TRH) isolated from ovine and porcine hypothalamic extract has the structure: pyro-glutamate-histidine-proline amide (Burgus gt_g1., 1969, 1970; Nair gt g1., 1970); Luteinizing hormone releasing factor (LRH) is the decapeptide pro- glutamine-histidine-tryptophan-serine-tyrosine-g1ycine- leucine-arginine-proline-g1ycine—amide (Matsui gt g1., 1971; Baba gt g1., 1971; Burgus gt g1,, 1971); Growth hormone 31 release inhibiting factor (somatostatin) has been identified as the tetradecapeptide: H-alanine-g1ycine-cystine-lysine- asparagine-phenylalanine-phenylalanine-tryptophan-lysine- threonine-phenylalanine-threonine-serine-cystine-OH (Brazeau gt 31., 1973; Burgus gt g1., 1973; Greibroth gt g1., 1974). A. Distribution of Hypothalamic Hormones The microdissection technique of Palkovits (1973) coupled with radioimmunoassay of hypothalamic hormones, and immunohistochemistry of hypothalamic hormones have provided extensive information on their distribution. The highest concentration of TRH was found in the median eminence (Brownstein gt g1., 1975b; Krulich gt_g1,, 1974). Significant amounts were observed in the dorsomedial, ventromedial and arcuate nuclei and in the periventricular nucleus (Brownstein gt_g1., 1974b). Unexpectedly, TRH was also observed in extrahypothalamic sites in the CNS. Although concentration of TRH is lower outside the hypothal- amus, extrahypothalamic TRH content accounts for 80% of total brain TRH. Outside of the hypothalamus the posterior pituitary had the highest concentration of TRH (Jackson and Reichlin, 1974; Oliver gt g1., 1974). Significant concen- trations were found in the brain stem, mesencephalon, thalamus, preoptic area, septum, basal ganglion and cerebral cortex (Brownstein gt g1,, 1974b; Jackson and Reichlin, 1974; Oliver gt_g1., 1974; Winokur and Utiger, 1974). 32 The ovine, bovine and porcine (White gt g1., 1974), but not rat pineal gland (Reichlin gt g1., 1976), has high TRH con— centration. Cerebrospinal fluid also contains TRH (Knigge and Joseph, 1974). The distribution of luteinizing hormone releasing hor- mone (LRH) in the hypothalamus has been determined by bio- assay of LRH activity 1§_ytttg, by radioimmunoassay and by immunocytochemistry CHE hypothalamic slices. Each procedure indicated LRH concentration was highest in the median eminence (Crighton gt g1,, 1970; Palkovits gt g1., 1974; Barry gt g1., 1973). Immunochemical localization of LRH in the median eminence established that the highest concentration was in the zona externa (Barry gt_g1,, 1973; Baker gt g1,, 1974; King gt_g1., 1974; Kordon gt g1., 1974; Hdkfelt gt g1,, 1975; Kozlowski gt g1., 1975), and a high concentration was in the lateral part of the contact zone (Baker gt g1., 1974), the point of termination of arcuate dopaminergic neurons. LRH was also observed in the zona interna and the subependymal lining of the floor of the third ventricle (Kordon, 1974; Goldsmith and Ganong, 1974). In the median eminence LRH is contained within granules of the nerve terminals opposing hypophyseal portal vessels (Goldsmith and Ganong, 1974, 1975; Pelletier gt_g1., 1974) and tanocytes (Zimmerman gt_g1,, 1974; Zimmerman 1976). Low concentration of LRH was ob- served in the arcuate (Palkovits gt_g1., 1974; Zimmerman 33 gth1., 1974; Wheaton gt g1., 1975) and ventromedial nuclei (Palkovits gt g1., 1974). The catecholaminergic neurons of the basomedial hypo- thalamus apparently do not contain LRH since 6-hydroxydop- amine reduced basomedial hypothalamic catecholamines without altering basomedial LRH content (Kizer gt g1., 1975). In the rostral hypothalamus LRH was found in the suprachiasmatic nucleus by bioassay (Schneider gt_g1., 1970) and in the suprachiasmatic and supraoptic nuclei by radioimmunoassay (Palkovits gt_g1., 1974). Barry and colleagues were the first to observe LRH-containing cell bodies (Barry and Dubois, 1974; Barry gt g1,, 1974). After inhibiting axonal flow LRH was observed in cell bodies of the arcuate nucleus and pre0ptic nucleus, and a preoptico-infundibular pathway connecting the preOptic area with the arcuate nucleus was described. Other investigators reported that LRH in the preoptic area was located in the circumventricular organ, the organum vasculosum of the lamina terminalis (OVLT) (Barry gt_g1., 1974; Zimmerman gt g1., 1974; Kordon, 1975; Wheaton gt 31., 1975). The synthesis of LRH in the nerve terminals of the OVLT and the mediobasal hypothalamus are independently regulated. Isolation of the mediobasal hypothalamus reduced LRH content of the mediobasal hypothalamus but did not effect LRH con- tent of the OVLT. Also Araki gt_g1. (1975) reported 34 differential changes in the LRH content of the median eminence and the preOptic area (presumably the OVLT) during the estrous cycle and after castration. Reports of extrahypothalamic LRH are few. All the circumventricular organs contain LRH (Kizer gt_g1., 1976); highest concentration was in the median eminence and OVLT but significant amounts were observed in the area postrema, subfornical organ and subcommissural organ. LRH containing neurons project from the hypothalamus into the septum and paraolfactory cortex (Barry and Dubois, 1974; Barry gt_g1,, 1974). Hokfelt gt_g1. (1974) reported LRH-containing fibers in the amygdala. LRH is found in ovine, bovine and porcine pineal glands by radioimmunoassay (White gt_g1., 1974) but not in ovine monkey or rat pineal glands by immunocytochem- istry (Araki gt_g1,, 1975). Controversy likewise exists concerning the presence of LRH in the cerebrospinal fluid; some investigators find it there (Joseph gt g1,, 1975; Morris and Knigge, 1975) while others do not (Cramer and Barraclough, 1975). Somatostatin (growth hormone release inhibition hor- mone, SRIF) was the most recently identified hypothalamic hormone, consequently its distribution is still under study. Radioimmunoassay of hypothalamic nuclei indicates SRIF is present to some extent in all nuclei, but the highest con- centration is found in the median eminence with moderate 35 amounts in the ventromedial and arcuate nuclei, the peri- ventricular nucleus and the ventral premammillary nucleus (Brownstein gt g1., 1976). A similar pattern of distribu- tion was found by bioassay for SRIF activity except for the ventromedial nucleus (Vale gt g1., 1974). However, SRIF activity in the ventromedial nucleus could be low if growth hormone releasing factor were also present. By immunohisto- chemistry SRIF was found in the zona externa of the median eminence near portal vessels (Hekfelt gt g1., 1974; Pelletier gt_g1,, 1975). Recently Alpert gt g1. (1976) reported a few SRIF containing fibers in the preoptic area and the anterior periventricular area, extending from the region between the anterior commissure and optic chiasm to the anterior ventral medial nucleus. Outside the hypothalamus, the amygdala has a high content of SRIF (Hokfelt gt 31,, 1974), while the cerebral cortex (Patel gt_g1., 1975) and posterior pitui- tary contain lesser amounts. The circumventricular organs, the OVLT and the subcommissural organ, contain SRIF as does the pineal gland (Pelletier gt_g1., 1975). Human cerebro- spinal fluid had measureable amounts of SRIF (Patel gt g1,, 1975). In addition SRIF is distributed in the periphery: stomach, pylorus, duodenum, jejunum and pancreatic islets (Luft gt_g1., 1974; Arimura gt g1., 1975; Patel EE.El-r 1975). 36 The distribution of other hypothalamic hormones is not known since sensitive procedures for their measurement are not available. B. Action of Hypothalamic Hormones Prior to identification and synthesis, hypothalamic hormones were presumed to stimulate or inhibit release of a single anterior pituitary hormone, but this has not proven to be true. TRH does induce synthesis and release of pitui- tary TSH. In addition, Tashjian EE.E£: (1971) reported that TRH stimulated prolactin release from cultured GH3 cells, a cell line derived from an anterior pituitary tumor. TRH stimulation of prolactin release from rat (Hill-Samli and MacLeod, 1974) and bovine (Smith and Convey, 1975) pitui- taries i2.YlE£2 and human (Jacobs gt_g1,, 1971; Bowers gt_g1., 1973) and rat (Mueller gt g1,, 1973; Takahara gt_g1., 1973) pituitaries 12_yttg_indicated a similar action on normal pituitary tissue. Some investigators have suggested that TRH is the agent inducing prolactin release under physio- logical conditions. However, prolactin and TSH release are not always parallel. Stress and administration of high amounts of estrogen induce prolactin release (Neill, 1972; Chen and Meites, 1970) while decreasing TSH—thyroid function (Reichlin, 1966; Fisher and D'Angelo, 1972). Cold exposure increases TSH release and decreases prolactin release (Mueller gt g1,, 1974). The release of other hypothalamic 37 neurohumors along with TRH may explain nonparallelism of TSH and prolactin release. SRIF inhibited TRH induced release of TSH but not prolactin (Siler gt g1., 1974; Udeschine gt g1., 1976; Chihara gt g1., 1976), whereas catecholamines prevented TRH induced release of prolactin but not TSH (Takahara gt_g1., 1974). Thus, the role of TRH as a prolac- tin releasing factor under physiological conditions remains unresolved. TRH was also reported to induce growth hormone release (Takahara gt_g1., 1974). The extrahypothalamic distribution of TRH suggests ad- ditional effects of this compound. Based on its phylogenic distribution in invertebrates, Reichlin gt g1. (1976) Specu- lated that TRH regulation of TSH release is an instance in evolution where a pre-existing molecule (TRH) acquired a new function (regulation of TSH release).. TRH does affect the central nervous system directly. Iontophoretic application of TRH decreased the Spontaneous firing rate of neurons in the hypothalamus (Renaud and Martin, 1974; Dyer and Dyball, 1974). Depression of neuronal firing rate by TRH was also observed in the cerebrum and cerebellum by Renaud and Martin (1974) but not by Dyer and Dyball (1974). TRH was reported to have antidepressant effects in humans (Prange gt 31,, 1972; Kastin gt g1,, 1972; Van der Ves Melren and Weiner, 1972), to have a "relaxing and mild euphoric effect" (Wilson gt g1., 1973) and to ameliorate symptoms of 38 schizophrenia (Wilson gt g1., 1973). Later trials did not confirm the antidepressant effect of TRH (Takahashi gt g1., 1973; COOper gt_g1., 1974; Mountjoy gt g1., 1974; Dimitri- houdi gt g1., 1974). In rats TRH is a hypothermic agent (Metcalf, 1974). Spontaneous motor activity is increased by TRH (Segal and Mandell, 1974) and when TRH is injected into the medial mesencephalon, shaking, shivering, paw tremor and lacrimation result (Weis gt_g1,, 1975). Brown and Vale (1975) suggested that TRH may be a general brain excitant since it raised the LD of pentobarbital and 50 decreased the LD of strychine. Earlier reports revealed 50 TRH antagonized the behavioral and temperature-reducing effects of pentobarbital (Prange gt g1., 1974) and the narcosis and hypothermia induced by ethanol (Breese gt g1., 1974). However, Nemenoff gt g1. (1975) found no change in electroshock-induced seizure activity after TRH and an en- hancement of the anticonvulsant properties of pentobarbital. TRH also blocked morphine sulfate or pentobarbital-induced hormone release (Brown and Vale, 1975). The mechanism of TRH action in the central nervous sys- tem is unknown, but preliminary reports indicate multi- faceted actions. TRH has been reported to increase nor- epinephrine turnover and release by some researchers (Keller gt_g1,, 1974; Horst and Spirit, 1974) but not by others (Reigle gt_g1., 1974). The effects of 1-dopa on motor 39 activity are enhanced by TRH. A direct central action of TRH is indicated since ablation of the pituitary or thyroid did not diminish TRH enhancement of 1-dopa action (Plotnikoff, 1972, 1974). Behavioral changes following increased 5- hydroxytryptophan accumulation are also potentiated by TRH (Green and Grahame-Smith, 1974). In evaluating the central actions of TRH, the discrepancy in pituitary and CNS sensi- tivity is noteworthy. A thousand-fold greater dose is neces- sary to produce central effects than to induce pituitary hormone release. This discrepancy may reflect poor penetra- tion of TRH into the brain (Stumpf and Sar, 1973). In sum- mary, the effects of TRH on the Pituitary are well- established while those on the CNS remain to be confirmed. The hypothalamic hormone LRH induces synthesis and release of LH and FSH. The magnitude of LH release is much greater than FSH following a single injection of LRH 1t_gtyg. If a similar dose of LRH is slowly infused the release of FSH is greatly increased (Schally gt_g1,, 1973). Since LRH induces release of both, no separate FSH releasing hormone may exist (Schally gt g1,, 1973). Yet the non-parallelism between LH and FSH release after preoptic area stimulation (Kalra gt g1., 1971) during the proestrous surge (Gay gt_g1., 1970) and during testosterone treatment of castrated male rats, suggests separate regulation of these hormones. Whether the separate control of FSH and LH release consists 40 of a difference in pituitary response to LRH for FSH or LH release or a separate FSH-RF remains unresolved. In addi- tion to inducing hormone release LRH also primes the anteri- or pituitary so that subsequent LRH induces greater LH release (Aiyer gt_31., 1971). A direct effect of LRH on neuronal firing in the preoptic and arcuate-median eminence areas has been reported (Moss gt 31., 1975; Kawakami and Sakuma, 1974). Most neurons are uneffected while the remain- ing neurons are either inhibited or stimulated. Such direct neural effects of LRH may explain the ability of LRH to induce lordosis behavior in female rats (Moss and McCann, 1973, 1975; Pfaff, 1973). LRH also potentiates the effect of 1-dopa on motor activity and slightly enhances the effects of serotonin (Plotnikoff gt 31., 1976). As with TRH the amount of LRH required for central effects is much greater than that for hormone release. The distribution of SRIF is wider than that of LRH or TRH and its range of effects is greater. SRIF reduces basal blood growth hormone concentration and the GH release follow- ing stimulation (Reichlin gt 31., 1976). SRIF inhibits TRH induced TSH release in rats (Borgeat gt 31,, 1974; Vale gt_31., 1974) and humans (Hall gt 31., 1973; Siler gt 31., 1973, 1974; Hall gt 31., 1974). Release of prolactin is reduced by SRIF in vitro (Vale gt 31,, 1974; Drouin gt 31,, 1976) but not 13 vivo (Drouin gt 31., 1976). These effects 41 of SRIF on pituitary hormone release may result from its ability to reduce anterior cyclic AMP content (Borgeat 2E,2l°r 1974) although other mechanisms are involved. Within the CNS, SRIF altered neuronal firing rates (Renaud gt 31., 1975). Several observations suggest SRIF has an inhibitory effect on the CNS. Thus, SRIF reduced spontaneous motor activity (Segal and Mandell, 1974), increased the length of pentobarbital anesthesia (Prange gt 31., 1974) and reduced the LD50 of pentobarbital (Brown and Vale, 1975). Also, duration of strychine induced seizures is reduced and the LD50 of strychine is increased (Brown and Vale, 1975) by SRIF. In the periphery blood concentration of gastrin, insulin and glucagon is reduced by SEIF (Alberti gt 31., 1973; Bloom gt 31., 1974; Koerber gt_31,, 1974; Gerish gt 31., 1975). Other hypothalamic hormones induce anterior pituitary release, but since they have not been identified and synthe- sized, their full range of activities remains unknown. IV. Hypothalamic Regulation of Pituitary Prolactin, Luteinizing Hormone (LHLLand Thyroid Stimulating Hormone (TSH) Release A. Prolactin l. Catecholamines The release of prolactin, unlike the release of other anterior pituitary hormones, is tonically inhibited by the 42 hypothalamus. Thus, pituitary transplantation or median eminence lesion increased prolactin release (Everett, 1954; Chen gt_31,, 1970; Sud gt_31,, 1970; Welsch gt 31., 1971). Extract of hypothalamic tissue reduced prolactin release from pituitary tissue £2.21EEQ during short term incubation (Talwalker gt 31., 1963) and in organ culture (Pasteel, 1961; Gala and Reece, 1963) and also 13_yttg_(Grosvenor gt_31,, 1965). These findings demonstrated the existence of a prolactin release inhibiting factor (PIF) in the hypo- thalamus. Subsequently catecholamines were found to increase hypothalamic content of PIF and reduce blood concentration of prolactin. Dopamine injected into the third ventricle or its precursor, l-dopa, given systemically, reduced blood concentration of prolactin (Kamberi gt 31., 1971; Lu and Meites, 1971, 1972). Inhibition of catecholamine metabolism likewise reduced blood prolactin concentration (Lu and Meites, 1971; Donoso gt 31., 1971; Quadri and Meites, 1973). On the other hand inhibition of catecholamine synthesis (Lu gt_31,, 1970; Lu and Meites, 1971; Carr gt_31,, 1975), depletion of catecholamine stores (Ratner gt_31,, 1965) or blockade of catecholamine receptors by drugs (Lu gt 31., 1970; Danon and Sulman, 1970; MacLeod gt 31., 1970; Dicker— man gt 31., 1972; Meites and Clemens, 1972; Quijada gt_31,, 1973), increased blood concentration of prolactin. 43 Initial observations indicated physiological concentra- tions of catecholamines did not directly inhibit prolactin release from the pituitary (Talwalker gt 31., 1963; Koch gt 31., 1970; Kamberi gt 31., 1971) although at high pharma- cological concentrations they did (Jacobs gt 31., 1968; MacLeod, 1969; Birge gt 31., 1970; Koch gt 31., 1970; MacLeod gt_31,, 1970). It was therefore concluded that catecholamines induced PIF secretion to reduce prolactin , release. ngever, some investigators have proposed that dOpamine is a PIF. Recent evidence showed catecholamines act directly on the anterior pituitary to inhibit prolactin release at concentrations lower or equivalent to hypothala- mic content. Low concentrations of dOpamine and norepin- ephrine significantly reduced the spontaneous release of prolactin 13_21ttg_(8haar and Clemens, 1974; Dibbet gt 31,, 1974) and 13_ytyg_(Takahara gt_31., 1974; Schally gt 31., 1974). Higher concentrations of epinephrine were required to produce comparable inhibition. Furthermore, Shaar and Clemens (1974) observed that passage of hypothalamic extract through an aluminum oxide column removed PIF activity along with the catecholamines. Elution of these columns with acid, washed out the catecholamines and PIF activity to- gether. Monoamine oxidase, an enzyme, catabolized catechol- amines and destroyed PIF activity of hypothalamic extract as well. Parallel changes in the hypothalamic content of 44 PIF activity and catecholamines after various drug treatments have been observed (Shaar and Clemens, 1976). The effects of systemically administered catecholamines can result from direct action on the pituitary. In hypophysectomized rats with a pituitary grafted beneath the kidney capsule, an in- jection of l-dopa reduced serum prolactin (Lu and Meites, 1972; Donoso, 1973). In the periphery 1-dopa can be con- verted to dopamine and directly effect the pituitary. Donoso gt 31. (1974) observed 1-dopa suppression of prolac- tin release in pituitary grafted rats with a median eminence lesion. Finally dopamine, the catecholamine most potent in inhibiting prolactin release has recently been reported to be present in hypophyseal portal blood using a sensitive enzyme assay (Ben Jonathan gt 31., 1976). However, before dopamine is assumed to be a physiological PIF, confirmation of its presence in the portal blood is required. Also dopamine release into the portal vessels must be shown to correlate with reduced pituitary prolactin release. In fact the reverse has been observed so far, portal blood concentra- tion of dopamine is highest when prolactin release is great- est (Ben Jonathan gt 31., 1976). Other evidence points to a PIF separate from catechol— amines. Small polypeptides with PIF activity have been puri- fied from hypothalamic extract (Dular gt 31., 1974; Greibrok gt 31., 1974; Takahara gt 31., 1974; Schally gt 31., 1975). 45 Although catecholamines may contaminate some of these extracts, others are free of them (Schally gt 31,, 1976). When dopamine is injected into the third ventricle, it reduced serum prolactin (Kamberi gt_31., 1971); yet no tritiated dopamine could be extracted from hypophyseal portal blood following administration into the third ventri- cle (Ben Jonathan gt 31., 1975). Levo-dopa reduced prolac- tin release even after its peripheral conversion to dopamine was blocked by MK468 (Jaminez gt 31., 1976). In this instance inhibition of prolactin release resulted from the effects of 1-dopa on the hypothalamus rather than on the pituitary. Although norepinephrine acts on the pituitary to in- hibit prolactin release, norepinephrine injected into the third ventricle induced a small increase in blood prolactin concentration (Kamberi gt 31., 1971). The norepinephrine agonist clonidine also induced increased prolactin release (Lawson and Gala, 1975). After inhibition of catecholamine synthesis, raising brain norepinephrine concentration with 3,4-dihydroxyphenylserine (DOPS) increased serum prolactin concentration (Donoso gt_31., 1971). Disulfiram inhibits dopamine-B-hydroxylase and reduces brain norepinephrine; it has also been found to decrease blood concentration of prolactin (Clemens and Meites, 1972). 46 In summary, d0pamine inhibits prolactin release; yet its physiological site of action is still undetermined. The effect(s) of norepinephrine on prolactin release are not clearly established. 2. Serotonin In addition to tonic inhibition, the hypothalamus in- duces or allows surges of prolactin release. While prolac- tin surges may result from removal of tonic inhibition, secretion of prolactin releasing factor has been suggested as a stimulus for release. When the inhibitory influence of catecholamines on prolactin is eliminated by giving reserpine, stress was still able to induce a further rise in blood prolactin concentration (Valverde gt_31., 1973; Marchlewski-Kaj and Kurlich, 1975). In this instance prolactin release is not due to lessening of inhibition. During lactation suckling produces a rapid release of pro- lactin such that the prolactin concentration is twenty times basal levels. Treatment of lactating dams with the cate- cholamine synthesis inhibitor, alpha-methyl-para-tyrosine, prior to pup suckling did not prevent suckling-induced prolactin release (Voogt and Carr, 1975). In addition suckling-induced prolactin release is not accompanied by a change in hypothalamic catecholamine turnover until maximal blood prolactin concentration is reached. Then dopamine turnover is, in fact, increased (Voogt and Carr, 1974). 47 These data suggest that stimulation rather than disinhibi- tion is the mechanism producing increased blood prolactin concentration after stress and suckling. These surges of prolactin release may involve stimulation by serotonergic neurons. Kamberi gt_31. (1971) found that serotonin injected into the third ventricle stimulated prolactin release. Lu and Meites (1973) showed that systemic injection of the sero- tonin precursors tryptophan or S-hydroxytryptophan also increased blood prolactin concentration. Tryptophan in feed increased brain serotonin and serum prolactin of rats on a tryptophan-free diet (Gid Ad gt_31,, 1976). Parachloro- phenylalanine (PCPA) and 5,7-dihydroxytryptamine reduced both brain serotonin and serum prolactin of adult rats (Gid Ad gt 31., 1976). In addition PCPA and methysergide, a serotonin receptor blocker, inhibited surges of prolactin release. Thus, the suckling-induced rise in serum prolactin was inhibited by PCPA (Kordon gt 31., 1973) while methyl- sergide blocked suckling-induced prolactin release (Gallo gt 31., 1975) and prevented the afternoon rise in serum prolactin of estrogen-primed ovariectomized rats (Subramanian and Gala, 1976). The elevated basal concentration of prolac— tin in estrogen-primed ovariectomized rats was reduced by PCPA, methysergide (Caligaris and Taleisnak, 1974) and by parachloroamphetamine, a specific depletor of serotonin 48 (Chen and Meites, 1975). From their study Caligaris and Taleisnak (1974) concluded serotonergic neurons participate in estrogen-stimulated prolactin release. Recently Mueller gt 31. (1976) showed that the rise in prolactin after stress paralleled the increase in serotonin turnover induced by stress. Krulich (1975) found that the serotonin reuptake inhibitor Lilly 11040 potentiated prolactin release after stress. Taken together these data suggest serotonergic neurons stimulate prolactin release. 3. Acetylcholine Cholinergic neurons also have been implicated in the hypothalamic regulation of prolactin release. Atropine, a muscarinic receptor blocker, prevented depletion of pro— lactin from the pituitary after suckling (Grosvenor and Turner, 1958). At a high dose atropine inhibited the pro- estrous surge of prolactin (Libertun and McCann, 1973) and the nocturnal surge during pseudOpregnancy (MacLean and Nikitovitch-Winer, 1975). In contrast atropine implanted in the median eminence induced pseudopregnancy (Gala gt 31., 1970) and when injected into the third ventricle enhanced prolactin release following hypothalamic stimulation (Gala gt 31., 1972). Yet given over a wide dose range, atropine had no effect on basal prolactin release (Grandison and Meites, 1976). In contrast, the reports on the effects of cholinergic agonists on prolactin release are consistent. 49 Prolactin release was reduced by intraventricular injection of acetylcholine or carbachol (Grandison gt 31., 1974; Kuhn and Lens, 1975), and systemic injection of physostigmine and the muscarinic agonist pilocarpine (Grandison gt_31,, 1974; Libertun and McCann, 1974). Hall and Meites (unpub- lished) recently found that acetylcholine augmented inhibi- tion of prolactin release from pituitaries co-incubated with hypothalamic fragments. In support of a physiological role for cholinergic neurons, pilocarpine blocked the stress- induced release of prolactin (Grandison and Meites, 1976; Krulich gt_31,, 1976; Meltzer gt_31,, 1976). A nicotinic agonist blocked suckling-induced prolactin release and the afternoon surge of prolactin in estrogen-primed ovariectom- ized rats (Blake and Sawyer, 1972; Subramanian and Gala, 1976). The proestrous afternoon surge of prolactin was delayed by nicotine (Blake gt 31., 1973). The reported effects of cholinergic drugs on prolactin release are few and at this time no conclusions can be made about the physio— logical role of cholinergic neurons in regulating prolactin release. 4. GABA, Histamine, and Glycine Both GABA and histamine are found in high concentration in the hypothalamus. The effect of these putative trans- mitters on prolactin release is currently being studied. When injected into the ventricles, GABA and histamine each 50 induce prolactin release (Mioduszewski gt 31., 1976; Ondo and Pass, 1976; Libertun and McCann, 1974; Donoso gt 31., 1976). Blockade of histidine decarboxylase, the enzyme catalyzing the synthesis of histamine, reduced blood pro- lactin concentration. The histamine receptor blocker diphenohydramine blocked stress-induced prolactin release (Libertun and McCann, 1974) but this report has not been confirmed (Meltzer gt 31., 1976). Another putative trans— mitter glycine has recently been reported to stimulate prolactin release (Ondo and Pass, 1976). B. Luteinizing_Hormone l. Catecholamines Sawyer gt 31. (1947) were the first to suggest that catecholamines stimulated gonadotropin release. They ob- served that dibenamine, an adrenergic receptor blocker pre- vented ovulation in the rabbit. Subsequent studies revealed that the ovulation during the estrous cycle or the induced ovulation in PMS treated immature rats was blocked by another catecholamine receptor blocker, chlorpromazine (Barraclough and Sawyer, 1957; Zarrow and Brown-Grant, 1964), by the depletion of brain catecholamines with reserpine (Barra— clough and Sawyer, 1957; Hopkins and Pincus, 1963) and by inhibition of normal catecholamine synthesis with alpha— methyl-para-tryosine (Lippman gt_31., 1967) or methyl dopa (Coppola, 1969). Interpretation of these early studies was 51 complicated by the effects of catecholamines at the ovarian level. France (1970) reported that reserpine blocked ovula- tion in hypophysectomized rats given exogenous gonadotropins (PMS and HCG). Only a few early reports showed that re— serpine or alpha methyl-para-tyrosine decreased gonadotropin activity in the blood (Gronoos gt_31., 1965; Labhsetwar, 1967; Donoso and Santolaya, 1969). With the development of radioimmunoassays for LH, the effects of catecholamines on LH release were then directly observed. It has been found that both dopamine and norepinephrine influence LH release. Studies reporting the effects of norepinephrine are generally consistent and indicate norepinephrine stimulates LH release. Rubenstein and Sawyer (1970) found norepineph- rine given centrally induced ovulation in pentobarbital blocked proestrous rats. Likewise, after intraventricular injection into rabbits norepinephrine induced LH release (Sawyer gt 31., 1974). The rise in blood LH concentration during proestrus, or following estrogen and/or progesterone treatment is inhibited by alpha-methyl-para-tyrosine, an inhibitor of catecholamine synthesis. However, a rise in LH will take place under the above conditions if brain nor- epinephrine levels are restored by giving dihydroxyphenyl~ serine (DOPS) (Kalra gt_313, 1972; Kalra and McCann, 1972, 1974). The preovulatory and estrogen induced LH surge are blocked by diethyldithiocarbamate (DDC) and U-l4624. 52 These compounds inhibit dOpamine-B—hydroxylase, the enzyme catalyzing dopamine conversion to norepinephrine. Again restoration of norepinephrine levels with DOPS permits the LH surge to occur. In male rats inhibition of norepineph- rine synthesis but not dopamine blocked the postcastration increase in blood LH concentration (Ojeda and McCann, 1973). Similarly blockade of dopamine receptors did not inhibit postcastration LH release but blockade of dopamine and nor- epinephrine receptors did. Thus, according to the above studies LH release is stimulated by norepinephrine on pro- estrous afternoon, after estrogen treatment or after castra- tion. In addition, measurement of changing hypothalamic norepinephrine content and synthesis also suggest norepi- nephrine stimulates LH release. The anterior hypothalamic content of norepinephrine increases during the estrous cycle to a peak at proestrus (Stefano and Donoso, 1976; Donoso gt 31., 1971). Likewise turnover of norepinephrine and its synthesis from H3 tyrosine increased at proestrus (Donoso and De Gulierrez-Mozunno, 1970; Zscheck and Wurtman, 1973). After castration LH release increases as does anterior hypothalamic content of norepinephrine (Anton Tay and Wurt- man, 1968; Wurtman gt_31,, 1969, Anton Tay gt 31., 1970). Similar parallel increases in norepinephrine content and turnover and LH release occur at puberty (Coppola, 1968, 1969). 53 \ Still unsettled is the role of dopamine in the release I I of LH. Both stimulatory and inhibitory effects have been reported. Initial investigation indicated dopamine stimu- \ \ lated LRH and LH release. Injection of dopamine into the \ third ventricle increased LRH activity in the systemic blood i of hypophysectomized rats (Schneider and McCann, 1970) and I in hypophyseal portal blood of intact rats (Kamberi gt 31., 1969). Such treatment increased LH and FSH release also (Kamberi gt 31., 1971; Schneider and McCann, 1970a,b). Norepinephrine and epinephrine were less effective in alter- ing LH release. Dopamine caused LH release 13_21ttg from anterior pituitaries co-incubated with hypothalamic frag— ments (Schneider and McCann, 1969; Kamberi gt 31., 1970). These experiments could not be repeated (Miyachi gt 31., 1973) even in the original laboratories (Quijada gt 31., 1973; Porter gt 31., 1972; Cramer and Porter, 1973). McCann and Moss (1975) suggested that tg'ytttg, dopamine may have been converted to norepinephrine by the hypothalamic fragments. Reserpine, which destroys synaptic vesicles where dopamine is converted to norepinephrine, blocked the stimulatory effect of dopamine on LH release 12.Y£E£2. (Schneider and McCann, 1969). In these early studies nor- epinephrine itself was ineffective, perhaps due to rapid metabolism. However, a stimulatory role for dopamine in regulation of LH release has not been conclusively disproven. 54 The dopaminergic receptor blocker pimozide inhibited ovula- tion in pregnant mare serum (PMS) treated immature rats (Corbin and Upton, 1973; Fuxe gt 31., 1976). Indirect evi- dence indicates that dopamine synthesis in the median eminence is increased after gonadectomy. The activity of tyrosine hydroxylase in the whole hypothalamus and in the median eminence increased after castration (Beattie gt 31., 1972; Kizer gt_31., 1974). Sectioning the ventral nor- adrenergic bundle destroys noradrenergic neurons in the hypothalamus; yet it did not reduce the activity of tyrosine hydroxylase in the median eminence nor diminish the increase in tyrosine hydroxylase after castration (Kizer gt 31., 1976a). Also, electrochemical stimulation of the preoptic area induced LH release and at the same time increased fluor— escence of dopaminergic neurons in the median eminence (Lichtensteiger and Keller, 1974). In addition to a stimulatory effect of dopamine on LH release, an inhibitory influence has been reported as well. Administration of testosterone or antifertility steroids to inhibit ovulation increased the fluorescence of dopamine in the median eminence (Klawson gt 31., 1971; Fuxe gt 31., 1972; Fuxe gt 31., 1976). The dopamine agonists, ergotamine and apomorphine, blocked ovulation in proestrous rats and in pregnant mares serum-treated immature rats (Madhwa and Greep, 1973; Fuxe gt 31., 1976). Injection of dopamine into 55 the third ventricle of rabbits prevented norepinephrine from stimulating LH release (Sawyer gt 31., 1974). If dopamine were inhibitory to LH release, dopaminergic neurons might become less active during LH release. A decline in dOpamine turnover was observed using histochemical fluorescence during proestrus, the critical period of PMS induced ovulation and after castration (Fuxe and Hokfelt, 1969; Hokfelt and Fuxe, 1972; Fuxe gt 31., 1973). In summary, the involvement of catecholamines in regulating LH release has been established. Norepinephrine appears to stimulate LH release while the effect(s) of dOpamine still require further study. 2. Serotonin It is generally assumed that serotonin reduces LH release. Stimulation of brain sites containing serotonergic neurons reduced LH release and blocked ovulation (Carrier and Taleisnak, 1970, 1972). In the ewe serotonin content of the hypothalamus fell just before the LH surge (Wheaton gt 31., 1972). In ovariectomized rats an injection of estrogen reduced LH release and increased tryptophan in the hypo- thalamus (Bapna gt_31., 1971) and serotonin in the dien- cephalon midbrain region (Tonge and Greengross, 1971). When brain serotonin concentration was elevated by giving its precursor 5-hydroxytryptophan or by reducing its catabolism with a monoamine oxidase inhibitor, PMS-induced ovulation was blocked (Kordon gt 31., 1968). Serotonin given 56 intraventricularly reduced LH and FSH release in intact and gonadectomized rats (Kamberi gt 31., 1970, 1971, 1973; Schneider and McCann, 1970) and blocked spontaneous and progesterone-induced ovulation (Kamberi, 1973; Zolovick and Labhsetwar, 1973). Serotonin implanted in the median eminence lowered the pituitary content of LH (Fraschine, 1970). However, there are some reports in which serotonin given intraventricularly failed to block spontaneous ovula- tion (Rubenstein and Sawyer, 1970; Schneider and McCann, 1970; Wilson and McDonald, 1974). In addition, two reports suggest serotonin may stimulate LH release (Wilson gt 31., 1974; Cramer and Porter, 1973). Yet the majority of evidence indicates serotonin inhibits LH release. 3. Acetylcholine The few studies investigating the effects of acetyl- choline on LH release generally suggest a stimulatory in- fluence. Acetylcholine injected intraventricularly stimu- lated progesterone secretion (Ehdroczi and Hillard, 1965). During co-incubation of pituitaries and hypothalamic frag- ments acetylcholine facilitated LH and FSH release (Simonovic gt 31., 1974; Fiorindo and Martini, 1975). There was no direct effect of acetylcholine on the pituitary. In estrogen- primed ovariectomized rats the cholinergic agonist pilocar- pine or physostigmine, an inhibitor of acetylcholine catabolism produced a biphasic effect on LH release 57 (Libertun and McCann, 1974). After injection, LH release was temporarily decreased while at six hours release in- creased. A similar immediate reduction of LH release fol— lowed the elevation of brain acetylcholine concentration with oxotremorine in estrogen primed ovariectomized rats (Marks, 1973). Atropine, a muscarinic receptor blocker, inhibited ovulation (Everett gt_31,, 1949; Benedetti gt_31., 1971), the proestrous rise in LH and FSH (Libertun and McCann, 1973) and reduced LH in ovariectomized rats (Dickey and Marks, 1971; Libertun and McCann, 1972, 1973). Atropine also prevented ovarian compensatory hypertrophy (Monti gt_31., l970)_and LH release after electrochemical stimulation of the preoptic area (Lichtensteiger and Keller, 1974). In con- trast the nicotinic type of cholinergic receptors appears inhibitory to LH release since nicotine delayed the preovu- latory surge of LH (Blake gt_31., 1972). No definitive con— clusion can be made at this point concerning the role of acetylcholine in regulating LH release since the activity of cholinergic neurons has not been correlated with LH release. 4. Other Putative Transmitters Isolated reports indicate other neuroactive compounds effect LH release. Histamine stimulated progesterone secre— tion (Endroczi and Hillard, 1965), induced ovulation (Sawyer, J1955) and increased LH release (Libertun and McCann, 1974; 58 Donoso gt_31., 1976). LH release was also increased by GABA (Ondo, 1974), glutamate and lysine (Ondo and Pass, 1976). CL Thyroid Stimulating Hormone The regulation of TSH release by neurotransmitters in the hypothalamus is poorly understood. Few studies have considered this problem and little concensus exists. Initial investigation indicated that intraventricular injection of dopamine, norepinephrine or serotonin had no effect on TSH release in rats or rabbits (Greer gt 31., 1960; Harrison, 1961). Recent studies using more sensitive measures of TSH or TRH release demonstrate monoamines can alter TSH release, yet agreement on effect is lacking. Reichlin gt 31. (1972) described an elegant 13_vitro procedure for measuring TRH synthesis by murine hypothalamic fragments. Norepinephrine stimulated TRH synthesis (Grimm and Reichlin, 1973). Dopamine also stimulated TRH production, but not when its conversion to norepinephrine was inhibited by disulfiram. In agreement, cold-induced TSH release in rats was inhibited by the alpha-adrenergic blockers phentolamine and phenybenza— mine or disulfiram (Kotandi gt 31., 1973; Tuomisto gt 31., 1975). Other studies suggest dopamine rather than norepin- ephrine stimulates TSH release. Dopamine stimulated TRH release from ovine median eminence synaptosomes (Bennett gt_31,, 1975). There appears to be an increase in dopamine 59 synthesis and TSH release after thyroidectomy, although a causal relationship has not been established. After thyroidectomy tyrosine hydroxylase activity increased in the median eminence, arcuate, ventromedial, and periventricular nuclei (Kizer gt 31., 1974, 1976). This increase in tyro- sine hydroxylase activity still occurs after elimination of noradrenergic input into the hypothalamus, suggesting dopaminergic neurons are responsible. In disagreement with the above, alpha—adrenergic receptor blockers had no effect on basal or cold induced TSH release in humans (Fisher gt 31., 1971; Woolf gt 31., 1972) and the inhibitor of catecholamine synthesis alpha methyl-para-tyrosine did not alter TSH release in rats (Chen and Meites, 1975). Levo-dopa did not change TSH release in rats nor in euthyroid human subjects (Eddy gt 31., 1971; Chen and Meites, 1975). While the re- duction of TSH release by reserpine is cited as evidence that catecholamines stimulate TSH, reserpine depletes brain stores of serotonin as well as catecholamines. After re- serpine, restoration of catecholamines by administering 1- dopa did not increase TSH whereas restoration of serotonin by administering 5—HTP did increase TSH (Chen and Meites, 1975). Still other evidence suggests that dopamine inhibits TSH release. Mueller gt 31. (1976) found the dopamine agonists apomorphine and peribidil to reduce TSH release. 60 The reported effects of serotonin on TSH release are equally conflicting. Grimm and Reichlin (1973) reported that serotonin decreased TRH synthesis 12.YEE£23 The sero— tonin precursor tryptophan increased brain serotonin and reduced TSH serum concentration 13.ytyg_(Mueller gt 31., 1976). On the other hand 5-HTP increased TSH in estrogen- primed ovariectomized rats (Chen and Meites, 1975). Drugs which deplete brain serotonin such as parachloramphetamine and parachlorophenylalanine reduced blood TSH concentration in estrogen-primed ovariectomized female, and male rats (Shenkman gt 31., 1973; Shapman gt_31., 1974; Chen and Meites, 1975). Cholinergic agonists did not alter TRH synthesis or TSH release (Grimm and Reichlin, 1973; Chen and Meites, 1975). However, atropine did inhibit cold-induced TSH release (Kotandi gt_31., 1973). Based on the above reports one concludes further research on TSH regulation is warranted. V. Effects of Prolactin on the Hypothalamus Neurotransmitters regulate secretion of hypothalamic i hormones and thus pituitary hormone release. In addition, hypothalamic hormones, anterior pituitary hormones, and hormones from end-organs each can modulate the synthesis and ) release of neurotransmitters in the hypothalamus. J// 61 This circular relationship between the hypothalamus and hormone concentration forms a feedback control loop. The effects on the hypothalamus of end-organ, anterior pituitary, and hypothalamic hormones are independent of one another, although the hypothalamus integrates this type of input along with others such as sensory and extrahypothalamic. Hypothalamic activity is modified by end-organ hormones in 'long-loop feedback', by anterior pituitary hormones in 'short-100p feedback' or autoregulation, and by hypothalamic hormones in 'ultra-short-loop—feedback'. The CNS-anterior pituitary-ovarian axis is an example where each type of feedback loop has been reported. In other cases such as the regulation of prolactin release, long and 'ultra-short-loop feedback' have not been established. No end-organ hormone is specifically associated with prolactin, thus making 'long-loop feedback' difficult to interpret. The existence of a PIF separate from dopamine is controversial, thus 'ultra-short loop feedback' in this instance can not be resolved. These factors make the well documented 'short loop feedback' effect of prolactin even more significant. Furthermore, no other anterior pituitary hormone or other hormone, with the exception of steroids, has such potent or diversified effects on hypothalamic activity. 62 A. Effects of Prolactin on Prolactin Secretion The first evidence for autoregulation of prolactin secretion came from anterior pituitary tumor-bearing rats. In rats carrying the pituitary tumor MtTWS which secretes large amounts of prolactin and growth hormone, or tumor 7315a which secretes prolactin and ACTH, the pituitary weights per 100 grams body weight were significantly reduced (MacLeod gt 31., 1966, 1968). Also the pituitary content of prolactin was significantly lower than in non—tumor bearing rats. Chen gt_31. (1968) found that rats bearing the pro— lactin secreting tumors MtTW5 or MtTW15 had pituitaries of lower weight and containing less prolactin. The hypothalamic content of PIF was also reduced. In these cases prolactin acted directly on the CNS since feedback inhibition was observed in ovariectomized, adrenalectomized tumor bearing rats. The pituitary tumors secrete great amounts of prolac- tin such that the blood concentration of prolactin is well above the physiological range. Thus, the physiological implications of these observations is unknown. Later auto- regulation of prolactin was observed in rats given anterior pituitary (AP) grafts or exogenous prolactin. Pituitary grafts secrete mainly prolactin and at a concentration well ‘Mithin physiological limits (Meites and Nicoll, 1965). FHKDlactin content of 13331t3_pituitaries was reduced by AP gréifts in ovariectomized rats but unchanged or increased in 63 intact female rats (Sinha and Tucker, 1968; Welsch gt 31., 1968). In the intact female rat prolactin secreted from the AP grafts caused luteinization of the ovaries. The ovarian secretions then prevented depletion of pituitary prolactin content. Reduction in pituitary weight and prolactin con- tent in rats with AP grafts indicated prolactin synthesis was reduced (Sinha and Tucker, 1968). Decreased mammary gland develOpment following median eminence implantation of prolactin demonstrated release was decreased as well (Welsch gt 31., 1968; Minkhinsky, 1970). Clemens and Meites (1968) were the first to establish that prolactin acted on the hypothalamus to reduce pituitary prolactin secretion. Implantation of ovine prolactin into the median eminence caused a decrease in pituitary weight, and pituitary prolactin content and concentration. Atrophy of mammary glands and ovaries indicated release was decreased also. Averill (1969) implanted anterior pituitary tissue into the hypothalamus and found corpora lutea were not main- tained, another indication of reduced prolactin release. The physiological consequences of prolactin autoregula- tion is demonstrated by the effects of median eminence implants of prolactin on several reproductive states. Prolactin implanted into the median eminence one day after cervical stimulation shortened the duration of pseudopreg— nancy from 14 days to 10 and significantly inhibited the 64 deciduomata response (Chen gt 31., 1968). Pregnancy was terminated when median eminence prolactin implants were made during the first eight days (Clemens gt 31., 1969a). During lactation prolactin implants reduced litter weight gain (Clemens gt_31., 1969b). The mammary glands of these rats weighed less and vaginal estrous cycles were initiated earlier. These three reproductive states are dependent on prolactin secretion for their continuance. Implants of prolactin in the median eminence also can prevent acute surges in prolactin release. Prolactin implants at 1000 hours on proestrus day prevented the preovulatory prolactin surge. When prolactin was implanted on day four of lacta- tion, it prevented suckling-induced prolactin release on days 6, 8 and 10 (Voogt and Meites, 1973). Recently Advis gt 31. (1976) found ovine prolactin given intraperitoneally four hours prior reduced the rise in serum prolactin follow- ing restraint stress. Estrogen is one of the most potent stimuli for prolactin secretion, yet prolactin implants prevented increases in pituitary weight and pituitary pro- lactin content and concentration after daily injection on 1 ug estradiol benzoate for five days (Welsch gt 31., 1968). At higher doses of estrogen, autoregulation was less effec- tive. Autoregulation is specific for molecules with lacto— genic or prolactin-like action. Thus, human growth hormone 65 and human placental lactogen which have lactogenic proper- ties can reduce prolactin release in rats (Voogt gt 31., 1971; Clemens and Meites, 1972). B. Effects of Prolactin on Gonadotropin Secretion In addition to autoregulation, prolactin also influ- ences release of other anterior pituitary hormones. Clemens gt 31. (1969c) reported that systemic injections or median eminence implants of prolactin advanced vaginal open- ing in female rats from an average of 37 days after birth to 30 days. It was suggested prolactin induced precocious puberty by increasing FSH release. Subsequently Voogt gt 31. (1969) found pituitary FSH concentration was reduced in immature female rats implanted with prolactin in the median eminence suggesting an enhanced release of FSH. In mature female rats, prolactin implants four days after cervical stimulation caused resumption of vaginal estrous cycling in these pseudopregnant rats while LH and FSH concentration in the blood was increased two-fold compared to control pseudo- pregnant rats. Although these data indicate that prolactin acting centrally can stimulate gonadotropin release, there is an inverse relationship in several reproductive states between blood prolactin and gonadotropin concentration. During pseudopregnancy, pregnancy and lactation blood pro— lactin concentration is periodically elevated while 66 gonadotropin concentratin remains at low, diestrous levels. The effects of the nocturnal surges of prolactin on gonado- tropin release have not been examined. During lactation ovarian steroids are not responsible for cessation of estrous cycles. Ovariectomy is not followed by an increase in LH or FSH release in dams nursing six or more pups (Ford and Melampy, 1973; Hammons gt 31., 1973) in contrast to the dramatic postcastration rise in gonadotropin secretion occurring during the estrous cycle (Gay and Midgley, 1969). Inhibition of estrous cycles and gonadotropin release during lactation is associated with pup contact. Physical contact between pups and dam is sufficient to maintain postpartum diestrum (Moltz gt_31., 1969; Zarrow gt 31., 1973). The sight and smell of pups causes an increase in prolactin and suckling is one of the most potent stimuli for prolactin release. Prolactin appears to be at least partially responsi- ble for the inhibition of gonadotropin release during lac— tation. If prolactin release is suppressed by administering ergocornine, serum FSH concentration increases in lactating rats (Lu gt 31., 1976) and humans (Seke gt 31., 1974). Although LH release did not increase soon after ergocornine treatment, it must be noted that ergocornine is a dopaminerg- ic agonist and has been found to inhibit LH as well as prolactin release albeit at a higher dose (Fuxe gt 31., 1976). However, the suckling stimulus alone did prevent 67 resumption of estrous cycles for several days in ergocornine treated rats (Lu gt 31., 1976). In contrast, removal of pups at the end of lactation is followed almost immediately by proestrus as indicated by vaginal smears. In conclusion, during lactation prolactin and suckling together inhibit gonadotropin release. Prolactin and suckling during lacta- tion decreased hypothalamic LRH and PIF activity (Minaguchi and Meites, 1967). In addition, prolactin may act at other sites to inhibit the release and action of gonadotropins. The response of the pituitary to LRH is reduced during lac- tation in rats (Lu gt 31., 1976; Mougdal gt_31., 1976) and in humans (Tolis E£.§l3r 1973, 1974; Lemaire gt 31., 1974). Also it has been suggested that the ovaries are less respon- sive to gonadotropins during lactation (Keeltel and Bradburg, 1961; Zarate gt 31., 1972; Weiss gt 31., 1973; Erysthe gt_31., 1973). A more direct relationship between high prolactin and inhibition of gonadotrOpin release is observed in some patho- logical conditions. Galactorrhea often is associated with ammenorrhea. Forbes—Albright and Chiari—Frommel syndromes are conditions where hyperprolactinemia is associated with infertility. Reduction of prolactin in these circumstances leads to resumption of menstrual cycles and increased gona— dotropin release (Turkington, 1972; Varga gt 31., 1973; Zarate gt 31., 1973; Seki and Seki, 1974). The mechanism 68 by which hyperprolactinemia reduced fertility has not been determined but it has been reported that hyperprolactinemia prevented the stimulation of LH release by estrogen (Aono gt 31., 1976). C. Effects of Prolactin on ACTH Secretion The secretory pattern of ACTH is altered during lacta— tion and it appears that prolactin is at least partially responsible for this change. The serum concentration of corticosterone during the morning is higher in lactating as compared to non-lactating rats as reported by some (Voogt gt 31., 1969) but not by others (Zarrow gt_31., 1972; Endroczi and Nayakas, 1974). The elevated morning values may result from suckling since suckling has been reported to stimulate ACTH release (Voogt gt_31., 1969). There is no diurnal rhythm in the release of ACTH (Endroczi and Nyakas, 1974) and the release of corticosterone, ACTH, and CRF in response to stress is significantly less in lactating than in nonlactating rats (Thoman gt 31., 1968; Kamoun and Haberg, 1969; Endroczi and Nyaka, 1972a, 1974). The buffer- ing of stress-induced corticosterone release during lacta- tion appears to result from elevated blood prolactin since prolactin implanted in the median eminence of nonlactating rats depressed the release of corticosterone following stress (Endroczi and Nyaka, 1972b). Prolactin may affect ACTH 69 release by lowering the threshold for corticosterone nega- tive feedback. The dose of dexamethasone required to inhibit stress induced corticosterone release is sixteen- fold higher in nontreated than in prolactin treated female rats (Zarrow gt 31., 1972). D. Effects of Prolactin on Hypothalamic Activity Prolactin may reach the hypothalamus through the peri- pheral circulation or perhaps via the few hypophyseal portal vessels that carry blood from the pituitary to the hypo- thalamus (Torok, 1954). Clemens and Sawyer (1974) found prolactin in the cerebrospinal fluid of the rabbit and its concentration there paralleled blood concentration during the estrous cycle and after administration of exogenous prolactin. Following intravenous infusion of prolactin, the activ- ity of hypothalamic neurons is altered: some activated, others inhibited (Clemens gt 31., 1972). The effects of prolactin are directly on hypothalamic neurons as was shown by iontophoretic application (Yamada, 1975). Fuxe and H5kfelt (1969) found that prolactin increased the turnover of dopamine in the median eminence. The increase in dOpa- mine may explain the higher PIF concentration observed in prolactin treated rats. The reduction in LH and ACTH re- lease may also result from increased dopamine since release of both is reportedly inhibited by catecholamines. 70 E. Physiological Role for the Central Effects of ProIactin Although prolactin can modulate other hormone secretion, its physiological significance in this respect is undeter— mined. The majority of reports dealing with the central effects of prolactin concern experiments where prolactin is given over periods of days. In these cases results are con- sistent. However, during acute studies lasting hours the reported effects of prolactin are less dramatic or absent. Implantation of prolactin in the median eminence on pro- estrous morning blocked the afternoon surge (Voogt and Meites, 1972), whereas injection of prolactin four hours prior to stress reduced but did not abolish the stress in- duced release of prolactin (Advis gt_31., 1976). In con- trast, injection of prolactin prior to suckling did not reduce depletion of pituitary prolactin stores (Grosvenor gt 31., 1965). Infusion of bovine prolactin in cows for two hours prior to milking did not prevent prolactin release (Tucker gt 31., 1973). However, infusion of prolactin which raised basal concentration eight-fold diminished the peak concentration of prolactin after milking. More recently it was found that TRH infusion for twelve hours increased endogenous prolactin release, yet it failed to block pro- staglandin F alpha stimulated release (Tucker gt 31., 2 1975). Perhaps prolonged exposure to high concentrations of prolactin is required for modulation of hypothalamic 71 functioning. Stimulation of dopamine turnover by prolactin first occurred twelve hours after prolactin administration (Gudelsky gt 31., 1976). Thus, the physiological effect of prolactin on the central nervous system might be most sig- nificant during periods when prolactin is elevated for days, as in lactation. The physiological function for the central effects of prolactin is as yet unknown. The central action of prolac- tin during lactation buffers stress induced corticosterone release and may thus protect the developing nervous system of the pups from excessive exposure to corticosterone in milk (Stern gt 31., 1973). During lactation the autoregula- tion of prolactin may prevent depletion of pituitary prolac— tin stores, thus conserving prolactin for future release. However, further research is required to establish the sig- nificance of the central action of prolactin. MATERIALS AND METHODS I. Animals Mature male or female Sprague Dawley rats were pur- chased from Spartan Research Animals (Haslett, MI), and mature female Wistar Furth rats were purchased from Micro- biological Associates (Bethesda, MD). All rats were kept in a ventilated temperature controlled room (24°C) illumi- nated for 14 hours daily (lights on from 0500 to 1900 hours). Lactating rats were housed with 8 pups in large individual plastic cages. Rats implanted with cannulae were placed in individual wire suspension cages. All other rats were housed 3 to 5 per wire suspension cage. Purina Rat Chow (Ralston Purina Company, St. Louis, MO) and tap water were provided 33 libitum. To minimize problems with infection, rats received antibiotics after surgical procedures. After castration or implantation of pituitary grafts underneath the kidney capsule, 0.2 ml of Longicil (60,000 units of penicillin G; Fort Dodge Laboratories, Fort Dodge, IA) was given intramuscularly. After implantation of steel cannulae into the median eminence or plastic cannulae into the later— al ventricle, terramycin (oxytetracycline HCL, Pfizer, New York, NY) was supplied in the drinking water. 72 73 II. Cannulation of the Lateral Ventricle of the Rat For injection of GABA or bicuculline methyliodide into the lateral ventricle, cannulae were implanted into the skull according to the description of Verster gt_31. (1971). The cannulae were constructed from PE#10 polyethylene tubing (Clay Adams, Parsippany, NJ). A piano wire was treaded through the lumen before heating the tubing over a soldering gun. When the plastic was soft, the two ends were pushed toward the middle forming a bulb. After cooling one end was cut diagonally so that there was a 1 mm bevelled point and a total length of 4 mm between tip and bulb. The other end was approximately 5 cm from bulb to end. In preparation for implantation, rats were anesthetized with ether and placed in a stellar stereotaxic instrument (C. H. Stoelting Company, Chicago, ILL). The hair on top of the skull was shaved, and the skin cut and retracted. The underlying fascia was retracted and 3 holes were drilled into the skull. One hole was drilled 2 mm lateral to and 1 mm behind bregma. The dura mater was punctured with a syringe needle and the cannula was then placed so that the 4 mm end projected down through the brain into the right lateral ventricle. The other end was sealed by heating it. Two other holes were drilled 3 mm lateral and 5 mm behind bregma on either side. After the skull was dried, metal screws (browline anchor 74 screws; Shuron/Continental, Rochester, NY) were placed into the latter 2 holes to serve as support. Dental cement (Nu Weld Caulk; L. D. Caulk Company, Milford, DL) was dusted over the screws and the bulb of the plastic cannula. Caulk liquid (Nu Weld liquid; L. D. Caulk Company, Milford, DL) was applied to harden the powder. After the powder was completely dried, the skin was sewn together. Three days were allowed for recovery and materials were injected from a 10 pl microsyringe (Glenco Scientific Ind., Houston, TX). Following blood collection, an aqueous solution of methylene blue dye was injected into the cannulae and the brain was cut along a coronal plane through the hypothalamus. Data was accepted only from those rats with stain in the median eminence. III. Radioimmunoassay After collection, blood was left to clot overnight in a cold room (4°C). Serum was separated by centrifugation (4000 x G, 20 min.) and stored at -20°C until assayed. Prolactin was measured by the radioimmunoassay procedure and reagents of Niswender gt_31. (1969). LH was measured by the radioimmunoassay of Niswender gt_31. (1969). In one in- stance, basal serum LH concentration of intact and intact anterior pituitary—grafted male rats (see EXperiment IV) was determined by a micro-modification of the standard LH 75 radioimmunoassay (Marshall, Bruni, Campbell, and Meites, in press). Serum TSH measurements were made by the procedures and reagents of the NIAMDD kit. Serum concentration of hormones was expressed as ng/ml of NIAMDD rat prolactin-RP—l, NIAMDD rat LH RP-l or NIAMDD rat TSH-RP-l. Only when all samples of ant experiment were assayed in the same radio- immunoassay, were comparisons of mean hormone concentration made. EXPERIMENTAL DATA I. Evidence for Adrenergic Mediation of Cholinergic Inhibithn of Prolactin Release A. Introduction The hypothalamus contains hypophysiotroPic hormones and biogenic amines that can either inhibit or stimulate release of prolactin from the anterior pituitary (Clemens and Meites, 1972; Meites gt 31., 1972). Under most condi- tions the mammalian hypothalamus tonically inhibits prolac- tin release but in some states (e.g., stress, suckling) it can stimulate prolactin release. It is believed that catecholamines, particularly dopamine, induce release of a prolactin inhibiting factor (PIF) from the hypothalamus and/or act directly on the anterior pituitary to inhibit prolactin release (Shaar and Clemens, 1974; MacLeod, 1969; Birge gt 31., 1970; Dular EE.Elr' 1975; Dhariwal gt 31., 1969). Serotonin, on the other hand, has been shown to stimulate prolactin release, perhaps by promoting secretion of a prolactin releasing factor (PRF). Recently cholinergic neurons have been implicated in the regulation of prolactin release (Grandison gt 31., 1974; 76 77 Libertun and McCann, 1973, 1974; Kuhn and Lens, 1974) and we have found that acetylcholine, pilocarpine and physo— stigmine each can significantly reduce serum prolactin in rats. It was of interest to determine whether cholinergic inhibition of prolactin release is mediated via adrenergic neuron S o B. Materials and Methods Vaginal smears were obtained daily from female rats and only those showing at least two regular, consecutive 4 or 5 day estrous cycles were used. Male rats were handled daily for 3 days prior to experimentation in order to reduce possible stress effects. Blood was collected by cardiac puncture under light ether anesthesia. Estradiol benzoate (EB) (Nutritional Biochemicals Corporation, Cleveland, OH) was dissolved in absolute ethanol and diluted with corn oil for subcutaneous injection. Pilocarpine nitrate, atropine sulfate and methyl-atropine bromide (Nutritional Biochemicals Corporation, Cleveland, OH), chlorpromazine hydrochloride (Smith, Kline, and French, Philadelphia, PA) and haloperidol (McNeil Laboratories Incorporated, Fort Washington, PA) were dissolved in 0.85% NaCl for intraperitoneal injection. Pimozide (McNeil Laboratories, Fort Washington, PA) was dissolved in 2% tartaric acid. Reserpine (Serpasil, Ciba, Summit, NJ) was given in soluble form (mg/ml). 78 To determine whether cholinergic inhibition of prolac- tin release involved activation of adrenergic neurons, the effect of pilocarpine was observed in animals pretreated with drugs known to inhibit catecholamine activity. The doses of drugs and time intervals used previously were shown to elevate prolactin release (Meites gt_31., 1972; Clemens and Meites, 1972; Lu gt 31., 1970). Reserpine a depletor of brain catecholamines and serotonin (Sheppard and Zimmerman, 1960), was given at a dose of 10 mg/kg to 16 male rats (350- 400 9). Three hours later blood samples were collected: 8 of the rats were then given 5 mg pilocarpine/kg and the remaining 8 were given 0.85% NaCl. At 15 and 45 min after injection of pilocarpine or 0.85% NaCl, blood samples were collected. In a separate experiment chlorpromazine, a catecholamine receptor blocker (McGeer, 1971), was given at a dose of 25 mg/kg to male rats (350-400 9). Blood samples were collected 45 min later, and 8 rats then received 5 mg pilocarpine/kg while the other 8 were given 0.85% NaCl. Blood samples were collected 15, 45 and 90 min after pilo- carpine or its vehicle was injected. In a third experiment haloperidol also a catecholamine receptor blocker (Janssen, 1967) was given to 16 male rats (350-425 g) in a dose of 0.5 mg/kg. Forty-five min later blood samples were collected and 8 rats were given 5 mg pilocarpine/kg and 8 other rats were given 0.85% NaCl. Blood samples were collected 15 and 79 45 min after pilocarpine or 0.85% NaCl injection. Pimozide, a specific dopamine receptor blocker (Janssen gt 31., 1968) was injected subcutaneously at a dose of 2.5 mg/kg to 16 male rats (330-400 9). Four hours after pimozide injection, blood samples were collected and 8 rats then received 5 mg pilocarpine/kg and 8 other rats were given 0.85% NaCl. At 15, 45, and 90 min after pilocarpine or 0.85% NaCl, blood samples were collected. Since reserpine, chlorpromazine, haloperidol and pimo- zide increase the release of prolactin, the effects of pilo- carpine were examined in female and male rats with high rates of prolactin release for purposes of comparison. Twenty- four proestrous female rats were bled during the afternoon surge at 1730 hours and immediately 12 were injected intra— peritoneally with 10 mg pilocarpine/kg while 12 others were injected with 0.85% NaCl (controls). Blood samples were collected from all rats 20 min after injection of pilocar- pine or 0.85% NaCl. In addition 16 estrogen primed (5 pg EB/rat for 10 days) male rats were used since serum prolac- tin levels are low in normal male rats. On the 11th day, 8 of these rats were given 5 mg pilocarpine/kg while the other 8 received 0.85% NaCl. Blood samples were collected from all male rats 15 and 45 min after pilocarpine or 0.85% NaCl injection. An attempt also was made to determine whether cholinerg- ic inhibition of prolactin was exerted centrally or 80 peripherally. Thirty-two male rats were divided into four groups of 8 each.‘ One group received atropine, a central and peripheral cholinergic (muscarinic) receptor blocker, at a dose of 10 mg/kg. The rats were bled 20 min after injec- tion, given 5 mg pilocarpine/kg, and bled 45 min later. A second group received methyl-atrOpine, a peripheral cholinergic receptor blocker, at a dose of 10 mg/kg. Twenty min after methyl-atropine injection these rats were bled, then given 5 mg pilocarpine/kg and bled again 45 min later. For comparison 16 rats were injected with 0.85% NaCl and bled 20 min later. Eight of these rats were given 5 mg pilocarpine/kg, the other 8 received 0.85% NaCl and the rats were bled 45 min after pilocarpine or 0.85% NaCl injection. In addition, the effect of atropine sulfate on serum prolactin was examined. A pretreatment blood sample was collected from 48 male rats (350-500 9). Thirty min after the initial blood sample separate groups of 8 each received 0.85% NaCl or 3, 10, 30, 90, or 250 mg atropine sulfate/kg. At 30, 60, 120 and 240 min after injection of atropine sul- fate or 0.85% NaCl, a 0.7 ml blood sample was collected from each rat. Comparisons between treatment and control groups were analyzed by using Student's t test. 81 C. Results 1. Effects of Pilocarpine on Proestrous Female Rats and Estrogen Primed Male Rats At 0900 hours on the morning of proestrus the serum prolactin concentration was approximately 40 ng/ml but by 1730 hours a surge had occurred and the level averaged 293:23 ng/ml. A single injection of pilocarpine (10 mg/kg) during the proestrous surge reduced prolactin concentration in 20 min to 109:72 ng/ml (p<=0.005) whereas an injection of 0.85% NaCl had no significant effect (230136 ng/ml) (Figure 4A). In male rats given 5 mg of EB for 10 days, serum pro- lactin was increased to 245:15 ng/ml relative to controls (42:5). An injection of 0.85% NaCl into estrogen-primed male rats increased prolactin release when blood was col- lected 15 min later 352:33 (ng/ml) but by 45 min the serum level (290:26 ng/ml) was not significantly different from the pretreatment value. The increase at 15 min may have been due to stress. An injection of pilocarpine (5 mg/kg) significantly (p‘<0.001) lowered serum prolactin by 15 min to 202:22 ng/ml and to 165:17 ng/ml by 45 min (p< 0.005) (Figure 4B). 2. Effects of Pilocarpine in Drug- treated Rats Reserpine at a dose of 10 mg/kg increased serum prolac- tin from 43:6 ng/ml in untreated male rats to 146:11 ng/ml Figure 4. 82 Effects of pilocarpine on prolactin release in proestrous female and estrogen-primed male rats. B3rs represent serum prolactin concentration (X : SOEOM.) A. ** Female rats on proestrus at 0900 hours (hori— zontally striped), at 1730 hours (solid) and at 1750 hours 20 min after 0.85% NaCl (solid) or pilocarpine injection (diagonally striped). Untreated male rats (horizontally striped or estrogen primed male rats given 0.85% NaCl (solid) or pilocarpine injection (horizontally striped). p< 0.05 vs. 0.85% NaCl at same time after injection p< 0.01 83 300 SERUM pm. n91 ml 200 I00 - 0900 1730 1750, sal pulo 300 St mm pm Mg "II 200 ‘00 swab arm 15 min 45 mm Figure 4 84 by 3 hours after injection. Fifteen and 45 min after the injection of 0.85% NaCl the reserpine-treated rats the prolactin level was unchanged. Pilocarpine failed to sig- nificantly reduce serum prolactin at 15 min (105:17 ng/ml) or 45 min (169:13 ng/ml) after injection (Figure 5A). In male rats an injection of chlorpromazine increased serum prolactin from 38:6 ng/ml in untreated rats to 135:7 ng/ml by 45 min after injection. A subsequent injection of pilocarpine or saline failed to reduce serum prolactin by 15, 45 or 90 min after injection (Figure SB). A similar inability to reduce prolactin by pilocarpine was observed in rats first treated with haloperidol or pimozide, although each of the latter two drugs by themselves produced signifi- cant increases in serum prolactin levels (Figure 6). 3. Effects of Cholinergic Receptor Blockade Atropine is known to block the effect of acetylcholine and pilocarpine at muscarinic receptors (Innes and Nickerson, 1970). When atrOpine sulfate was given alone at doses of 3 to 250 mg/kg intraperitoneally, no significant difference was noted in serum prolactin levels as compared with those in 0.85% NaCl injected rats (Figure 7). However, atropine sulfate prevented pilocarpine from decreasing prolactin release (Figure 8). Methyl-atropine, which does not easily penetrate the blood brain barrier (Innes and Nickerson, 1970), did not prevent pilocarpine from reducing prolactin release. Figure 5. 85 Effects of pilocarpine on prolactin release in male rats given reserpine or chlorpromazine. B3rs represent serum prolactin concentration (x : S.E.M.) A. Untreated male rats (horizontally striped) or reserpinized rats given 0.85% NaCl (solid) or pilocarpine (diagonally striped). B. Untreated male rats (horizontally striped) or chlorpromazine treated rats given 0.85% NaCl (solid) or pilocarpine (diagonally striped). 86 200 SERUM PRL ng/nn mm untrealm RSP RSP RSP RSP RSP sal pulo sal pilo 15 mun 45 min 200 SF RUM PRL nq /"n “’"l untreated CF’Z CPZCPZ CPZCPZ CPZCPZ sal plo sal orb sal pdo 15 mm 45 min a) min Figure 5 Figure 6. 87 Effects of pilocarpine on prolactin release in male rats given haloperidol or pimozide. Bars represent serum prolactin concentration (x : S.E.M.) A. 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