u. I? 3.1,: M; ; . .‘Y‘ . i ‘.. ”'5‘; "T‘T'T‘ it. :;’§:' .51 \LL‘. I“. H: U: :53?! ‘33 C? 1 F Deg; a... eréati-sn f0? tin «r» r ,3 n :1 I L '2: 2.6a A a, It", ix=”-;.z.;:;::-z~21 9.43%? n .. ., . .. ”I". . I . I;"fl.' y . _ . L..-\t . .1 This is to certify that the thesis entitled RELATION OF BIOGENIC AMINES, TEMPERATURE AND STRESS TO THE RELEASE OF ANTERIOR' PITUITARY HORMONES presented by Gregory Paul Mueller has been accepted towards fulfillment of the requirements for Ph . D . degree in Physiology Date 7/7/76 0-7639 manna n "7 HMS & SNIS' 800K HINDU" INC. ueum moms mm'on, "my . .'f. 4 Fins—v—mV ABSTRACT RELATION OF BIOGENIC AMINES, TEMPERATURE AND STRESS TO THE RELEASE OF ANTERIOR PITUITARY HORMONES By Gregory Paul Mueller 1. A single injection of synthetic thyrotropin-releasing hormone (TRH) significantly elevated serum levels of prolactin and thyroid- stimulating hormone (TSH) in proestrous female and in normal and estrogen- primed male rats by l0 minutes after injection. Graduated doses of TRH in proestrous female rats stimulated TSH release in a dose-related fashion whereas no definite dose-response relationship in the prolactin response was observed. Estrogen-priming in male rats significantly ele- vated serum prolactin, reduced TSH and had little influence on the pro- lactin and TSH responses by 10 or 60 minutes after injection of 1 pg TRH. It is concluded that synthetic TRH can significantly increase serum pro- lactin as well as TSH in proestrous female, and in normal and estrogen- primed male rats. 2. When mature male rats were placed in a chamber at 40°C for 30 minutes, there was a significant decrease in serum TSH (0.46 3.0.08 pg/ml vs. 0.l2 :_0.02 ug/ml) and a fivefold elevation of serum prolactin. A temperature of 4°C for 120 minutes increased TSH and resulted in a significant fall in serum prolactin (25 1.3 ug/ml vs. 6 :_l ng/ml). Gregory Paul Mueller Plasma levels of growth hormone were not altered under these conditions. Removal of the pituitary from hypothalamic influence by transplantation under the kidney capsule eliminated the ability of warm or cold tempera- ture to influence the release of TSH and prolactin. Thyroidectomy (l0 days) maximally elevated TSH but had no influence on the cold-induced suppression of prolactin. Pimozide, a dopamine receptor blocker, significantly elevated resting levels of TSH and prolactin and prevented cold temperature from further increasing TSH or depressing prolactin. Plasma levels of growth hormone tended to be reduced by pimozide; however, this effect was not significant due to animal variation. These findings indicate that TSH and prolactin respond oppositely to the same temperature changes. The effects of temperature on TSH and pro- lactin release are mediated by the hypothalamus and are not due to associated changes in thyroid function. Dopaminergic neurons tonically inhibit TSH and prolactin secretion and appear to be involved in the cold-induced suppression of prolactin release. 3. The dose response effects of apomorphine and ET-495 (piribedil), two specific dopamine receptor stimulators, and halo- peridol, a dopamine receptor blocker, were tested on the secretion of prolactin, TSH, growth hormone and luteinizing hormone (LH) in male rats. Both apomorphine and piribedil reduced serum levels of prolactin and TSH, stimulated growth hormone release at low but not at high doses and either had no effect or tended to reduce serum LH levels. Gregory Paul Mueller The minimal effective dose of apomorphine for reducing prolactin by 30 min was 0.01 mg/kg, whereas TSH inhibition was observed at a dose of 0.l mg/kg - 0.3 mg/kg. The inhibitory effects of apomorphine (l.0 mg/kg) on prolactin and TSH levels were maximal by l5 min and diminished by l20 min, whereas plasma growth hormone was highest by l20 min after injection. Thyroidectomy (l0 days) markedly elevated serum TSH, but had no effect on serum prolactin and inhibited the ability of apomorphine (0.1 mg/kg or 0.3 mg/kg) to reduce TSH but not prolactin levels. These observations may indicate the existence of separate dopaminergic control mechanisms for TSH and prolactin secretion. Administration of halo- peridol elevated serum prolactin, tended to lower TSH, dramatically reduced growth hormone and had no effect on LH levels. Haloperidol pre- treatment blocked the effects of apomorphine on prolactin, TSH and growth hormone secretion. The overall results of this study indicate that dopamine agonists, and thus dopaminergic mechanisms, inhibit pro- lactin and TSH, stimulate growth hormone and do not alter release of LH in male rats. 4. The dose response and time course effects of L- and D- tryptophan, 5-hydroxytryptophan (5-HTP) and restraint stress on the metabolism of serotonin and release of TSH, prolactin and growth hormone were tested in male rats. All treatments increased serotonin levels in the hypothalamus and remaining brain tissue minus the cerebellum (brain) and significantly elevated brain levels of 5-hydroxyindoleacetic acid Gregory Paul Mueller (5-HIAA), indicating stimulation of serotonin turnover. L-tryptophan and restraint stress increased serotonin turnover in the hypothalamus and brain as determined by the accumulation of serotonin after monoamine oxidase inhibition. Both L-tryptophan and restraint stress inhibited TSH and growth hormone and stimulated prolactin release. The effects of D-tryptophan and S-HTP on TSH, prolactin and growth hormone release generally followed a similar pattern although changes in hormone levels were not as marked, presumably due to the reduced potency and multiple actions of the two drugs. respectively. These findings indicate that enhanced rates of serotonin turnover produced by administration of serotonin precursors and physical restraint are associated with inhibi- tion of TSH and growth hormone and stimulation of prolactin release from the anterior pituitary. RELATION OF BIOGENIC AMINES, TEMPERATURE AND STRESS TO THE RELEASE OF ANTERIOR PITUITARY HORMONES By Gregory Paul Mueller 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 I dedicate this thesis to my parents Alice and Gerald C. Mueller and to this institution, Michigan State University. ii ACKNOWLEDGMENTS I wish to thank Dr. Joseph Meites for his constant guidance, advice and support throughout my stay at Michigan State University. Dr. Meites has worked to create and maintain an academic environment for which he asks only that his students use this setting wisely--to benefit both themselves and the laboratory. His efforts on a personal level are always directed towards the well-being of his students. For all these reasons I think Dr. Meites is truly a teacher of graduate students. I am honored to have had the opportunity to be a student under his guidance, and have developed a deep and lasting respect for him. I would like to thank the fellow members of our laboratory for their help and friendship, and Dr. K. E. Moore who was essential in designing and carrying out some of the work presented in this thesis. TABLE OF CONTENTS LIST or TABLES................................................... LIST OF FIGURES .................................................. INTRODUCTION ..................................................... LITERATURE REVIEW ................................................ I. Hypothalamic Control of Anterior Pituitary Hormone Secretion ................................................ A. Early Observations on the Functional Relationship Between the Hypothalamus and Anterior Pituitary Gland ................................................. B. Hypophyseal Portal Vessels ............................ C. Chemotransmission and Hypothalamic Releasing Factors.. 0. General Physiology of Hypothalamic Releasing Hormones. E. Anatomy of the Hypothalamus and Location of Hypo- thalamic Hormones..... ........ .... ..... .......... ..... II. Hypothalamic Neurotransmitters ........................... A. General ............................................... B. Catecholamines ........................................ C. Serotonin ...................... . ...... . ................ D. Monoamine Pathways Innervating the Hypothalamus.. ..... E. Catecholamine and Serotonin Synthesis and Turnover.... iv Page vii viii 11 12 16 16 17 18 19 20 Page III. Hypothalamic Control of Prolactin......... ..... .......... 24 A. General.. ...... .... .............. ..................... 24 B. Hypothalamic Hormones ................................. 25 C. Effects of Catecholamines on Prolactin Secretion ...... 29 D. Serotonin and Prolactin Release ....................... 38 E. Putative Brain Neurotransmitters and Prolactin Release 40 F. Inhibitory Feedback of Prolactin on Pituitary Prolac- tin Secretion ......................................... 4l IV. Current Views of the Hypothalamic Control of Thyroid Stimulating Hormone (TSH), Growth Hormone and Luteinizing Hormone (LH) ............................................. 45 A. TSH ................................. . ................. 45 B. LH .................................................... 49 C. Growth Hormone .............................. . ......... 54 V. Effects of Environmental Temperature and Physical Stress on Pituitary Hormone Secretion and Brain Biogenic Amines. 58 A. General... ....................... . .......... . ..... .... 58 8. Effects of High and Low Temperature and Physical Stress on Anterior Pituitary Hormone Secretion........ 59 C. Effects of High and Low Temperature and Physical Stress on Brain Catecholamines and Serotonin .......... 63 MATERIALS AND METHODS ....... . .................................... 66 1. Animals, Treatments and Blood Collection ................. 66 II. Radioimmunoassays of Blood Hormones ................ . ..... 68 III. Brain Tryptophan, Serotonin and S-Hydroxyindoleacetic Acid (S-HIAA) Assays ..................................... 70 EXPERIMENTAL ..................................................... 71 I. Effects of Thyrotropin-Releasing Hormone (TRH) on the Ig.Vivo Release of Prolactin and TSH in Proestrous Female, Male and Estrogen-Primed Male Rats.... ........... 7l A. Objectives ............................... . ............ B. Materials and Methods .......................... . ...... C. Results ............................................... D. Conclusions ........................................... II. Effects of Heat and Cold on the Release of TSH, Growth Hormone and Prolactin in Male Rats.... ............. . ..... A. Objectives ........................................ .... B. Materials and Methods ................................. C. Results ...................... . ........................ 0. Conclusions ..................... . ....... .............. III. Effects of Dopaminergic Drugs on the Release of Prolactin, TSH, Growth Hormone and LH in Male Rats.. ....... ......... A. Objectives ............................................ B. Materials and Methods ................................. C. Results .............................................. . D. Conclusions ........................................... IV. Effects of Tryptophan, S—HTP and Restraint Stress on Hypothalamic and Brain Serotonin Turnover and Pituitary Hormone Release ..... . ........ . ................ . ....... ... A. Objectives ............................................ B. Materials and Methods ................................. C. Results ....................... . ............... . ....... D. Conclusions ........................................... GENERAL DISCUSSION ............................................... BIBLIOGRAPHY .................................................. ... APPENDIX A. Serotonin and S-HIAA Assay Procedures ............... APPENDIX B. Tryptophan Assay Procedure ................... . ...... CURRICULUM VITAE ................................................. vi Page 71 72 73 76 77 77 73 73 84 87 87 87 100 105 105 106 106 116 123 129 170 173 175 TABLE 10. LIST OF TABLES . Effects of TRH on Prolactin Release in Proestrous Female and in Untreated and Estrogen-Primed Male Rats..... ............. . Effects of TRH on TSH Release in Proestrous Female and in Untreated and Estrogen-Primed Male Rats ..................... . Effects of Heat and Cold on Blood Concentrations of TSH, GH and PRL in Male Rats ........................................ . Effects of Heat and Cold on Blood Concentrations of TSH, GH and PRL in Male Hypophysectomized-AP Transplanted Rats...... . Effects of Apomorphine on Serum Prolactin and TSH Concentra- tions in Intact and Thyroidectomized Rats................... . Minimally Effective Doses of Dopaminergic Agonists Required to Alter the Blood Concentration of Pituitary Hormones and to Cause Stereotyped Sniffing in Male Rats ...... . ........... . Effects of L-Tryptophan on Concentrations of Cerebellum Tryptophan,Hypothalamic and Brain Serotonin, Brain S-HIAA, and Serum TSH and Prolactin in Male Rats.. ..... ............. . Effects of D- and L-Tryptophan on Concentrations of Cerebel- lum Tryptophan, Hypothalamic and Brain Serotonin, Brain 5-HIAA, and Blood Hormones in Male Rats..................... . Effects of Pargyline, Pargyline Plus L-Tryptophan, and 5-HTP on Concentrations of Hypothalamic and Brain Serotonin and Serum TSH and Prolactin in Male Rats .......... .... ...... Effects of Restraint Stress on Concentrations of Cerebellum Tryptophan, Hypothalamic and Brain Serotonin, Brain S-HIAA, and Blood Hormones in Male Rats............................. vii Page 73 75 79 80 99 110 111 112 115 FIGURE 10. LIST OF FIGURES . Effects of thyroidectomy on cold induced changes in blood concentrations of TSH, growth hormone (GH) and prolactin (PRL) in male rats......................................... . Effects of pimozide on cold induced changes in blood con- centrations of TSH, growth hormone (GH) and prolactin (PRL) in male rats.......... ..... ........ ...... .................. Time course of the effects of apomorphine on blood content of pituitary hormones in male rats ..................... .... . Dose-related effects of high doses of apomorphine on blood content of pituitary hormones in male rats .......... . ...... Effects of low doses of apomorphine on blood content of pituitary hormones in male rats.... ..... ............ ....... . Dose-related effects of piribedil on blood content of pituitary hormones in male rats ......... . ....... .... ....... . Effects of increasing doses of haloperidol alone or in com- bination with apomorphine on blood content of pituitary hormones in male rats ............ ....... ....... . ...... ..... . Time course of the effects of pargyline on hypothalamic and brain concentrations of serotonin in male rats............. . Dose-related effects of S-HTP on concentrations of hypo- thalamic and brain serotonin, brain SvHIAA, and blood hormones in male rats...................................... Time course of the effects of restraint stress on the accumulation of serotonin in hypothalamus and brain of pargyline treated male rats......... ........ ......... ..... . viii Page 82 83 89 91 92 95 97 107 114 117 INTRODUCTION Neural regulation of hormone secretion by the anterior pituitary gland (AP) appears to involve specific relationships between neuronal activity in the hypothalamus and the release of hypophysiotrophic hor- mones into the hypophyseal portal circulation. These hypothalamic hor- mones originate within specialized neurosecretory cells and act directly on the AP to modify the synthesis and release of pituitary hormones. The work presented in this thesis was carried out to further investigate hypothalamic mechanisms controlling release of prolactin, thyroid stimu- lating hormone (TSH), growth hormone and luteinizing hormone (LH) in the rat. Extensive investigation into the biochemical nature of hypo- thalamic hormones has revealed the structure of three of these agents; thyrotropin-releasing hormone (TRH), luteinizing hormone-releasing hor- mone (LRH) and somatostatin. All of these substances have proven to be small peptides. Synthetic and natural TRH and LRH stimulate the release of TSH and the gonadotropins (LH and follicle stimulating hormone) respectively whereas, somatostatin inhibits the release of growth hor- mone. Under experimental conditions, TRH also stimulates the release of prolactin and somatostatin blocks TRH-induced release of TSH but not prolactin. One aspect of this thesis is an investigation of the pos- sible role TRH may have in mediating the prolactin and TSH responses to changes in ambient temperature and physical restraint. Findings presented here and reports of others indicate that TRH is not involved in the physiological control of prolactin release in the rat whereas, the possible role of somatostatin in regulating TSH secretion has not been determined. Several neurotransmitters including the catecholamines, dopamine and norepinephrine, and the indoleamine, serotonin, are found in high concentration in the hypothalamus. Recent 1g_yjtgg_evidence indicates that dopamine may be the prolactin release-inhibiting factor (PIF), or possibly one of several PIFs present in crude hypothalamic extracts. However, the ability of hypothalamic extracts to inhibit prolactin release jg_yiyg_cannot be accounted for solely on the basis of the small amount of dopamine present in the extracts. This difference suggests that another substance, presumably a small peptide (like TRH, LRH and somatostatin), is responsible for the jg_yiyg_inhibition of prolactin release by hypothalamic extracts. There is general agreement that dopamine neurons located in the median eminence region of the hypothala- mus act to inhibit prolactin secretion by stimulating the release of PIF. Recent evidence clearly demonstrates that various drugs and physio- logical conditions which enhance hypothalamic dopamine activity, also elevate PIF activity in the hypothalamus and inhibit the release of pituitary prolactin. Treatments that reduce dopamine activity have the opposite effects on PIF and prolactin. Accumulating evidence indicates that serotonin neurons mediate stimulation of prolactin release; however, a PIF and/or prolactin-releasing factor (PRF) mechanism for this stimulation has not been elucidated. Thus, prolactin release in the rat is under stimulatory control by serotonin and inhibitory control by dopamine. By contrast, the possible role of other neurotransmitters in control of prolactin, and neurotransmitter regulation of TSH, growth hormone and LH release are less clearly understood. The findings of some, but not all investigators suggest that in the rat, TRH¥TSH and LRH~gonadotr0pin release may be stimulated by nor- epinephrine and inhibited by serotonin and possibly dopamine. Stimula- tion of growth hormone release appears to be mediated by a dopaminergic mechanism. Development of specific and highly potent dopamine receptor agonists and antagonists has made it possible to more carefully define the influence of dopamine neurons on the release of hypothalamic and AP hormones. In work presented here, several of these agents were used alone and in combination to determine the effects of dopamine receptor activity on the release of prolactin, TSH, growth hormone, and LH in male rats. The influence of serotonin on the release of AP hormones was evaluated by correlating changes in brain serotonin metabolism as pro- duced by the administration of serotonin precursors and physical restraint with associated changes in the release of prolactin, TSH and growth hormone. The results of these studies support the established view on neurotransmitter control of prolactin release and provide new evidence on the roles of dopamine and serotonin in the control of TSH, growth hormone and LH release. Findings presented here and reports of many others indicate that exteroceptive and pharmacological stimuli act through the hypothalamus to selectively alter the release of AP hormones. The objective of this thesis was to clarify and extend current understanding of mechanisms in the hypothalamus controlling the release of prolactin, TSH, growth hormone and LH in the rat. LITERATURE REVIEW 1. Hypothalamic Control of Anterior Pituitary Hormone Secretion A. Early Observations on the Functional Relationship Between the Hypothala- mus and Anterior Pituitary Gland Control of anterior pituitary hormone secretion is mediated by the central nervous system (CNS) and influenced by blood concentrations of target gland hormones. In 1797 Haigton observed that mating induces ovulation in rabbits, subsequently this phenomena was determined to be a neuroendocrine reflex involving relay of sensory information to the brain which led to the release of pituitary luteinizing hormone (LH) and subsequent ovulation. Today many exteroceptive stimuli (e.g., temperature, light, odor and touch) are known to affect pituitary hor- mone secretion by acting through the CNS (Marshall, l942; Harris, 1955). Study of the functional relationship between the brain and pituitary has developed as the field of neuroendocrinology. The pituitary gland lies immediately below yet connected to the hypothalamus by the hypophyseal or pituitary stalk. The hypothalamus constitutes a region of densely clustered nuclei located in the ventral- most portion of the diencephalon (Netter, l968; Jenkins, l972). The pituitary stalk consists of: l) nerve tracts which transport oxytocin and vasopressin from their site of origin in the anterior hypothalamus to the posterior pituitary; 2) structural elements; and 3) blood vessels which communicate between the hypothalamus and anterior pituitary gland. Virtually no nerve fibers pass directly from the hypothalamus to the anterior pituitary. Although the anterior pituitary does receive some fibers which arise from outside the hypothalamus, this innervation is thought to function solely in vasomotion (Harris, 1955; Szentagothai gt_al,, 1972). Early investigations demonstrated that the hypothalamus was of major importance in the regulation of pituitary hormone secretion. In 1912 Aschner reported that localized lesions in the anterior hypothala- mus caused gonadal atrophy in dogs. Similar observations were made in rats (Camus and Roussy, 1920) and guinea pigs (Day, 1943). Hypothalamic lesions were also reported to produce atrophy of the thyroid (Cahane and Cahane, 1938; Greer, 1952; Bogdenove and Hamli, 1953) and adrenal cortex (de Groot and Harris, 1950). By contrast electrical stimulation of the hypothalamus (Harris, 1937; Haterius and Derbyshire, 1937) but not the pituitary (Markee gt.al,, 1946; Harris, 1948a) was found to in- duce ovulation in rabbits. Prolonged hypothalamic stimulation enhanced thyroid (Harris, 1948b) and adrenal cortical (de Groot and Harris, 1950) activity in rabbits. These observations indicate that experimental alterations of hypothalamic function can dramatically affect pituitary hormone-target organ physiology. Removing the pituitary gland from the direct influence of the brain by stalk section or transplantation to a distant site in the body decreased whole body metabolism and caused atrophy of pituitary hormone target blands with the exception of corpora lutea (Harris, 1948b and 1955; Everett, 1954, 1956). Pituitary histology shifts from a normal makeup of acidophils, basophils and chromophobes to a predominantly chromophobe cell type with maintenance of a substantial number of acidophils (Harris, 1955; Everett, 1956). By contrast transplantation of pituitary hormone target organs (gonads, thyroid and adrenal cortex) does not dramatically affect the histology or function of the trans- plants (Harris, 1948b, 1955), indicating that the pituitary is under a trophic influence by the CNS which usually is not carried to the systemic circulation. Some of the first pituitary stalk section experiments were carried out by Dott (1923) who observed that placement of small platinum plates between the pituitary and hypothalamus resulted in lowered body temperature and degeneration of the thyroid and gonads in dogs. Later Mahoney and Sheehan (1936) made similar observations after placing silver clips on the pituitary stalk. Harris (1937) found that stalk section caused gonadal atrophy in rabbits. Contrary to these findings many others reported that stalk transection did not appreciably affect the endocrine status of their experimental animals (see Harris, 1955). This conflict was resolved when Harris (1948b) demonstrated that the portal blood vessels transcending the pituitary stalk must be intact for hypo- thalamic regulation of anterior pituitary hormone secretion. Following stalk section these vessels often regenerated to restore the functional connection between the pituitary and hypothalamus (Harris, 1948b; Harris and Jacobsohn, 1950). B. Hypophyseal Portal Vessels Popa and Fielding (1930) first observed that the vessels trans- cending the human pituitary stalk are true portal vessels connecting a capillary bed in the median eminence region of the basal hypothalamus to sinusoids in the anterior pituitary. The anatomy of the portal vessels was confirmed by Wislocki and King (1936) who (contrary to Papa and Fielding) concluded on the basis of morphological evidence that blood flow was directed from the hypothalamus into the pituitary. The presence of this vascular connection, termed the hypophyseal portal vessels, was observed to be a common feature among vertebrates (Green and Harris, 1947, 1949; Harris, 1972). Direct microscopic observation demonstrated that blood flow in amphibians (Houssay gt_al,, 1935) and in rats (Green and Harris, 1949) was from the hypothalamus to the pituitary. Two types of portal vessels, long and short, have been described (Adams 23.21,, 1965; Daniel, 1966) on the basis of the relative dis- tances they transcend from the hypothalamic plexus to the pituitary, the region of the pituitary they supply and the origin of their arterial blood flow. Arterial blood to the hypothalamic plexus is carried by the superior and inferior hypophyseal arteries which arise from the internal carotid and posterior communicating arteries and supply the long and short portal vessels, respectively. Venous flow from the pituitary is by way of small venules which drain into sinuses lying adjacent to the anterior pituitary gland (Adams §t_al,, 1965; Daniel, 1966). C. Chemotransmission and Hypothalamic ReTeasing_Factors The possibility that specialized hypothalamic neurons might secrete hormones into blood was first considered by Scharrer and Scharrer (1940). Subsequent investigations in several vertebrate species demon- strated the phenomenon of neurosecretion (Bargmann and Scharrer, 1951; Scharrer, 1952; Scharrer and Scharrer, 1954). These workers observed that oxytocin and vasopressin are synthesized in cell bodies of neurons located in the anterior hypothalamus and transported down long axons to the posterior pituitary from which these hormones are released into the general circulation. Based on the anatomy and the importance of the hypophyseal portal system on the control of anterior pituitary hormone secretion, Harris (1948b) proposed the "chemotransmitter hypothesis". He suggested that nervous stimuli induced release of hypothalamic humors into the capillary plexus of the median eminence. These humors were then transported via the hypophyseal portal vessels to the anterior pituitary, where they either stimulate or inhibit hormone secretion. Subsequent demonstrations that extracts of hypothalamic tissue contained substances which acted directly on the pituitary to alter hormone re- lease supported this hypothesis. Saffran and Schally (1955) and Guillemin £3.31, (1957) reported that rat, bovine and ovine hypothalamic extracts contained a cortico- tropin-releasing factor (CRF) which stimulated the jn_yjtrg.release of adrenocorticotropic hormone (ACTH). Other laboratories demonstrated that mammalian hypothalamic extracts stimulated release of thyroid stimulating hormone (TSH) (Shibusawa gt_al,, 1956, 1959; Guillemin gt_al., 10 1963), LH (McCann gt_al,, 1960), prolactin (Meites gt_gl,, 1960), follicle stimulating hormone (FSH) (Igarashi and McCann, 1964; Mittler and Meites, 1964) and growth hormone (Deuben and Meites, 1964). Hypothalamic extracts were also reported to inhibit jg_!jtrg_release of prolactin (Talwalker gt_al,, 1961, 1963; Pasteels, 1961) and growth hor- mone (Krulich gt_al,, 1968). Presently there is general agreement that specific substances, probably small polypeptides and perhaps some cate- cholamines, are responsible for each of the above activities attributed to hypothalamic extracts (McCann and Dhariwal, 1966; Burgus and Guille- min, 1970; Schally 23.31,, 1973; Vale et.a1,, 1973b; Rippel, 1975; Shaar and Clemens, 1974). Three hypothalamic hormones have been isolated, structurally analyzed and synthesized. Thyrotropin-releasing hormone (TRH) which stimulates TSH release was first isolated from porcine hypothalami and reported to contain the amino acids histidine, proline and glutamic acid in equimolar ratios (Schally gt_al,, 1966). In 1969 the laboratories of Schally (Schally gt_al,, 1969; Folkers gt a1,, 1969) and Guillemin (Burgus et_al:, 1969, 1970) working on porcine and ovine TRH, respective- ly, reported the structure of TRH to be the tripeptide amide, (pyro)- Glu-His-Pro-NHZ. Shortly thereafter the structure of porcine luteinizing hormone-releasing hormone (LRH) which stimulates the release of both LH and FSH was proposed (Matsuo gt_al,, 1971a) and the decapeptide was syn— thesized (Matsuo gt_al,, 1971b). Full activity for both TRH and LRH requires the C-terminal of these peptides to be present as the amide and the N-terminal present as the cyclic pyroglutamyl residue (Schally gt_al,, 1973). 11 Recently ovine somatotropin release-inhibiting factor (SRIF) was isolated, determined to be a tetradecapeptide and named somatostatin (Brazeau gt_al,, 1973). Synthetic somatostatin was first prepared by Rivier gt_al, (1973). The capacity of synthetic somatostatin, TRH and LRH to influence release of their respective hormones was found to be dose-related and equipotent to purified native materials (Schally gt_al,, 1973; Vale gt_al,, 1975). D. General Physiology of Hypothalamic Releasing Hormones Synthetic releasing hormones as well as hypothalamic extracts do not appear to be species specific in their actions on pituitary hormone secretion. However, a single hypothalamic hormone can influence the release of more than one pituitary hormone (McCann and Dhariwal, 1966; Schally gt_al,, 1973; Vale gt 91,, 1975; Rippel 93 91,, 1975; Convey gt_al,, 1975). Initially it was thought that LH and FSH were under con- trol of separate releasing factors (Schally gt_gl,, 1968). However, the isolation and synthesis of LRH was followed by many demonstrations that this agent stimulates the release of both LH and FSH (see Schally gt_al,, 1973; Convey gt_al,, 1975; Yen gt_al,, 1975). Separate hypothalamic hormones which selectively stimulate LH and FSH release have not been isolated and LRH is presently thought to be the physiological releaser for both gonadotropins (Schally gt_al:, 1976). The occasional divergence of LH and FSH release observed in humans and other mammals may be due in part to modification of their secretion by sex steroids and/or different biological half lifes of LH and FSH (Schally $3.21,, 1973; Yen gt_al,, 1975). 12 Synthetic TRH was shown to rapidly stimulate the 1n.yjtrg_and ig_yjyg_release of prolactin as well as TSH in many experimental animals and humans (Jacobs gt_al,, 1971; Tashjian gt_al,, 1973; Meites, 1973; Convey gt.al,, 1973). In addition TRH has been reported to stimulate growth hormone release in the rat (Takahara gt_al,, 1974b; Kato 93.21:: 1975), bovine (Convey gt_a1,, 1973) and in humans (Schalch gt_al,, 1972; Maeda gt_al,, 1975). Somatostatin is the most diverse hypothalamic hormone in terms of its varied biological actions. Somatostatin was reported to inhibit TRH induced release of TSH jn_yjtrg_and jn_yjyg_in the rat (Vale gt_a1,, 1973a, 1975) and in humans (Hall gt_gl,, 1974). By contrast somato- statin did not inhibit TRH stimulation of prolactin release (Vale gt_al,, 1974a, 1975). In addition to having multiple effects on pituitary hor- mone secretion, somatostatin was reported to act directly on the pan- creas to inhibit secretion of both insulin and glucagon (Fujimoto gt 91,, 1974; see Vale 23.21,, 1975) and in the gut to inhibit gastrin secretion (Blood gt_al,, 1974). The physiological significance of the ability of TRH to stimulate prolactin and growth hormone release and for somato- statin to block TRH induced TSH release and to inhibit the endocrine pancreas and gut gastrin secretion remain to be determined. E. Anatomy_of the Hypothalamus and Location of Hypothalamic Hormones Further consideration of hypothalamic function requires a more detailed description of the anatomy of the hypothalamus. This review is based on the works of Harris (1955), Netter (1968), Szentagothai gt_al, (1972), and Jenkins (1972). 13 From the ventral aspect of the brain the hypothalamus extends from the anterior border of the optic chiasm to the caudal border of the mammillary bodies and medial from the optic tracts. Located in the middle of this region is the tuber cinereum which gives rise to the hypo- physeal stalk. The tuber cinereum contains the primary plexus of the hypophyseal portal system and the median eminence. The dorsal border of the hypothalamus is marked by the anterior commissure rostrally and hypothalamic sulcus caudally. In a rostral-caudal sequence there are three major gray regions termed anterior, intermediate and posterior hypothalamic areas. Hypo- thalamic nuclei are bilaterally located on each side of the third ventricle with the exception of the arcuate nucleus which lies in the midline below the ventricle and above the median eminence. The hypothal- amus receives afferent fibers from the fornix, medial forebrain bundle, thalamus, stria terminalis, and mammillary peduncle. Major efferents leave by way of the hypophyseal, periventricular and manmillary tracts. The median eminence is a region of densely packed nerve terminals which surrounds the capillaries of the primary plexus. This area con- tains very high concentrations of hypothalamic hormones (Harris, 1955, 1972; Szentagothai gt_al,, 1972; McCann and Moss, 1975; Brownstein gt_al,, 1976) and neurotransmitters, especially dopamine (Brownstein gt_al,, ‘ 1976). The median eminence has a profound influence on the secretion of all anterior pituitary hormones and represents the site at which chemo- transmission occurs (Harris, 1948b, 1955, 1972). 14 The preoptic-suprachiasmatic area of the anterior hypothalamus regulates the ovulatory release of gonadotropins. Electrolytic lesion (Hillarp, 1949) or isolation of this region by a Halasz knife cut (Halasz, 1969) resulted in failure of ovulation and loss of estrous cycles in rats. By contrast, electrical stimulation of this area in- duced release of LH and FSH and thereby caused ovulation (Harris, 1937; Haterius and Derbyshire, 1937; Markee gt_al,, 1946; McCann, 1974; Sawyer, 1975). Other hypothalamic sites which function to control individual pituitary hormones have not been clearly defined (Szentagothai gt_gl,, 1972) and the influence of higher brain centers, e.g., brain stem, limbic system, cortex) on hypothalamic-pituitary function is largely unknown (Szentagothai §t_al,, 1972; McCann, 1974; Reichlin, 1974; Neill, 1974). Hypothalamic Hormones Hypothalamic hormones are concentrated in the median eminence although measurable amounts of immunoreactive TRH, LRH and somatostatin were found to be present throughout the hypothalamus (Brownstein gt_al,, 1976) and in other regions of the rat brain (Oliver §t_al,, 1974; Jackson and Reichlin, 1974). Recently Burt and Snyder (1975) demon- strated the presence of specific high affinity TRH receptors in membrane fractions prepared from a variety of brain regions indicating a possible physiological role for TRH in neuronal function. High concentrations of LRH were measured in the medial basal hypothalamus of the rat (Palkovits gt_al,, 1974a; McCann and Moss, 1975; Setalo gt_al,, 1975). LRH was found by some (White gt_al,, 1974; 15 Barry and Dubois, 1975) but not by others (Winters gt_a1,, 1974; Setalo gt.al,, 1975) to be located in brain regions outside of the hypothalamus. By contrast 96% of bioassayable somatostatin present in rat brain was reported to be located outside of the hypothalamus (Vale gt_al,, 1974b, 1975). It should be noted that bioassay of somatostatin measures a composite of both GH inhibitory and stimulatory-release activity and thus does not provide an accurate measure of somatostatin. Radioimmunoassayable somatostatin was found to be concentrated in the median eminence and present to a lesser extent throughout the hypothalamus, diencephalon and mesencephalon (Brownstein §t_al,, 1975a, 1976). sggthetic TRH (Prange gt.al,, 1972), LRH (Moss and McCann, 1973; Pfaff, 1973) and somatostatin (Vale §t_al,, 1975) were reported to pro- duce behavioral effects in rats and humans (also see McCann and Moss, 1975). Iontophoretic application of LRH was reported to alter firing rate of central neurons (Kawakami and Sakuma, 1974; McCann and Moss, 1975) and intraperitoneal injections of TRH enhanced brain (central) norepinephrine turnover (Keller gt_al,, 1974) in rats. The possibility that hypothalamic hormones and other small peptides may act as neuro- transmitters or neuromodulators is under investigation (Plotnikoff gt_gl,, 1975). Another hypothesis proposed for the wide spread central distribu- tion of hypothalamic hormones is that cerebral spinal fluid may func- tion in the transport of these agents to the pituitary (L6fgren, 1959). Specialized ependymal cells (tanycytes) lining the floor of the third 16 ventricle were observed to send foot processes to the vessel walls of the hypophyseal plexus and to contain granules which appear to serve a secretory function (Bleier, 1971; Mitchell, 1975). Hypothalamic hor- mones have been reported to be present in cerebral spinal fluid (Shambanch gt_al,, 1975) and localized within tanycytes (Zimmerman gt_gl,, 1974). Radiolabeled TRH (Oliver gt.al,, 1975), dopamine (Ben- Jonathan gt_al,, 1975a) and steroid and protein hormones (Ondo gt 31,, 1972) were found to emerge in hypophyseal portal blood shortly after their injection into the third ventricle of rats. Together these reports suggest that tanycyte transport of substances between the cerebral spinal fluid and hypophyseal portal circulation may be in- volved in regulation of the anterior pituitary. However, the physio- logical importance of this mechanism has not been determined. 11. Hypothalamic Neurotransmitters A. General Neurotransmitters are chemically active substances which mediate the transmission of nerve impulses across synapses. In this respect, these agents are directly involved in neuroendocrine regulation. Drugs and other treatments which affect the function of neurotransmitters can have a profound effect on pituitary hormone secretion. Several estab- lished and putative neurotransmitters are reported to be concentrated in different regions of the hypothalamus. 17 B. Catecholamines The catecholamines dopamine and norepinephrine are present in the hypothalamus at concentrations which are among the highest found in the brain (Palkovits gt_al,, 1974b). Vogt (1954) first demonstrated the presence of norepinephrine in the hypothalamus of dogs and cats. Following development of the Falck-Hillarp histofluorescence method (Flack gt 91,, 1962) for identifying brain catecholamines and serotonin, high concentrations of norepinephrine were demonstrated in the anterior hypothalamus and internal layer of the median eminence of rats (Carlsson gt_al,, 1962; Dahlstrom and Fuxe, 1964; Fuxe, 1965). Dopamine was found to be highly concentrated in the external layer of the median eminence, arcuate nucleus, dorsomedial nucleus and zona incerta of the hypothala- mus (Carlsson gt,al,, 1962; Fuxe, 1963, 1964, 1965; Fuxe and H6kfelt, 1966, 1969; Jonsson gt__l,, 1972; ijrkland gt_al,, 1973). Recent development of sensitive enzymatic isotopic assays for catecholamines (Cuello gt_al,, 1973; Coyle and Henry, 1973) and a method for removal of individual nuclei (Palkovits, 1973) has made possible the precise mapping of hypothalamic catecholamines. However, one drawback in these assays is that norepinephrine cannot be distinguished from epinephrine. This may be of potential importance since both epinephrine (Vogt, 1954) and the enzyme, phenylethanolamine-N-methyltransferase, which converts norepinephrine to epinephrine have been found in the hypothalamus (H6kfelt, 1974; Hdkfelt gt_al,, 1974). Using these tech- niques the highest concentration of brain dopamine was found in the median eminence (65 ng/mg protein). Dopamine was also concentrated in 18 the arcuate nucleus (28 ng/mg) and to a lesser extent (4-15 ng/mg) in several nuclei located in the anterior and intermediate hypothalamic areas. Norepinephrine-epinephrine content was highest in the retrochias- matic area of the anterior hypothalamus (48 ng/mg) and high in the dorso- medial nucleus (39 ng/mg), periventricular nucleus (34 ng/mg) and in the median eminence (30 ng/mg). Measurable amounts of dopamine and nor- epinephrine-epinephrine were present throughout the hypothalamus; however, nuclei in the posterior area contained the lowest concentra- tions (Palkovits £3 31., 1974b; Brownstein gt_al,, 1976). C. Serotonin Regional distribution of brain serotonin was first reported by Amin gt_gl, (1954) who found concentrations of this indoleamine to be highest in the hypothalamus and lowest in the cerebellum of the dog. Histofluorescence techniques revealed a dense population of serotonin nerve terminals located in the suprachiasmatic nuclei of the anterior hypothalamus whereas little serotonin fluorescence was observed in other portions of the rat hypothalamus (Fuxe, 1965; Loizou, 1972). Saavedra £3.21, (1974a) have reported the regional distribution of serotonin in individual hypothalamic nuclei as measured by an enzymatic-isotopic technique. Their report confirms the earlier observations of high serotonin concentrations in the suprachiasmatic nuclei (Fuxe, 1965; Loizou, 1972). In addition, many nuclei of the basal and posterior hypothalamus, including the arcuate nucleus and median eminence con- tained relatively large amounts of serotonin which had not been detected by histofluorescence (Fuxe, 1965; Loizou, 1972). 19 These findings indicate that dopamine, norepinephrine, serotonin and possibly epinephrine are concentrated in the hypothalamus; however, they are not evenly distributed throughout the hypothalamic nuclei. The median eminence is rich in dopamine and also contains significant amounts of norepinephrine-epinephrine and serotonin. Recently Brownstein gt_gl, (1974) reported that the hypothalamic concentration of histamine (a puta- tive neurotransmitter) was highest in the median eminence-arcuate nucleus region. Enzymes which synthesize epinephrine (Saavedra gt 21,, 1974b) acetylcholine (Brownstein gt_al,, 1975b) and gamma aminobutyric acid (Kuryama and Kimura, 1976) were also present in the hypothalamus. Little is known about the roles these putative brain neurotransmitters (epine- phrine, histamine, acetylcholine and gamma aminobutyric acid) play in neuroendocrine regulation. 0. Monoamine Pathways Innervating the Hypothalamus Norepinephrine containing cell bodies in the medulla oblongata and serotonin containing cell bodies in the midbrain raphae give rise to axons which ascend in the medial forebrain bundle and terminate in the hypothalamus (Fuxe gt 31,, 1968; Fuxe and kufelt, 1969; Ungerstedt, 1971; Fuxe and Jonsson, 1974). Brainstem cell bodies containing phenyl- ethanolamine-N-methyltransferase also appeared to project onto the hypothalamus (H6kfelt, 1974), indicating possible adrenergic innervation of this region by extrahypothalamic loci. By contrast extrahypothalamic dopamine systems (nigro-striatal and mesolimbic) do not innervate the hypothalamus (Ungerstedt, 1971). The tubero-infundibular dopamine 20 system has cell bodies located in the arcuate nucleus and terminals in the external layer of the median eminence (Carlsson 35 31,, 1962; Fuxe, 1965; Fuxe and H6kfe1t, 1966, 1969; ijrklund, 1973). Recently a new hypothalamic dopamine system, the incerta-hypothalamic, was observed to have cell bodies located in the posterior hypothalamus and medial zona incerta with diffuse terminal projections to anterior and dorsal hypo- thalamic areas (ijrklund gt 31,, 1975). Hypothalamic deafferentation with a Halasz knife resulted in a dramatic reduction of norepinephrine and serotonin but not dopamine con- centrations in the rat hypothalamus. Choline acetyl-transferase and phenylethanolamine-N-methyltransferase activities were also reduced following deafferentation (Brownstein et_al,, 1976). These findings sug- gest that only dopamine neurons have their cell bodies located within the hypothalamus; whereas, norepinephrine, epinephrine, serotonin, and acetylcholine are for the most part present in terminals of neurons having cell bodies located outside of the hypothalamus. E. Catecholamine and Serotonin Synthesis and Tfirnover Neurotransmitters are continuously being synthesized, released and metabolized and the rate at which these processes occur is termed turnover. The biosynthesis of catecholamines occurs by the following steps: tyrosine "L’- dopa "3-, dopamine «3-» norepinephrine "9-) epinephrine. The enzymes involved are l. tyrosine hydroxylase, 2. L-aromatic amino acid decarboxylase, 3. dopamine beta hydroxylase and 4. phenylethanolamine-N-methyltransferase (PNMT). Tyrosine hydroxy- lase is the rate-limiting enzyme for catecholamine synthesis and its 21 activity is regulated by feedback inhibition of dopamine and norepine- phrine (Levitt gt.al,, 1965, 1967). This enzyme which requires a re- duced pteridine cofactor and molecular O2 and Fe++ for activity (Nagatsu, 1964; Levitt, 1967), is stereOSpecific and under normal condi- tions is saturated by its substrate L-tyrosine (Nagatsu §t_al,, 1964; Cooper gt_al,, 1971). There is considerable evidence that treatments which increase the firing rate of catecholamine neurons also stimulate tyrosine hydroxylase activity acutely (Sedvall gt_al:, 1968; Dairman gt_al,, 1968) and elevate neuronal concentrations of this enzyme when given chronically (Jon et_al,, 1973). L-aromatic amino acid decarboxylase displays little substrate specificity and catalyzes decarboxylation steps in the synthesis of catecholamines, and serotonin (Carlsson gt_al,, 1972; Goldstein §t_a1:, 1974). This enzyme is widely distributed throughout the brain and other tissues and requires pyridoxal phosphate as a cofactor (see Nagatsu, 1973, Cooper et’al,, 1971). Dopamine is converted to norepinephrine by dopamine beta hydroxylase. This enzyme, a copper containing protein, is not highly substrate specific and can oxidize most phenylethylamines in the presence of ascorbic acid (Kaufman, 1966; Levitt 93.31,, 1969; Cooper $3.21,, 1971). Epinephrine is mainly present in the adrenal medulla and is synthesized from norepinephrine by PNMT. Although the brain contains measurable amounts of epinephrine (Vogt, 1954; Gunne, 1962) and PNMT (Hfikfelt gt.al,, 1974) the function of this monoamine as a central neurotransmitter remains to be demonstrated. 22 Serotonin synthesis occurs in a two step process beginning with the conversion of L-tryptophan to 5-hydroxytryptophan (5-HTP) by the enzyme tryptophan hydroxylase. Serotonin is formed from 5-HTP by L-aro- matic amino acid decarboxylase. Tryptophan hydroxylase is the rate limiting enzyme in the synthesis of serotonin and its activity is mainly dependent on the intraneuronal concentration of its substrate L-tryp- tophan (Lovenberg gt_al,, 1968; Fernstrom and Wurtman, 1971, 1972; Héry gt'al,, 1972) and on the firing rate of serotonin neurons (Aghajanian and Weiss, 1968; Weiss and Aghajanian, 1971). Brain concentrations of tryptophan were reported to be below the saturation point for tryptophan hydroxylase (Lovenberg gtnal., 1968; Lovenberg and Victor, 1974) and increasing brain tryptophan enhanced formation of central serotonin (Fernstrom and Wurtman, 1971, 1972; Graham-Smith, 1971). It is general- 1y accepted that tryptophan hydroxylase activity is not subject to feed- back inhibition by serotonin under normal conditions (Lin 95:21:» 1969; Héry §t_al,, 1972; Hillard gt_al,, 1972). This enzyme appears to be located only in serotonin neurons and requires molecular 02 and a re- ducing agent, probably tetrahydopteridine, for activity (Friedman gt_al,, 1972; Lovenberg and Victor, 1974). The physiological actions of central neurotransmitters appear to be terminated mainly by the active process of reuptake into the pre- synaptic terminal (Glowinski gt_al,, 1965; Glowinski and Axelrod, 1966; Wurtman, 1972; Wilson, 1974; Kuhar gt_al,, 1974). Catecholamines are to a minor extent metabolized by catechol-O-methyl transferase (COMT) in the synaptic cleft (Axelrod, 1959; Acton-lay and Wurtman, 1971). 23 Following reuptake, monoamines (catecholamines and serotonin) may be incorporated into synaptic vesicles or metabolized by monoamine oxidase (MAO). MAO is widely distributed throughout the brain and other tissues and is located on the outer surface of mitochondria (Schnaitman gt_al,, 1967; Wurtman, 1972). Administration of MAO inhibitors markedly ele- vated brain concentrations of serotonin and to a much lesser extent con- centrations of catecholamines (Lin gt_al,, 1969), supporting the concept that the synthesis of catecholamines but not serotonin is subject to feedback inhibition. There are many methods for estimating turnover rates of brain catecholamines (Anton-Tay and Wurtman, 1971; Wurtman, 1972) and sero- tonin (Horot-Gaudry $3.21,, 1974) and each has inherent virtues and draw- backs (Wurtman, 1972; Weiner, 1974). Moreover, estimating changes in brain neurotransmitter turnover by current methods can only be assumed to reflect changes in synaptic activity. Catecholamines and probably serotonin exist in two or more intraneuronal pools; release and storage pools, and each may differ in their respective rates of turnover. Release pools are of primary interest with respect to transsynaptic com- munication; however, turnover measurements represent a composite of amine activity within multicompartmentalized pools. Thus, turnover studies provide estimates for comparative overall turnover rates under different experimental conditions (Wurtman, 1972; Weiner, 1974). Three of the most widely used methods for estimating monoamine turnover rates are: 1) measuring the rate of decline of monoamine concentrations or histofluorescence following drug treatments which inhibit their synthesis, 24 2) measuring the rate at which tritium labeled tyrosine and tryptophan accumulate into catecholamines and serotonin, respectively, and 3) measuring the accumulation of serotonin following drug treatments (MAO inhibitors) which block its metabolism. The third method was used in some of the experiments presented in this thesis. III. Hypothalamic Control of Prolactin A. General Prolactin is best known for promoting mammary growth and lacta- tion (Lyons gt 21,, 1958; Meites, 1966). In addition, it may be important for development and growth of manmary tumors in humans (Jick gt_al,, 1974; Frantz gt,al,, 1972, 1973) and has been shown to stimUlate growth and development of mammary tumors in rats (Meites, 1972, 1973). There also is evidence that prolactin is involved in development (Negro- Vilar gt_al,, 1973) and function (Hafez gt_al,, 1972; Bartke ££“21:’ 1975) of the male reproductive system and may have a role in cancer of the prostate (Farnsworth, 1972). In rats prolactin maintains luteal function during pregnancy and pseudopregnancy (Neill, 1974), and during the estrous cycle causes luteolysis of old corpora lutea prior to ovula- tion (Wuttke and Meites, 1971). In addition prolactin was reported to have a role in the control of adrenal, kidney and liver functions (Nicoll and Bern, 1972). 25 B. Hypothalamic Hormones Prolactin Release-Inhibiting Factor Under physiological conditions the mammalian hypothalamus exerts a predominantly inhibitory influence over prolactin secretion and a predominantly stimulatory influence over other anterior pituitary hor- mones (Meites, 1973). Everett (1954, 1956) first proposed that prolactin release in the rat is inhibited by the hypothalamus although prior ob- servations had suggested this (Desclin, 1950). Following hypophysectomy and autotransplantation of the pituitary under the kidney capsule, Everett (1954, 1956) observed that these autografts maintained luteal function for several months as determined by ovarian histology and the formation of deciduomata induced by uterine traumatization. Similarly, pituitary stalk section was observed to induce luteal function and pseudopregnancy whereas other pituitary hormone-target tissues (ovarian follicles, adrenal cortex, thyroid) atrophied (Everett and Nikitovitch- Winer, 1963). Pituitary stalk section also was reported to enhance prolactin secretion in humans (Turkington, 1972a). Transplantation of a pituitary under the renal capsule resulted in prolonged elevation of blood prolactin concentrations in rats as measured by radioimmunoassay (Chen £3 21,, 1970; Lu and Meites, 1972). Electrolytic lesions of the median eminence or basal hypothala- mus stimulated prolactin secretion as measured by enhanced mammary development (Sud et_al,, 1970), onset of lactation (McCann and Friedman, 1960; Nikitovitch-Winer, 1960) and high concentrations of circulating prolactin (Chen et 21,, 1970, Welsch gt_al,, 1971). When placed in_vitro 26 rat anterior pituitaries spontaneously secreted large amounts of prolac- tin (Meites §t_al,, 1961; Talwalker gt_al,, 1962; Meites and Nicoll, 1966). All of these observations indicate that removing a pituitary from hypothalamic control results in a marked increase in prolactin secretion. Based on his early findings, Everett (1956) suggested that tonic inhibition of prolactin release may be mediated by a hypothalamic in- hibitory hormone. This hormone was later named prolactin release- inhibiting factor (PIF) (Talwalker gt a1,, 1961). Subsequent studies demonstrated that crude acid extracts of rat hypothalami inhibited the jn_vitro release of prolactin by rat pituitaries (Talwalker §t_al,, 1961, 1963; Pasteels, 1961). Similarly, extracts of sheep, bovine and porcine hypothalami were observed to inhibit prolactin release jn_yitrg (Schally gt_al,, 1965). Injections of hypothalamic extracts into rats were found to inhibit prolactin based on measurements of pituitary prolactin content (Grosvenor gt_al,, 1964, 1965) and reduced serum concentrations of pro- 1actin (Amenomori and Meites, 1970; Watson gt_al,, 1971; Kuhn gt_al,, 1974). Infusion of hypothalamic extract directly into hypophyseal portal vessels of rats inhibited prolactin release in a dose related fashion (Kamberi gt_al,, 1971c; Schally gt 31,, 1974; Takahara 25.51,, 1974a). There is widespread agreement on the existence of PIF; however, the structure of this substance has not been elucidated. Prolactin-Releasing Factor and Thyrotropin- ReTeasing Hormone Prolactin-releasing factor (PRF) activity was first reported in extracts of hypothalamus and cerebral cortex from both estrogen—primed 27 and lactating rats. Injection of these extracts, but not those of liver or kidney initiated lactation in estrogen-primed female rats (Meites et_al,, 1960; Mischkinsky gt_al,, 1968). However, these workers could not exclude the possibility that agents other than PRF may have been involved since initiation of lactation is not a specific measure of pro- lactin release (Meites gt_al,, 1963). Subsequently PRF activity was found in hypothalamic extracts of several avian species (Assenmacher and Texier-Vidal, 1964; Kragt and Meites, 1965; Meites and Nicoll, 1966; Chen gt_al,, 1968). Based on the observation that rat hypothalamic extracts initially inhibited and later stimulated jg_yjtrg_release of prolactin by rat pituitaries, Nicoll gt_al, (1970) suggested the presence of both PIF and PRF in hypothalamic extracts. Valverde gt_al, (1972) reported chroma- tographic separation and partial purification of separate PIF and PRF fractions from bovine hypothalamus. By a similar method Dular gt_al, (1974) demonstrated PRF activity in porcine hypothalamus. In both cases the PRF activity was reported to be free of TRH. Recently the in_yitrg. synthesis of PRF by rat hypothalamus was reported by Mitnick gt.al, (1973). The ability of TRH to stimulate prolactin as well as TSH release is well established; however, under most conditions, TSH and prolactin are not released together. Cold temperature, the classical stimulus for TSH secretion, inhibited prolactin release in rats (Mueller gt_al,. 1974) and bovine (Wettemann and Tucker, 1974; Tucker and Wettemann, 1976); whereas, warm temperature increased serum prolactin concentrations and 28 decreased serum TSH concentrations. Blake (1974) observed in rats that on the afternoon of proestrus when serum prolactin concentrations were dramatically elevated, serum TSH concentrations were unchanged as com- pared to values on the morning of proestrus. In humans, nursing stimu- lated prolactin but not TSH release (Gautvik gt_al,, 1973); whereas, in rats, suckling stimulated the release of both hormones (Blake, 1974). However, the rise in serum prolactin preceded that of TSH by 5 min indicating that separate stimulatory mechanisms were operating. Adminis- tration of physical stress or L-tryptophan elevated serum prolactin concentrations and inhibited TSH release in normal male rats (Mueller gt 51,, 1976a). Pituitary TSH secretion is inversely related to circu- lating concentrations of thyroid hormones (Reichlin gt_al,, 1972); whereas, serum prolactin concentrations were little affected by thyroid status in rats (Lu gt 31,, 1972) and humans (Jacobs st 31,, 1971; Bowers gt_al,, 1971; Refetoff gt_al,, 1974). High doses of estrogen were also reported to inhibit TSH release (D'Angelo, 1968) and to stimulate pro- lactin release (Chen and Meites, 1970) in rats. Taken together these observations that release of prolactin and TSH do not occur together strongly indicate that TRH is not involved in the physiological release of prolactin. Little is known about how the above conditions effect the synthesis and release of TRH. Reichlin gt.al, (1972) reported that jn_vivo cold exposure or administration of thyroxine enhanced the jn_vitro synthesis of TRH by rat hypothalami. Serum prolactin concentrations were decreased (Mueller $3.31,, 1974; Wettemann and Tucker, 1974; Tucker and Wettemann, 1976) or unchanged 29 (Lu gt_al,, 1972) by these treatments. Hypothyroidism reduced TRH syn- thesis (Reichlin gt_al:, 1972) but had no effect on serum prolactin levels (Lu gt.al,, 1972) in the rat. Under certain experimental condi- tions, e.g., following the administration of TRH, dopaminergic drugs (Mueller gt 21,, 1976b) or serotonergic drugs (Chen and Meites, 1975a) changes in prolactin and TSH release occurred in parallel. However, there are no physiological data which directly suggest that TRH is PRF. In pituitary incubations somatostatin was observed to inhibit the TRH induced release of TSH but not prolactin (Vale gt_al,, 1975) and dopamine blocked TRH induced prolactin but not TSH release (Dibbett et_al,, 1974; Takahara gt_al,, 1974c). These findings present the pos- sibility that TRH, somatostatin and dopamine may act together in regu- lating pituitary TSH, prolactin, and growth hormone secretion. At present, this relationship must be considered when evaluating hypo- thalamic control of these three hormones. C. Effects of Catecholamines on Prolactin Secretion General Central acting drugs have long been known to influence pituitary hormone secretion. Adrenergic and cholinergic drugs were reported to induce ovulation; whereas, respective blocking agents inhibited ovulation in rabbits (Sawyer eta]... 1947; Markee 331., 1948; Sawyer $139.1.” 1949). Similar observations also were made in the rat (Everett 33.91,, 1949; Markee £31., 1952). 30 Chlorpromazine, a catecholamine receptor blocker (Carlson and Lindquist, 1963; Janssen gt_al,, 1968) and reserpine, a drug which depletes neurotransmitters (Holzbauer and Vogt, 1956; Sheppard and Zimmerman, 1960) were reported to initiate lactation in humans (Sulman and Hinnik, 1956; Rabinowits and Freedman, 1961) and laboratory animals (Meites, 1957; Kanematsu gt_al,, 1962; Meites §t_al,, 1963) indicating a possible involvement of neurotransmitters in the control of prolactin. Development of sensitive and specific radioimmunoassays for prolactin has made possible the evaluation of pituitary secretion rate based on changes in serum prolactin concentrations. Catecholamines and PIF The role of catecholamines in the control of prolactin secretion has been the subject of extensive investigation and many reviews (Meites gt_al,, 1972; Meites and Clemens, 1972; McCann gt_al,, 1972; Meites, 1973; MacLeod, 1974; Neill, 1974). Dopamine and to a lesser extent norepine- phrine and epinephrine acted directly on the rat pituitary to inhibit prolactin release jg_yjtrg_(MacLeod, 1969; Koch §t_al,, 1970; MacLeod et_al,, 1970; Birge gt_al,, 1970; MacLeod and Leymeyer, 1974; Quijada et_al,, 1973/74; Shaar and Clemens, 1974). Contrariwise other early studies indicated that catecholamines either had no effect (Talwalker §t_al,, 1963) or (depending on dose) stimulated (Gala and Reece, 1965; Kock e311,, 1970) pituitary prolactin release 3133331. Recently Shaar and Clemens (1974) clearly demonstrated that both dopamine and norepine- phrine in concentrations at or below those found in rat hypothalamus significantly reduced the jn_vitro release of prolactin by rat 31 pituitaries. Dopamine was found to be much more effective than norepine- phrine. Apomorphine, a potent dopamine receptor stimulating drug (Andén et_gl,, 1967) also was observed to inhibit prolactin release ig_vitro (MacLeod and Leymeyer, 1974; Smalstig and Clemens, 1974; Smalstig gt a1., 1974) and this effect was antagonized by pimozide (Smalstig and Clemens, 1974; Smalstig gt_al,, 1974), a dopamine receptor blocking drug (Janssen gt_al,, 1968). Kamberi gt_al, (1971a) reported that saline solutions of dopamine, norepinephrine and epinephrine had no effect on serum prolactin concen- trations when infused directly into the anterior pituitary of male rats via a hypophyseal portal vessel. More recently both dopamine and nor- epinephrine dissolved in 5% glucose were observed to inhibit prolactin release in the same experimental model (Takahara gt_al,, 1974a; Schally gt 21,, 1974). This discrepancy has been explained on the basis of vehicles used, the latter workers finding that saline solutions of cate- cholamines quickly lost PIF activity as compared to 5% glucose solutions. Intravenous infusions (Shaar, 1975; Blake, 1976) but not single injec- tions (Lu gt_al,, 1970) of either dopamine, norepinephrine or epine- phrine reduced basal serum concentrations and blocked the proestrous surge of prolactin in female rats. Dopamine is more effective than either norepinephrine or epinephrine (Blake, 1976). Removal of catecholamines from rat hypothalamic extracts by aluminum oxide absorption or treatment with monoamine oxidase resulted in a complete loss of PIF activity jn_yltrg, indicating that endogenous catecholamines accounted for all the PIF activity in crude hypothalamic 32 extracts (Shaar and Clemens, 1974). The possibility that catecholamines, particularly dopamine, may be PIF is under investigation. However, initial attempts to measure naturally occurring dopamine in hypophyseal portal blood were unsuccessful (Eskay gt_al,, 1974; Ben-Jonathan gt_al,, 1975b). In 1975 Porter and co-workers reported the presence of norepine- phrine but not dopamine in hypophyseal portal blood of urethane anes- thetized proestrous female rats (Ben-Jonathan gt_al,, 1975a). Contrari- wise, the same group subsequently reported detectable amounts of dopamine but not norepinephrine in portal blood (Ben-Jonathan gt,al,, 1976). The difference between the two reports had not been explained and suggests that additional evidence is required before dopamine can be considered to be a natural and varying constituent of hypophyseal portal blood. Several laboratories have reported the isolation and partial purification of hypothalamic peptides with PIF activity (Takahara gt.§l,, 1974a; Schally 23.91,, 1974; Dular 25.31,, 1974; Greibrokk gt_al,, 1974). The possibility that PIF may be a catechol-peptide complex does exist, although failure of proteolytic enzymes to abolish the jn_yjtrg_PIF activity of rat hypothalamic extracts (Shaar and Clemens, 1974) argues against a peptide PIF. However, it should be noted that small peptides are not always cleaved by proteolytic enzymes (Lehninger, 1970). Quijada gt_al, (1973/74) reported that pharmacological blockade of pituitary dopamine receptors only partially counteracted the inhibitory influence of hypothalamic tissue on prolactin release in pituitary- hypothalamus co-incubations. These results suggest that action of one or more factors other than dopamine. Their experimental design did not, 33 however, preclude the possible involvement of norepinephrine. The abil- ity of hypothalamic extracts to inhibit prolactin release jn_vivo cannot be explained on the basis of catecholamines present in the extracts. Shaar and Clemens (1974) reported a single rat hypothalamus contains about 0.03 pg of dopamine and 0.05 pg norepinephrine. Extracts of a single rat hypothalamus significantly reduced serum prolactin concentra- tions in male rats by 8 min after injection (Watson gt_al,, 1971) and also prevented the suckling and stress-induced decrease in pituitary prolactin content (Grosvenor gt_al,, 1965). Amenomori and Meites (1970) reported that extracts of eight rat hypothalami (which presumably con- tained about 0.24 pg of dopamine and 0.40 pg of norepinephrine) depressed serum prolactin concentrations by l and 4 hours after injection into normal male and female rats. By contrast, Lu gt_al, (1970) found that injections of 5 pg and 10 pg of either dopamine or norepinephrine had no effect on serum prolactin levels in female rats by 30, 60 and 120 min af- ter injection. However, continuous infusions of very large amounts of dopamine (80 pg/hr) were reported to reduce basal prolactin concentra- tions by 1 hour and to prevent the spontaneous rise in serum prolactin on the afternoon of proestrus in female rats (Blake, 1976). These find- ings indicate that the relative 1n_vivo PIF activity of crude rat hypo- thalamic extract is greater than that of pure dopamine or norepinephrine. This relationship is in direct opposition to the 15.!itrg_findings of Shaar and Clemens (1974) showing that catecholamines accounted for all of the PIF activity of rat hypothalamic extract. The difference between the jn_vivo and jn_vitro PIF activities of hypothalamic extracts 34 suggests that catecholamines are not PIF; however, the biochemical nature of PIF may be closely related to that of catecholamines. The chemical structure of PIF(s) remain to be determined although present evidence indicates that dopamine and dopamine-like compounds must be carefully considered. Drugs which specifically stimulated dopamine activity inhibit prolactin; whereas, dopamine antagonists enhance prolactin release in rats and humans (see Meites, 1973). Both apomorphine and piribedil (ET 495) which act to stimulate dopamine receptors (Andén gt.al,, 1967; Corrodi et_al,, 1971a) decreased serum prolactin concentrations in rats (Smalstig gt.al,, 1974; Ojeda $3.21,, 1974b; Lawson and Gala, 1975; Mueller gt_al,, 1976b). Apomorphine also was reported to inhibit pro- lactin release in humans (Martin gt_gl,, 1974). Conversely, pimozide, haloperidol and Chlorpromazine which act to block dopamine receptors (Janssen §t_al,, 1968) dramatically stimulated prolactin release in rats (Meites and Clemens, 1972; Clemens gt_al,, 1974; Dickerman gt_al,, 1974; Ojeda and McCann, 1974; Ojeda gt,al,, 1974a; Lawson and Gala, 1975). Haloperidol and chlorpromazine also stimulated prolactin release in humans (Apostokalis gt_al,, 1972; Turkington, l972a,b). Haloperidol was reported to decrease hypothalamic PIF activity in rats (Dickerman gt_al,, 1974) and ewes (Bass gt_al,, 1974). Pretreatment with halo- peridol (Mueller 93.21,, 1976b) or Chlorpromazine (Smalstig gt_al,, 1974) blocked the inhibitory effect of apomorphine on prolactin release in rats and similarly, haloperidol counteracted the inhibitory effect of piribedil (Mueller 23.31,, 1976b). The interaction between these 35 dopaminergic drugs on prolactin release indicates that dopamine receptors probably have a role in the physiological control of prolactin secretion. Like dopamine (Shaar, 1975; Blake, 1976), apomorphine was reported to block the suckling-induced and proestrous rise in serum pro- lactin concentrations in rats (Smalstig §t_al,, 1974). Injection of either dopamine (Kamberi gt 31,, 1971a) or apomorphine (Ojeda gt_al:, 1974b) into the third ventricle of the brain depressed serum concentra- tions of prolactin and similar injections of dopamine were reported to elevate PIF activity in hypophyseal portal blood (Kamberi et_al,, 1970a, 1971b). Dopamine (Ben-Jonathan et_al,, 1975a) and other compounds (Ondo gt 31., 1972; Oliver et_§l:, 1975) were observed to pass from the third ventricle into the hypophyseal portal blood, suggesting that the inhibi- tory effects of centrally administered dopamine and apomorphine on pro- lactin release may be due in part to a direct action on the pituitary. The observation that median eminence implants of pimozide were much more effective in elevating serum prolactin concentrations as compared with pituitary implants (Ojeda gt_gl,, 1974a) indicates that central dopamine receptors are the principle site at which dopaminergic drugs act to influence prolactin release. Administration of L-dihydroxyphenylalanine (L-Dopa) dramatically elevated brain dopamine concentrations in rats (Everett and Borcherding, 1970; Hyyppfi §t_al,, 1971) and inhibited prolactin release in rats (Lu and Meites, 1971; Donoso et_al,, 1971; Smythe and Lazarus, 1974a; Chen gt_ 1., 1974; Chen and Meites, 1975a) and humans (Malarkey et_al,, 1971; Friesen gt_al,, 1972; Frantz gt_al., 1972, 1973). L-Dopa was reported 36 to block the suckling-induced release of prolactin (Chen gt__l,, 1974) and to depress elevated serum prolactin concentrations produced by estro- gen (Chen and Meites, 1975a) median eminence lesions (Donoso gt_al,, 1974; Shaar, 1975), pituitary transplants (Lu and Meites, 1972) and transplants of pituitary tumor tissue (Malarky and Daughaday, 1972) in rats. A single intraperitoneal injection of L-Dopa increased PIF activ- ity in hypothalami and serum of intact and hypophysectomized rats (Lu and Meites, 1972). L-Dopa was reported to inhibit mammary tumor growth in rats (Quadri §t_al,, 1973) and humans (Frantz et_al,, 1973), presumably by decreasing blood prolactin concentrations. Ergot alkaloids and their derivatives are well-known for their ability to inhibit the jg_vivo (Lu gt 31,, 1971; Malven and Hoge, 1971; Shaar and Clemens, 1972; Smith gt_al,, 1974) in jg_yitrg_(Lu gt al,, 1971; MacLeod and Leymeyer, 1974; Clemens gt 21,, 1975) release of pro- lactin, and these agents have been successfully used to reduce mammary cancer growth in rats (Cassell et_al,, 1971; Quadri gt_al,, 1971). Recently ergocornine, ergocryptine and 2 bromo-alpha ergocryptine were shown to stimulate central dopamine receptors (Corrodi gt_al,, 1973; Stone, 1974; Fuxe gt_al,, 1974a), and ergot drugs were reported to ele- vate hypothalamic PIF activity in rats (Wuttke gt_al,, 1971). In vitro inhibition of prolactin release by lergotrile mesylate, an ergot deriva- tive, was found to be antagonized by pimozide but not by adrenergic blocking drugs, indicating that pituitary as well as central dopamine receptors may mediate the inhibitory effects of ergot drugs on prolactin release (Clemens gt_al,, 1975; Shaar, 1975). 37 Together, the above reports demonstrate that under experimental conditions dopamine inhibits prolactin secretion by a central mechanism involving PIF release and by a direct action on the pituitary. Most of the recent evidence indicates, but does not prove, that dopamine is the PIF. The natural occurrence of dapamine in hypophyseal portal blood has not been clearly demonstrated and hypothalamic peptides, presumably free of catecholamines, were reported to have PIF activity. There is no agreement on the role of norepinephrine in the con- trol of prolactin. As mentioned earlier, norepinephrine acted directly on the pituitary to inhibit the jg.yjyg_(lakahara §t_al,, 1974a; Schally g_t__a_l_., 1974; Blake, 1976) and jam (MacLeod, 1969; Birge e_t_§_1_., 1970; MacLeod and Leymeyer, 1974; Shaar and Clemens, 1974) release of prolactin in rats. Kamberi gt_alL_(l97la) reported that only very high doses of norepinephrine (100 pg) significantly lowered serum prolactin concentrations when injected into the third ventricle of the rat brain. Similarly Ojeda gt_al: (1974b) found that intraventricular injection of 2 pg norepinephrine had no effect on prolactin release. Quijada gt_al, (1973/74) concluded that norepinephrine was without effect on the in. yitrg_release of PIF by rat hypothalami based on results obtained from pituitary-hypothalamus co-incubations. Relatively high doses of clonidine (O.2-5.0 mg/kg, I.P.), an alpha adrenergic stimulating drug (Andén‘gt_al,, 1970; Haeusler. 1974) were reported to elevate prolactin levels in ovariectomized estrogen- primed rats (Lawson and Gala, 1975). By contrast we observed that lower doses of clonidine (0.3-0.30 mg/kg, I.P.) inhibited prolactin release in normal male rats; whereas, higher doses (1—3 mg/kg) tended to elevate serum prolactin and appeared to cause physical stress (Mueller, Simpkins, Meites, Moore, unpublished). Stresses were reported to stimulate pro- lactin release in rats (Krulich gt_al,, 1974) suggesting that high doses of clonidine may induce prolactin release by a nonspecific stress effect. L-Dihydroxyphenyl serine (L-DOPS) which increases brain nor- epinephrine but not dopamine content (Fuxe and H6kfelt, 1969) was shown to stimulate prolactin release in rats (Donoso gt_al,, 1971). Conversely, blockade of norepinephrine synthesis by disulfiram which acts to inhibit dopamine beta hydroxylase (Turner 95.21,, 1974), was reported to depress serum concentrations of prolactin in rats (Meites and Clemens, 1972). These findings suggest that norepinephrine may stimulate prolactin release. 0. Serotonin and Prolactin Release There is general agreement that serotonin stimulates prolactin release. Kamberi £3.21, (l971e) reported that injection of serotonin into the third ventricle of male rats evoked a prompt rise in serum pro- 1actin concentrations. Systemic administration of serotonin precursors, tryptophan or 5-hydroxytryptophan, stimulated prolactin release in rats (Lu st 31,, 1972; Chen and Meites, 1975; Mueller gt_al,, 1976a) and humans (MacIndoe and Turkington, 1973; Kato §t_al,, 1974). Para-chloro- phenylalanine, a drug which inhibits serotonin synthesis by blocking tryptophan hydroxylase (Miller st 91,, 1970), lowered serum prolactin concentrations in estrogen-primed rats (Chen and Meites, 1975) and blocked the suckling-induced release of prolactin (Kordon 35.91:, 1974). 39 Caligaris and Taleisnik (1974) reported that para-chloroamphetamine, another tryptophan hydroxylase inhibitor (Miller gt_al:, 1970), blocked the estrogen-induced release of prolactin in rats. Methysergide, an ergot derivative which is reported to block serotonin receptors (Douglas, 1971), inhibited the stress--(Koj and Krulich, 1975) and estrogen (Caligaris and Taleisnik, l974)--induced release of prolactin in rats. Blockade of serotonin re-uptake by Lilly 110140 (3-[p-triflouromethyl- phenony-3-pheny1-N-methyl-propy1amine hydrochloride), which presumably prolongs the action of serotonin on its post synaptic receptor (Wong gt_al,, 1974), potentiated the stimulatory effects of 5-hydroxytrypto- phan and restraint stress on prolactin release in male rats (Krulich, 1975). Serotonin was reported to have no effect on the jn_yitrg_release of prolactin (Smalstig and Clemens, 1974) and did not alter serum pro- lactin levels in rats following infusion into the pituitary gland by way of a cannulated portal vessel (Kamberi gt_al,, 1971e). These find- ings indicate that serotonin has no direct influence on pituitary pro- lactin secretion and that the actions of serotonergic drugs on prolactin release are mediated by a hypothalamic PIF and/or PRF mechanism. Systemic administration of serotonin was reported to either have no effect (Lu gt_al:, 1970) or to stimulate prolactin release in rats (Lawson and Gala, 1975). Entry of serotonin into the brain is blocked by the blood-brain barrier (Douglas, 1970) indicating that the stimula- tion of prolactin release produced by intravenous administration of serotonin (Lawson and Gala, 1975) may be due to a nonspecific stress effect of the drug. 40 E. Putative Brain Neurotransmitters and Prolactin‘Rélease The influence of acetylcholine on prolactin release is unclear. Grandison g§_213 (1974) reported that lateral ventricle injections of acetylcholine or systemic injections of pilocarpine and physostigmine reduced serum prolactin concentrations in rats. Pilocarpine stimulates cholinergic receptors and physostigmine prevents acetylcholine metabol- ism by blocking choline esterase (Koelle, 1970). Similarly Kuhn and Lens (1974) and Libertun and McCann (1974a) reported that cholinergic drugs acutely inhibited prolactin release in rats. Recently Grandison £3.31, (unpublished) found that cholinergic agonists blocked the stress- induced release of prolactin and that pre-treatment with atropine had no effect on basal serum prolactin concentrations but blocked the pilocar- pine induced-inhibition of prolactin release in male rats. Furthermore, pimozide also was observed to prevent the cholinergic-induced suppres- sion of prolactin release indicating that acetylcholine may act to inhibit prolactin by stimulating a dopamine-PIF system. On the other hand, Libertun and McCann (1973) reported that sub- cutaneous or third ventricle injections of large doses of atropine (1/4 to 1/2 L050) blocked the proestrous rise in serum prolactin and gonadotropin concentrations in rats. Atropine was also reported to pre- vent the nocturnal rise in serum prolactin in pseudopregnant rats and‘ this effect was reversed by physostigmine (McLean and Nikitovitch-Winer, 1975). Lawson and Gala (1975) found that both cholinergic and anti- cholinergic drugs generally had no effect on serum prolactin concentra- tions in estrogen-primed rats. 41 These apparentJy contradictory findings indicate that acetyl- choline inhibits, stimulates or has no effect on prolactin release, and these differences may be due in part to drug doses and experimental animals used and possibly because of a dual function of cholinergic neurons on both PIF and PRF release mechanisms. It is too early to assign a specific role to cholinergic receptors in the physiological con- trol of prolactin secretion. Gama-aminobutyric acid (Mioduszewski, 1976; 0ndo, in press), melatonin (Kamberi ethal,, l97le), cyclic adenosine monophosphate (Ojeda gt_gl,, 1974), and histamine (Libertum and McCann, 1974b; Donoso gt_a1,, 1976) all were reported to stimulate prolactin release in rats when administered directly into the brain. Methyl-histidine which blocks the formation of histamine, lowered serum prolactin concentrations and di- phenhydramine, an antihistiminic drug, blocked the stress-induced release of prolactin (McCann and Mass, 1975). Some prostaglandins also were reported to stimulate prolactin release in rats (Harms 9_t_ _a_1_., 1973; Odeda‘gthal,, 1974c) and to block the inhibitory effects of third ventricle injections of dopamine and apomorphine (Ojeda 33.91,, 1974c). The physiological significance of these observations remain to be determined. F. %gh%blt%ry§geedback of Prolactin on Pituitary, ro act n cretion easel. Physiological control of prolactin secretion may involve a hypo- thalamic autoregulatory mechanism (Sgouris and Meites, 1953; Meites and 42 Clemens, 1972). Under normal conditions prolactin target tissues (mam- mary gland, corpora lutea, etc.) do not appear to exert hormonal feedback control on the secretion of prolactin (Meites and Clemens, 1972). High serum prolactin concentrations produced by transplants of mammotrophic pituitary tumor tissue were associated with decreased prolactin content in the in.§jtg_pituitary (MacLeod gt.al,, 1966; Chen gt_al,, 1967) and elevated PIF activity in the hypothalamus of rats (Chen ££“21:: 1967). Daily injections of purified prolactin or pituitary transplants under the kidney capsule also were reported to reduce the prolactin content of the 1g_§13g_pituitary (Sinha and Tucker, 1968). Hypothalamic grafts of pituitary tissue inhibited prolactin secretion as determined by mammary gland regression in estrogen-primed rats (Averill, 1969). Prolactin implants into the median eminence of rats were found to depress the con- centration of prolactin in the pituitary and blood (Clemens and Meites, 196B; Mishkinsky gt_al,, 1969; Voogt g§_al,, 1971, 1973), increase hypo- thalamic PIF activity (Clemens and Meites, 196B), shorten the duration of pregnancy and pseudopregnancy (Clemens and Meites, 1969a; Clemens gt_al,, 1968), and inhibit lactation (Clemens et_al,. 1969b). Voogt and Meites (1973) reported that median eminence implants of prolactin re- duced basal serum levels and blocked the proestrous and suckling induced release of prolactin in rats. Recently, Advis, Hodson and Meites (un- published) found that pre-treatment with ovine prolactin inhibited the stress-induced release of prolactin in male rats. By contrast, prolactin did not act directly on the pituitary to alter the jn_yltgg_release of prolactin in rat pituitaries (Nicoll, 1971; Voogt and Ganong, 1974) indicating a brain site of action. 43 Tubero-Infundibular Dopamine Ne_urons and’PIF The inhibitory effect of prolactin on its own secretion appears to be mediated by a hypothalamic PIF system. Fuxe and co-workers (Fuxe and H6kfelt, 1969; Ahrén gthgl,, 1971; H6kfelt and Fuxe, l972a,b) first proposed that the tubero-infundibular dopamine neurons operate as a com- ponent in this mechanism. These workers observed that the rate of median eminence dopamine turnover as measured by histofluorescence techniques was directly related to circulating concentrations of prolac- tin. Under conditions when serum prolactin or lactogenic activity was known to be elevated (e.g., during pregnancy, lactation or after pitui- tary transplantation or the administration of exogenous prolactin) median eminence dopamine turnover was enhanced whereas in states associ- ated with low serum prolactin concentrations (e.g., after hypophysectomy or treatment with ergot drugs), dopamine turnover was reduced (Ahren £31., 1971; Hdkfelt and Fuxe, l972a,b; Fuxe 31.9.1." 1974b). During the rat estrous cycle, median eminence dopamine activity was reduced on proestrous afternoon at the time when surges of prolactin, LH and FSH normally occur. Following this period, dopamine turnover was enhanced and this may explain the low concentrations of serum prolactin seen dur- ing diestrus (Ahrén‘gt,al,, 1971; Hdkfelt and Fuxe, 1972a,b; Fuxe gt_al,, 1974b). Administration of LH, FSH, TSH, ACTH or vasopressin had no effect on the activity of the tubero-infundibular neurons. Furthermore, prolactin stimulated dopamine turnover only in median eminence neurons; whereas, other dopaminergic systems (nigro-striatal and mesolimbic) were not affected (Hfikfelt and Fuxe, l972a, 1972b; Gudelsktrgtwal:, in 44 preparation). Thus, only the activity of tubero-infundibular dopamine neurons are selectively sensitive to the influence of prolactin. Dopamine injections into the third ventricle of rats were reported to elevate PIF activity in hypophyseal portal blood (Kamberi §t_al,, 1970a, 1971b) and to inhibit prolactin release (Kamberi §t_al,, 1971a; Ojeda et_al,, 1974b). 0n the basis of these observations it appears that prolactin acts on the hypothalamus to stimulate median eminence dopamine activity, resulting in increased PIF release and de- creased prolactin release. In support of this hypothesis, Clemens and Sawyer (1974) reported the natural occurrence of prolactin in the cerebral spinal fluid of rats and Gelato and Wuttke (unpublished) have observed the presence of specific prolactin binding activity in hypothalamic membrane preparations. Prolactin also was found to influence the firing rate of hypothalamic neurons in the rabbit (Clemens ££“21:» 1971) and rat (Yamada, 1975). On the other hand, a prolactin-dopamine-PIF short loop feedback model does not explain the control of prolactin secretion under all con- ditions. Dramatic increases in serum prolactin concentrations regularly occurred twice daily during early pregnancy, and suckling induced a prompt release of prolactin in lactating rats (see Neill, 1974). In each state (pregnancy and lactation) median eminence dopamine turnover and presumably PIF release was enhanced (H6kfelt and Fuxe, l972a, 1972b), indicating that a separate stimulatory mechanism may be operating to release prolactin under these conditions. The physiological significance of prolactin regulating its own secretion remains to be determined 45 although present experimental evidence indicates that this phenomenon may be involved in the normal control of prolactin secretion. IV. Current Views of the Hypothalamic Control of Thyroid StimulatingtggzmgngiéTSflgimgggwthngormone and 9- A. I§H_ General Pituitary TSH secretion is primarily controlled by two opposing mechanisms: stimulation by TRH and inhibition by thyroid hormones. This relationship represents a classical neuroendocrine control system containing both neurogenic and target gland hormone-feedback regulation. The pituitary is the principal site for feedback inhibition by thyroid hormones; whereas, TRH secretion is under neural control by the brain (Reineke and Soliman, 1953; D'Angelo, 1963; Reichlin, 1966; Reichlin et.al,, 1972; Florsheim, 1974). Circulating concentrations of TSH con- trol the synthesis and release of thyroid hormones which, in turn, have a profbund influence on many metabolic systems. Thyroid hormones func- tion in the control of oxygen consumption and heat production, growth and development, nerve function, the metabolism of lipids, carbohydrates. proteins, nucleic acids, vitamins, and inorganic ions, as well as the metabolism and effects of other hormones (Hoch, 1974). Effects of Neurotransmitters on TRH-TSH Reléase Relatively little is known about how specific neurotransmitters influence the release of TRH-TSH. Hypothalamic deafferentation (Hefco 45 gt 91,. l975a,b) and reserpine treatment (Reichlin gt_al,, 1972; Tuomisto gt;al,, 1973; Chen and Meites, 1975) were reported to induce basal serum concentrations and to block the cold-induced release of TSH in rats. In_gjyg_administration of reserpine depressed the jn_yitrg_ synthesis of TRH by isolated rat hypothalamic fragments (Reichlin gt_al,, 1972). Both norepinephrine and dopamine stimulated the jg_!1trg.release of pulse labeled TRH by mouse hypothalamus. However, dopamine was not effective when its conversion to norepinephrine was blocked pharmaco- logically (Grimm and Reichlin, 1973) indicating that norepinephrine but not dopamine acts directly on the hypothalamus to stimulate TRH release. In agreement with these in_gitgg_results, disulfiram and phentolamine which act to block norepinephrine synthesis and receptors, respectively (Turner gt.al,, 1974; Nickerson, 1970), were reported to inhibit TSH release in rats (Tuomisto gt_al,, 1973). Administration of L-dopa consistently depressed elevated serum TSH levels in patients with long standing primary hypothyroidism (Rapoport _e_t__a_l_., 1973; Refetoff 391., 1974; Minozzi _e_ta_]_., 1975) but had little effect on basal serum TSH concentrations in euthyroid humans (Eddy g_t_a_l_., 1971; Simonin _e_i_:.3_1_., 1972; Minozzi _e_t_gl., 1975) or rats (Chen and Meites, 1975). Recently we observed that apomorphine and piribedil (dopamine receptor stimulators) inhibited TSH release; whereas, blockade of dopamine receptors by pimozide markedly elevated'TSH release. Further, pre-treatment with haloperidol blocked the inhibitory effect of apomorphine on TSH secretion in male rats (Mueller gt_al,, 1976b, Thesis). These findings indicate that dopamine receptors have an inhibitory influence on TRH-TSH release. 47 Serotonin appears to inhibit TSH release. Grimm and Reichlin (1973) reported that serotonin inhibited the jn_yjtrg_release of pulse labeled TRH by mouse hypothalamus. Administration of either tryptophan, 5-hydroxytryptophan or restraint stress to male rats elevated hypothala- mic serotonin and reduced serum TSH concentrations in dose-related manners (Mueller gt_al,, 1976a). Similarly, hypothalamic implants of serotonin depressed pituitary-thyroid activity and reduced the content of TRH in the hypothalamus of rats (Mess and Peter, 1975). The activity of brain serotonin neurons was directly related to environmental tempera- ture (Corrodi $.21” 1967; Aghajanian and Weiss, 1968; Reid _e_t_al_., 1968; Weiss and Aghajanian, 1971; Squires, 1974); whereas, TSH secretion was inversely related to temperature (see Reichlin, 1966; Reichlin et.al:, 1972; Florsheim, 1974). Associated with reduced brain serotonin turn- over, cold exposure was reported to enhance hypothalamic TRH synthesis (Reichlin gt_aly, 1972; Reichlin and Mitnick, 1973; Hefco ££;21:9 1975c) and release (Montoya gt;al,, 1975) in the rat. Furthermore, the stimu- latory effects of thyroid hormones on brain serotonin turnover (Engstrom 3391., 1974, 1975; Rastogi e_t_a_1_., 1974; Jacoby 3591., 1975) is just opposite to their inhibitory effects on TSH secretion. This relation- ship suggests a possible brain mechanism for feedback inhibition of thyroid hormones on TSH release. Together these reports indicate that increased activity of serotonin neurons reduces pituitary TSH release by inhibiting hypothalamic TRH secretion. In apparent disagreement with these observations, Chen and Meites (l975a) reported that high doses of S-hydroxytryptophan (30 pg/rat) stimulated; whereas, blockade of serotonin synthesis with para chloro- amphetamine (3 mg/rat, given 16 hr. prior to blood collection), inhibited TSH release in ovariectomized estrogen-primed rats. Similarly, Shopsin gtgal, (1974) found that inhibition of serotonin synthesis by para chloro- phenyalanine (150 mg/kg daily for 5 days) depressed serum TSH and prolac- tin concentrations in male rats. These differences may be due to an effect of estrogen on the action of the serotonergic drugs used by Chen and Meites (1975a) and to the relatively high drug doses used in both studies. High doses of 5-hydroxytrptophan were fbund to reduce brain catecholamine concentrations and to induce the formation of serotonin in neurons which normally do not contain this transmitter (Butcher gt_gl,, 1972). The acute actions of para chloramphetamine and para chlorophenyl- alanine are not selective to inhibition of tryptophan hydroxylase in serotonin neurons. Both drugs were reported to alter the metabolism of catecholamines for the first 24 to 48 hours after their administration (Miller g_t_a_l_., 1970; Strada £11., 1970; Sanders-Bush 51391., 1974). In both studies (Chen and Meites, l975a; Shopsin et_al,, 1974), the tryptophan hydroxylase inhibitors were administered less than 24 hours before blood collection. ' On the whole, the present data suggest that control of TRH-TSH release is under the stimulatory influence of norepinephrine and the inhibitory influence of dopamine and serotonin. Virtually nothing is known about the influence of other neurotransmitters (e.g., acetyl- choline, epinephrine, histamine, gamma aminobutyric acid) on TRH-TSH release. 49 General Pituitary gonadotropin secretion is stimulated by the hypothal- amic hormone LRH and is subject to the feedback influence of gonadal steroids. Thus, control of LH and FSH secretion involves both neurogenic and hormonal-feedback mechanisms. In the adult female, cyclic release of gonadotropins regulates estrous and menstrual cycles. There is gener- al agreement that FSH stimulates the initial growth of the ovarian fellicle beyond the stage of early antrum formation and then facilitates the action of LH to promote follicular maturation and estrogen produc- tion. The ovulatory surge of LH induces ovulation and formation of the corpus luteum. In the male, LH stimulates androgen secretion and FSH is required for spermatogenesis (see McCann, 1974). Central acting drugs have long been known to effect gonadotropin secretion (Sawyer et._1,, 1947, 1949; Everett gt_al,, 1949); however, the influence of neurotransmitters on LRH-gonadotropin release remains unclear and highly controversial. Effects of Catecholamines on the Release of’GBnadotropins Dopamine injections into the third ventricle of rats were re- ported to elevate LRH activity in hypophyseal portal blood (Kamberi gt_al,, 1969, 1971b) and stimulate the release of LH and FSH (Kamberi gt.al,, 1970b, 1971d; Schneider and McCann, 1970a,b). Injections of norepinephrine and epinephrine also stimulated the release of gonado- tropins but to a much lesser extent as compared with dopamine (Kamberi ‘gt_al,, 1970b, l97ld). In hypophysectomized female rats, 50 intraventricular injections of dopamine elevated LRH activity in the general circulation and this effect was blocked by the prior administra- tion of estradiol (Schneider and McCann, 1970b). Ojeda and co-workers (Ojeda and McCann, 1973; Ojeda gt_a1,, 1974a) observed that pimozide (a dopamine receptor blocker) tended to reduce serum gonadotropin con— centrations in rats. Catecholamines had no direct effect on pituitary gonadotropin secretion in_yjtrg; however, dopamine stimulated the release of LRH by rat hypothalami as determined in pituitary-hypothala- mus co-incubations (Schneider and McCann, 1969; Kamberi gt_al,, 1970c). Subsequently, Schneider and McCann (1970c) showed that estradiol also counteracted the i vitro stimulatory effect of dopamine on LRH release. Although these earlier reports indicated that dopamine stimulates LRH- gonadotropin release, more recent work suggests that the action of nor- epinephrine may be more important in this respect. Recently Quijada gt_al, (1973/74) found that dopamine had no effect and Miyachi gt_al, (1973) reported that dopamine inhibited the jn_!itrg_release of LRH by rat hypothalamus. Similarly, Cramer and Porter (1973) reported that they were unable to consistently stimulate LRH-gonadotrOpin release with intraventricular injections of dopamine. The apparent disagreement between these reports and earlier work showing that dopamine stimulated LRH-LH release (Kamberi gt_a1,, 1969, 1970b, 1970c, 1971b, 1971d; Schneider and McCann, 1969, 1970a,b,c) has not been adequately explained. Drugs which stimulate dopamine receptors (apomorphine and piri- bedil) did not evoke LH release in normal male rats (Mueller et_al,, 51 1976b). Rubinstein and Sawyer (1970) found that norepinephrine but not dopamine induced ovulation in proestrous rats anesthetized with pento- barbital. Recently Sawyer's group (Sawyer gt_al,, 1974; Sawyer, 1975) reported that intraventricular injections of norepinephrine but not dopamine stimulated LH release in the rabbit, and that dopamine given in combination with norepinephrine completely inhibited stimulation of LH release by norepinephrine. Administration of dopamine beta hydroxylase inhibitors to block norepinephrine synthesis, reduced basal serum LH concentrations and blocked the proestrous, post-castration and steroid- induced rises in gonadotropins. Reinitiation of norepinephrine synthesis by dihydroxyphenyl serine generally restored LH release under these con- ditions (Donoso §t_al,, 1971; Kalra §t_a1,, 1972; Kalra and McCann, 1973, 1974; Ojeda and McCann, 1973; Terasawa gt_al,, 1975). Interest- ingly, alpha receptor blockers (phentolamine and phenoxybenzamine) were reported to inhibit the in_yitrg_($chneider and McCann, 1969; Kamberi Sfliéfl:: 1970c) and in_yiyg_(5chneider and McCann, 1970a) stimulation of LRH release by dopamine, suggesting that dopamine may be acting through a noradrenergic mechanism. Phentolamine, an alpha receptor blocker, was reported to inhibit the post-castration rise in gonadotropins in rats (Ojeda and McCann, 1973) and to block the pulsatile release of LH which occurs in the ovariectomized monkey (Bhattacharya gt_gl,, 1972). Alpha- methyl-para-tyrosine, which inhibits the synthesis of catecholamines also was reported to block the post-castration rise in gonadotropins. This effect was partially counteracted by reinitiating norepinephrine synthesis with dihydroxyphenyl serine (Kalra gt_al,, 1972; Kalra and McCann, 1973, 1974). 52 Fuxe and co-workers (Fuxe and H6kfelt, 1969; Ahrén et_al:, 1971; H6kfe1t and Fuxe, l972a, l972b) have proposed that the tubero-infundibu- 1ar dopamine neurons inhibit LRH release. These neurons showed reduced dopamine turnover on the afternoon of proestrus when the ovulatory surge of gonadotropins was known to occur. By contrast, estrogen treatment was associated with enhanced median eminence dopamine activity and re- duced gonadotropin secretion (see H6kfelt and Fuxe, 1972b). Donoso and Moyano (1970) observed that in contrast to median eminence dopamine, content and turnover of hypothalamic norepinephrine was elevated on the afternoon of proestrus. Similarly Zachaeck and Wurtman (1973) reported that the rate whole brain catecholamine synthesis was markedly enhanced on the afternoon of proestrus as compared with the synthesis rates ob- served on estrus or diestrus. Thus, present data indicates that the ovulatory release of gonadotropins is associated with enhanced synthesis of hypothalamic norepinephrine and reduced turnover of median eminence dopamine, indicating that norepinephrine may stimulate LRH release under physiological conditions. Effects of Serotonin and Other Agents on the—Release ofTGonadotropins There is general agreement that serotonin and melatonin inhibit the release of gonadotropins. Both agents were found to reduce serum gonadotropin concentrations after injection into the third ventricle of rats (Kamberi gt_a1,, 1970b, l97le; Schneider and McCann, 1970a). By contrast, serotonin and melatonin had no effect on pituitary gonadotropin release when infused directly into a hypophyseal portal vessel indicating a hypothalamic site of action (Kamberi gt 21,, 1970b, 1971c). 53 Hypothalamic implants of serotonin (Wilson, 1974) or electrical stimula- tion of the midbrain raphae (serotonin nuclei) (Carrer and Taleisnik, 1970, 1972) both were reported to inhibit ovulation in rats. Systemic injections of 5-hydroxytrypt0phan inhibited; whereas, blockade of sero- tonin synthesis by para chlorOphenylalanine facilitated ovulation in rats (Kordon and Glowinski, 1972). However, para chlorophenylalanine had no effect on serum LH and FSH concentrations in normal or castrated male rats, indicating that serotonin probably does not exert a tonic inhibitory influence on gonadotrOpin secretion in the male rat (Donoso gt_al,, 1971). Acetylcholine, gamma aminobutyric acid, histamine and some prostaglandins may stimulate the release of LRH. Subcutaneous and intra- ventricular injections of atropine were reported to inhibit the release of gonadotropins in rats (Markee et 21., 1952; Libertun et_al,, 1974a) and acetylcholine stimulated the release of LRH when added to hypothala- mus-pituitary co-incubations (Simonovic et_al,, 1973; Kamberi and Bacleon, 1973; Fiorindo and Martini, 1975). Consistent with these find- ings, Kamberi and Bacleon (1973) reported that atropine blocked the proestrous surge of gonadotropins and inhibited ovulation in rats. Ovulation was restored by the administration of either LH or crude hypo- thalamic extract indicating that atropine was acting through a brain mechanism. Intraventricular injections of carbachol (McCann and Moss, 1975), a colinergic receptor agonist (Koelle, 1970), or systemic admin- istration of two other cholinergic agonists, physostigmine and pilocar- pine (Libertun and McCann, 1974a), initially inhibited but later 54 stimulated the release of gonadotropins in ovariectomized estrogen- primed rats. Intraventricular injection of gamma aminobutyric acid (Ondo, 1974), histamine (Libertun and McCann, 1974b; Donoso et_gl,, 1976) and some prostaglandins (Harms gt_al,, 1974) also were reported to stimu- late LH release in rats. Presently it is too early to assign specific roles to cholinergic, histaminergic and gabaergic receptors in the control of gonadotropin secretion. However, these preliminary reports indicate that each of these receptors may be involved in the physiological control of the anterior pituitary. Under many conditions it appears that serotonin and probably dopamine inhibit; whereas, norepinephrine stimulates the release of LH and FSH. C. Growth Hormone General The secretion of growth hormone is under the control of hypothal- amic somatotropin release-inhibiting hormone (somatostatin) and growth hormone releasing-factor (GRF). In addition, a "short loop" feedback mechanism may operate (as with prolactin) since a classic target tissue- feedback relationship appears to be lacking. Preliminary evidence indi- cates that elevated concentrations of plasma growth hormone may stimu- late the secretion of somatostatin (Root e__al,, 1973; Reichlin, 1974). Growth hormone release in humans also is stimulated by hypoglycemia and some amino acids, especially arginine. These effects are thought to be mediated through GRF and somatostatin and are not considered to be of primary importance in the physiological control of growth hormone 55 secretion (see Reichlin, 1974; Martin, 1976). Growth hormone is essen- tial for normal growth and development, and functions in the control of protein, carbohydrate and fat metabolism. Effects of Neurotransmitters on the ReTease of Growth Hormone Dopamine, norepinephrine and serotonin each have been implicated in the control of growth hormone secretion. Stimulation of dopamine receptors by apomorphine elevated plasma growth hormone concentrations in rats (Mueller at 21,, 1976b) and humans (Lal gt_al,, 1972; Brown 33.31,, 1973; Maany §t_al,, 1975). Piribedil, another dopamine receptor agonist, also stimulated the release of growth hormone; whereas, blockade of dopamine receptors with pimozide or haloperidol generally reduced plasma growth hormone concentrations in male rats. Furthermore, pre- treatment with haloperidol blocked the stimulatory effect of apomorphine on growth hormone (Mueller gt__l,, 1976b, Thesis) indicating that dop- amine receptors function in the stimulatory control of growth hormone. The direct effects of these drugs on growth hormone release from the pituitary can probably be excluded since catecholamines and blockers of catecholamine action were shown to have no effect on the jn_vitro re- lease of growth hormone by rat pituitaries (MacLeod, 1969; MacLeod gt_§1,, 1970). Simon and George (1975) observed that diurnal variation in plasma growth hormone concentrations paralleled changes in hypothalamic dopamine content. Intraventricular injections of dopamine were reported to induce depletion of pituitary growth hormone content indicating stimulation of growth hormone release (Mfiller gt_ 1., 1968). Systemic 56 administration of L-dopa stimulated growth hormone release in the rat (Chen gt_al,, 1974; Smythe et_gl,, 1975), dog (Lovinger gt_al,, 1974) and human (Boyd gt_al,, 1970; Boden gt_al,, 1972; Silver gt__1,, 1974). By contrast, Collu gt_al, (1972) reported a fall in plasma growth hor- mone concentrations following intraventricular injection of dopamine into urethane-anesthetized rats. Other workers found that L-dopa either inhibited (Mfiller gt_al,, 1973) or had no effect (Kato gt_al,, 1974) on blood growth hormone concentrations in rats. These differences from the findings of others (Mfiller st 21,, 1968; Chen and Meites, 1975) may be due to the urethane-anesthesis and drug doses used (see Martin, 1976). On the whole, reports generally indicate that dopamine stimulates growth hormone release. Efforts to determine the effect of norepinephrine on growth hor— mone secretion have produced several conflicting reports. Using pharma- cological agents which stimulate or block alpha (phenylephrine and phentolamine) and beta (isoproterenol and pr0panolal) adrenergic recep- tors Kato gt_al, (1973) proposed that beta adrenergic receptors stimu- late; whereas, alpha adrenergic receptors inhibit growth hormone secre- tion in the urethane-anesthetized rat. It should be noted there is sub- stantial evidence indicating that the opposite situation exists in humans (see Reichlin, 1974; Wilson, 1974; Martin, 1976). In a single experiment we observed that clonidine, a central acting alpha adrenergic stimulator, evoked the release of growth hormone in male rats. Intra- ventricular injections of norepinephrine were reported to either stimu- late (Mfiller §t_al,, 1968), or to have no effect (Collu $3.31,, 1972) on 57 the release of growth hormone in rats. Blockade of norepinephrine syn- thesis (by inhibiting dopamine beta hydroxylase with FLA-63) produced a fall in plasma growth hormone concentrations in male rats (Mfiller gt_al,, 1973). Although these reports generally show norepinephrine to stimulate growth hormone release, the mechanism of its action is unclear. There is some evidence that serotonin may be involved in the physiological control of growth hormone. Administration of tryptophan (MOller gt 91,, 1974) or 5-hydroxytryptophan (Imura gt 31,, 1973; stimu- lated growth hormone release in humans. Similarly, 5-hydroxytryptophan elevated plasma growth hormone concentrations in rats (Smythe and Lazarus, 1973b; Smythe 35 31., 1975). Pre-treatment with either cyproheptadine or methysergide, proposed serotonin antagonists, tended to reduce basal serum concentrations and partially blunted the insulin-induced release of growth hormone in humans (Bivens et_al:, 1973). Cyproheptadine also was reported to block the 5-hydroxytryptophan-induced release of growth hormone in rats (Smythe gt_al,, 1975). Collu 95:91, (1972) found that large doses of serotonin (1 pg/rat) stimulated growth hormone release when injected into the lateral ventricle of urethane anesthetized rats. By contrast, MUller 33 31, (1973) reported that intra-ventricular admin- istration of serotonin (1 pg/rat) or intraperitoneal injections of 5-hydroxytryptophan both elevated hypothalamic serotonin content but had no consistent affect on plasma concentrations of growth hormone. However, para chloroamphetamine reduced brain serotonin and slightly increased blood growth hormone concentrations in rats (Miiller 5311., 1973) indicating that serotonin may inhibit growth hormone. 58 Also, Martin (1976) observed a fall in growth hormone following electric- al stimulation of the midbrain raphae (serotonin nuclei), and we found that a single injection of L-tryptophan reduced plasma growth hormone concentrations in male rats (Thesis). Thus, the roles of serotonin and norepinephrine in the control of growth hormone remain to be established; whereas, there is general agreement that dopamine stimulates growth hormone release. The possible involvement of other neurotransmitters in growth hormone control has not been investigated. Just recently Dunn §t_al, (1974/74) and Martin (1976) observed that under normal conditions growth hormone is released from the rat pituitary in dramatic pulsatile bursts. These events occurred regularly, about every three hours within a given animal. The possibil- ity that this normal oscillation of plasma growth hormone may have been interpreted as a drug effect in earlier studies is apparent. This may be the basis for many of the contradictions between reports on the con- trol of growth hormone. V. Effects of Environmental Temperature and Physical Stress on Pituitary Hormone Secretion and Brain BiogenicAmines A. General Environmental temperature and physical stress can have a pro- fbund influence on pituitary hormone secretion. Although ambient temperature is an easily definable and reproducible condition, the term "stress" encompasses a wide variety of noxious stimuli ranging from animal handling to severe physical and psychological trauma. Extreme 59 temperature conditions in this respect can be stressful. Temperature is best known for its effect on TSH-thyroid activity; whereas, stresses are the classical stimuli for ACTH release. However, both stress and temperature have direct and/or indirect affects on the secretion of other anterior pituitary hormones. Temperature and stresses are thought to evoke changes in pituitary function primarily by neuroendocrine reflex mechanisms involving sensory perception and central integration which lead to alterations in the release of hypothalamic hormones. Little is yet known about the specific roles neurotransmitters play in this se- quence of events. 8. Effects of High ahd Low Temperature and Physical Stress on AnteriorIPituitary Hormone Secretion The rate of TSH secretion is inversely related to environmental temperature (see Reichlin gt_al,, 1972); whereas, both high and low temperature stimulate ACTH release (see Harris, 1955). It should be noted that very severe and prolonged cold was observed to inhibit TSH- thyroid activity (Williams gt_al,, 1949; Brown-Grant gt-gl,, 1954a) pre- sumably because of a nonspecific stress effect on TSH release. Stresses were consistently associated with inhibition of TSH release in labora- tory animals and humans (Brown-Grant gt 31,, 1954b; Reichlin, 1966; Ducommun 25.31,, 1966; Florsheim, 1974; Mueller et_al,, 1976a). Exposure of rats to low environmental temperature was reported to stimu- late hypothalamic TRH synthesis (Reichlin gt_al,, 1972; Hefco gt,al,, l975c) and release (Montoya gt_al,, 1975) although Jobin gt_al, (1975) were unable to demonstrate an effect of low temperature on the content 60 of TRH in the rat hypothalamus. Virtually nothing is known about the effects of stress on TRH release or of temperature and stress on the secretion of other hypothalamic hormones. In contrast to TSH, pituitary prolactin release was stimulated by warm temperature; whereas, cold temperatures inhibited prolactin in the rat (Mueller gt_al,, 1974; Chen and Meites, 1975b; Simpkins gt_al,, in preparation and bovine Wettemann and Tucker, 1974; Tucker and Wettemann, 1976). Stresses, however, profoundly elevated serum prolac- tin concentrations in rats (Krulich and Illner, 1973; Krulich gt.al,, 1974; Euker £91., 1975; Mueller £11-” 1976a). Jobin eta]: (1975) reported a brief elevation in serum prolactin concentrations by 5 and 15 minutes (but not later) after placing rats in 5°C. This transient prolactin response to cold exposure is probably due to the accompanying stress associated with the novel environment (Krulich gt_al,, 1974). Nicoll §t_al, (1960) observed that chronic cold exposure (0° for 5 days) initiated lactation in estrogen-primed female rats suggesting that cold temperature may stimulate prolactin release. However, cold-induced initiation of lactation may be due to activation of the ACTH-adrenal system since injections of glucocorticoids were reported to initiate lactation in estrogen-primed female rats (Meites gt_al,, 1963). Consistent with this view Wettemann and Tucker (1974) found that chronic cold (4.5°C for up to 9 days) resulted in a sustained reduction in serum prolactin concentrations in the bovine. At present, all reports show warm temperature to stimulate prolactin release (Mueller gt.al,, 1974; Chen gt 31., l975b; Wettemann and Tucker, 1974; Tucker and Wettemann, 61 1976). However, it remains to be determined if this effect is due sole- ly to temperature, or is part of a nonspecific stress response. Many exteroceptive stimuli influence the secretion of growth hormone in mammals (see Reichlin gt_al,, 1974; Martin, 1976). Prolonged exposure to low ambient temperature (4.5°C) reduced; whereas, warm temperature (32°C) slightly elevated serum growth hormone levels in the bovine (Tucker and Wetteman, 1976). Both warm and cold temperatures were reported by several laboratories to inhibit growth hormone release in rats (Schalch and Reichlin, 1968; Collu gt_al,, 1974; Strosser gt_al,, 1974). By contrast, we reported that acute changes in environmental temperature had no significant effect on growth hormone release in male rats (Mueller ££“21:- 1974). The conditions of our study were different and may not have been as stressful as in the experiments cited above. There is widespread agreement that stresses inhibit growth hormone release in rats (Schalch and Reichlin, 1968; Collu $3.31,, 1973, 1974; Dunn gt.al,, 1973/74; Krulich gt_al,, 1974; Thesis) and many other non- primate mammals (see Reichlin gt_al,, 1974; Martin, 1976). Interestingly, stresses stimulated growth hormone release in monkeys (Brown ££“21:7 1971) and humans (Baylis gt_a1,, 1968; Noel gt_al,, 1972), indicating an important species difference in hypothalamic control of growth hormone secretion. The effects of stress and temperature on gonadotropin secretion have not been extensively studied. Acute stresses were reported to stimulate gonadotropin release in rats (Ajika gt_al,, 1972; Euker £5 91,, 1975). Krulich §t_al, (1974) observed a biphasic response in which LH 62 release was initially stimulated and later inhibited by prolonged stresses. In agreement with these findings, Nicoll et_gl, (1960) observed that chronic stresses reduced ovarian and uterine weights in female rats, indicating a suppression of gonadotropin secretion. Although Neill (1970) reported that surgical stress had no effect on LH release in rats, this difference may be due to the time at which blood samples were collected fbllowing onset of the stress. There also is some evidence that temperature affects pituitary-gonadal function in rats. Female rats maintained at low ambient temperature exhibited lengthened estrous cycles (Lee, 1926; Bohanan, 1939) and tended to have reduced ovarian and uterine weights (Nicoll gt_al,, 1960). Both of these conditions suggest decreased gonadotropin secretion. The adrenal glands appear to have little, if any, role in medi- ating the acute effects of stress and temperature on the release of pituitary hormones other than ACTH. The inhibitory effect of stress on the release of thyroid hormones in rabbits was not altered by bilateral adrenalectomy in combination with constant cortisone replacement (Brown-Grant gtual,, 1954b). Similarly, Krulich (personal communica- tion) found that adrenalectomy had no affect on the stress-induced inhibition of TSH release in rats. Pre-treatment with high doses of dexamethasone (50 pg/lOO mg for 8 days), a synthetic glucocorticoid, only slightly reduced the release of prolactin and LH, but completely blocked the adrenal response associated with acute stress in rats (Euker gt_al,, 1975). Both high and low temperatures stimulate adrenal cortex function but had opposite effects on the secretion of TSH and prolactin 63 (see above). Thus, acute changes in TSH and prolactin release produced by temperature occur independently from, and appear not to be directly related to, stimulation of ACTH-adrenal hormone release. Likewise, adrenal hormones probably have no major influence on changes in TSH, prolactin, growth hormone and gonadotropin release produced by acute stresses. C. Effects of High and Low Temperature and * Physical Stress on Brain Catecholamines and Serotonin Brain neurotransmitters are thought to mediate the neuroendo- crine responses to temperature and stress by specifically influencing the release of hypothalamic hypophysiotropic hormones. The effects of stress and temperature on central catecholamines and serotonin has just recently come under investigation. Various forms of stresses were reported to decrease brain nor- epinephrine concentrations (Bliss gt_al,, 1968; Carr and Moore, 1968; Stone, E. A., 1973) and increase norepinephrine turnover (Gordon gt 21,, 1966; Thierry gt.al,, 1968a; Corrodi gt_al,, 1971b; Librink gt a1,, 1972) in rats. Thus, there is general agreement that stresses enhance the activity of brain noradrenergic neurons. By contrast, the effect of stressful conditions on dopamine is not clearly understood. Stresses in rats were found to have no effect on brain concentrations of dopamine (Gordon gt_al,, 1966; Bliss gt_al,, 1968; Carr and Moore, 1968; Corrodi et.al,, 1971b). Dopamine turnover was reported to be increased (Bliss gt 31,, 1968), decreased (Corrodi gt 31,, 1971b; Lidbrink gt_al,, 1972) or unchanged (Gordon gt_al,, 1966; Thierry gt_al,, 1968a) by various 64 stresses. Recently, Palkovits gt_al, (1975) reported that stresses re- duced dopamine and norepinephrine concentrations, and increased tyrosine hydroxylase activity in the arcuate nucleus of rats; whereas, catechol- amines and tyrosine hydroxylase activity in other hypothalamic nuclei and brain regions were little affected by stress. These findings sug- gest that catecholamines in the medial basal hypothalamus are probably involved in the neuroendocrine changes associated with stress. There is some agreement that stresses stimulate brain serotonin turnover and lead to elevated concentrations of brain serotonin and 5-HIAA in rats (Thierry gt_al,, 1968b; Bliss 33.21,, 1972; Ladish, 1975). Recently we found that acute restraint stress enhanced the accumulation of serotonin in the hypothalamus of rats following MAO inhibition (Mueller gt.al,, 1976a). These findings indicate that stresses stimu- late hypothalamic serotonin turnover and this effect may be involved in stress-induced changes in pituitary hormone secretion. Variations in environmental temperature also influence the syn- thesis and release of brain neurotransmitters. High temperature (32°C) was reported to produce a threefold increase in hypothalamic norepine- phrine turnover while it had no effect on norepinephrine in the rest of the rat brain (Iversen and Simonds, 1969). Cold temperature (4°C) stimulated median eminence dopamine turnover (Lichtensteiger, 1969) but had no effect on hypothalamic norepinephrine metabolism (Iversen and Simonds, 1969). Brain serotonin turnover is directly related to ambient temperature. High temperatures stimulated (Corrodi gt_al,, 1967; Aghajanian gt_gl,, 1968; Reid gt_al,, 1968; Weiss and Aghajanian, 1971; 65 Squires, 1974); whereas, low temperatures reduced (Corrodi gt_al,, 1967; Mueller, unpublished) brain serotonin turnover in rats. Taken together these findings demonstrate that both temperature and stress influence the activity of central catecholamine and serotonin neurons. The relationship of these changes to the secretion of specific hypothalamic hormones remains to be determined. MATERIALS AND METHODS 1. Animals, Treatment and Blood Collection Mature male and female Sprague-Dawley rats (Spartan Research Animals, Haslett, Mich.) and male hypophysectomized rats (Hormone Assay Labs, Chicago, Ill.) were housed in a temperature (24°C) and light con- trolled environment (lights on from 6:00 AM to 8:00 PM) for at least 4 days prior to each experiment. Rats were provided with Purina Rat Chow (Ralston Purina Company, St. Louis, Mo.) and tap water ag,libitum. Hypophysectomized rats received orange slices and sugar cubes as a daily food supplement. Thyroidectomy (THX) and anterior pituitary (AP) trans- plantation were performed under deep ether anesthesia. All hypophysec- tomized rats were given a single AP graft from a male donor rat under the left renal capsule. Surgically treated rats were given 0.2 ml Longicil (60,000 units of penicillin G; Fort Dodge Laboratories, Fort Dodge, IA) post-Operatively to prevent infection and THX rats were given 0.1% calcium lactate solution in their drinking water for 5 days after surgery. All experiments with the exception of Experiment I were car- ried out using male rats. Synthetic tryrotropin-releasing hormone (TRH; provided by Dr. K. Folkers, Institute for Biomedical Research, University of Texas, Austin, Tx.), pargyline hydrochloride (Sigma Chemical Co., St. Louis, Mo.), 66 67 piribedil mesylate (ET 495; provided by Dr. M. Derome-Tremblay, Les Laboratoires Servier, Neuilly, France) and DL-S-hydroxytryptophan, ethyl ester, hydrochloride (5-HTP; Sigma Chemical Co., Morton Grove, Ill.) were dissolved in 0.9% NaCl. Pimozide (obtained from Dr. P. A. J. Janssen, Janssen Pharmaceutical Research Laboratories, Beerse, Belgium) and haloperidol (obtained from Dr. Kleis, McNeil Laboratories, Inc., Fort Washington, Pa.) were dissolved in 0.3% tartaric acid. Apomorphine hydrochloride (Eli Lilly and Co., Indianapolis, Inc.) was dissolved in 0.1% sodium metabisulfite. D- and L-tryptophan (Sigma Chemical Co., St. Louis, Mo.) was suspended in 0.9% NaCl containing 1% carboxymethyl- cellulose. Estradiol benzoate (Nutritional Biochemicals Corp., Cleveland, Ohio) was dissolved in corn oil. Temperature and restraint stress experiments were carried out using mature male rats. Control rats were maintained at room tempera- ture (24 i_l°C). For high temperature the rats were placed in a venti- lated drying oven at 40 :_2°C and for cold temperature the rats were placed in individual cages in a room at 4 1.1°C. Restraint stress was administered by taping rats to wire test tube racks and then placing the rats on their backs. Route, dose and time of treatments (drugs or ap- propriate vehicles, temperature and restraint stress) are given in the section entitled "Experimental". Blood samples were collected either by decapitation or orbital sinus cannulation under light ether anesthesia (see Experimental). Plasma samples for growth hormone assay were obtained by mixing 0.1 ml of 100 mg% sodium heparin (Sigma Chemical Co., St. Louis, Mo.) with 1.0 68 ml blood prior to clotting. Plasma and serum were separated by centrifu- gation and stored at ~20°C until assayed for hormone content. II. Radioimmunoassays of Blood Hormones Determination of blood hormone concentrations were made by standard radioimmunoassay procedures. Serum prolactin was measured by the method of Niswender g__gl, (1969), serum TSH by the method of Dickerman gt 31, (1972). Serum LH was measured by a sensitive micro- radioimmunoassay (Marshall, Bruni, Campbell and Meites, in press) developed as a minor modification of the method of Niswender et 91, (1968). Values are expressed in terms of NIAMDD-rat prolactin-RP-l, NIAMDD-rat TSH-RP-l, NIAMDD-rat GH-RP-l and NIAMDD-rat LH-RP-l, respec- tively. All blood samples from an individual experiment were assayed in the same radioimmunoassay. III. Brain Tryptophan, Serotonin and 5-Hydroxyindole- acetic Acid*(5-HIAA) Assays After decapitation the brain was immediately removed and the pineal gland discarded. The cerebellum was separated from the brain stem by sectioning the cerebellar peduncles. The hypothalamic area removed constituted the region lying between the rostral borders of the optic chiasm and mammillary bodies and medial from the optic tracts which was removed to a depth of about 3 mm. These two structures and remaining tissue (brain) were frozen on Dry Ice subsequent to weighing (tissues' were weighed while still frozen) and biochemical analysis. Average 69 tissue weights (mean :_1 standard error) based on 50 samples were cerebellum = 234.1 :_2.7 mg, hypothalamus = 47.3 :_0.6 mg and brain = 1.454 :_0.007 gm. Serotonin and 5-HIAA in the brain and serotonin in the hypothal- amus were assayed by the solvent extraction methods of Cruzon and Green (1968) as modified by Hyyppfi £3 21,, (1973). A detailed description of reagents used and assay procedures is presented in Appendix A. In brief, this method involved homogenization of brain tissue in acidi- fied butanol. Following centrifugation an aliquot of the supernatant was transferred to a test tube containing heptane and 0.1 N HCl. Organic and inorganic phases were mixed by shaking and separated by centrifugation. The inorganic phase containing serotonin was mixed with O-phthaldehyde (OPT)-hydrochloric acid solution and then heated to 100°C to form a highly fluorescent serotonin-OPT complex. The 5-HIAA present in the organic phase was extracted into 0.5 M phosphate buffer (pH 7.5) by shaking and centrifugation and the aqueous phase was then mixed with OPT and heated to 100°C. After cooling to room temperature sample fluorescence was read in an Aminco-Bowman spectrophotofluorim- eter (American Optical Comp., Silver Spring, Ma.) at 355 nm excitation and 480 nm emission wave lengths. Recoveries of pure 5-HT and S-HIAA standards by this method averaged 95% for serotonin and 70-80% for 5-HIAA. Cross extraction of serotonin into the 5-HIAA fraction and visa versa were less than 1%. Tryptophan in the cerebellum was assayed by the method of Denckla and Dewey (1967) within 24 hours after the samples were 70 collected. Because the extraction procedure for tryptophan is not com- patible with that of serotonin and 5-HIAA, the effects of various treat- ments on concentrations of cerebellum tryptophan were determined presum- ing that these would reflect the relative changes in tryptbphan concen- trations occurring in other regions of the brain. Changes in cerebellum tryptophan concentrations as affected by diet were found to parallel changes in tryptophan content which occurred in other regions of the brain (Colmanares and Wurtman, unpublished). A detailed description of the tryptophan assay is presented in Appendix B. This method involved tissue homogenization and tricholoroacetic acid protein precipitation. After centrifugation tryptOphan in the supernatant was converted to the fl uorophore .norharman by condensation with formaldehyde and oxidation with ferric chloride (FeCl3) at 100°C. Sample fluorescence was read at 371 nm excitation and 443 nm emission wavelengths. Recovery of pure tryptophan standard was 65%. IV. Methods of Statistical Analysis All statistical analysis was carried out as described by Sokal and Rohlf (1969). The specific tests used are indicated under Experimental. EXPERIMENTAL I. Effects of Thyrotropin-Releasing Hormone (TRH)on the In Viyg Release of Prolactin and—TSH in roestrous Female;_Male and Estrogen-PrimedMaleRats A. Objectives Synthetic TRH, pyroglutamylhistidylproline amide, stimulates release of TSH in several species ig_yiyg_and 13.31359 (see Reichlin gt_al,, 1972; Vale et_gl,, 1973b; Schally £3,213, 1973; Florsheim, 1974). Tashjian gt_al, (1971) observed that TRH increased prolactin release when added to incubations of clonal cells from pituitary tumor. However, prior to the present study TRH had not been clearly shown to stimulate jg_yitrg_release of prolactin from normal rat hemi-pituitaries (Bowers, 1971; Lu 35.91,, 1972) or from incubated bovine pituitary tissue (LaBella and Vivian, 1971; Convey $5.91,, 1973). Similarly TRH had not been demonstrated to stimulate 1g_yiyp_release of prolactin in rats (Lu gt_gl,, 1972) although it was active in the human (Jacobs e£_gl,, 1971; Bowers gt_gl,, 1971) and bovine (Convey gt_gl,, 1973). This study was under- taken to further explore the possibility that TRH may induce ig_yiyg, release of prolactin in the rat, and to examine the relation of estrogen to TRH induced prolactin release. In addition the effect of TRH on TSH release under these conditions was examined. 71 72 B. Materials and Methods Mature Sprague-Dawley virgin female and male rats were used in this experiment. Daily vaginal smears were taken from female rats for at least two recurrent 4- or 5-day estrous cycles to insure they were undergoing regular cycles. On the morning of proestrus, a pre-treatment blood sample was collected from the orbital sinus under light ether anesthesia at about 11:00 AM, and immediately thereafter the animals were injected intravenously with 0.2, 0.5, l, 5 or 25 pg TRH. In all cases the injection volume was 0.2 m1 of 0.8% NaCl and the controls were given NaCl alone. Post-treatment samples were collected 10 and 60 min after injection. The male rats were given subcutaneous injections of 10 pg estradiol benzoate dissolved in 0.2 m1 corn oil or corn oil alone for 5 days. On the sixth day a pre-treatment blood sample was collected at about 11:00 AM and the animals were immediately injected intravenous- ly with either 0.2 ml NaCl or 1 pg TRH dissolved in 0.2 m1 NaCl. Post- treatment samples were collected after 10 and 60 min. Student's t test was used to determine significance of differences between mean prolactin or TSH values of any 2 groups. Pre-treatment serum samples from the non- estrogen-primed male rats were pooled in order to obtain enough serum for the assay of TSH (see Table 1). C. Results All doses of TRH evoked a 2 to 3.5 fold increase in serum pro- 1actin by 10 min after injection into proestrous rats as compared with control rats (Table l). The 1 pg dose produced the greatest increase in serum prolactin 10 min after injection when compared to control values, .mo.o vac “screen we?» mean an qsocm paucoewcmaxm m> upocpcou 73 58H on... a: H «mm m u 8.— 3: Sasha .5: on _ : .... a: m H m: m .... 8. E 53:3 .3228 A H 8 am .... Q s H 8 E 5833 2. £5 9. _ e .H mm m .H me N .H m— Amy :muocumo o: .mpoeucou mum; ope: 5% ..n 5 as H N: 2 ..H S 8: s: as 3 2 H 8 5...: H ex 2 M Ne 23 5: an m 2 M Q new.“ NNN : n S E 5: a; _ «N H 2: mm H a: 2 H 8 8: =5 9. 8m 2an ENE :ums :5 5: 9. 8m 2 M Na NF H 8 a n 3 3: £828 mace o_oeoe mzocumooca cps on :we op pcmeummeuoea Among .ocv acmeuoocp Agape mo mmiflifiawmmv cwuuupocn Eseom $3. 25. wattage use vmamocucn c? use apnea; maocumooca cw ammopom :vuuopoca :6 xx» mo muummmm .p open» 74 and was slightly more effective than the other doses of TRH when com- pared with pre-treatment values. By 60 min after TRH injection, serum prolactin fell from peak concentrations but still tended to be higher than pre-treatment values. There appeared to be no definite dose- response relationship observed between the doses of TRH given and increases in serum prolactin. In both untreated and estrogen—primed male rats, significant increases in serum prolactin were observed after injection of 1 pg TRH. The pre-treatment control, non-estrogen-primed male rats had less serum prolactin than the control female rats (p‘:0.05) and estrogen markedly increased serum prolactin concentration (p<:0.05). All doses of TRH used dramatically increased serum TSH by 10 min after injection into proestrous female rats as compared with pre- treatment control values (Table 2). A clear dose-response relationship was observed over a range of doses from 200 ng to 5 pg TRH; whereas, the highest dose (25 pg) was less effective than the 5 pg dose in elevating serum TSH concentration by 10 min after injection. By 60 min after injection serum TSH concentrations fell but were still higher than pre- treatment values. Injection of 1 pg TRH profoundly elevated serum TSH concentra- tions in both normal and estrogen-primed male rats by 10 min after injection as compared with control values. Estrogen appeared to have no affect on TSH release induced by this dose of TRH. Sequential blood sampling at 10 and 60 min was associated with significant reductions in serum TSH concentrations in both normal and estrogen-primed, 75 .mo.ouva3 “mm:_m> pocucou newspmmeuuoea Ease vacate; appchvacmvm mm:_u> ewe on can ewe o— .mo.o vac "vowema mew» meow Hm naoem Poucmepcmaxm m> mpoeucou 53 H 82 some H 32 com H a: E 58:0 é: a: p 3% H mm as H mm a? H a: 2: 59:5 .3228 .88 H «2 .03: H a: 3 H men A. 3 53:8 8 .5: as _ 3m .H mm cm .H mpm Amv :mmocumo o: .mpocacou mung msz new H E. as: H 2: on 5 :E 9. mm 53” H 8m 52” H as 8 5 5: as m sum H 3N saw H we: on E 5: a; _ .03 H 8. com: H 3.: on 5 IE 9. 8m .02 H 8 cm: H a: 8 8: IE 9. com on v on V cm v Amy upocucoo mace mpoeme mzocpmmoca ewe om ewe op ucoeummeamcg Amume .oev «casumoch (demos we mmJH Fawmcve Imp Escom mama m_m: cos_ca-:omoeamm U20 twumguca cw Ucm w—MEmm magpmmOLm E. mmmmpmm Ink :0 I”: ..vo muumntm .N mpnuh 76 saline-injected male rats as compared with pre-treatment control values. Pre-treatment serum TSH concentrations were highest in normal male rats, lowest in proestrous female rats, and estrogen-priming significantly reduced serum TSH concentrations in male rats. 0. Discussion This study shows that a single injection of synthetic TRH can stimulate a rapid increase in serum prolactin and TSH in proestrous female rats and in normal and estrogen-primed male rats. Previously our laboratory had reported that a single intravenous injection of 7.5 pg TRH or intracarotid infusion of 5 pg TRH failed to increase serum prolac- tin by 15 or 30 min after administration (Lu gt_al,, 1972). This differ- ence may be due to the different preparation (obtained from Abbott Labs, Chicago, Ill. and Merck, Sharp and Dohme Research Labs, Rahway, N.J.) of TRH used. Also, Valverde §t_al, (1972) observed no increase in serum prolactin in estrogen-primed male rats after injection of 100 ng TRH, but tnis dose may have been insufficient to cause prolactin release. The TRH dose employed in the present study in male rats was 10 times as great, and was effective in both estrogen—primed and normal male rats. These observations that TRH stimulated TSH release are con- sistent with many earlier reports (see Schally et_gl,, 1973). However, the finding that 5 pg TRH was more effective than 25 pg TRH in stimu- lating TSH release in proestrous female rats cannot presently be explained. Estrogen-priming reduced serum TSH concentrations in male rats, in agreement with an earlier report by D'Angelo (1968) but 77 appeared to have no affect on TRH induced TSH release indicating that estrogen does not change the sensitivity of the pituitary to 1 pg TRH. The reduction in serum TSH and increase in serum prolactin in the NaCl injected control rats with time is believed to reflect the stress associated with multiple blood collections and anesthesia (Mueller et_al,, 1976a). These observations indicate that in the rat, synthetic TRH stimulates the ig_vivo release of prolactin as well as TSH. II. Effects of Heat and Cold on the Release of TSH, Growth Hormone andTrolactin in Male Rats A. Objectives Pronounced alterations in temperature evoke rapid changes in release of TSH in rats, with cold producing an increase and heat a decrease in TSH release. Cold was reported to decrease plasma growth hormone concentrations in rats (Schalch and Reichlin, 1968; Collu gt_gl,, 1974 and an inverse relationship between environmental temperature and circulating prolactin and growth hormone concentrations was reported in the bovine (Wettemann and Tucker, 1974; Tucker and Wettemann, 1976). The effects of warm temperature on growth hormone and of warm or cold temperature on prolactin release in rats had not been previously report- ed. The present investigation was undertaken to determine effects of warm and cold temperatures on blood concentrations of TSH, growth hor- mone and prolactin in normal and hypophysectomized-AP transplanted male rats. Also, the possible roles of thyroid hormones and dopamine receptors in cold induced changes in blood TSH, growth hormone and prolactin concentrations were investigated. 78 B. Materials and Methods Nature Sprague-Dawley intact and hypophysectomized male rats were used in this study. Thyroidectomy (THX) was performed ten days, and AP transplantation into hypophysectomized rats seven days prior to the temperature experiments. Pimozide (2.0 mg/kg) or 1% tartaric acid vehicle (0.2 ml/lOO gm B.W.) were injected subcutaneously 2 hours before decapitation. Warm (40°C for 30 min) or cold (4°C for 120 min) were administered as discussed under the general Material and Methods section. All blood samples were collected by decapitation. Student's t test was used to determine significance of difference between control and experi- mental serum hormone concentrations: p<=0.05 was chosen as the level of significance. The term “blood concentrations“ used in tables and figures refers to the concentrations of prolactin and TSH in the serum and of growth hormone in the plasma. cm A temperature of 40°C for 30 min decreased serum TSH to 27% of control values and plasma GH to 45% of control values, and produced about a five-fold increase in serum prolactin (Table 3). The growth hormone difference was not significant due to a large variation in the control group. A temperature of 4°C for 120 min evoked almost a two-feld in- crease in serum TSH, and a significant fall in serum prolactin from 25 ng/ml to 6 ng/ml. Plasma growth hormone concentrations were not altered by cold exposure. Table 4 shows the effects of heat and cold on blood TSH, growth hormone and prolactin concentrations in male hypophysectomized-AP 79 Table 3. Effects of Heat and Cold on Blood Concentrations of TSH, GH and PRL in Male Rats Treatment and Serum TSH Plasma GH Serum PRL no. of rats pg/ml ng/ml ng/ml Controls, 24°C (8) 0.46 t. 0.08 152 1 55 25 2‘. 3 Heat, 40°C (8) 0.12 1 0.02“ 59 i. 4 123 1 8“ Cold 4°C (7) 0.84 1 0.15“ 192 i 55 6 i 1“ Heat was for 30 min and cold was for 120 min. Valuesasignificantly different from room temperature (24°C) control value p < 0.05. 80 Table 4. Effects of Heat and Cold on Blood Concentrations of TSH, GH and PRL in Male Hypophysectomized-AP Transplanted Rats Treatment and Serum TSH Plasma GH Serum PRL no. of rats ng/ml ng/ml ng/ml Controls, 24°C (7) < 30 74 1_4 l9 :_6 Heat, 40°C (7) < 30 66 :_7 32 :_6 Cold 4°C (7) < 30 59 :_7 23‘:_4 Heat was for 30 min and cold was for 120 min. 81 transplanted rats. Neither heat nor cold had a significant effect on blood concentrations of any of these three hormones. Both cold and to a much greater extent thyroidectomy (THX) increased serum TSH concentrations as compared with room temperature intact control (Veh) values (Figure 1). Cold in combination with THX did not further elevate serum TSH above the value produced by THX alone. Neither THX nor cold had a significant effect on plasma growth hormone concentrations under these conditions. serum prolactin concentrations at room temperature were significantly elevated by ten days THX as com- pared with intact control values (14 :.2 ng/ml vs. 23 :_1 ng/ml). Cold exposure significantly reduced serum prolactin in both intact and THX groups as compared with room temperature control values. Pre-treatment with pimozide (2 mg/kg, 2 hr.) at room temperature produced almost a three-fold elevation in serum TSH content as compared with vehicle injected (Veh) control values (Figure 2). Cold signifi- cantly elevated blood TSH in Veh control animals but cold in combination with pimozide did not produce a further elevation in serum TSH above that evoked by pimozide alone. Growth hormone tended to be reduced by both cold and pimozide; however, neither treatment given alone or in combination had a significant effect on plasma GH concentrations. Serum prolactin values were reduced by cold and markedly elevated by pimozide. Cold was ineffective in reducing serum prolactin in animals pre-treated with pimozide. TSH GH PRL 4000. '1’ ...... I g -1 a i ‘ -aoo ~30 fl 2 .81. a: 800« a g i 9 )200 ~20 a 3 2 a 2 .. a a 2 r 2 2 7° -'° I z a I 1 I I 9 2 I 5 fl 5 ‘ _ _ _, .. _ 7 ,.'_‘__._. _..._.._._ Figure 1. Effects of thyroidectomy on cold induced changes in blood concentrations of TSH, growth hormone (GH) and prolactin (PRL) in male rats. Rats were either left intact (Veh) or thyroidectomized (THX) 10 days prior to temperature exposure. Each bar represents the mean of 8 determinations. Verticle lines projected on each bar represent 1_l standard error. Room temperature (RT) = 24°C; cold = 4°C for 120 min. TSH and GH ‘nglml 800-1 2001 83 TSH GH PRL -8 i .140 } ~12o ; g g .100 PRL 2 2 2 "°’"" 2 2 2 1 2 2 2 '1 é a a 40 I 2 2 2 2 2 2 - 2 2° 2 2 a 2 2 2 I 2 2 I I I Figure 2. Effects of pimozide on cold induced changes in blood concens trations of TSH, growth hormone (GH) and prolactin (PRL) in male rats. ‘ Rats were injected subcutaneously with pimozide (2.0 mg/kg) or vehicle (Veh) 2 hours prior to sacrifice. Room tempera— ture = 24°C; cold = 4°C for 120 min. Each bar represents the mean of 8 determinations. The verticle lines projected on each bar represent :_1 standard error. 84 0. Conclusions A warm temperature of 40°C produced a rapid decrease in circu- lating TSH and a marked increase in prolactin. Conversely, cold tempera- ture (4°C) stimulated TSH and inhibited prolactin release. Neither warm nor cold temperature had a significant effect on plasma growth hormone concentrations under these conditions. Whether these results are due solely to changes in temperature or also to the possible stress experi- enced by these animals is unknown at present. Stresses have been reported to inhibit TSH and to stimulate prolactin release in rats (see Florsheim, 1974; Neill, 1974; Mueller, 1976a). In the present study, cold temperature was observed to have the opposite effect on the release of these two hormones indicating that changes in pituitary hormone secre- tion associated with acute cold exposure (4°C for 120 min) are primarily due to the affect of temperature and can not be explained on the basis of a stress response. Further, stresses were reported to inhibit growth hormone release in the rat (Schalch and Reichlin, 1968; Collu et_gl,, 1973; Krulich gt_al,, 1974; Mueller gt_§l,, 1976a). In the present study neither warm nor cold temperature had an affect on growth hormone secretion. ' Removal of the pituitary from hypothalamic influence blocked both heat and cold induced changes in pituitary hormone secretion (Table 4) indicating that changes in TSH and prolactin release associ- ated with variations in ambient temperature are mediated by hypothalamic control mechanisms which are not effective by the general circulation. 85 Serum TSH concentrations were elevated more than nine-fold by 10 days T1X and cold in combination with THX failed to further elevate serum TSH as compared with room temperature THX values (Figure 1). These findings indicate that THX is a maximal stimulus for the secretion of TSH. Cold exposure reduced serum prolactin concentrations to a simi- lar extent in both intact and THX animals as compared with room tempera- ture control values. Thus, inhibition of prolactin secretion by two hours cold exposure is not mediated by the cold induced stimulation of TSH-throid activity. Pimozide alone dramatically elevated serum TSH and prolactin concentrations (Figure 2) indicating that TSH as well as prolactin may normally be under a dopamine-mediated tonic inhibitory influence by the hypothalamus. Cold temperature in combination with pimozide failed to further elevate serum TSH or to reduce prolactin as compared room temperature pimozide control values. These findings suggest that dop- amine is involved in the physiological regulation of TSH and prolactin; however, the mechanism is unclear. Cold was reported to enhance the fluorescence of median eminence dopamine neurons (Lichtensteiger, 1969) and to stimulate tyrosine hydroxylase activity in the arcuate nucleus (Palkovits e§_al,, 1975). These findings suggest that enhanced tubero- infundibular dopamine activity may be responsible for the cold induced inhibition of prolactin release. This model does not, however, explain cold stimulation of TSH release since the effect of pimozide and other dopaminergic drugs on TSH (Mueller 53.91,, 1976b, Thesis) indicate that enhanced dopamine activity inhibits TSH release. Most likely another neurotransmitter, probably serotonin, is involved in the cold-induced stimulation of TSH. Cold temperature has been reported to reduce brain serotonin turnover (Corrodi gt_al,, 1967; and unpublished findings); whereas, high temperature stimulated brain serotonin turnover (Corrodi gt_al,, 1967; Aghajanian 25.91,, 1968; Reid 23 21,, 1968; Weiss and Aghajanian, 1971; Squires, 1974), and inhibits TSH release. Under experimental conditions serotonin inhibited TRH-TSH release in rats (Grimm and Reichlin, 1973; Mess and Peter, 1975; Mueller g§_al,, 1976a). Together these findings suggest that the rise in serum TSH produced by cold may be due to reduced brain serotonergic activity; whereas, the opposite situation may exist in the case of warm temperature. Roles that other neurotransmitters may play in mediating the effects of temper- ature on pituitary hormone secretion remain to be determined. Little is known about the effects of temperature on the hypothal- amic hypophysiotrophic hormones. TRH induces prolactin as well as TSH release in many species, and cold was observed to increase TRH synthesis (Reichlin et_gl,, 1972; Hefco gt.gl,, 1975c) and release (Montoya gt_gl,, 1975) by the rat hypothalamus. However, the observation that cold increased TSH but decreased serum prolactin; whereas, heat produced the opposite effects on the release of these two hormones, indicates that TRH is not responsible fer the changes observed on prolactin release. 87 III. Effects of Dopaminergic Drugs 0n the Release of Prolactifi, TSH, Growth Hormone, and LH in Male Rats A. Objectives It is well-established that conditions which elevate brain dop- aminergic activity also inhibit prolactin secretion, presumably because release of PIF is increased or by a direct action of dopamine on the pituitary. By contrast, the influence of dopaminergic neurons on release of TSH, growth hormone and LH is unclear (see Literature Review). Development of specific and highly potent dopamine receptor agonists has made it possible to more carefully evaluate the influence of dopamine receptor stimulation on secretion of pituitary hormones. Apomorphine, the prototype dopamine agonist (Andén et_gl,, 1967), piribedil, a long- acting dopamine agonist (Corrodi $5.31!, 1971) and haloperidol, a dopa- mine receptor blocker were used in this study to determine the effects of dopamine receptor activity on secretion of prolactin, TSH, growth hormone and LH in male rats. 8. Materials and Methods Male Sprague-Dawley rats weighing 200-225 gm were used in this study. All drugs or appropriate vehicles (see general Materials and Methods section) were given as single subcutaneous injections in a volume of 0.2 m1/100 gm body weight at the dose and time schedules indi- cated in Results. All blood samples were collected by decapitation between 10:00 AM-1:00 PM. Effects of drug treatments on behavioral activity were determined by Dr. K. E. Moore. Student's t test was used to test significance between control and experimental blood hormone concentrations; the level of significance was chosen as p<:0.05. Abbreviations on figures are prolactin (PRL) and growth hormone (GH) and the term "blood concentrations“ refers to serum concentrations of pro- lactin, TSH and LH, and plasma concentrations of growth hormone. C. Results Time Course Effects of Apomorphine on Ejood Concentrations—6? Prolactin, TSH, Growth Hormone and LH An initial experiment was carried out to determine the time course of the effects of a large dose of apomorphine on blood content of four pituitary hormones. Groups of 8 rats each were injected sub- cutaneously with 1 mg/kg apomorphine and killed at various times there- after (Figure 3). Both serum prolactin and TSH concentrations were reduced at 15, 30 and 60 min after injection. By 120 min prolactin values were beginning to return toward normal but remained significantly lower than the control mean; whereas, TSH concentrations had returned to control values. The time course of these effects are consistent with the short duration of action of apomorphine. Since maximal effects were observed 30 min after the administration of apomorphine, subsequent dose- response effects were examined at this time interval. Plasma growth hormone rose progressively throughout the later time intervals to 355 :_41 ng/ml at 120 min as compared with 71 :_2 ng/ml in the control group. The reason for this unexpected response to apo- morphine was not understood at the time of the experiment, but became clear when the dose-response relationships of this hormone were 89 so, PRL .00. GH , l 300- r//+- 40- - 3 3 == 7510(1- 5 20 - /+- 2. 1 3 5 . 45 g o L I l A l l 4.: J .1 3 0L l 1 I l I 1IIfi j _ 8 0 3O 60 120 o O 30 60 12 u 300 r TSH '1 u 60 ' LH 1 z: :z o O z 2 a: a: o 200- . 0 40- - :1 :: 10°F 20- d 1 ' Gk I l n n a i: J _ o a a a a a .2 g _ 0 30 60 120 0 3O 60 120 MINUTES AFTER APOMORPHINE Figure 3. Time course of the effects of apomorphine on blood content of pituitary hormones in male rats. Rats were injected subcutaneously with apomorphine (1 mg/kg) and sacrificed at various times thereafter. Each symbol rep— resents the mean of 8 determinations. Vertical lines pro- jected on each symbol represent :_1 standard error; where not shown the standard error is less than the radius of the sym- bol. Solid symbols indicate values that are significantly different (p< 0.05) from vehicle (0.5% sodium metabisulfide) treatment (zero-time value). 90 determined. Serum LH was elevated at 15 min but was slightly reduced at 60 and 120 min after apomorphine administration. Effects of Graduated Doses of Apomorphine on Blood:Concentratian 0f'Prolactin, TSH, Growth Hormone and LH Apomorphine reduced both serum prolactin and TSH concentrations by 30 min after injection, the lowest dose tested (0.03 mg/kg) producing a near maximal depression of serum prolactin (Figure 4). Increasing doses of apomorphine caused serum TSH values to fall in a dose-related manner with the 0.3 mg/kg dose producing the first significant reduction. Plasma growth hormone was elevated by lower doses (0.03, 0.1 and 0.3 mg/kg) but was unchanged by the higher doses (1, 3 and 10 mg/kg) of apomorphine. No clear pattern in LH response was observed. In order to determine the threshold of the prolactin response to apomorphine and to repeat, in part, the observations on TSH, growth hor- mone and LH, a second dose-response experiment was done using a lower range of doses (Figure 5). Serum prolactin was not altered by 0.003 mg/kg but was maximally reduced by 0.01 mg/kg apomorphine. Serum TSH values were not significantly altered by the lower doses of apomorphine used in this experiment. Plasma growth hormone concentrations were markedly increased by the 0.03 mg/kg dose, as in Figure 4, but was not altered by the two lower doses of apomorphine. Again, no consistent LH response was observed during this time interval. Effects of Apomorphine on Serum TSH and Pro- lactififiCBncentrations in Hypotherid’Rats In light of the pronounced inhibitory effects of apomorphine on TSH and prolactin secretion, it was of interest to test this drug in rats with elevated TSH values 10 days after thyroidectomy. The effects 91 0‘. I: GOFPRL a 400 ”“1 A I 404 - zoo 3' a 'E' ‘ 3 20- 4 3100 - 5 5 . ' .5 4: z 0_ PL 0 a L a J J 2 J 3 0.03.1.31310 8° l-o..0:1"'—"'_“_“_'1.10 TSH 2:30!) 1 g: 301L0 <3 <> 3 E 3200- - g 20 1 ICKJ' ' I0) ‘ ob w l o W " 10 (N! I .3 I 3 ND 10 4MB 4 43 I 3 N) DOSE OF APOMORPHIME (mo/k0) Figure 4. Dose-related effects of high doses of apomorphine on blood content of pituitary hormones in male rats. Rats were injected subcutaneously with various doses of apo- morphine or vehicle and sacrificed 30 min. later. Each symbol represents the mean of 8 determinations. Vertical lines projected on each symbol represent + 1 standard error; where not shown the standard error is less than the radius of the symbol. Solid symbols indicate values that are sig- nificantly different (P<=O. 05) from vehicle treatment (zero- dose of drug). ' 1:141.w 92 so) 001 20Nl~ . , 4t) - LE '3 gomyt -. a 20- - a. 5 5 es es 2 o _ a a a 1 J J z o a 1 a g fiJ g o .003 .01 .03 .1 3 o .003 .01 .03 I TSH LH “.0021 . |u 60h. . z: :z c: c: a. a 3400 - g 40 - 4 '1:‘ 1: ZCNJ - 2&1- - o L a a J 4- J o b a j a 0 JJ <0 .JIOS .0! 403) .I I) .CNNB ADI 403) .l DOSE OF APOMORPMINE (mo/t0) Figure 5. Effects of low doses of apomorphine on blood content of pituitary hormones in male rats. Rats were injected subcutaneously with various doses of apo- morphine or vehicle and sacrificed 30 min. later. Each symbol represents the mean of 8 determinations. Vertical lines projected on each symbol represent :_1 standard error; where not shown the standard error is less than the radius of the symbol. Solid symbols indicate values that are sig- nificantly different (P<:0.05) from vehicle treatment (zero- dose of drug). 93 of a single dose of apomorphine on blood TSH and prolactin concentrations in intact and thyroidectomized rats by 30 min after injection are given in Table 5. Thyroidectomy produced a greater than 8-fold rise in serum TSH and had no significant effect on blood prolactin. A dose of 0.1 mg/kg apomorphine significantly reduced serum prolactin values by 30 min both in the intact and thyroidectomized animals. The 0.1 mg/kg dose also reduced serum TSH in the intact rats, but neither the 0.1 mg/kg nor 0.3 mg/kg significantly altered TSH in the thyroidectomized rats. Dose-Response Effects of Piribedil on Blood Prolactip,_TSH, Growtthormone and LH Concentrations Like apomorphine, piribedil stimulates dopamine receptors in the central nervous system but the latter drug has a much longer duration of action (Corrodi et_gl,, 1971; Thornburg and Moore, 1973). Since these two drugs share similar actions on the brain their effects on pituitary hormone release also would be expected to be similar. Graded doses of piribedil were injected subcutaneously and blood samples were collected 1 hr later (Figure 6). Piribedil caused dose-related reductions both in serum prolactin and TSH. Similar to apomorphine (Figure 4) the dose of piribedil required to reduce serum TSH was approximately 30 times the dose needed to reduce blood prolactin concentrations. Consistent with the effect of apomorphine on growth hormone release, the lower doses of piribedil (0.3, 1, 3 and 10 mg/kg) increased; whereas, the highest dose (30 mg/kg) was without effect on plasma growth hormone concentrations. Serum LH was significantly reduced to about 60% of control values by the l, 3, 10 and 30 mg/kg doses; however, due to the low basal LH concentra- tions no clear dose-response pattern was observed. Table 5. Effects of Apomorphine 0n Serum Prolactin and TSH Concentra- tions in Intact and Thyroidectomized Rats Prolactin TSH Treatment ng/ml pg/ml Intact Vehicle 28 :_9 0.50 :_0.08 0.1 mg/kg Apomorphine 6 :_1* 0.33 :_0102* Ihyroidectomized Vehicle 30 :_7 3.77 :_0.12 0.1 mg/kg Apomorphine 7 i 13* 3.93 :2 0.82 0.3 mg/kg Apomorphine 6 i. 13* 3.71 2‘. 0.21 Intact 0r thyroidectomized (10 days) rats were injected with vehicle or apomorphine and sacrificed 30 min later. of 8 determinations :_1 standard error. Each value represents the mean *Values significantly reduced from intact vehicle controls, p<=0.05. **Va1ues significantly different from thyroidectomized vehicle controls, p<0.05. 2C1 5 MOR wow: " cowc. ing/ml) 0 Figure 6. 95 ‘ 200. q 5100- - ‘ E h—l—I—d—d—l-J O h—I__—a_.|_|.1 131030 go‘oflalsloao T8143. 0 - g 2|]:g.‘ 1 O - I! 1 I: O I a n n a 444 1 0.3131030 i 10 36 DOSE OF PIRIBESIL (mg/3913 Dose-related effects of piribedil on blood content of pitui- tary hormones in male rats. Rats were injected sc with various doses of piribedil mesylate or vehicle and sacrificed 1 hr. later. Each symbol represents the mean of 8 determinations. Verticle lines pro- jected on each symbol represent + 1 standard error; where not shown the standard error is less than the radius of the symbol. Solid symbols indicate values that are significantly different (p¢:0.05) from vehicle treatment (zero-dose of drug . 96 Effects of Haloperidol Pre-treatment on Apomorphine InducedfChanges_in Blood Hormone’Concentrations To demonstrate that the effects of apomorphine on pituitary secretion were due to the dopamine agonist properties of this drug, apomorphine was administered to animals pre-treated with haloperidol, a dopamine receptor blocker (Janssen g£_al,, 1968). The effects of a standard dose of apomorphine (0.3 mg/kg) on the content of blood hor- mones in rats pre-treated with increasing doses of haloperidol are shown in Figure 7. Haloperidol was administered 3 hr and apomorphine 30 min before blood collection. The larger doses of haloperidol produced a dose-related increase in serum prolactin; whereas, apomorphine signifi- cantly reduced serum prolactin to about 50% of vehicle injected control values. The ability of apomorphine to reduce serum prolactin was pro- gressively diminished by increasing doses of haloperidol and completely inhibited by the highest dose of this drug (1.0 mg/kg). Haloperidol produced only a modest reduction of serum TSH values. Apomorphine alone depressed serum TSH to about 30% of control levels and the higher doses of haloperidol (0.1-1 mg/kg) blocked this apomorphine-induced reduction in serum TSH. Plasma growth hormone was dramatically reduced by haloperidol, the two highest doses (0.3 and 1 mg/kg) causing concentrations to fall to less than 20% of control values. Apomorphine alone (0.3 mg/kg) did not alter resting values of plasma growth hormone. This 0.3 mg/kg dose of apomorphine previously was shown (Figure 4) to be 10 times greater than the dose which maximally elevated plasma growth hormone. - HORMONE couc. (II) 89'" g (I 0‘ o 8 O PRL 97 Figure 7. 0L n g; 4.) 0 .03-151 0.03.131‘ DOSE OF HALOPERIDOL(mg/kg) Effects of increasing doses of haloperidol alone or in combi- nation with apomorphine on blood content of pituitary hormones in male rats. Various doses of haloperidol or vehicle were injected sc 3 hours prior to sacrifice. Thirty minutes prior to sacrifice the same animals received sc injections of apomorphine (0.3 mg/kg) (0 ----- 0) or its vehicle (o——-——o). Each symbol represents the mean of 8 determinations and the vertical line represents i_l standard error. When given in combination with an intermediate dose of haloperidol (0.3 mg/kg), apomorphine elevated plasma growth hormone. Presumably this dose of haloperidol reduces the effective concentration of apomorphine at dopamine receptors into a range that stimulates growth hormone release. However, in combination with the highest dose of haloperidol (1.0 mg/kg), apomorphine had no effect on plasma growth hormone. Serum LH was not altered by either haloperidol, apomorphine or by treatment with the two drugs in combination (not shown). Drug:induced Behavioral Responses Dopaminergic agonists cause a variety of motor responses which are collectively referred to as stereotyped behaviors. In rats these behaviors have been described as a series of dose-related events char- acterized at low doses by sniffing, licking and rearing, and at higher doses by gnawing, biting and restricted locomotor activity. The be- haviors of drug-treated animals in the foregoing experiments were re- corded and the minally effective doses of apomorphine and piribedil necessary to cause stereotypes and to alter blood concentrations of hor- mones are summarized in Table 6. The reduction of prolactin values and the increase in growth hormone values occurred at doses of apomorphine and piribedil that were 1/10 to 1/30 of those necessary to produce stereotyped sniffing. The doses of both dopaminergic agonists that reduced TSH levels also caused stereotypes. Thus, the latter, but not the former drug-induced hormonal responses may be influenced by or associated with the stereotyped behaviors. The failure of the higher doses of apomorphine (l-lO mg/kg) and piribedil (30 mg/kg) to elevate Table 6. 99 Minimally Effective Doses (mg/kg, sc) of Dopaminergic Agonists Required to Alter the Blood Concentration of Pituitary Hormones and to Cause Stereotyped Sniffing in Male Rats Reduce Increase Reduce Stereotyped Prolactin Growth Hormone TSH Sniffing Apomorphine 0.01 0.03 0.3 0.3 Piribedi1 0.30 0.30 10.0 3.0 100 blood content of growth hormone may have resulted indirectly from stress associated with the stereotyped behavior, which then counteracted the direct ability of the drug to release growth hormone. A At low doses neuroleptics selectively block the behavioral ef- fects of dopaminergic aponists and at higher doses these drugs cause catalepsy. In the experiment depicted in Figure 7, the stereotyped sniffing caused by 0.3 mg/kg of apomorphine was blocked by 0.1 but not by 0.03 mg/kg haloperidol; only at the highest dose (1 mg/kg) did halo- peridol cause the characteristic cataleptic response. 0. Conclusions The results of this study demonstrate that pharmacological stimu- lation of brain dopamine receptors produce differential effects on release of prolactin, TSH, growth hormone and LH. Both apomorphine and piribedil evoked dramatic reductions in serum prolactin and TSH which are consistent with the relative dopaminergic properties (potencies and durations of action) of these two drugs. The sensitivity of the prolac- tin response to these agents was about 30-fold greater than that observed for TSH. Blockade of dopamine receptors by haloperidol resulted in dose-related increases in serum prolactin concentrations and tended to reduce serum TSH but blocked the ability of apomorphine to inhibit the release of either hormone. These observations support the view that dopamine mediates on inhibitory influence over prolactin release (Hiikfelt and Fuxe, l972a,b; Meites, 1973; Ojeda 3391., 1974; Clemens gt_gl,, 1974), and also demonstrate that dopamine exerts an inhibitory action on release of TSH. The ability of apomorphine (at doses tested) 101 to suppress TSH release in euthyroid animals was eliminated when TSH was highly elevated by thyroidectomy, but the capacity of apomorphine to reduce serum prolactin was not affected by thyroidectomy. These observ- ations suggest that the lack of feedback inhibition by thyroid hormones is such a powerful stimulus for TSH release that it cannot be overcome by a dopaminergic inhibitory mechanism. Release of growth hormone was stimulated both by apomorphine and piribedil, although this effect was observed only over a lower range of doses for both drugs. The observation that plasma growth hormone was markedly increased at the latest time interval (2 hr, Figure 3) after administration of a relatively large dose of apomorphine (1.0 mg/kg) indicates again that small doses of dopamine agonists stimulate growth hormone release. The ability of haloperidol to reduce plasma growth hormone concentrations suggest that growth hormone may be tonically released by a dopaminergic stimulatory system. The specificity of the growth hormone response to dopamine receptor stimulation is demonstrated by the blockade of this response by the highest dose of haloperidol; whereas, apomorphine prevented the inhibitory action of intermediate doses of haloperidol on growth hormone release. L-dopa, a dopamine precursor, also has been reported to increase blood growth hormone con- centrations in rats (Chen e£_gl,, 1974; Smythe e§_gl,, 1975) and humans (Boyd gt_gl,, 1970; Boden gt_gl,, 1972; Silver g£_gl,, 1973). These results do not agree with the findings of Collu et_al, (1972) who in- jected dopamine (1 pg) intraventricularly and found that this agent reduced plasma growth hormone concentrations in urethane—anesthetized 102 male rats, nor with the observations of MOller et_gl, (1973) who re- ported that L-dopa lowered plasma growth hormone values in rats pre- treated with alpha-methyl-para-tyrosine. However, Martin et_gl, (1975) has shown that the episodic release of growth hormone is inhibited by alpha-methyl-para-tyrosine, a drug which inhibits the synthesis of catecholamines. Possible explanations for these differences are that the drug combinations they employed modified the effect of dopamine or that the doses used were stressful. Stresses were reported to inhibit growth hormone secretion (Schlach and Reichlin, 1968; Collu 93.21:: 1973, 1974; Dunn et_gl,, 1973/74; Krulich et_gl,, 1974; Thesis). Serum LH concentrations were generally reduced by apomorphine and piribedil. These observations are in agreement with the reports that dopamine does not stimulate and may inhibit gonadotropin release. Fuxe and co-workers (Fuxe and H6kfelt, 1969; Ahrén e£_gl:, 1971; Hfikfelt and Fuxe, l972a,b) pr0posed that median eminence dopamine neurons inhib- it LH release in rats. Recently Sawyer (1975) observed that intraven- tricular injections of norepinephrine but not dopamine stimulated LH release in the rabbit, and that dopamine given in combination with nor- epinephrine completely inhibited stimulation of LH release by norepine- phrine. By contrast, Kamberi e;_§1, (1969, 1970b) reported that injec- tions of dopamine into the third ventricle of rats markedly elevated blood concentrations of LH and increased the concentration of LRH in hyp0physea1 portal blood. ig_yitgg.release of LRH by rat hypothalamic fragments also was observed to be stimulated by dopamine (Schneider and McCann, 1970c). Our results on LH are not in agreement with those of 103 Kamberi g; 31, (1969, 1970b) or of Schneider and McCann (1970c), although their experimental designs did not preclude the possible con— version of dopamine to norepinephrine, the latter acting to stimulate LRH-LH release (Sawyer et.al,, 1974; Sawyer, 1975). Last year our laboratory reported (Chen and Meites, 1975a) that drugs which modify catecholamines had little or no effect on TSH secre- tion in the rat. However, the drugs used did not differentiate dopamine from norepinephrine activity. The present results show that stimulation of dopamine receptors inhibited TSH release in the male rat. Grimm and Reichlin (1973) reported that norepinephrine stimulated TRH release from the mouse hypothalamus. These observations suggest that TSH release may be inhibited by dopamine but stimulated by norepinephrine. It has been reported that L—dopa reduces serum TSH in human subjects (Rapoport gt_al,, 1973; Refetoff g§_al,, 1974; Minozzi $3.21,, 1975). The tenden- cy for haloperidol to reduce serum TSH in the present study may be due to the weak noradrenergic blocking properties of this drug (Janssen £51., 1968). The role of the hypothalamic hypophysiotropic hormones in medi- ating the actions of the dopamine agonists and haloperidol on prolactin, TSH, growth hormone and LH release remain to be elucidated. Stimulation of dopaminergic activity is believed to inhibit prolactin release by increasing PIF activity (Meites, 1973) and possibly by a direct action of dopamine on the pituitary (Koch 33-31,, 1970; Shaar and Clemens, 1974). Apomorphine also has been shown to act directly on the pituitary to inhibit the ig_vitro release of prolactin (Smalstig e§_gl,, 1974; 104 Smalstig and Clemens, 1974). Thus, the observed difference in response to prolactin and TSH to both dopamine agonists used in the present study may be due to a direct action of these drugs on the pituitary to inhibit prolactin release; whereas, inhibition of TSH release may be mediated by neuronal mechanisms which require higher doses. TRH has been shown to induce prolactin release in animals (Mueller et_al,, 1973; Convey et.al,, 1973) and man (Jacobs et_gl,, 1971). Our findings demonstrate parallel reductions in both prolactin and TSH by the dopamine agonists, but do not prove that these are mediated via reduction of TRH release. The dose-dependent stimulation of growth hormone secretion by apomor- phine and piribedil may reflect an alteration in the balance of hypo- thalamic GRF and somatostatin release. Low doses of dopamine agonists may stimulate GRF and/or inhibit somatostatin release; whereas, higher doses may not disturb the balance of release between these two hypothala- mic hormones. The dopaminergic agonists produced either no change or minimal decreases in serum LH, suggesting that dopamine does not stimu- late LRH release in male rats. The decrease in prolactin and increase in growth hormone pro- duced by dopaminergic agonists are probably not due to the behavioral effects of these drugs. The minimally effective dose of these drugs for reducing prolactin and increasing growth hormone is 1/10 to 1/30 of that required to induce stereotyped sniffing. However, the minimally effective dose of apomorphine needed to reduce TSH was the same as that which induced stereotyped sniffing, and, therefore, a relationship between the drug induced behavior and the TSH reduction is possible. 105 IV. Effects of Tryptpphan 5-HTP and Restraint Stress on Hypothalamic andIBrain Serotonin Turnover and Pituitary Hormone Release A. Objectives Little is yet known about how alterations in brain serotonin metabolism affect release of TSH and prolactin. Recently Grimm and Reichlin (1973) f0und that serotonin inhibited ig.yjtgg_release of pulse labeled TRH from mouse hypothalamic tissue, suggesting that serotonin may inhibit TSH release ig_!iyg, However, Chen and Meites (l975a) reported that large doses of 5-hydroxytryptophan (S-HTP), the immediate precursor to serotonin, stimulated TSH release in ovariectomized, estro- gen-primed rats. Reports from other laboratories indicated that sero- tonin may either stimulate (Shopsin e§_gl,, 1974) or inhibit (Mess and Peter, 1975; Mueller et_al,, 1976a, Thesis) TRH-TSH release. There is general agreement that serotonin stimulates release of prolactin (see Meites, 1973). Stresses were reported to either inhibit (Ducommun gta_l_., 1966) or stimulate (Krulich and Illner, 1973) release of TSH; whereas, acute stress profoundly increased prolactin release (Krulich gt_gl:, 1974; Euker 22.21:: 1975). Serotonin metabolism was reported to be enhanced by stress (Thierry gt_al,, 1968; Ladisich, 1975). The purpose of this study was to attempt to correlate alterations in brain serotonin metabolism produced by administration of tryptophan, 5-HTP, pargyline and restraint stress with the release of pituitary TSH and prolactin in rats. 106 B. Materials and Methods Male Sprague-Dawley rats weighing 225-250 gm each were used in this study. 0- and L-tryptophan, 5-HTP, pargyline or appropriate vehicle were given as single intraperitoneal injections in a volume of 0.2 m1/ 100 gm at dose and treatment times indicated under Results. Animals were killed by decapitation and biochemical and hormone assays were performed as described in the general Materials and Methods section. Data were analyzed statistically using analysis of variance and the Least Signifi- cant Difference test (Sokal and Rohlf, 1969); the level of significance was chosen as p<:0.05. The time course effects of pargyline on accumu- lation of central serotonin (Figure 8) were analyzed by a least squares regression analysis (Sokal and Rohlf, 1969). The slope of the regression line calculated for data points at 0, 15, 30 and 60 minutes post- treatment was used to determine the turnover rate of brain and hypothala- mic serotonin in nanomoles/gram/hour. When possible, serum concentra- tions of growth hormone were measured following a minor modification in assay reagents which eliminated the need to use plasma samples for growth hormone determinations. Abbreviations used in figures and tables are prolactin (PRL), growth hormone (GH) and 5-hydroxytryptophan (5-HTP). C. Results Time Course Effects of Pargyline on Con- gentrations of HypothalaMic and Brain Serotonin The time course effects of a single injection of pargyline (75 mg/kg, i.p.) on the accumulation of hypothalamic and brain serotonin are shown in Figure 8. Pargyline at a dose of 75 mg/kg was found in our NG\GM 60° '1 Figure 8. 107 _ .- , __ ’ 2400a HYPOTHALAMUS 2000" 1600- I200-L ‘I . 1 l l l ' 0 IS GO GO 120 MINUTES .00.) BRAIN 400‘ , b l 1 I ' I T o ”5 3° 6° ‘20 ‘Time course of the effects of pargyline on hypothalamic and brain concentrations of serotonin in male rats. Rats were injected i.p. with pargyline (75 mg/kg) and killed at various times thereafter. Each symbol represents the mean of 6-7 determinations. Verticle lines projected on each symbol represent :_1 standard error. Zero-time control values represent the serotonin levels of animals treated with vehicle 60 min. prior to decapitation. 108 laboratory (W. Chen, Mueller and Meites, unpublished) and reported by others (Tozer‘gt_gl,, 1966; Lin gt_al,, 1969; Morot-Gaudry e§_al,, 1974) to effectively inhibit brain monoamine oxidase (MAO) activity in rats. A higher dose of pargyline (250 mg/kg) produced a slightly greater in— hibition of MAO (as determined by the accumulation of serotonin after injection) as compared with a dose of 75 mg/kg, but also produced marked behavioral effects and appeared to be toxic to the animals (Chen, Mueller and Meites, unpublished). Pargyline (75 mg/kg, i.p.) caused hypothala- mic and brain serotonin concentrations to rise at linear rates for 60 minutes after injection. The turnover of serotonin in the hypothalamus and brain was calculated to be 4.4 nm/gm/hr and 1.7 nm/gm/hr, respec- tively. ' Time Course Effects of L-tryptophan on Concentrations ofTCerebellUm Tryptophan, Hypofllaljmic and Brain Serotonin, Brain S-HIAA, and Serum’TSH and Prelactin A single injection of L-tryptophan (200 mg/kg, i.p.) produced rapid and prolonged increases in concentrations of cerebellum trypto- phan, hypothalamic and brain serotonin, and brain 5-HIAA in male rats. Serum concentrations of TSH were decreased by 30, 60 and 120 min after injection as compared with vehicle injected control values. Serum pro- lactin concentrations were significantly increased by 30 min but re— turned to control values by 60 and 120 min after L-tryptophan injection. Changes in hypothalamic and brain serotonin, brain 5-HIAA and serum TSH concentrations were maximal by 60 min after injection; whereas, concen- trations of cerebellum tryptophan rose progressively throughout the 2-hour treatment period. 109 Effects of D- and L- t_yptophan on Concentrations of Cerebellum‘Tryptophan, ,flypothalamic andiBrain Serotonin, Brain15- HIAA afidiBlood Hormones A single injection of D-tryptophan (200 mg/kg, i.p.) increased concentrations of cerebellum tryptophan, hypothalamic and brain sero- tonin and brain 5-HIAA in a time related manner (Table 8). These changes were not as great as those produced by the same dose (200 mg/kg) of L-tryptophan by 120 min after injection. Both 0- and L-tryptophan tended to reduce serum TSH and growth hormone concentrations, the L- isomer being more effective than D-tryptophan by 120 min after injection. Serum prolactin was slightly elevated by 30 min after injection of D-tryptophan. Effects of L-tryptOphan in Combination With Pargyiine on Concentrations of Hypothaiamic and Brain Serotonin, and Serum TSH andiPFolactin The effects of increasing doses of L—tryptophan given in combi- nation with pargyline (75 mg/kg) on hypothalamic and brain concentra- tions of serotonin and serum concentrations of TSH and prolactin are shown in Table 9. In addition the effects of 5-HTP (30 mg/kg) alone on the above parameters was tested. All treatments were given as single i.p. injections 30 min prior to decapitation and control rats received appropriate vehicle injections. Pargyline alone significantly increased hypothalamic and brain concentrations of serotonin, reduced serum TSH and had no significant effect on serum prolactin concentrations. Increasing doses of L-tryptophan given in combination with pargyline produced dose-related increases in hypothalamic and brain concentrations of serotonin but did not significantly reduce serum TSH values below .Amo.ou.a .mpocpcoo vmuomncw m-owgm> ease Hemsoemwu appceupw_=m_m moapm>n .coecm weeucmpm P.“ cowumcweeouou sum we came on» mucomocamc m=Fe> seam .emummoeoeu nose» vowewomqm Hm uwHeHFQeumu ecu A.q.w .mx\me oowv cecnouazguuo Lo mpuwno> cur: vapomwcp 0L0: mung 110 F + e SH 3: ..em H an ..2 H :3 48 H 3: ..o; H 3.3 ...E or _H m «a H m: .90. 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Interestingly, 5-HTP markedly increased concentrations of serotonin in the hypothalamus and brain but had no effect on serum TSH as compared to the vehicle injected control mean. This dose of S-HTP produced about a two-fold increase in serum prolactin concentrations. Dose-response Effects of 5-HTP on Concen- trations of’Hypothalamic andlBrain Serotonin. Brain 5-HIAA, and Blood Hormones Graduated doses of 5-HTP dramatically increased concentrations of hypothalamic and brain serotonin and brain S-HIAA by 30 min in a manner which was exponentially related to the log of the S—HTP dose (Figure 9). Serum prolactin concentrations were increased by the 30 mg/kg and lOO mg/kg doses of S-HTP but not by lower doses. All doses of S-HTP significantly reduced serum concentrations of TSH. Serum growth hormone concentrations were not consistently affected by increasing doses of S-HTP. Effects of Restraint Stress on Concentrations of CerEBellum Try tophan, Hypothalamic and’ Brain serotonin rain 5-HIAA, andlBlood' Hormones To further investigate the relationship between brain serotonin metabolism and pituitary TSH, prolactin and growth hormone secretion, effects of stress on these processes was examined. Restraint stress for periods from 5 to l50 min increased concentrations of hypothalamic and brain serotonin, brain S-HIAA and reduced serum TSH and growth hormone concentrations in a time related fashion (Table l0). Serum prolactin was maximally elevated (ll-feld) by 5 and l5 min of restraint but returned towards but not to non-stress control values by 45 and l50 min. Figure 9. 114 " HYPOTHAL 75" ‘ 3mm S-HT S-HY 0. ~ . "Wm WW an a. . Y I V I fir ‘ fi 0 3 IO 30 oo o a to 30 00 (0086 5""? WW) 3mm TSH s—HIAA H 4 1. "9’9” WIN ~ - u 1 1* v o J 10 )0 100 o a 0 :0 oo PRL GH I. m . "04ml Wm! S a. . r v 30 IN ___#\.r .f. __ o o 8 éi o Dose-related effects of S-HTP on concentrations of hypo- thalamic and brain serotonin, brain S-HIAA, and blood hormones in male rats. Rats were injected i.p. with various doses of S-HTP or vehicle and killed 30 min. thereafter. Each symbol repre- sents the mean of 7-8 determinations. Verticle lines pro- jected on each symbol represent 1_l standard error; where not shown standard error is less than the radius of the symbol. Solid symbols indicate values that are significantly differ- ent (P<:0.05) from vehicle treatment (zero-dose of drug). 115 mg» mucmmmgamg mapm> comm .mo.o Va .mpepcou mmmafiwmlco: 59C. flcgmhwwfi Apucmuwmwcmwm mmzpm?’ .gogem veaucmum P.“ meowuozwsgmumu sum mo some .cowumuwamuov weapon eve om— Lo me .mp .m .o co. no:_ngummg use: mama .m H .2 .m H 2 .2 H 2 .3 H 2H .2 Ham .2 H 82 :5 H N: 5... o2 .2 H 22 .m H S ...:H m: .m H SN .2 H mm... .3 H x: 25H 3.. Se 3 .m H :2 .2 H 8 .2H 2: ..2 H 93 2 H a... 8 H ...N: 2.oH 3.... 5:. 2 .2 H mm .2 H cm .8 H Sm .m H mm .2 H mum 8 H :: 25H 2... 5... m mmmgum 2H .8 2 H m 2H :m N H SN m H 8. EH R2 ...EH 8.. 22:8 mmmgumucoz ~E\m= Fs\mc Fs\m: su\m: Em\m: smxuc Saxon acmEummg» Ia age :me <: .cmcqopaxu» s=—_mnmgmu yo meowangucmucou co mmmgpm u=_msumu¢ mo manommm .o_ mpaah 116 Tryptophan concentrations in the cerebellum were not significantly altered by restraint stress. Effect of Restraint Stress on the Accumulation of CentraTFSerotonin in Rats Pre-treatédfwithfi Pargyline The effects of restraint stress on the accumulation of serotonin in the hypothalamus and brains of pargyline treated animals is shown in Figure l0. All animals received pargyline (75 mg/kg) l5 min prior to decapitation. Restraint was administered for the last 5 min or for the entire l5 min pargyline treatment period. Restraint stress produced rapid and time related increases in hypothalamus and brain concentra- tions of serotonin which were significant by l5 min as compared to non— stress control values. D. Conclusions The results of this study demonstrate that stimulation of sero- tonin turnover by administration of either tryptophan or physical restraint is associated with inhibition of TSH and growth hormone, and stimulation of prolactin release. A single injection of L-tryptophan (200 mg/kg) increased concentrations of hypothalamic and brain serotonin and brain S-HIAA indicating stimulation of serotonin synthesis. These findings are in agreement with the earlier work of Grahme-Smith (197l), Carlsson and Lindquist (1972) and Colmenares gt_al, (l976). Correspond- ing with enhanced serotonin metabolism in the hypothalamus and brain, serum TSH and growth hormone were reduced and serum prolactin was increased. D-tryptophan tended to produce similar changes in concen- trations of hypothalamic and brain serotonin and blood hormones but to 1900 E c» \ c» c .E 1800 c o .p 2 a: m U 1700 E P to .c 4.: o E; a: Figure 10. 117 Brain Serotonin ng/gm i a a l 4 a 3 a a [‘4 ‘4 ‘1 Time course of the effects of restraint stress on the accumulation of serotonin in hypothalamus (solid bars) and brain (striped bars) of pargyline treated male rats. All animals received pargyline (75 mg/kg, i.p.)lS min prior to decapitation. Restraint was administered fbr the last 5 min or for the entire l5 min of pargyline treatment. Control animals received pargyline alone. Values are the means of 6 determinations :_l standard error. *Values significantly different from non-stressed controls; p< 0.05. 118 a much lesser extent as compared to an equal dose of L-tryptophan. This difference is probably due to the required conversion of D- to Lu tryptophan in the liver prior to incorporation into brain S-hydroxy- indoles (Yuwiler, 1973). The biosynthetic pathway allows for enhanced metabolism of D-tryptophan as compared to the L-isomer. The turnover rates of hypothalamic and brain serotonin were calculated to be 4.4 nm/gm/hr and l.7 nm/gm/hr, respectively. The dif- ference in rates is probably due to the relatively high content of serotonin nerve terminals located in the hypothalamus as compared with most other brain regions (see Fuxe and Jonsson, l974). Presumably terminal regions on neurons turnover neurotransmitters faster than any other portion. The turnover rates reported here are in close agreement with those determined by many others (Tozer gt_gl,, 1966; Lin gt_gl,, 1969; Hery gt_gl:, l972; Millard gt_gl,, 1972; Carlsson gt_gl:, 1972), although lower than the rate reported by Morot-Gaudry gt_al, (l975). In combination with MAO inhibition by pargyline, L-tryptophan administration produced dose related increases in concentrations of hypothalamic and brain serotonin and in serum prolactin. Failure of L-tryptophan to reduce serum TSH concentrations below those produced by MAO inhibition alone suggest that pargyline causes a maximum inhibition of pituitary TSH release. L-tryptophan was reported to increase brain. tryptamine concentrations (Saavedra and Axelrod, 1973) and produce symptoms of stress (Grahme-Smith, 1971) in rats pre-treated with MAO inhibitors. The possibility that tryptamine or a nonspecific stress effect may be involved in the hormone changes observed following 119 tryptophan or combined pargyline-tryptophan treatment cannot be excluded. However, neither of these treatments produce behavioral responses indi- cating they were stressful to the rats. Injection of 5-HTP, the immediate precursor to serotonin, in- creased concentrations of hypothalamic and brain serotonin in a manner which appears to be exponentially related to the log dose of 5-HTP. Serum prolactin concentrations were generally elevated by 5-HTP. However, in one experiment a single dose of 5-HTP (30 mg/kg) had no effect on serum TSH (Table 9); whereas, in a subsequent dose-response experiment, this dose and others reduced serum TSH (Figure 9). Serum concentrations of growth hormone also were not consistently affected by increasing doses of S-HTP. These findings on TSH and growth hormone may be explained by the effect of exogenous 5-HTP on catecholamine neurons. Dopamine was re- ported to inhibit (Mueller £11.” 197.6b, Thesis) and norepinephrine to stimulate (Grimm and Reichlin, l973) TRH-TSH release. Butcher gt_al, (1972) reported that administration of DL-S-HTP resulted in the appear- ance of indoleamine fluorescence in catecholaminergic nerve cells corre- lated biochemically with increases in central serotonin and reductions in brain dopamine and norepinephrine concentrations. By contrast, administration of L-tryptophan selectively elevated serotonin only in serotonin containing neurons (Aghajanian and Asher. 197l). This suggests the possibility that 5-HTP but not L-tryptophan alters the function of both catecholamine and serotonin neurons, thus producing changes in pituitary secretion which may not be specific to the initial activation of serotonin neurons. This may explain the earlier report of Chen and Meites (l975a) who found that large doses of 5-HTP stimulated TSH 120 release in estrogen-primed ovariectomized rats. A similar situation may exist in the case of growth hormone since serun growth hormone concentra- tions were not consistently altered by 5-HTP but were reduced by adminis- tration of L-tryptophan (Table 8). Restraint stress produced time related increases in concentra- tions of hypothalamic and brain serotonin and brain 5-HIAA (Table 10). In combination with pargyline, restraint significantly increased hypo- thalamic and brain serotonin concentrations by 15 min (Figure 10) indi- cating that stimulation of serotonin turnover is associated with physical restraint was very rapid. Equally rapid were the changes in pituitary TSH, pro1act'in and growth hormone release. Serum TSH and growth hormone fell to 35% and 29% of control values respectively by l5 min of restraint. Prolactin was maximally elevated ll-fold by 5 and l5 min of restraint but returned towards non-stress control values by 45 and 150 min re- straint. The decline in pituitary prolactin release at the longer restraint periods (45 and 150 min) as compared to the shorter periods (5 and 15 min) may be due to the negative feedback of prolactin on its own secretion. Elevated levels of circulating prolactin were reported to enhance median eminence dopamine turnover (Hdkfelt and Fuxe, 1972a,b; Fuxe gt 31,, l974b) and stimulation of dopamine activity is believed to inhibit prolactin release by increasing PIF activity (see Meites, l973) and possibly by a direct action on the pituitary (Shaar and Clemens, l974). Another possible explanation for the decline in serum prolactin concentrations at the longer restraint periods as compared to the short- er periods is that the clearance of this hormone from the serum may be 121 gradually increased to a rate above that at which prolactin is being released from the anterior pituitary. The probable involvement of noradrenergic and other types of neurons in mediating the neuroendocrine changes observed in this study cannot be overlooked. Althouth L-tryptophan appears to be specific for the activation of serotonin neurons (Aghajanian and Asher, 197l), stresses were reported to alter turnover of both catecholamines (Corrodi gt;al:, 1971; Palkovits §t_gl,, l975) and serotonin (Thierry gt 31,, l968; Ladisich, 1975). Cerebellum tryptophan concentrations were not significantly altered by stress suggesting that restraint induced stimulation of sero- tonin metabolism was not due to an increase in precursor availability. This presumes that changes in cerebellum tryptophan reflect the avail- ability of tryptophan within serotonin neurons. Changes in cerebellum tryptophan concentration as affected by diet were reported to generally parallel the changes in tryptophan concentration which occurred in other regions of the brain (Colmenares gt_al:, 1976). The role of the hypothalamic hypophysiotrophic hormones in mediating the effects of stress on TSH prolactin and growth hormone remain to be elucidated. These findings demonstrate an inverse rela- tionship between central serotonin turnover and the release of TSH and growth hormone. These TSH results are in agreement with the findings of Grimm and Reichlin (1973) who reported serotonin inhibited the 1g_!itgg_ release of TRH from mouse hypothalamus. Exogenous TRH has been 122 reported to induce prolactin release in animals (Convey 23.31,, 1973; Mueller §t_al,, 1973) and man (Jacobs gt_al,, 1973). However, the observation that L-tryptophan and stress have opposite effects on TSH and PRL do not support a physiological role for TRH induced prolactin release. Serotonin may inhibit GRF release and/or stimulate the release of somatostatin. Conversely, serotonin may inhibit PIF and/or stimulate the release of prolactin releasing factor. GENERAL DISCUSSION The data presented in this thesis show that both exteroceptive and pharmacological stimuli have differential effects on the secretion of anterior pituitary hormones. Thyrotropin-releasing hormone (TRH) rapidly stimulated the jg_vivg_release of prolactin as well as TSH, sug- gesting that TRH may release prolactin under physiological conditions in the rat. Consistent with this view, Noel gt a1, (1974) reported that the smallest dose—of TRH required to evoke TSH release in humans also produced an increase in serum prolactin concentrations. However, in rats, the pronounced rise in serum prolactin as a result of being placed in a warm temperature, and fall in serum prolactin produced by cold were just opposite to the TSH responses to these temperature changes. The findings presented here and reports of others indicate that the effects of temperature on TSH and prolactin secretion are mediated by the hypo- thalamus and occur independently from associated changes in thyroid and adrenal function. There is considerable evidence that cold-induced TSH release is mediated by enhanced TRH secretion (Reichlin §t_al,, 1972; Hefco 23.91:, 1975c; Montoya gt_gl , 1975). The observation that cold decreased serum prolactin indicates that TRH is not responsible for the temperature-induced changes in prolactin release. This suggests that different mechanisms in the hypothalamus are activated by temperature changes to alter the secretion of TSH and prolactin. Thus, temperature changes provide an interesting approach for studying the differential control of these two hormones. 123 124 Physical restraint stress increased serum prolactin and de- creased serum TSH and growth hormone concentrations. This also suggests that prolactin release is not dependent on TRH release during physical restraint, but may be due to a mechanism involving increased serotonin turnover as shown here. Enhanced rates of hypothalamic serotonin turn- over as produced by administration of L-tryptophan and physical restraint were associated with stimulation of prolactin release and inhibition of TSH and growth hormone release. These results provide evidence that serotonin neurons are involved in the neuroendocrine responses to phy- sical restraint and may act to inhibit the release of prolactin inhibit- ing factor (PIF) and TRH, and to stimulate prolactin releasing factor (PRF) and somatostatin. The decrease in both TSH and growth hormone as a result of physical restraint may be due in part to increased release of hypothalamic somatostatin, since somatostatin can inhibit growth hor- mone release and also can depress TRH-induced release of TSH (Vale gt_gl,, 1975). Serotonergic mechanisms also may be involved in the neuroendo- crine responses to temperature changes. Brain serotonin turnover was reported to be increased by high ambient temperature (Corrodi gt_al,. l968; Aghajanian gt_al,, l968; Reid gt_al,, l968; Weiss and Aghajanian, l97l; Squires, l974) and reduced by low temperature (Corrodi gt_gl,, T967). These findings taken together with hormone results presented here show that brain serotonin turnover and pituitary prolactin release are directly related to ambient temperature; whereas, TSH release is inversely related to ambient temperature and serotonin turnover. This comparison is in agreement with the view that serotonin stimulates 125 prolactin and inhibits TSH and suggests that serotonin neurons mediate certain neuroendocrine responses to temperature changes. The probable involvement of other neurotransmitters in mediat- ing temperature and stress-induced changes in pituitary hormone release cannot be overlooked. Blockade of dopamine receptors by pimozide pre- vented the fall in prolactin release normally produced by cold exposure. Lichtensteiger (1969) reported that low ambient temperature stimulated median eminence dopamine activity as determined by histofluorescence techniques. Together these findings suggest that dopamine neurons also are involved in the cold-induced inhibition of prolactin release. However, this dopaminergic activity must be isolated to a specific pro- lactin control system since low temperatures enhanced TSH secretion; whereas, general pharmacological stimulation of brain dopamine receptors inhibited TSH release. Median eminence (tubero-infundibular) dopamine neurons are believed to function mainly in the inhibitory control of prolactin secretion (Fuxe and kufelt, l969; Ahrén 33 21:, 1971; Hdkfelt and Fuxe, 1972a,b); whereas, virtually nothing is known about function of other hypothalamic dopamine neurons in the control of pituitary hor- mone release. The major portion of hypothalamic dopamine (about 80%) appears to be located outside the arcuate nucleus-median eminence com- plex and recently the presence of a new hypothalamic dopamine system, the incerta—hypothalamic was described (see Bjdrklund gt_al,, 1975). The possibility exists that the incerta-hypothalamic system is involved in the inhibition of TRH-TSH release. It would be interesting if dop- amine turnover in the anterior hypothalamus, the terminal projection (126 area of the incerta-hypothalamic neurons, correlated with physiological changes in TRH-TSH release. Presumably cold temperature would reduce dopamine turnover in this area; whereas, warm temperature and stresses may stimulate dopamine turnover. Similar relationships between the function of incerto-hypothalamic dopamine neurons and the release of other pituitary hormones also may exist and these too await further investigation. Hypothalamic norepinephrine turnover was reported to be stimu- lated by high ambient temperature (Iversen and Simonds, 1969) and physical stress (Lidbrink gt_al,, l972). Similar conditions of tempera- ture and stress stimulated prolactin release and inhibited TSH release (Thesis). Although these few findings suggest that noradrenergic neurons stimulate prolactin and inhibit TSH, other reports indicate that the opposite situation may exist. Grimm and Reichlin (1973) found that norepinephrine stimulated the jg_vitrg_release of TRH from mouse hypo- thalamus, suggesting that norepinephrine stimulates TSH release jg_vjxg, Disulfiram and phentolamine, which block norepinephrine synthesis and receptors, respectively, were reported to inhibit TSH release in rats (Tuomisto gt_gl,, 1973). He observed that low doses of clonidine (an alpha receptor agonist) stimulated TSH release and inhibited prolactin release (Mueller, Simpkins, Meites and Moore, unpublished). Others found that injections of norepinephrine into the third ventricle had little effect on prolactin release in rats (Kamberi gt.al,, l97lc; Ojeda gt_al,, l974b); whereas, other reports showed that norepinephrine may stimulate prolactin release (Donoso gt_gl,, l97l; Meites and Clemens, 127 1972; Lawson and Gala, 1975). At present the physiological role(s) of noradrenergic neurons in the control of prolactin and TSH is unclear. Apparent differences between conclusions made in various reports prob- ably arise from the use of nonspecific drugs, different experimental designs and possibly to multiple functions of noradrenergic neurons on the control ofa single pituitary hormone. Development and application of specific agonists and antagonists to central norepinephrine receptors, measurement of norepinephrine turnover in individual hypothalamic nuclei, and careful electrical stimulation and lesioning of the ascending nor- adrenergic fibers which innervate the hypothalamus used in conjunction with endocrine measurements should further clarify the role of norepine- phrine in the control of prolactin and TSH secretion. In contrast to the effects of norepinephrine on these two hormones, there is accumulat- ing evidence that norepinephrine stimulates LH release and that dopamine can inhibit the stimulatory effect of norepinephrine (Sawyer gt_al,, 1974; Sawyer, 1975). This may explain why in the present study, the two dopamine agonists used, apomorphine and piribedil, either reduced or had no effect on LH release. The effect of norepinephrine on growth hormone release is not clear at present, in contrast to the considerable evidence that dopamine stimulates growth hormone release, as also shown in the present study. Several of the experiments presented in this thesis were designed to investigate the influence of serotonergic and dopaminergic neurons on the release of TSH, prolactin and growth hormone. In the case of prolac- tin and growth hormone, the action of these two neurotransmitters were 128 found to be antagonistic. Dopamine inhibited prolactin and stimulated growth hormone; whereas, serotonin had the opposite effects on the secre- tion of these two hormones. By contrast, both d0pamine and serotonin inhibited TSH release in male rats. These findings are consistent with current understanding of neurotransmitter control of prolactin release (see Meites, l973) and provide new evidence on the regulation of TSH and growth hormone. BIBLIOGRAPHY BIBLIOGRAPHY Adams, J. H., P. M. Daniel, and M. M. L. Prichard: Obwervations on the portal circulation of the pituitary gland. Neuroendocrinology, 1:193-213, 1965. Aghajanian, G. K. and B. L. Weiss: Block by LSH of the increase in brain serotonin turnover induced by elevated ambient temperature. Nature 220:795-796, 1968. Aghajanian, G. K. and I. M. Asher: Histochemical fluorescence of raphe neurons: selective enhancement by tryptophan. Science 172: 1159-1161, 1971. Ahrén, K., K. Fuxe, L. Hamberger, and T. kufelt: Turnover changes in the tubero-infundibular dopamine neurones during the ovarian cycle of the rat. Endocrinology_88:1415-1425, 1971. Ajika, K., S. P. Kalra, C. P. Fawcett, L. Krulich, and S. M. McCann: The effect of stress and nembutal on plasma levels of gonadotrop- ins and prolactin in ovariectomized rats. Endocrinology_90: 707-715, 1972. Amenomori, Y. and J. Meites: Effects of a hypothalamic extract on serum prolactin levels during the estrous cycle and lactation. Proc. Soc. Exp. Biol. Med. 134:492-495, 1970. Amin, A. H., T. B. 8. Crawford, and J. H. Gaddum: The distribution of substance P and 5-hydroxytryptamine in the central nervous sys- tem of the dog. J. Physiol. 126:596-618, 1954. Andén, N. E., A. Rubenson, K. Fuxe, and T. Hdkfelt: Evidence for dopamine receptor stimulation by apomorphine. J. Pharm. Pharmacol. 19:627-629, 1967. Andén, N. E., H. Corrodi, K. Fuxe, B. H6kfe1t, T. Hfikfelt, C. Rydin, and T. Svensson: Evidence for a central noradrenaline receptor stimulation by clonidine. Life Sci. 9:513-523, 1970. Anton-Tay, F. and R. J. Hurtman: Brain monoamines and endocrine func- tion. In; Frontiers in Neuroendocrinolo , 1971, edited by L. Martini and H. F. Ganong, pp. 45-66, Oxfor University Press, New York, 1971. 129 130 Apostolakis, H., S. Kapetanakis, G. Lazos, and A. Madena-Pyraki: Plasma prolactin activity in patients with galactorrhea after treatment with psychotropic drugs. In; LactogenicJHormones, edited by G. E. H. Holstenholme and J. Knight, pp. 349-554, Churchill Livingston, London, 1972. Aschner, B. Uber die Funktion der Hypophyse. Pflfigers Arch. ggs, Physiol. 146:1-146, 1912. Assenmacher, I. and Tixier-Vidal: Repercussions de la section des veines porte hypophysaires sur la prehypophyse du canard pekin male, entier ou castre. Arch. Anal. Microbio. Morphol. Expertl. 53: 83-108, 1964. Averill, R. L. w.: Failure of luteotrophic function due to pituitary grafts in the rat hypothalamus. Neuroendocrinology 5:121-131, 1969. Axelrod, J.: Metabolism of epinephrine and other sympathomimetic amines. Physiol. Rev. 39:751-776. 1959. Bargmann, w. and E. Scharrer: The site of origin of the hormones of the posterior pituitary. Amer. Scientist 39:255-259. 1951. Barry, J. and M. P. Dubois: Immunofluorescence study of LRF-producing neurones in the cat and dog. Neuroendocrinology_18:290-298, 1975. Bartke, A., B. T. Croft, and S. Dalterio: Prolactin restores plasma testosterone levels and stimulates testicular growth in hamsters exposed to short day-length. Endocrinology 97:1601-1604, 1975. Bass, E., J. Shani, Y. Givant, R. Yagil, and F. G. Sulman: The effect of psychopharmacologic prolactin releasers on lactation in sheep. Arch. Int. Pharmacodyn. 211:188-192, 1974. Baylis, E. M., F. Greenwood, V. James, J. Jenkins, J. Landon, V. Marks, and E. Samols: An examination of the control mechanisms postu- lated to control growth hormone secretion in man. In: Growth Hormones, edited by A. Pecile and E. E. Mfiller, pp._BB-104, Exerpta Medica Foundation, Amsterdam, 1968. Ben-Jonathan, H., R. S. Mical, and J. C. Porter: Transformation of 3N—dopamine during transport from CSF to hypophysial portal blood. Endocrinology 96:375-383. 1975a. Ben-Jonathan, H., R. S. Mical, and J. C. Porter: Dopamine (DA) and nor- epinephrine and/or epinephrine (NE-E) in hypophyseal portal plasma, arterial plasma, and hypothalamic tissue. The Endocrine Society, 57th Ann. Meeting. Abst. 291. pp. 196, 1975b. 131 Ben-Jonathan, H., C. Oliver, R. S. Mical, and J. C. Porter: Hypo- thalamic secretion of dopamine into hypophyseal portal blood. Fed. Proc. 35:305. 1976. Bhattacharya, A. N., D. J. Dierschke, T. Yamaji, and E. Knobil: The pharmocologic blockade of the circhoral mode of LH secretion in the ovariectomized rhesus monkey. Endocrinology_90:778-786, 1972. Birge, C. A., L. S. Jacobs, C. T. Hammer, and H. H. Daughaday: Catechol- amine inhibition of prolactin secretion by isolated rat adeno- hypophysis. Endocrinology 86:120-130, 1970. Bivens, C. H., H. E. Lebovitz, and J. M. Feldman: Inhibition of hypo- glycemia-induced growth hormone secretion by the serotonin antagonists cyproheptadine and methysergide. New Egg. J. Med. 289:236-239, 1973. Bj6rk1und, A., R. Y. Moore, A. Nobin, and U. Stenevi: The organization of tuber-infundibular and reticulo-infundibular catecholamine neuron systems in the rat brain. Brain Res. 51:171-191, 1973. Bj6rklund, A., 0. Lindval, and A. Nobin: Evidence of an incerto- hypothalamic dopamine neurons system in the rat. Brain Res. 89:29-42, 1975. Blake, C. A.: Stimulation of pituitary prolactin and TSH release in lactating and proestrous rats. Endocrinology 94:503-508. 1974. Blake, C. A.: Effects of intravenous infusion of catecholamines on rat plasma luteinizing hormone and prolactin concentrations. Endocrinology_98:99-104, 1976. Bleier, R.: The relations of ependyma to neurons and capillaries in the hypothalamus: A golgi-Cox study. J. Comp. Neur. 142:439-463, 1971. Bliss, E. L., J. Ailion, and J. Zwanziger: Metabolism of norepinephrine, serotonin and dopamine in rat brain with stress. J. Pharmacol. Exp. Ther. 164:122-134, 1968. Bliss, E. L., H. Thatcher, and J. Ailion: Relationship of stress to brain serotonin and 5-hydroxyindoleacetic acid. J. Psychiat. figs, 9:71-80, 1972. Gomez-Pan, V. M. Roy, R. C. G. Russell, D. H. oy, A. J. Kastin, and A. V. Schally: Inhibition of gastrin and astric-acid secretion by growth-hormone release-inhibiting hormone. Lancet 2:1106-1109, 1974. Blood, 5. R., C. H. Mortimer, M. 0. Thorner, G. M. BejEer, R. Hall, A. 132 Boden, G., L. E. Lundy, and 0. E. Owen: Influence of Levodopa on serum levels of anterior pituitary hormones in man. Neuroendocrin- ology 10:309-315. 1972. Bogdanove, E. M. and N. S. Halmi: Effects of hypothalamic lesions and subsequeht propylthiouracil treatment on pituitary structure and function in the rat. Endocrinology 53:274-292. 1953. Bohanan, E. H.: Effects of environmental factors on the length of estrous cycles in rats. Amer. J. Hyg. 29:1-10, 1939. Bowers, C. Y.: Studies on the role of cyclic AMP in the release of anterior pituitary hormones. Ann. N. Y. Acad. Sci. 185:263-290, 1971. Bowers, C. Y., H. G. Friesen, P. Hwang, H. J. Guyda, and K. Folkers: Prolactin and thyrotropin release in man by synthetic pyroglu- tamyl-histidyl-prolinamide. Biochem. Biophys. Res. Comm. 45: 1033-1041, 1971. Boyd, A. E., H. E. Lebovits, and J. B. Pfeiffer: Stimulation of growth hormone secretion by L-dopa. New Engl. J. Med. 283:1425-1429, 1970. Brazeau, P., H. Vale, R. Burgus, N. Ling, M. Butcher, J. Rivier, and R. Guillemin: Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone. Science 179:77-79, 1973. Brown, G. H., D. S. Schalch, and S. Reichlin: Patterns of growth hormone and adrenal stress response in the squirrel monkey. Endocrin- ology 88:956-963. 1971. Brown, M. A., M. H. Van Hoert, and L. M. Ambani: Effect of apomorphine bn growth hormone release in humans. J. Clin. Endocrinol. Metab. 37:463-465. 1973. Brown-Grant, K., C. van Euler, G. N. Harris, and S. Reichlin: The measurement and experimental modification of thyroid activity in the rabbit. J. Physiol. 126:1-28, 1954a. Brown-Grant, K., G. N. Harris, and S. Reichlin: The effect of emotional and physical stress on thyroid activity. J. Physiol. 126:29-40, 1954b. Brownstein, M. J., J. M. Saavedra, M. Palkovits, and J. Axelrod: Histamine content of hypothalamic nuclei of the rat. Brain Res. 77:151-156, 1974. 133 Brownstein. M., A. Arimura, H. Sato. A. V. Shally, and J. S. Kizer: The regional distribution of somatostatin in the rat brain. Endocrinology_96:1456-1461, 1975a. Brownstein. M., R. Kobayashi, M. Palkovits. and J. M. Saavedra: Choline acetyltransferase levels in diencephalic nuclei of the rat. J. Neurochemistry 24:35-38. 1975b. Brownstein, M. J., M. Palkovits. J. M. Saavedra. and J. S. Kizer: Distribution of hypothalamic hormones and neurotransmitters within the diencephalon. In; Frontiers in Neuroendocrinology, 1016 4, edited by H. F. Ganong, pp.‘1-2§. Raven Press, New York, 97 . Burgus. R., T. F. Dunn, D. Desidiero, and R. Guillemin: Structure mole- culaire du facteur hypothalamique hypophysiotrope TRF d' origine ovine: mise en evidence par spectrometrie de masse de la séquence PCA-His-Pro-NHZ. C.r.- 'hebd. Séanc. Acad. Sci. 268:2116- 2118, 1969. Burgus, R. and R. Guillemin: Hypothalamic releasing factors. BEE: Rex, Biochem. 39:499-525. 1970. Burgus, R., T. F. Dunn. D. Desiderio. 0. M. Ward. M. Vale, and R. Guillemin: Characterization of ovine hypothalamic hypophysio- tropic TSH-releasing factor. Nature Lond. 226:321-325. 1970. Burt, 0. R. and S. Snyder: Thyrotropin releasing hormone (TRH): apparent receptor binding in rat brain membranes. Brain Res. 93:309-328. 1975. Butcher, L. L., J. Engel, and K. Fuxe: Behavioral. biochemical, and histochemical analysis of the central effects of monoamine precursors after peripheral decarboxylase inhibition. Brain Res. 41:387-411. 1972. Cahane, M. and T. Cahane: Sur l'existence des centres nerveux infundibu- laris reglant la fonction du corps thyroide. Acta. Med. Scand. 94:320-327. 1938. Caligaris, L. and S. Taleisnik: Involvement of neurones containing 5- hydroxytryptamine in the mechanism of prolactin release induced by oestrogen. J. Endocr. 62:25-33, 1974. Camus. J. and G. Roussy: Experimental researches on the pituitary body. Endocrinology.4:507-522, 1920. Carlsson. A., A. Dahlstrom. K. Fuxe. and N. A. Hillays: Cellular locali- zation of brain monoamines. Acta Physiol. Scand. 56:Supp1. 196, 1—28. 1962. 134 Carlsson. A. and M. Lindquist: Effects of chlorpromazine and haloperidol on formation of 2 methoxytyramine and normethanephrine in mouse brain. Acta. Pharmacol. Toxicol. 20:371-374. 1963. Carlsson, A. and M. Lindquist: The effect of L-tryptophan and some psy- chotropic drugs on the formation of 5-hydroxytryptophan in mouse brain jfl_vivo. J. Neural. Trans. 33:23-43. 1972. Carlsson. A. and M. Lindquist: Ig_vivo measurements of tryptophan and tyrosine hydroxylase activities in mouse brain. J. Neural. Transm. 34:79-91. 1973. Carr. L. A. and K. E. Moore: Effects of reserpine and alpha-methyltyro- sine on brain catecholamines and pituitary-adrenal responses to stress. Neuroendocrinology_3:285-302. 1968. Carrer, H. F. and S. Taleisnik: Effect of mesencephalic stimulation on the release of gonadotropins. J. Endocrinol. 48:527-539. 1970. Carrer, H. F. and S. Taleisnik: Neural pathways associated with the mesencephalic inhibitory influence on gonadotropin secretion. Brain Research 38:299-313. 1972. Cassell, E. E., J. Meites. and C. H. Helsch: Effects of ergocornine and ergocryptine on growth of 7.12-dimethylbenzanthracene-induced mamnary tumors in female rats. Cancer 23:601-607. 1971. Chen. C. L., H. Minaguchi. and J. Meites: Effects of transplanted pituitary tumors on host pituitary prolactin secretion. Proc. Soc. Exp. Biol. Med. 126:317-320, 1967. Chen. C. L., E. J. Baker. A. I. Weber, and J. Meites: Hypothalamic stimulation of prolactin release from the pituitary of turkey hens and poults. Gen. Comp. Endocrinol. 11:489-494. 1968. Chen. C. L. and J. Meites: Effects of estrogen and progesterone on serum and pituitary prolactin levels in ovariectOmized rats. Endocrinology_86:503-505. 1970. Chen. C. L., Y. Amenomori, K. H. Lu, J. L. Voogt, and J. Meites: Serum prolactin levels in rats with pituitary transplants or hypo- thalamic lesions. Neuroendocrinology_6:220-227. 1970. Chen. H. J., G. P. Mueller. and J. Meites: Effect of L-dopa and somato- statin on suckling induced release of prolactin and GH. Endocrine Res. Comm. 1:283-291. 1974. Chen. H. J. and J. Meites: Effect of biogenic amines and TRH on release of prolactin and TSH in the rat. Endocrinology 96:10-14. l975a. 135 Chen. H. J. and J. Meites: Effects of TRH and somatostatin (SRIF) on TSH and prolactin release at high and low temperature. Fed. Proc. 34:342. l975b. Clemens. J. A. and J. Meites: Inhibition by hypothalamic prolactin implants of prolactin secretion, mammary growth and luteal func- tion. Endocrinology 82:878-881. 1968. Clemens. J. A., M. Sar, and J. Meites: Termination of pregnancy in rats by a prolactin implant in median eminence. Proc. Soc. Exper. Biol. Med. 130:628-630. 1969a. Clemens, J. A., M. Sar. and J. Meites: Inhibition of lactation and luteal function in postpartum rats by hypothalamic implantation of prolactin. Endocrinology 84:868-872. 1969b. Clemens, J. A., R. V. Galla. D. I. Hhitmoyer, and C. H. Sawyer: Prolac- tin responsive neurones in the rabbit hypothalamus. Brain Res. 25:371-379. 1971. Clemens. J. A. and B. D. Sawyer: Identification of prolactin in cerebro- spinal fluid. ExpL Brain Res. 21:399-402. 1974. Clemens, J. A., C. J. Shaar, E. B. Smalstig. and C. Matsumoto: Effects of some psycoactive agents on prolactin secretion in rats of different endocrine states. Horm. Metab. Res. 6:187-190. 1974. Clemens. J. A., E. B. Smalstig. and C. J. Shaar: Inhibition of prolactin secretion by lergotrile mesylate: mechanism of action. Acta. Endocrin. 70:230-237. 1975. Collu. R., F. Fraschini, P. Visconti, and L. Martini: Adrenergic and serotonergic control of growth hormone secretion in adult male rats. Endocrinology_90:1231-1237. 1972. Collu, R., J. C. Jéquier. J. Letarte, G. Leboeuf. and J. R. Ducharme: Effect of stress and hypothalamic deafferentation on the secre- tion of growth hormone in the rat. Neuroendocrinology_11:183- 190. 1973. Collu. R., G. Leboeuf. J. Letarte, and J. R. Ducharme: Mechanism of the inhibitory effect of stress on rat growth hormone secretion. EndocrinologyD94zA227. 1974. Colmenares. J. L., R. J. Hurtman. and J. D. Fernstrom: Effect of ingest- ing a carbohydrate-fat meal on the levels and synthesis of 5-hydroxyindoles in various regions of the rat central nervous system. J. Pharmacol. Exp. Ther.: In press, 1976. 136 Convey. E. M., H. A. Tucker. V. G. Smith, and J. Zolman: Bovine prolac- tin. growth hormone. thyroxine and coritcoid response to thyrotropin-releasing hormone. Endocrinology 92:471-476. 1973. Convey. E. M., V. G. Smith, K. Mongkonpunya. J. Zolman. and H. D. Hafs: Clinical applications of hypothalamic hormones in animals. In: Hypothalamic Hormones. edited by E. S. E. Hafex and J. R. ReET. ppkw - nn r or Science Publishers Inc. . Ann Arbor, 1975. C00per. J. R., F. E. Bloom. and R. H. Roth: Catecholamines. In: The Biochemical Basis of Neuropharmacology. pp. 80-141. Oxford -__' University Press. Inc., London. 1971. Corrodi. C. . K. Fuxe, and T. H6kfe1t: A possible role played by central monoamine neurones in thermoregulation. Acta. Physiologica Scandinavica 71: 224-232. 1967. Corrodi. H., K. Fuxe. and V. Ungerstedt: Evidence fbr a new type of dopamine receptor stimulating agent. J. Pharm. Pharmacol. 12:989-991. 1971a. Corrodi, H., K. Fuxe. P. Lidbrink, and L. Olson: Minor tranquilizers, stress and central catecholamine neurons. Brain Res. 29:1-16. l97lb. Corrodi. H., K. Fuxe. T. H6kfe1t. P. Lidbrink. and U. Ungerstedt: Effect of ergot drugs on central catecholamine neurons: evidence for stimulation of central dopamine neurons. J. Pharm. Pharmacol. 25:407-411. 1973. r Coyle. J. T. and 0. Henry: Catecholamines in fetal and newborn rat brain. J. Neurochem. 21:61-67. 1973. Cramer, O. M. and J. C. Porter: Input to releasing factor cells. In: ___gress in Brain Research. edited by E. Zimmerman, H. H. G1spen, B. H. ‘Marks and D. DeHied, pp. 72- 85, Elsevier Scientific Pub- lishing Company. New York,1973. Cuello, A. C.. R. Hiley. and L. L. Iversen: Use of catechol O-methyl- transferase for the enzyme radiochemical assay of dopamine. J. Neurochem. 21:1337-1340. 1973. Curzon, G. and A. R. Green: Rapid method for the determination of S-hydroxytryptamine and 5-hydroxy-indoleacetic acid in small regions of rat brain. Br. J. Pharmacol. 39:653-655. 1970. Dahlstrom, A. and K. Fuxe: Evidence for the existence of monoamine- containing neurons in the central nervous system. I. Distribu- tion of monoamines in cell bodies of brain stem neurons. Acta. Physiol. Scand. 62:Supp1. 232, 1-55. 1964. 137 Dairman, H., R. Gordon. S. Spector, A. Sjoerdsma. and S. Undenfriend: Increased synthesis of catecholamines in the intact rat follow- ing administration of a-adrenergic blocking drugs. 591: Phanmacol. 4:457-464. 1968. D'Angelo. S. A.: Central nervous regulation of the secretion and re- lease of thyroid stimulating hormone. In: Advances in Neuro- endocrinology, edited by A. V. NalbanddV: pp. ISB-ZOS. Univer- s ty 0 1nois Press. Urbana, 1963. D'Angelo. S. A.: Simultaneous effects of estradiol on TSH secretion and adrenocortical function in male and female rats. Endocrinology_ 82:1035-1041. 1968. Daniel. P. H.: The anatomy of the hypothalamus and pituitary gland. In: Neuroendocrinolo , Vol. I, edited by L. Martini and N. F. Ganong, pp. 15-80. Aca emic Press, New York. 1966. Denckla. H. D. and H. K. Dewey: The determination of tryptophan in plasma, liver and urine. J. Lab. Clin. Med. 69:160-169. 1967. Desclin. L.: A propos du mecanisme d‘action des oestrogenes sur le lobe anterieur de l'hypophyse chez le rat. Ann. Endocr. 11:656-659. 1950. Deuben, R. R. and J. Meites: Stimulation of pituitary growth hormone release by hypothalamic extract ig_vitro. Endocrinology_74:404- 414. 1964. Dey. F. L.: Evidence of hypothalamic control of hypophyseal gonadotropic function in the female guinea pig. Endocrinology_33:75-82. 1943. Dibbet. J. A., M. J. Boudreau. J. F. Bruni. and J. Meites: Possible role of dopamine in modifying prolactin (PRL) response to TRH. Endocrinology_94:A-186, 1974. Dickerman, E., S. Dickerman. and J. Meites: Influence of age. sex and estrous cycle on pituitary and plasma GH levels in rats. In: Growth and Growth Hormone. edited by A. Pecile and E. E. MUT1er. pp. 252-265, Exerpta MEEica Foundation. Amsterdam. 1972. Dickerman, S.. G. Kledzik, M. Gelato, H. J. Chen. and J. Meites: Effects of haloperidol on serum and pituitary prolactin, LH and FSH and hypothalamic PIF and LRF. Neuroendocrinology_15:10-20. 1974. Donoso. A. O. and M. B. DeGutierrez-Mayano: Adrenergic activity in hypothalamus and ovulation. Proc. Soc. Exp. Biol. Med. 135:633- 641. 1970. 138 Donoso. A. 0., N. Bishop, C. P. Fawcett, L. Krulich. and S. M. McCann: Effect of drugs that modify brain monoamine concentrations on plasma gonadotropin and prolactin levels in the rat. Endocrinology 89:774-784. 1971. Donoso. A. 0.. H. Bishop. and S. M. McCann: The effects of drugs which modify catecholamine synthesis on serum prolactin in rats with median eminence lesions. Proc. Soc. Exp. Biol. Med. 143:360- 363, 1973. Donoso. A. 0., A. M. Banzan. and M. I. Borzino: Prolactin and luteiniz- ing hormone release after intraventricular injection of histamine in rats. J. Endocr. 68:171-172. 1976. Dott. N. M.: An investigation into the functions of the pituitary and thyroid glands. Part 1. Technique of their experimental sur- gery and summary of results. Quart. J. Exp. Physiol. 13:241- 282. 1923. Douglas. H. H.: Histamine and antihistamines. S-hydroxytryptamine and antagonists. In: The Pharmacolotical Basis of Therapeutics. 4th Edition. ed1tedby L. S. Goodman and A. Gilman. pp. 621-662. Macmillan Co., London, 1970. Ducommun. P., E. Sakiz, and R. Guillemin: Lability of plasma TSH levels in the response to nonspecific exteroceptive stimuli. Proc. Soc. Exp. Biol. Med. 121:921-923. 1966. Dular, R., F. LaBella. S. Vivian. and L. Eddie: Purification of prolac- tin-releasing and inhibiting factors from beef. Endocrinology_ 94:563-567. 1974. Dunn, J. D.. H. J. Schindler. M. D. Hutchins. L. E. Scheving, and C. Turpen: Daily variation in rat growth hormone concentration and effect of stress on periodicity. Neuroendocrinology.13:69- 78, l973/74. Eddy, R. L., A. L. Jones. Z. H. Chakmakjian, and M. C. Silverthorne: Effect of Levodopa (L-dopa) on human hypophyseal trophic hormone release. J. Clin. Endocrinol. Metab. 33:709-712. 1971. Engstrom, G., T. H. Svensson. and B. Haldeck: Thyroxine and brain cate- cholamines: increased transmitter synthesis and increased receptor sensitivity. Brain Res. 77:471-483. 1974. Engstrom, G., U. Strfimbom. T. H. Svensson. and B. Naldeck: Brain mono- amine synthesis and receptor sensitivity after single or re— peated administration of thyroxine. J. Neural Transm. 37:1-10. 1975. I ' 139 Eskay, R. L., C. Oliver. N. Ben-Jonathan. and J. C. Porter: Hypothalamic hormones in portal and systemic blood. Hypothalamic Hormones: Chemistry._Physiology. Pharmacology and Clinical Uses. Internal. Soc. Neuroendo. Serono Symposia, Milano, Italy. 1974. Euker. J. S.. J. Meites. and G. D. Reigle: Effects of acute stress on serum LH and prolactin in intact. castrate and dexamethasone- treated male rats. Endocrinology 96:85-92. 1975. Everett. G. M. and J. N. Borcherding: L-Dopa: effect on concentrations of dopamine. norepinephrine. and serotonin in brains of mice. Science 168:849-850, 1970. Everett, J. H.: Luteotrophic function of autographs of the rat hypophy- sis. Endocrinology 54:685-690. 1954. Everett, J. H.: Functional corpora lutea maintained for months by auto- graphs of rat hypophysis. Endocrinology 58:786-796. 1956. Everett, J. H., C. H. Sawyer. and J. E. Markee: A neurogenic timing factor in control of the ovulating discharge of luteinizing hormone in the cyclic rat. Endocrinology 44:234-250. 1949. Everett, J. H. and IH..Nikitovitch-Niner: Physiology of the pituitary gland as affected by transplantation or stalk transection. IQ; Advances in Neuroendocrinolo . edited by A. V. Nalbandov, pp. 2893304. Univ. 111. Press. Ur ana, 1963. Falck, B.. N. A. Hillarp. G. Thieme, and A. Tharp: Fluorescence of cate- cholamines and related compounds condensed with formaldehyde. J. Histochem. Cytochem. 10:348-354. 1962. Farnsworth, H. E.: Prolactin and the prostate. In: Prolactin and Carcino enesis. edited by A. R. Boyns and‘Kl Griffiths. pp. 217- 224. Aipfia 0559a Alpha Pub., Cardiff. Hales, 1972. Fernstrom. J. D. and R. J. Wurtman: Brain serotonin content: physio- logical dependence on plasma tryptophan levels. Science 173: 149-152. 1971. Fernstrom. J. D. and R. J. Hurtman: Brain serotonin: physiological regulation by plasma neutral amino acids. Science 178:414-416, 1972. “““" Fiorindo, R. P. and L. Martini: Evidence for a cholinergic component in the neuroendocrine control of luteinizing hormone (LH) secretion. Neuroendocrinology_18:322-332. 1975. Florsheim. H. H.: Control of thyrotropin secretion. _I_p_: Handbook of Ph siolo : Section 7._Endocrinolo Vol. IV. Part 2, edited by R. O. Breep and‘E. B. Astwood. pp. 449-468. Williams and Wilkins. Baltimore. 1974. 140 Folkers, K., F. Enzmann. J. Boler. C. Y. Bowers, and A. V. Schally: Discovery of modification of the synthetic tripeptide-sequence of the thyrotropin releasing hormone having activity. Biochem. Biophys. Res. Commun. 37:123-126. 1969. Frantz. A. G., D. V. Habif. G. A. Hyman. and H. K. Suh: Remission of metastatic breast cancer after reduction of circulating prolac- tin in patients treated with L-Dopa. Clin. Res. 20:864. 1972. Frantz. A. G., D. V. Habif, G. A. Hyman, H. K. Suh. J. F. Sassin. E. A. Zimmerman. G. L. Noel. and D. L. Kleinberg: Physiological and pharmacological factors affecting prolactin secretion. including its suppression by L-Dopa in treatment of breast cancer. In; Human Prolactin. edited by J. L. Pasteels, C. Robyn, and F. J. G. Ebling. pp. 273-290. American Elsevier Pub. Comp. Inc., New York. 1973. ' Friedman. P. A., A. H. Kappelman, and S. Kaufman: Partial purification and characterization of tryptophan hydroxylase from rabbit hind brain. J. Biol. Chem. 237:4167-4173. 1972. Friesen, H., H. Guyda, P. Hwang, J. E. Tyson, and A. Barreau: Func- tional evaluation of prolactin secretion: a guide to therapy. J. Clin. Invest. 51:706-709. 1972. Fujimoto. W., J. Ensinck. and R. Williams: Somatostatin inhibits insulin and glucagon release by monolayer cultures of rat endo- crine pancreas. Life Sci. 15:1999-2004. 1974. Fuxe, K.: Cellular localization of monoamines in the median eminence and infundibular stem of some mammals. Acta. Physiol. Scand. 58:383-384. 1963. Fuxe, K.: Cellular localization of monoamines in the median eminence and infundibular stem of some mammals. Z. Zellforsch. 61:710- 724. 1964. Fuxe. K.: Evidence of the existence of monoamine neurons in the central nervous system. IV. Distribution of monoamine nerve terminals in the central nervous system. Acta. Physiol. Scand. 64:Supp1. 247:37-85. 1965. Fuxe, K. and T. Hokfelt: Further evidence for the existence of tubero- infundibular dopamine neurons. Acta. Physiol. Scand. 66:243- 244. 1966. Fuxe. K., T. H6kfelt. and U. Ungerstedt: Localization of indolealkyl- amines in CNS. Advan. Pharmacol. 6A:235-251. 1968. 141 Fuxe. K. and T. Hdkfelt: Catecholamines in the hypothalamus and pitui- tary gland. IQ: Frontiers in Neuroendocrinology, edited by L. Martini and W. F. Ganong, pp. 47-96. Oxford University Press. London, 1969. Fuxe. K. and G. Jonsson: Further mapping of central 5-hydroxytryptamine neurons: studies with the neurotoxic dihydroxytryptamines. lg; Advances in Biochemical Psychopharmacology. Vol. 10. edited by E. Costa, G.BL.fiGessa. and M. Sandler. pp. 1-12. Raven Press, New York, 1974. Fuxe. K., H. Corrodi. T. Hdkfelt. P. Lidbrink. and U. Ungerstedt: Ergocornine and 2-Br-a- Ergocryptine. Evidence for prolonged dopamine receptor stimulation. Medical Biol. 52:121-132. l974a. Fuxe, K., M. Goldstein. T. H6kfelt. G. Jonsson. and P. Lidbrink: Dopaminergic involvement in hypothalamic function: extrahypo- thalamic and hypothalamic control. A neuroanatomical analysis. Advances in Neurology 5:405-419, 1974b. Gala. R. R. and R. P. Reece: Influence of neurohormones on anterior pituitary lactogen production jn_vitro. Proc. Soc. Exp. Biol. Med. 120:220-222. 1965. Gautvik. K. M., B. D. Weintraub. C. T. Graeber, F. Maloof. J. E. Zucker- man. and A. H. Tashjian, Jr.: Serum prolactin and TSH: effects of nursing and pyroGlu-His-ProNH2 administration in postpartum women. J. Clin. Endo. Metab. 37.135-139. 1973. Glowinski, J. and J. Axelrod: Effects of drugs on the uptake, release. and metabolism of H3-norepinephrine in the rat brain. J. Phar- macol. Exp, Ther. 149:43-49. 1966. Glowinski, J., I. Kopin, and J. Axelrod: Metabolism of H3-norepine- phrine in the rat brain. J. Neurochem. 12:25-30. 1965. Goldstein. M., A. F. Battista. B. Anagnoste, P. M. Ceasar, K. Fuxe. and T. Hokfelt: The effect of ventromedial tegmental lesions on the disposition and biosynthesis of dopamine and serotonin. In: Advances in Biochemical Psychopharmacology. Vol. 10. editea'by E. Costa. GT’L.FGéssa. ahd”MT"Sendler. pp. 4553. Raven Press. New York, 1974. Gordon. R., S. Spector, A. Sjoerdsma. and S. Ubenfriend: Increased syn- thesis of norepinephrine and epinephrine in the intact rat dur- ing exercise and exposure to cold. J. Pharmacol. Exp. Ther. 153:440-447. 1966. 142 Grahame-Smith. D. G.: Studies jg_vivo on the relationship between brain tryptophan, brain 5-HT synthesis and hyperactivity in rats treated with a monoamine oxidase inhibitor and L-tryptophan. J. Neurochem. 18:1053-1066. 1971. Grandison. L., M. Gelato, and J. Meites: Inhibition of prolactin secre- tion by cholinergic drugs. Proc. Soc. Exper. Biol. Med. 145: 1236-1239. 1974. Green, J. D. and G. W. Harris: The neurovascular link between the neurohypophysis and adenohypophysis. J. Endocrinol. 5:136-146. 1947. Green. J. D. and G. W. Harris: Observation of the hypophysioportal vessels in the living rat. J. Physiol. 108:359-361, 1949. Greer. M. A.: The role of the hypothalamus in control of thyroid func- tion. J. Clin. Endocrinology_12:1259-1268. 1952. Greibrokk. T., B. L. Currie. K. N. Johansson. J. J. Hansen. and K. Folkers: Purification of a prolactin inhibiting hormone and the revealing of hormone DOGHIH which inhibits the release of growth hormone. Biochem. Biophys. Res. Commun. 59:704-709. 1974. Grimm. Y. and S. Reichlin: Thyrotropin-releasing hormone (TRH): neurotransmitter regulation of secretion by mouse hypothalamic tissue in vitro. Endocrinology 93:626-631. 1973. Groot. J. de. and G. W. Harris: Hypothalamic control of the anterior pituitary gland and blood lymphocytes. J. Physiol. 111:335-346, 1950. Grosvenor. C. E., S. M. McCann. and M. D. Nallar: Inhibition of suckling- induced release of prolactin in rats injected with acid extract of bovine hypothalamus. Pro ram 46th Meeting of the Endocrine Society, San Francisco. p 92. 1964. Grosvenor. C. E., S. M. McCann, and R. Nallar: Inhibition of nursing- induced and stress-induced fall in pituitary prolactin concen- tration in lactating rats by injection of acid extracts of bovine and rat hypothalamus. Endocrinology_76:883-889. 1965. Guillemin. R., W. R. Hearn. W. R. Cheek, and D. E. Housholder: Control of corticotrophin release: further studies with jg_vitro methods. Endocrinology 60:488-506. 1957. Guillemin. R., E. Yamazaki, D. A. Gard. M. Jutiza. and E. Sakiz: In vitro secretion of thyrotropin (TSH): stimulation by a hypo- thalamic peptide (TRF). Endocrinology_73:564-572. 1963. 143 Gunne. L. M.: Relative adrenaline content in brain tissue. Acta. Physiol. Scand. 56:324-333. 1962. Haeusler. G.: Clonidine-induced inhibition of sympathetic nerve activ- ity: no indication for central presynaptic or an indirect sym- pathomimetic mode of action. Naupyn-Schmiedebergs Arch. Pharmacol. 286:97-111. 1974. Hafiez. A. A., C. W. Lloyd. and A. Bartke: The role of prolactin in the regulation of testis function: the effects of prolactin and luteinizing hormone on the plasma levels of testosterone and androstenedione in hypophysectomized rats. J. Endocr. 52:325- 332. 1972. "“"“" Haighton, J.: An experimental enquiry concerning animal impregnation. Phil. Trans. P. Soc. 87:159-196. 1797. Halasz. 8.: The endocrine effects of isolation of the hypothalamus from the rest of the brain. IQ; Frontiers in Neuroendocrinology, edited by W. F. Ganong and L. Martini. pp. 307-342. Raven ress, New York, 1969. Hall, R., G. M. Besser, A. V. Schally. D. H. Coy. C. Evered. D. J. Goldie, A. J. Kastin, A. S. McNeilly. C. H. Mortimer, C. Phenekos. W. M. G. Tunbridge, and 0. Weightman: Action of growth hormone-release inhibitory hormone in healthy men and in acromegaly. Lancet 2:581-584. 1973. Harms. P. G., S. R. Ojeda. and S. M. McCann: Prostaglandin involvement in hypothalamic control of gonadotropin and prolactin release. Science 181:760-761, 1973. Harms. P. G., S. R. Ojeda. and S. M. McCann: Prostaglandin-induced release of pituitary gonadotropins: central nervous system and pituitary sites of action. Endocrinology 94:1459-1464, 1974. Harris, G. W.: The induction of ovulation in the rabbit by electrical stimulation of the hypothalamo-hypophyseal mechanism. Proc. Roy. Soc. B. 122:374-394. 1937. Harris. G. W.: Electrical stimulation of the hypothalamus and the mechanism of neural control of the adenohypophysis. J. Physiol., Lond. 107:418-429, 1948a. Harris, G. W.: Neural control of the pituitary gland. Physiol. Rev. 28:139-179. 1948b. Harris, G. W.: .Neural Control of the Pituitary_Gland. Edward Arnold. Lon don. 1955. 144 Harris. G. W.: Humours and hormones. J. Endocr. 53:ii-xxiii. 1972. Harris. G. W. and D. Jacobsohn: Proliferative capacity of hypophyseal portal vessels. Nature Lond. 165:819. 1950. Haterius, H. 0. and A. R. Derbyshire, Jr.: Ovulation in the rabbit following upon stimulation of the hypothalamus. Am. J. Physiol. 119:329-330. 1937. Hefco. E., L. Krulich, and J. E. Aschenbrenner: Effect of hypothalamic deafferentation on the secretion of thyrotropin in resting con- ditions in the rat. Endocrinology 97:1226-1233. 1975a. Hefco, E., L. Krulich. and J. E. Aschenbrenner: Effect of hypothalamic deafferentation on the secretion of thyrotropin during thyroid blockade and exposure to cold in the rat. Endocrinology 97:1234- 1240. 1975b. Hefco. E., L. Krulich. P. Illner, and P. R. Larsen: Effect of acute exposure to cold on the activity of the hypothalamic-pituitary- thyroid system. Endocrinology 97:1185-1195. l975c. Hery. F.. E. Rouer, and J. Glowinski: Daily variations of serotonin metabolism in rat brain. Brain Res. 43:445-455. 1972. Hillarp. N. A.: Studies on the localization of hypothalamic centers con- trolling the gonadotrophic function of the hypophysis. Acta. Endocrinol. 2:11-23, 1949. Hoch. F. L.: Metabolic effects of thyroid hormones. In: Handbook of Physiology, Sec. 1. Vol. III. edited by R. O. GFeep and E. B. Astwood. pp. 391-411. Williams and Wilkins. Baltimore, 1974. H6kfelt. 1.: Morphological contributions to monoamine pharmacology. Fed. Proc. 33:2177-2186. 1974. H6kfe1t. T. and K. Fuxe: Effects of prolactin and ergot alkaloids on the tubero-infundibular dopamine neurones. Neuroendocrinology 9:100-122. l972a. H6kfelt, T. and K. Fuxe: On the morphology and the neuroendocrine role of the hypothalamic catecholamine neurons. In: Brain-Endocrine Interaction. Median Eminence: Structure andFFunction. edited by K. M: Knigge, D. E. Scott. and A. Weindl, pp. 181-223, Karger. Basel. l972b. Hokfelt, T., K. Fuxe, M. Goldstein, and O. Johansson: Immunohistochem- ical evidence for the existence of adrenaline neurones in the brain. Brain Res. 66:235-251. 1974. 145 Holzbauer. M. and M. Vogt: Depression by reserpine of the noradrenaline concentration in the hypothalamus of the cat. J. Neurochem. 1:8-11. 1956. Houssay. B. A., A. Biasotti. and R. Sammartino: Modifications fonction- Hyyppfi. Hyyppfi. elles de l'hypophyse apres les lesions infundibulotuberiennes chez 1e crapaud. C.R. hebd. Seanc. Soc. Biol., Paris 120:725- 727. 1935. M., P. Lehtinen. and U. K. Rinne: Effect of L-Dopa on the hypo- thalamic, pineal and striatal monoamines and on sexual behavior of the rat. Brain Res. 30:265-272. 1971. M. T., D. P. Cardinali, H. G. Baumgarten. and R. J. Wurtman: Rapid accumulation of H3-serotonin in brains of rats receiving intraperitoneal H3-tryptophan: effects of 5.6-dihydroxytrypt- amine and female sec hormones. J. Neural Transm. 34:111-124. 1973. Igarashi, M. and S. M. McCann: A hypothalamic follicle stimulating hor- mone-releasing factor. Endocrinology 78:533-537. 1964. Imura. H., Y. Nakai, and T. Yoshimi: Effects of 5-hydroxytryptophan (5-HTP) on growth hormone and ACTH release in man. J. Clin. Endocrinol. Metab. 36:204-206. 1973. Iversen. L. L. and M. A. Simonds: Studies of catecholamine turnover in rat brain using 3H-noradrenaline. IQ; Metabolism of Amines in the Brain. edited by G. Hooper, pp. 48-57, Macmillan. New York. 69. Jackson, 1. M. D. and S. Reichlin: Thyrotropin-releasing hormone (TRH) Jacobs. Jacoby. distribution in hypothalamic and extra-hypothalamic brain tissues of mammalian and submammalian chordates. Endocrinology 95:854-862. 1974. L. S.. P. J. Snyder, J. F. Wilber. R. D. Utiger. and W. H. Daughaday: Increase in serum prolactin after administration of snythetic thyrotropin releasing hormone (TRH) in man. J. Clin. Endocrinol. Metab. 33:996-998. 1971. J. H., G. Mueller. and R. J. Wurtman: Thyroid state and brain monoamine metabolism. Endocrinology 97:1332-1335, 1975. Janssen. P., C. Niemegeers. K. Schellekens. A. Dresse. F. Lenaerts. A. Pinchard, W. Schaper, J. VanNueten. and F. Verbruggen: Pimozide. a chemically novel. highly potent and orally long- acting neuroleptic drug. Arzneimittel-Forsch. 18:261-287. 1968. 146 Jenkins. T. W.: Functional Mammalian Neuroanatomy. pp. 221-231. Lea and Febriger, Philadelphia. 1972. Jick. H., D. Slone. S. Shapiro. 0. P. Heinonen. S. C. Hartz. O. S. Mietien. M. P. Vessey. D. H. Lawson, and R. R. Miller: Reserpine and breast cancer. Lancet 7882:669-671. 1974.. Jobin. M., L. Ferland. J. Coté. and F. Labrie: Effect of exposure to cold on hypothalamic TRH activity and plasma levels of TSH and prolactin in the rat. Neuroendocrinology 18:204-212. 1975. Joh. T. H., C. Geghman, and D. Reis: Immunochemical demonstration of increased accumulation of tyrosine hydroxylase protein in sympa- thetic ganglia and adrenal medulla elicited by reserpine. Proc. Natl. Acad. Sci. 70:2767-2771, 1969. Jonsson. G., K. Fuxe. and T. H6kfelt: On the catecholamine innervation of the hypothalamuc with special reference to the median eminence. Brain Res. 40:271-281. 1972. Kalra. P. S.. S. P. Kalra. L. Krulich. C. P. Fawcett, and S. M. McCann: Involvement of norepinephrine in transmission of the stimulatory influence of progesterone on gonadotropin release. Endocrinology 90:1168-1176, 1972. Kalra. S. P. and S. M. McCann: Effect of drugs modifying catecholamine synthesis on LH release induced by preoptic stimulation in the rat. Endocrinology 93:356-362. 1973. Kalra. S. P. and S. M. McCann: Effects of drugs modifying catecholamine synthesis on plasma LH and ovulation in the rat. Neuroendocrinology 15:71-91, 1974. Kamberi. I. A., R. S. Mical, and J. C. Porter: Luteinizing hormone- releasing activity in hypophysial stalk blood and elevation by dopamine. Science 166:388-390. 1969. Kamberi, I. A., R. S. Mical, and J. C. Porter: Prolactin-inhibiting activity in hypophyseal portal blood and elevation by dopamine. Experientia 26:1150-1151. 1970a. Kamberi. I. A., R. S. Mical, and J. C. Porter: Effect of anterior pituitary perfusion and intraventricular injection of catechol- amines and indoleamines on LH release. Endocrinology 87:1-12, 1970b. Kamberi. I. A., H. P. G. Schneider, and S. M. McCann: Action of dopamine to induce release of FSH-releasing factor (FRF) from hypothalamic tissue jo_vitro. Endocrinology 86:278-284. 1970c. 147 Kamberi, I. A., R. S. Mical, and J. C. Porter: Effect of anterior pituitary perfusion and intraventricular injection of catechol- amines on prolactin release. Endocrinology 88:1012-1020. 1971a. Kamberi. I. A., R. S. Mical, and J. C. Porter: Hypophysial portal vessel infusion: in vivo demonstration of LRF. FRF and PIF in pituitary stalk plEEma. Endocrinology 89:1042-1046. 1971b. Kamberi, I. A., R. S. Mical, and J. C. Porter: Pituitary portal vessel infusion of hypothalamic extract and release of LH FSH and pro- lactin. Endocrinology 88:1294-1299. 1971c. Kamberi. I. A., R. S. Mical, and J. C. Porter: Effect of anterior pituitary perfusion and intraventricular injection of catechol- amines on FSH release. Endocrinology 88:1003-1011. 1971d. Kamberi. I. A., R. S. Mical, and J. C. Porter: Effects of melatonin and serotonin on the release of FSH and prolactin. Endocrinology 88:1288-1293. l97le. Kamberi, I. A. and E. S. Bacleon: Role of cholinergic synapses in neural circuits controlling gonadotropin secretion. Endocrinology 92:Al37. 1973. Kanematsu, S.. J. Hilliard, and C. H. Sawyer: Effects of reserpine and chlorpromazine on pituitary prolactin content and its hypo- thalamic site of action in rabbits. Acta. Endocrinol. 44:467- 474. 1963. Kato. Y., J. Dupre. and J. C. Beck: Plasma growth hormone in the anes- thetized rat: effects of dibutyryl cyclic AMP. prostaglandin E]. adrenergic agents. vasopressin. chlorpromazine. amphetamine and L-dopa. Endocrinology_93:135-146. 1973. Kato. Y., Y. Nakai, H. Imura. K. Chihara. and S. Ohago: Effect of S-hydroxytryptophan (5-HTP) on plasma prolactin levels in man. J. Clin. Endocrinol. Metab. 38:695-697. 1974. Kato, Y., K. Chihara. K. Maeda. S. Ohgo. Y. Okanishi, and H. Imura: Plasma growth hormone responses to thyrotropin-releasing hormone in the urethane-anesthetized rat. Endocrinology 96:1114-1118. 1975. Kaufman. S.: Coenzymes and hydroxylases: ascorbate and dopamine-B- hydroxylase; tetrahydopteridines and phenylalanine and tyrosine hydroxylases. Pharmacol. Rev. 18:61-70. 1966. Kawakami, M. and Y. Sakuma: Responses of hypothalamic neurons to the microiontophoresis of LH-RH, LH and FSH under various levels of circulating ovarian hormones. Neuroendocrinology_15:290-307. 1974. Keller. Koch. Y. Koelle. Koj, A. Kordon. Kordon s 148 H. H., G. Bartholini, and A. Pletscher: Enhancement of central noradrenaline rutnover by thyrotropin-releasing hormone. Nature 248:529. 1974. . K. H. Lu, and J. Meites: Biphasic effects of catecholamines on pituitary prolactin release jo_vitro. Endocrinology 87:673- 675, 1970. G. 8.: Anticholinesterase agents. parasympathomimetic agents. Io; The Pharmacological BAsis of Therapeutics. 4th edition, edited By L. S. Goodman and A. Gilman. pp. 442-477. Macmillan Co., London, 1970. M., and L. Krulich: The role of monoamines in the stress- induced prolactin in the rat. Fed. Proc. 34:252. 1975. C. and J. Glowinski: Role of hypothalamic monoaminergic neurones in the gonadotropin release-regulating mechanisms. Neuropharm. 11:153-162. 1972. C.. C. A. Blake, J. Terkel. and C. H. Sawyer: Participation of serotonin-containing neurones in the suckling-induced rise in plasma prolactin levels in lactating rats. Neuroendocrinology_ 13:213-223. 1974. Kragt. C. L. and J. Meites: Stimulation of pigeon pituitary prolactin Krulich, Kru1ich. Krulich, Krulich. release by pigeon hypothalamic extract jo_vitro. Endocrinology_ 76:1169-1176. 1965. - L.: The effect of a serotonin uptake inhibitor (Lilly 110140) on the secretion of prolactin in the rat. Life Sci. 17:1141- 1144, 1975. L., A. P. S. Dhariwal, and S. M. McCann: Stimulatory and in- hibitory effects of purified hypothalamic extracts on growth hormone release from rat pituitary jo_vitro. Endocrinology 83: 783-790. 1968. L. and P. Illner: Effects of stress on plasma levels of LH, FSH. prolactin, TSH and GH in normal male rats. Fed. Proc. 32: 281. 1973. L., E. Hefco. P. Illner, and C. B. Read: The effects of acute stress on the secretion of LH, FSH, prolactin and GH in the normal male rat. with comments on their statistical evaluation. Neuroendocrinology 16:293-311. 1974. 149 Kuhar. M. J., G. K. Aghajanian. and R. L. Roth: Serotonin neurons: a synaptic mechanism for the reuptake of serotonin. In: Advances in Biochemical ngchopharmacology, Vol. 10. edited 5? E. Costa. G. L. Gessa. andiMT'Sandler. pp. 287-296, Raven Press. New York. 1974. Kuhn, E., L. Krulich. C. P. Fawcett, and S. M. McCann: The ability of hypothalamic extracts to lower prolactin levels in lactating rats. Proc. Soc. Exp: Biol. Med. 146:104-108, 1974. Kuhn. E. R. and S. Lens: Studies on the cholinergic release mechanism for prolactin in the male rat. Internat. Res. Commun. Sept. 2:1532, 1974. Kuriyama, K. and H. Kimura: Distribution and possible functional roles of GABA in the retina. lower auditory pathway. and hypothalamus. In: GABA in Nervous System Function. edited by E. Roberts. T. N. Chase. and B. 0. Tower. pp. 203-216. Raven Press Books, Ltd.. New York, 1976. LaBella. F. S. and S. R. Vivian: Effect of synthetic TRH on hormone release from bovine anterior pituitary 1o_vitro. Endocrinology 88:787-789. 1971. Ladisich, W.: Influence of stress on regional brain serotonin metabolism after progesterone treatment and upon plasma progesterone in the rat. J. Neural Transm. 36:33-42. 1975. Lal. S.. C. E. DeLa Vega, T. L. Sourkes, and H. G. Friesen: Effect of apomorphine on human-growth-hormone secretion. Lancet 2:66. 1972. “"——' Lawson, D. M. and R. R. Gala: The influence of adrenergic, dopaminergic, cholinergic and serotonergic drugs on plasma prolactin levels in ovariectomized. estrogen treated rats. Endocrinology 96:313-318. 1975. Lee, 0. M.: Studies of the oestrous cycle in the rat. III. The effect of low environmental temperatures. Amer. J. Physiol. 78:246-253. 1926. Lehninger, A. L.: Proteins: covalent backbone and amino acid sequence. Io; Biochemistry, by A. L. Lehninger, pp. 97, Worth Publishers, Inc., New York. 1970. Levitt, M., S. Spector, A. Sjoerdsma. and S. Udenfriend: Elucidation of the rate limiting step in norepinephrine biosynthesis using the perfused guinea pig heart. J. Pharmacol. Exp. Ther. 148:1-8. 1965. 150 Levitt, M., J. W. Gibb, J. W. Daly. M. Lipman. and S. Udenfriend: A new class of tyrosine hydroxylase inhibitors and a simple assay of inhibition jo_vivo. Biochem. Pharmacol. 16:1313-1321. 1967. Libertun. C. and S. M. McCann: Blockade of the release of gonadotropins and prolactin by subcutaneous or intraventricular injection of atropine in male or female rats. Endocrinology 92:1714-1724, 1973. Libertun. C. and S. M. McCann: Further evidende for cholinergic control of gonadotropin and prolactin secretion. Proc. Soc. Exp. Biol. U29: 147:498-504. 1974a. Libertun. C. and S. M. McCann: Prolactin-releasing effect of histamine injected into the third ventricle. Endocrinology 94:A-266. 1974b. (Lichtensteiger. W.: The catecholamine content of hypothalamic nerve cells after acute exposure to cold and thyroxine administration. J. Physiol. 203:675-687. 1969. Lidbrink. P., H. Corrodi. K. Fuxe, and L. Olson: Barbiturates and meprobamate: decrease in catecholamine turnover of central dop- amine and noradrenaline neuronal systems and the influence of immobilization stress. Brain Res. 45:507-524. 1972. Lin, R. C.. N. H. Neff. S. H. Ngai. and E. Costa: Turnover rates of serotonin and norepinephrine in brain of normal and pargyline- treated rats. Life Sci. 8:1077-1084. 1969. L6fgren. F.: New aspects of the hypothalamic control of the adreno- hypophysis. Acta. Morph. Neerl. Scand. 2:220-229. 1959. Loizou, L. A.: The postnatal ontogeny of monoamine-containing neurons in the central nerbous system of the albino rat. Brain Res. 40:395-418. 1972. Lovenberg. W.. E. Jequier, and A. Sjoerdsma: Tryptophan hydroxylation in mammalian systems. Advan. Pharmacol. 6A:21-36, 1968. Lovenberg, W. and S. J. Victor: Tryptophan hydroxylase of the central nervous system: effect of intraventricular 5.6- and 5,7-di- hydroxytryptamine. Io; Advances in Biochemical Psychopharma- cology. Vol. 10. edited by E. Costa. G. L. Gessa. and M. Sandler, pp. 93-101, Raven Press. New York. 1974. Lovinger, R. 0., M. H. Connors. S. L. Kaplan. W. F. Ganong. and M. H. Grumbach: Effect of L-dihydroxyphenylalanine (L-Dopa), anes- thesis and surgical stress on the secretion of growth hormone in the dog. Endocrinology 95:1317-1321. 1974. 151 Lu. K. H., Y. Amenomori. C. L. Chen, and J. Meites: Effects of central acting drugs on serum and pituitary prolactin levels in rats. Endocrinology_87:667-672. 1970. Lu, K. H. and J. Meites: Inhibition of L-Dopa and monoamine oxidase inhibitors of pituitary prolactin release: stimulation by methyl dopa and d-amphetamine. Proc. Soc. Exp. Biol. Med. 137: 480-483. 1971. Lu. K. H., Y. Koch, and J. Meites: Direct inhibition by ergocornine of pituitary prolactin release. Endocrinology 89:229-233. 1971. Lu, K. H. and J. Meites: Effects of L-Dopa on serum prolactin and PIF in intact and hypOphysectomized. pituitary-grafted rats. Endocrinology 91:868—872. 1972. Lu. K. H., C. J. Shaar. K. H. Kortright, and J. Meites: Effects of synthetic TRH on jo_vitro and in vivo prolactin release in the rat. Endocrinology 9|:l540-1545. 1972. Lyons, W. R., C. H. Li. and R. E. Johnson: The hormonal control of mam- mary growth and lactation. Recent Prog. Hormone Res. 14:219- 254. 1958. Maany, I.. A. Frazer. and J. Mendels: Apomorphine: effect on growth hormone. J. Clin. Endocrinol. Metab. 40:162-163. 1975. MacIndoe. J. H. and R. W. Turkington: Stimulation of human prolactin secretion by intravenous infusion of L-tryptophan. J. Clin. Invest. 52:1972-1978. 1973. MacLeod. R. M.: Influence of norepinephrine and catecholamine-depleting agents on the synthesis and release of prolactin and growth hormone. Endocrinology 85:916-923. 1969. MacLeod. R. M.: Regulation of pituitary function by catecholamines. In: Mammar Cancer and Neuroendocrine Thera . edited by AC’Basil anfi E. §toe|. PP. 139-159. Butterworth and Company, New York, 1974. MacLeod, R. M., M. C. Smith, and G. W. Dewitt: Hormonal properties of transplanted pituitary tumors and their relation to the pitui- tary gland. Endocrinology 79:1149-1156. 1966. MacLeod. R. M., E. H. Fontham, and J. E. Lehmeyer: Prolactin and growth hormone production as influenced by catecholamines and agents that affect brain catecholamines. Neuroendocrinology 6:283-294, 1970. 152 MacLeod, R. M. and J. E. Lehmeyer: Studies on the mechanism of the dopamine-mediated inhibition of prolactin secretion. Endocrinology.94:1077-1985. 1974. Maeda. K., Y. Kato. S. Ohgo. K. Chihara. Y. Yoshimoto. M. Yamaguchi, S. Kuromaru, and H. Imura: Growth hormone and prolactin release after injection of thyrotrOpin-releasing hormone in patients with depression. J. Clin. Endocrinol. Metab. 40:501-505. 1975. Mahoney, W. and D. Sheehan: The pituitary-hypothalamic mechanism: experimental occlusion of the pituitary stalk. Brain 59:61-75, 1936. Malarkey. W. B.. L. S. Jacobs. and W. H. Daughaday: Levodopa suppres- sion of prolactin in nonpubertal galactorrhea. New Eogl. J. Med. 285:1160-1163. 1971. Malarkey. W. B. and W. H. Daughaday: The influence of levodopa and adrenergic blockade on growth hormone and prolactin secretion in the MS+TW15 tumor-bearing rat. Endocrinology 91:1314-1317. 1972. Malven. P. V. and W. R. Hoge: Effect of ergocornine on prolactin secre- tion by hypophyseal homografts. Endocrinology 88:445-449. 1971. Markee. J. E., C. H. Sawyer, and W. H. Hollinshead: Activation of anterior hypophysis by electrical stimulation in the rabbit. Endocrinology 38:345-357. 1946. Markee. J. E., C. H. Sawyer. and W. H. Hollinshead: Adrenergic control of the release of luteinizing hormone from hypophysis of the rabbit. Recent Progx. Hormone Res. 2:117-131. 1948. Markee. J. E., J. W. Everett. and C. H. Sawyer: The relationship of the nervous system to the release of gonadotropin and regulation of the sex cycle. Recent Progr. Hormone Res. 7:139-163. 1952. Marshall, F. H. A.: Exteroceptive factors in sexual periodicity. Biol. Rev. 17:68-70. 1942. Martin. J. 8.: Brain regulation of growth hormone secretion. Io: Frontiers in Neuroendocrinology, Vol. 4, edited by L. Martini and W. FT’Ganong. pp. 129-168. Raven Press. New York, 1976. Martin. J. B.. S. Lal. G. 10115, and H. G. Friesen: Inhibition by apo- morphine of prolactin secretion in patients with elevated serum prolactin. J. Clin. EndoCrinol. Metab. 39:180-182. 1974. Martin. J. B., J. O. Willoughby, and G. S. Tannenbaum: Evidence for an intrinsic central nervous system rhythm governing episodic GH secretion in the rat. Endocrinology 96:A 177. 1975. Matsuo. Matsuo. McCann. McCann, McCann, McCann, McCann, McCann, McLean. Meites. Meites, Meites. 153 H., Y. Baba, R. M. S. Nair. A. Arimura, and A. V. Schally: Structure of porcine LH and FSH-releasing hormone I. The pro- posed amino acid sequence. Biochem. Biophys. Res. Comm. 43: 1334-1339. 1971a. H., A. Arimura, R. M. G. Nair, and A. V. Schally: Synthesis of porcine LH and FSH releasing hormone by the solid-phase method. Biochem. Biophys. Res. Commun. 45:822-827. 1971b. 5. M.: Regulation of secretion of follicle-stimulating hormone and luteinizing hormone. In: Handbook of Physiology: Sect. 7. Vol. 4 Part 2. edited by RT 0. Greep and E. B. Astwood. pp. 489-51,, Williams and Wilkins Comp., Baltimore. 1974. S. M. and H. M. Friedman: The effect of hypothalamic lesions on the secretion of luteotropin. Endocrinology 67:597-608. 1960. S. M., S. Taleisnik. and H. M. Friedman: LH-releasing activity in hypothalamic extracts. Proc. Soc. Expx_Biol. Med. 104:432- 434. 1960. S. M. and A. P. S. Dhariwal: Hypothalamic releasing factors and the neurovascular link between the brain and the anterior pitui- tary. Io; Neuroendocrinolo . Vol. I. edited by L. Martini and W. F. Ganong. pp. 261-296, cademic Press. New York, 1966. S. M., P. S. Kalra. A. O. Donoso. W. Bishop, H. P. G. Schneider, C. P. Fawcett, and L. Krulich: The role of monoamines in the control of gonadotropin and prolactin secretion. Io; Brain- Endocrine Interaction. Median Eminence. Structure and Functioo, edited by K. M. Knigge,—D. E. Scott, andBW. WEindl. PP. 224-235. S. Karger. Basel. 1972. S. M. and R. L. Moss: Putative neurotransmitters involved in discharging gonadotropin-releasing neurohormones and the action of LH-releasing hormone on the CNS. Life Sci. 16:833-852. 1975. B. K. and M. B. Nikitovitch-Winer:. Cholinergic control of the nocturnal prolactin surge in the pseudopregnant rat. Endocrinology 97:763-770. 1975. J.: Induction of lactation in rabbits with reserpine. Proc. Soc. Exp. Biol. Med. 96:728-730. 1957. J.: Control of mammary growth and lactation. lo; Neuroendo- crinolo Vol. I, edited by L. Martini and W. F. Ganong. pp. 659-768, Academic Press. New York, 1966. J.: Relation of prolactin and estrogen to mammary tumorigene- sis in the rat. J. Nat. Cancer Inst. 48:1217-1224, 1972. 154 Meites. J.: Control of prolactin secretion in animals. In: Human Prolactin. edited by J. L. Pasteels, C. Robyn. and—F. J. G. EEling. pp. 105-118, American Elsevier Pub. Comp. Inc., New York, 1973. Meites. J., P. K. Talwalker. and C. S. Nicoll: Initiation of lactation in rats with hypothalamic or cerebral tissue. Proc. Soc. Exp. Biol. Med. 103:298-300, 1960. Meites, J., R. H. Hahn, and C. S. Nicoll: Prolactin production by rat pituitary jo_vitro. Proc. Soc. Exp. Biol. Med. 108:440-443, 1961. Meites. J., C. S. Nicoll, and P. K. Talwalker: The central nervous sys- tem and the secretion of prolactin. Io; Advances in Neuroendo- crinology. edited by A. V. Nalbandov, pp. 238-277.‘University of Illinois Press. Urbana. 1963. Meites. J. and C. S. Nicoll: Adenohypophysis: prolactin. Ann. Rev. Physiol. 28:57-88. 1966. Meites. J. and J. A. Clemens: Hypothalamic control of prolactin secre- tion. Vitamins and Hormones 30:165-221. 1972. Meites, J., K. H. Lu, W. Wuttke. C. W. Welsch, H. Nagasawa. and S. K. Quadri: Recent studies on functions and control of prolactin secretion in rats. Recent Progx, Hormone Res. 28:471-516. 1972. Mess. B. and L. Peter: Effect of intracerebral serotonin administration on pituitary-thyroid function. Endocrinologia Experimentalis 9:105-113, 1975. Millard. S. A., E. Costa, and E. M. Gal: On the control of brain sero- tonin turnover rate by end product inhibition. Brain Res. 40: 545-551. 1972. Miller. F. P., R. H. Cox. Jr., W. R. Snodgrass. and R. P. Maickel: Comparative effects of p-chlorophenylalanine. p-chloroampheta- mine and p-chloro-N-methylamphetamine or rat brain norepinephrine. serotonin and S-hydroxyindole-B-acetic acid. Biochem. Pharmacol. 19:435-442. 1970. Minozzi. M., M. Faggiano, G. Lombardi, C. Carella. T. Criscualo. and U. Scapagnini: Effect of L-Dopa on plasma TSH levels in primary hypothyroidism. Neuroendocrinology 17:147-153. 1975. Mioduszewski. R., L. Grandison, and J. Meites: Stimulation of prolactin release in rats by GABA. Proc. Soc. Exper. Biol. Med. 151:44- 46. 1976. 155 Mischkinsky. J., K. Khazan. and F. G. Sulman: Prolactin releasing activity of the hypothalamus of postpartum rats. Endocrinology_ 82:611-613. 1968. Mishkinsky. J., I. Mir. and F. G. Sulman: Internal feedback of prolac- tin in the rat. Neuroendocrinology 5:48-52. 1969. Mitchell. J. A.: Functional anatomy of the hypothalamus: [ossible role of the ependyma. In: Hypothalamic Hormones. edited by E. Hafez and J. Reel. pp. 1:16, Ann Arbor Science Publishers, Ann Arbor. 1975. Mitnick, M. A., C. Valverde. and S. Reichlin: Enzymatic synthesis of prolactin-releasing factor (PRF) by rat hypothalamic incubates and by extracts of rat hypothalamic tissue: evidence for “PRF" synthetase. Proc. Soc. Exp. Biol. Med. 143:418-421. 1973. Mittler, J. C. and J. Meites: Io_vitro stimulation of pituitary follicle- stimulating hormone release 5y hypothalamic extract. Proc. Soc. Exp, Biol. Med. 117:309-313, 1964. Miyachi, Y., R. S. Mecklenburg. and M. B. Lippsett: Io_vitro studies of pituitary-median eminence unit. Endocrinology 93:492-496. 1973. Montoya, E., M. J. Seibel. and J. F. Wilber: Thyrotropin-releasing hormone secretory physiology: studies by radioimmunoassay and affinity chromatography. Endocrinology 96:1413-1418. 1975. Morot-Gaudry. Y., M. Hamon. S. Bourgoin, J. P. Ley. and J. Glowinski: Estimation of the rate of 5-HT synthesis in the mouse brain by various methods. Naunyn-Schmiedeberg's Arch. Pharmacol. 282:223- 238. 1974. Moss. R. L. and S. M. McCann: Induction of mating behavior in rats by luteinizing hormone-releasing factor. Science 181:177-179. 1973. Mueller. G. P., H. T. Chen. J. A. Dibbet. H. J. Chen. and J. Meites: Effects of warm and cold temperatures on release of TSH. GH. and prolactin in rats. Proc. Soc. Exp. Biol. Med. 147:698-700. 1974. Mueller. G. P., C. P. Twohy. H. T. Chen. J. P. Advis, and J. Meites: Effects of L-tryptophan and restraint stress on hypothalamic and brain serotonin turnover. and pituitary TSH and prolactin release in rats. Life Sci. 18:715-724. 1976a. Mueller, G. P., J. Simpkins. J. Meites, and K. E. Moore: Differential effects of dopamine agonists and haloperidol on release of pro- lactin. thyroid stimulating hormone. growth hormone. and lutein- izing hormone in rats. Neuroendocrinology: in press. 1976b. 156 MUller. E. E., P. Dal Pra. and A. Pecile: Influence of brain neurohumors injected into the lateral ventricle of the rat on growth hormone release. Endocrinology 83:893-896. 1968. MUller. E. E., D. Cocchi. H. Jalando, and G. Udeschini: Antagonistic role for norepinephrine and dopamine in the control of growth hormone secretion in the rat. Endocrinology 92:A 248. 1973. MOller. E. E., F. Brambilla. F. Cavagnini. M. Peracchi. and A. Penerai: Slight effect of L-tryptophan on growth hormone release in normal human subjects. J. Clin. Endocrinol. Metab. 39:1-5. 1974. Nagatsu, T.: Biosynthesis of catecholamines. In: Biochemistry of Catecholamines. by T. Nagatsu. pp. 50-60: University Park Press. Baltimore,l973. Nagatsu, T., M. Levitt. and S. Udenfriend: Tyrosine hydroxylase: the initial step in norepinephrine biosynthesis. J. Biol. Chem. 239:2910-2917. 1964. Hegro-Vilar, A., L. Krulich. and S. M. McCann: Changes in serum prolac- tin and gonadotropins during sexual development of the male rat. Endocrinology 93:660-664. 1973. Neill. J. 0.: Effects of stress on serum prolactin and luteinizing hormone levels during the estrous cycle of the rat. Endocrinology 87:1192-1197, 1970. Neill. J. D.: Prolactin: its secretion and control. Io; Handbook of Physiology: SectionZ: Endocrinology, Vol. IV, Part 2. edited by R. O. Greep and E. B. Astwood. pp. - . W1 1ams and Wilkins. Baltimore. 1974. Hetter, F. H.: The Hypothtlamus. supp. to Vol. 1. Nervous System, The Ciba Collection of Medical Illustrations. Ciba Pharmaceutical Products, Inc., Summit, New Jersey. 1968. Nickerson, M.: Drugs inhibiting adrenergic nerves and structures inner- vated by them. In: The Pharmacological Basis of Therapeutics. edited by L. S. GEodman andlA. Gilman. pp. 549-584, The Macmillan Company. New York. 1970. Nicoll. C. S.: Aspects of neural control of prolactin secretion. Io; Frontiers in Neuroendocrinology. edited by L. Martini and W. F. Ganong, pp. 291-339, Oxford Un1versity Press. New York. 1971. Hicoll, C. S.. P. K. Talwalker, and J. Meites: Initiation of lactation in rats by nonspecific stresses. Am. J. Physiol. 198:1103-1106. 1960. 157 Nicoll. C. S.. R. P. Fiorindo. C. T. McKennee. and J. A. Parsons: Assay of hypothalamic factors which regulate prolactin secretion. Io; Hypophysiotrophic Hormones of the Hygothalamus: Assay and em1str . e 1te y . 1tes. pp. - , 1 1ams an Wilkins, Baltimore. 1970. Nicoll. C. S. and H. A. Bern: On the actions of prolactin among verte- brates: is there a common denominator? Io: Lacto enic Hormones. edited by G. E. W. Wolstenholme and J. Knight, pp. 99-324. Churchill Livingston. London. 1972. Niswender, G. 0., A. R. Midgley, Jr., S. E. Monroe. and L. E. Reichert: Radioimmunoassay for {at luteinizing hormone with antiovine LH serum and ovine LH-l3 I. Proc. Soc. Exp, 3101. Med. 128:807- 811, 1968. Niswender, G. 0., C. L. Chen. A. R. Midgley, Jr., J. Meites. and 5. Ellis: Radioimmunoassay for rat prolactin. Proc. Soc. Exp. Biol. Med. 130:793-797. 1969. Nikitovitch-Winer. M.: The influence of the hypothalamus on luteotropin secretion in the rat. Mem. Soc. Endocrinol. 9:70-72. 1960. Noel, G. L., H. K. Suh. J. G. Stone. and A. G. Frantz: Human prolactin and growth hormone release during surgery and other conditions of stress. J. Clin. Endocrinol. Metab. 35:840-851. 1972. Noel. G. L., R. C. Dimond. L. Wartofsky. J. M. Earll. and A. G. Frantz: Studies of prolactin and TSH secretion by continuous infusion of small amounts of thyrotropin-releasing hormone (TRH). J. Clin. Endocrinol. Metab. 39:6-17. 1974. Ojeda. S. R. and S. M. McCann: Evidence for participation of a cate- cholaminergic mechanism in the post-castration rise in plasma gonadotropins. Neuroendocrinology_12:295-315. 1973. Ojeda. S. R. and S. M. McCann: Development of dopaminergic and estro- genic control of prolactin release in the female rat. Endocrinology_95:1499-1505. 1974. Ojeda. S. R., P. G. Harms. and S. M. McCann: Effect of blockade of dopamine receptors on prolactin and LH release: median eminence and pituitary sites of action. Endocrinology_94:1650-1657. 1974a. Ojeda. S. R., P. G. Harms. and S. M. McCann: Possible role of cyclic AMP and prostaglandin E in the dopaminergic control of prolac- tin release. Endocrinology.95:1694-1703. 1974b. 158 Ojeda, S. R., P. G. Harms, and S. M. McCann: Central effect of prosta- glandin E] (PGEI) on prolactin release. Endocrinology 96:613- 618, 1974c. Oliver. C.. R. L. Eskay. N. Ben-Jonathan. and J. C. Porter: Distribution and concentration of TRH in the rat brain. Endocrinology 96: 540-546, 1974. Oliver. C.. N. Ben-Jonathan. R. S. Mical, and J. C. Porter: Transport of thyrotropin-releasing hormone from cerebrospinal fluid to hypo- physeal portal blood and the release of thyrotropin. Endocrinology_97:1138-1143. 1975. Ondo. J.: Gamma-aminobutyric acid effects on pituitary gonadotropin secretion. Science 186:738-739, 1974. Ondo. J. G., R. S. Mical, and J. C. Porter: Passage of radioactive sub- stances from CSF to hypophyseal portal blood. Endocrinology 91: 1239-1246, 1972. Palkovits. M.: Isolated removal of hypothalamic or other brain nuclei of the rat. Brain Res. 59:449-450. 1973. Palkovits. M., A. Arimura, M. Brownstein. A. V. Schally. and J. M. Saavedra: Luteinizing hormone-releasing hormone (LH-RH) content and hypothalamic nuclei in rat. Endocrinology,96:554-558. 1974a. Palkovits. M., M. J. Brownstein. J. M. Saavedra. and J. Axelrod: Nor- epinephrine and dopamine content of hypothtlamic nuclei of the rat. Brain Res. 77:137-149. 1974b. Palkovits. M., R. M. Kobayashi, J. S. Kizer, 0. M. Jacobowitz, and I. J. Kopin: Effects of stress on catecholamines and tyrosine hydrox- ylase activity of individual hypothalamic nuclei. ' Neuroendocrinology 18:144-153. 1975. Pasteel. J. L.: Secretion de prolactine par l'hypophyse en culture de tissue. Comp, Rend. Soc. Biol. 253:2140-2142, 1961. Pfaff, D. W.: Luteinizing hormone-releasing factor potentiates lordosis behavior in hypophysectomized ovariectomized female rats. Science 182:1148-1149. 1973. Plotnikoff. N. P., W. W. White. A. J. Kastin, and A. V. Schally: Gonadotropin releasing hormone (GnRH): neuropharmacological studies. Life Sci. 17:1685-1692. 1975. Popa. G. T. and U. Fielding: A portal circulation from the pituitary to the hypothalamic region. J. Anat. (London) 65:88-91. 1930. 159 Prange. A. J., Jr., C. 1. Wilson. P. P. Lara. L. B. Allt0p, and G. R. Breese: Effects of thyrotropin-releasing hormone in depression. Lancet 2:999-1007. 1972. Quadri, S. K. and J. Meites: Regression of spontaneous mammary tumors in rats by ergot drugs. Proc. Soc. Exp. Biol. Med. 138:999-1001, 1971. Quadri. S. K., G. S. Kledzik, and J. Meites: Effects of L-Dopa and Methyldopa on growth of mammary cancers in rats. Proc. Soc. Exper. Biol. Med. 142:759-761. 1973. Quijada, M., P. Illner, L. Krulich. and S. M. McCann: The effect of catecholamines on hormone release from anterior pituitaries and ventral hypothalami incubated jo_vitro. Neuroendocrinology 13: 151-163. 1973/74. Rabinowitz, P. and I. S. Friedman: Drug induced lactation in uremia. J. Clin. Endocrinol. Metab. 21:1489-1493. 1961. Rapoport. B.. S. Refetoff. V. S. Fang. and H. G. Friesen: Suppression of serum thyrotropin (TSH) by L-Dopa in chronic hypothyroidism: interrelationships in the regulation of TSH and prolactin secre- tion. J. Clin. Endocrinol. Metab. 36:256-262. 1973. Rastogi. R. B. and R. L. Singhal: Thyroid hormone control of 5-hydroxy- tryptamine metabolism in developing rat brain. J. Pharmacol. Exp. Ther. 191:72-81. 1974. Refetoff. S.. V. S. Fang. B. Rapoport. and H. G. Friesen: Relationships in the regulation of TSH and prolactin secretion in man: effects of L-Dopa. TRH and thyroid hormone in various combina- tions. J. Clin. Endocrinol. Metab. 38:450=457. 1974. Reichlin. S.: Control of thyrotropic hormone secretion. In: Neuro- endocrinolo Vol. 1, edited by L. Martini and W. F. Ganong. pp. 445-536. Academic Press. New York. 1966. Reichlin. S.: Regulation of somatotrophic hormone secretion. 19; Handbook of Physiology: Section 7. Endocrinology, Vol. IV. Part 2, edited'by R. 0. Greep andiEl B. Astwood. pp. 405-447. Williams and Wilkins, Baltimore. 1974. Reichlin. S.. J. B. Martin, M. Mitnick. R. L. Boshans. Y. Grimm, J. Bollinger. J. Gordon. and J. Malacara: The hypothalamus in pituitary-thyroid regulation. Recent Progr. Hormone Res. 28:229-277. 1972. 160 Reichlin. S. and M. Mitnick: Biosynthesis of hypothalamic hypophysio- trophic factors. Io; Frontiers in Neuroendocrinolo , 1973. edited by W. F. Ganong and’L. Martini. pp. 61-88, 0x ord Univer- sity Press, New York. 1973. Reid. W. D.. L. Volicer. H. Smookler, M. A. Beaven. and B. B. Brodin: Brain amines and temperature regulation. Biochem. Pharmacol. 20:329-344. 1968. Reineke. E. P. and F. A. Soliman: Role of thyroid hormones in the reproductive physiology of the female. Iowa State Collego_ Journal of Science 28:67-82. 1953. Rippel, R. H.: Chemical and biological properties of the synthetic hypo- thalamic-releasing and inhibiting hormones. In: H othalamic Hormones. edited by E. S. E. Hafez and J. R. Reel, pp. 53-75. n r or Science Publishers. Inc., Ann Arbor, 1975. Rivier, J., P. Brazeau, W. Vale. N. Ling. R. Burgus. C. Gilon. J. Yardley. and R. Guillemin: Synthese totale par phase solide d'un tetra- decapeptide ayant les proprietes chimiques et biologiques de la somatostatine. C. R. Acad. Sci. 276:2737-2740, 1973. Root. A. W.. P. J. Snyder, I. Revani, A. M. DiGeorge. and R. D. Utiger: Inhibition of tryrotropin-releasing hormone-mediated secretion in thyrotropin by human growth hormone. J. Clin. Endocrinol.‘ Metab. 36:103-107. 1973. Ruberstein, L. and C. H. Sawyer: The role of catecholamines in stimu- lating the release of pituitary ovulating hormones in the rat. Endocrinology 86:990-995. 1970. Saavedra. J. M. and J. Axelrod: Effects of drugs on the tryptamine con- tent of rat tissues. J. Pharmacol. Exp. Ther. 185:523-529, 1973. Saavedra. J. M., M. Palkovits, M. Brownstein. and J. Axelrod: Serotonin distribution in the nuclei of the rat hypothalamus and preoptic region. Brain Res. 77:157-165. 1974a. Saavedra, J. M., M. Palkovits. M. J. Brownstein. and J. Axelrod: Localisation of phenylethanolamine N-methyl-transferase in the rat brain nuclei. Nature Lond. 238:695-696, 1974b. Saffran. M. and A. V. Schally: Release of corticotrophin by anterior pituitary tissue jo_vitro. Canad. J. Biochem. 33:408-415. 1955. 161 Sanders-Bush. E., D. A. Gallager. and F. Sulser: On the mechanism of brain 5- -hydroxytryptamine depletion by p- -chloroamphetamine and related drugs and the specificity of their action. In: Advance in Biochemical Poychopharmacology. Vol.10, Edited by E. Costa. GTlL. Gessa. and M. Sandler, pp.1185- -l94. Raven Press. New York, 1974. Sawyer. C. H.: First Geoffrey Harris Memorial Lecture-~Some recent developments in brain-pituitary-ovarian physiology. Neuroendocrinology 17:97-124, 1975. Sawyer. C. H., J. E. Markee. and W. H. Hollinshead: Inhibition of ovu- lation in the rabbit by the adrenergic blocking agent dibenamine. Endocrinology 41:395-402. 1947. Sawyer. C. H., J. E. Markee. and B. F. Townsend: Cholinergic and adren- ergic components in the neurohumoral control of the release of LH in the rabbit. Endocrinology 44:18-37. 1949. Sawyer. C. H., J. Hilliard. S. Kanematsu. R. Scaramuzzi. and C. A. Blake: Effects of intraventricular infusions of norepinephrine and dopamine on LH release and ovulation in the rabbit. Neuroendocrinology 15:328-337. 1974. Schalch, D. S. and S. Reichlin: Stress and growth hormone release. In: Growth Hormone. edited by A. Pecile and E. E. Muller. pp. 211- 225. Exerpta Medica Foundation, Amsterdam, 1968. Schalch, D. S.. D. Gonzalez-Barcena, A. J. Kastin, A. V. Schally. and L. A. Lee: Abnormalities in the release of TSH in response to thyrotropin-releasing hormone (TRH) in patients with disorders of the pituitary. hypothalamus and basal ganglia. J. Clin. Endocrinol. Metab. 35:609-615. 1972. Schally. A. V., A. Kuroshima, Y. Ishida. T. W. Redding. and C. V. Bowers: The presence of prolactin inhibiting factor (PIF) in extracts of beef. sheep and pig hypothalami. Proc. Soc. Exp, Biol. Med. 118: 350- 352. 1965. Schally. A. V., C. Y. Bowers, T. W. Redding. and J. F. Barrett: Isola- tion of thyrotropin releasing factor (TRF) from porcine hypo- thalamus. Biochem. Biophys. Res. Commun. 25: 165-169. 1966. Schally. A. V., A. Arimura, C. Y. Bowers. A. J. Kastin, S. Sawano. and T. W. Redding: Hypothalamic neurohormones regulating anterior pituitary function. Recent Progr. Hormone Res. 24:497-581. 1968. Schally. A. V., C. Y. Bowers. T. W. Redding. and J. F. Barrett: Isola- tion and properties of porcine thyrotropin-releasing hOrmone. J. Biol. Chem. 244:4077-4087, 1969. 162 Schally. A. V., A. Arimura, and A. J. Kastin: Hypothalamic regulatory hormones. Science 179:341-350, 1973. Schally. A. V., A. Arimura, J. Takahara. T. W. Redding. and A. DuPont: Inhibition of prolactin release in vitro and jo_vivo by cate- cholamines. Fed. Proc. 33:237. 1974. Schally. A. V., A. Arimura, T. W. Redding. L. Debeljuk. W. Carter, A. DuPont. and J. A. Vilchez-Martinez: Re-examination of porcine and bovine hypothalamic fractions for additional luteinizing hormone and follicle-stimulating hormone-releasing activities. Endocrinology 98:380-391. 1976. Scharrer, E.: The general significance of the neurosecretory cell. Scientia 87:176-182. 1952. Scharrer. E. and B. Scharrer: Secretory cells within the hypothalamus. Res. Publ. Assoc. Res. Nervous Mental Disease 20:170-194. 1940. Scharrer, E. and B. Scharrer: Hormones produced by neurosecretory cells. Recent Progr. Hormone Res. 10:183-232. 1954. Schnaitman. C.. V. G. Erwin, and J. W. Greenawalt: The submitochondrial localization of monoamine oxidase. J. Cell. Biol. 32:719-735. 1967. Schneider, H. P. G. and S. M. McCann: Possible role of dopamine as transmitter to promote discharge of LH-releasing factor. Endocrinology 85:121-132. 1969. Schneider. H. P. G. and S. M. McCann: Mono- and indolamines and control of LH secretion. Endocrinology 86:1127-1133. 1970a. Schneider, H. P. G. and S. M. McCann: Release of LH-releasing factor into the peripheral circulation of hypophysectomized rats by dopamine and its blockade by estradiol. Endocrinology_87:249- 253, 1970b. Schneider, H. P. G. and S. M. McCann: Estradiol and the neuroendocrine control of LH release in vitro. Endocrinology 87:330-338. 1970c. Sedvall, G., V. K. Weiss. and I. J. Kapin: The rate of norepinephrine synthesis measured jo_vivo during short intervals: influence of adrenergic nerve 1mpu|ses. J. Pharmacol. Exp. Ther. 159:274- 282, 1968. Setalo, G., S. Vigh, A. V. Schally. A. Arimura, and B. Flerko: LH-RH- containing neural elements in the rat hypothalamus. Endocrinology 96:135-142. 1975. 163 Sgouris. J. T. and J. Meites: Differential inactivation of prolactin by mammary tissue from pregnant and parturient rats. Amer. J. Physiol. 175:319-321. 1953. Shaar. C. J.: The effects of catecholamines on anterior pituitary pro- lactin release. Thesis for Ph.D. Degree. Michigan State Univer- sity. pp. 137-144. 1975. Shaar. C. J. and J. A. Clemens: Inhibition of lactation and prolactin secretion in rats by ergot alkaloids. Endocrinology 90:285-288. 1972. Shaar, C. J. and J. A. Clemens: The role of catecholamines in the release of anterior pituitary prolactin jo_vitro. Endocrinology 95:1202-1212, 1974. Shambauch. G. E., J. F. Wilber. E. Montoya, H. Ruder. and R. Blonsky: Thyrotropin-releasing hormone (TRH) measurements in human spinal fluid. J. Clin. Endocrinol. Metab. 41:131-134. 1975. Sheppard. H. and J. H. Zimmerman: Reserpine and levels of serotonin and norepinephrine in the brain. Nature 185:40-41. 1960. Shibusawa. K., S. Saito, K. Nishi. T. Yamamoto. C. Abe. and T. Kawai: Effects of thyrotropin releasing principle (TRF) after section of the pituitary stalk. Endocrin. Jap. 3:151-157, 1956. Shibusawa. K., T. Yamamoto. K. Nishi. C. Abe. and S. Tomie: Studies on the tissue concentrations of TRF in the normal dog. Endocrinol. goo, 6:137-143. 1959. Shopsin. B.. L. Shenkman. I. Sanghui. and C. S. Hollander: Toward a relationship between the hypothalamic-pituitary-thyroid axis and the synthesis of serotonin. In: Advances in Biochemical Psychopharmacology, Vol. 10. Edited by E. Costa. GI L. Gessa. and'Ml Sandler, pp. 279-286. Raven Press. New York, 1974. Silver. T. N.. G. Vandenberg. and S. S. C. Yen: Inhibition of growth hormone release in humans by somatostatin. J. Clin. Endocrinol. Metab. 37:1202-1213. 1974. Simon, M. L. and R. George: Diurnal variations in plasma corticosterone and growth hormone as correlated with regional variations in norepinephrine. dopamine and serotonin content of rat brain. Neuroendocrinology 17:125-138. 1975. Simonin, R., H. Roux, C. H. Oliver. P. H. Jaquet. P. Argemi, and P. H. et Vague: Effects d'une prise orale de L-dopa sur les taux plasmatiques de TSH, ACTH et GH chex les sujets normaux. Ago, Endocrin. 33:294-296. 1972. 164 Simonovic, I.. M. Motta. and L. Martini: Acetylcholine and the release of the follicle-stimulating hormone-releasing factor. Endocrinology 95:1373-1377, 1974. Sinha. Y. N. and H. A. Tucker: Pituitary prolactin content and mammary development after chronic administration of prolactin. Proc. Soc. Exper. Biol. Med. 128:84-88. 1968. Smalstig, E. B. and J. A. Clemens: Pharmacological characterization of rat pituitary receptors mediating the inhibition of prolactin. Endocrinology_94zA-185, 1974. Smalstig, E. 8.. B. 0. Sawyer. and J. A. Clemens: Inhibition of rat prolactin release by apomorphine jo_vivo and 1o_vitro. Endocrinology_95:123-129. 1974. Smelik. P. G.: A dopaminergic innervation of the intermediate lobe of the pituitary. Acta. Physiol. Pharm. Neerl. 14:92-93, 1966. Smith. V. G., T. W. Beck, E. M. Convey and H. A. Tucker: Bovine serum prolactin. growth hormone. cortisol and milk yield after ergocryptine. Neuroendocrinology_15:172-181. 1974. Smythe, G. A. and L. Lazarus: Blockade of the dopamine-inhibitory con- trol of prolactin secretion in rats by 3.4-dimethoxyphenyleth- ylamine (3,4-di-O-methyldopamine). Endocrinology_93:147-151. 1973a. Smythe, G. A. and L. Lazarus: Growth hormone regulation by melatonin and serotonin. Nature 244:230-231. 1973b. Smythe, G. A., J. F. Brandstater. and L. Lazarus: Serotonergic control of rat growth hormone secretion. Neuroendocrinology 17:235- 257, 1975. Sokal. R. R. and F. J. Rohlf: Biometry. W. H. Freeman and Comp., San Francisco. 1969. Squires. R. F.: Hyperthermia and L-tryptophan-induced increases in serotonin in rat brain. In: Advances in Biochemical Psycho- harmacolo Vol. 10, edited by E. Costa. G. L. Gessa. and M. Eandler. pp. 267-212. Raven Press. New York. 1974. Stone. E. A.: Accumulation and metabolism of norepinephrine in rat hypothalamus after exhaustive stress. J. Neurochem. 21:589-601. 1973. Stone. T. W.: Studies on the central nervous system effect of agro- clavine, an ergot alkaloid. Arch. Int. Pharmacodyn. 202:62-65. 1973. . 165 Strada. S. J., E. Sanders-Bush. and F. Sulser: P-Chloroamphetamine: temporal relationship between psychomotor stimulation and metabolism of brain norepinephrine. Biochem. Pharmacol. 19: 2621-2629, 1970. Strosser, M. T., B. Bucher. B. Briand, B. Lutz. B. Koch, and C. Mialhe: Effet de la chaleur sur la secretion de l‘hormone de croissance et sur 1'activite du cortex surrenalien du rat. J. Physiol.. Paris 68:181-191. 1974. Sud, S. C.. J. A. Clemens, and J. Meites: Effect of median eminence lesions on mammary lobulo-alveolar development in hypophysectom- ized rats bearing one pituitary transplant. Ind. J. Exp. Biol. 8:81-83. 1970. Sulman. F. G. and H. Z. Winnik: Hormonal effects of chlorpromazine. Lancet 270: 161-162, 1956. Szentagothai. J., B. Flerko, B. Mess. and B. Halasz: Hypothalamic Con- trol of the Anterior Pituitary, pp. 22-155. Akademiai Kiado, Budapest, 1972. Takahara. J., A. Arimura, and A. V. Schally: Suppression of prolactin release by a purified porcine PIF preparation and catecholamines infused into a rat hypophysial portal vessel. Endocrinology. 95:462-465. 1974a. Takahara. J., A. Arimura, and A. V. Schally: Stimulation of prolactin and growth hormone release by TRH infused into a hypophyseal portal vessel. Proc. Soc. Exper. Biol. Med. 146:831-835, l974b. Takahara. J., A. Arimura, and A. V. Schally: Effects of catecholamines on the TRH-stimulated release of prolactin and growth hormone from sheep pituitaries jo_vitro. Endocrinology 95:1490-1494, 974C. Talwalker, P. K., A. Ratner. and J. Meites: In vitro inhibition of pro- lactin synthesis and release by hypotfidlamic extract. Symposium on Neuroendocrinology, Miami. Florida. 1961. Talwalker. P. K., A. Ratner. and J. Meites: Io_vitro inhibition of pituitary prolactin synthesis and release 5y hypothalamic ex- tracts. Am. J. Physiol. 205:213-218, 1963. Tashjian, A. H., N. J. Barowsky. and D. K. Jensen: Thyrotropin-releasing hormone: direct evidence for stimulation of prolactin production by pituitary cells in culture. Biochem. Biophys. Res. Commun. 43:516-523. 1971. 166 Terasawa, E., W. E. Bridson. J. W. Davenport, and R. W. Goy: Role of brain monoamines in release of gonadotropin before proestrus in the cyclic rat. Neuroendocrinology 18:345-358. 1975. Thierry. A. M., F. Javoy. J. Glowinski, and S. S. Kety: Effects of stress on the metabolism of norepinephrine. dopamine and sero- tonin in the central nervous system of the rat. I. Modifica- tions of norepinephrine turnover. J. Pharmacol. Exp. Ther. 163:163-171. l968a. Thierry. A. M., M. Fekete. and J. Glowinski: Effects of stress on the metabolism of noradrenaline. dopamine and serotonin (5 HT) in the central nervous system of the rat. II. Modifications of serotonin metabolism. Europ: J. Pharmacol. 4:384-389, l968b. Thornburg. J. E. and K. E. Moore: A comparison of effects of apomor- phine and ET-495 on locomotor activity and circling behavior in mice. Neuropharmach 13:189-197. 1974. Tozer. T. H., N. H. Neff. and B. B. Brodie: Application of steady state kinetics to the synthesis rate and turnover in the brain of normal and reserpine treated rats. J. Pharmacol. Exp. Ther. 153:177-182. 1966. Tucker. H. A. and R. P. Wettemann: Effects of ambient temperature and relative humidity on serum prolactin and growth hormone in heifers. Proc. Soc. Exp. Biol. Med. 151:623-626, 1976. Tuomisto. J., T. Ranta, A. Saarineu. P. Mannisto. and J. Leppaluoto: Neurotransmission and secretion of thyroid-stimulating hormone. Lancet 2:510-511. 1973. Turkington. R. W.: Measurement of prolactin activity in human serum by the induction of specific milk proteins jo.vitro: results in various clinical disorders. Io: Lactogenic Hormones. edited by G. E. W. Wolstenholme and J. Knight.'Churchil Livingstone. London. pp.l69-184. l972a. Turkington, R. W.: Phenothiazine stimulation test for prolactin reserve: the syndrome of isolated prolactin deficiency. J. Clin. Endocrinol. Metab. 34:247-249. l972b. Turner. A. J., J. A. Illingworth, and K. F. Tipton: Stimulation of bio- genic amine metabolism in the brain. Biochem. J. 144:353-360. 1974. Ungerstedt, U.: Stereotaxic mapping of the monoamine pathways in the rat brain. Acta. Physiol. Scand. 367-1-48, 1971. 167 Vale. W.. P. Brazeau, C. Rivier. J. Rivier, G. Grant. R. Burgus. and R. Guillemin: Inhibitory hypophysiotropic activities of somato- statin. Fed. Proc. 32:211. l973a. Vale, W.. G. Grant, and R. Guillemin: Chemistry of the hypothalamic releasing factors: Studies on structure-function relationships. In: Frontiers in Neuroendocrinology, 1973. edited by W. F. Gdhong and L. Martini. pp. 375-413, Oxford University Press. New York. 1973b. Vale, W.. C. Rivier. P. Brazeau, and R. Guillemin: Effects of somato- statin on the secretion of thyrotropin and prolactin. Endocrinology 95:668-677. 1974a. Vale, W.. C. Rivier. M. Palkovits. J. M. Saavedra. and M. Brownstein: Ubiquitous brain distribution of inhibitors of adenohypophysial secretion. Endocrinology 94:A-128, 1974b. Vale, W.. P. Brazeau, C. Rivier. M. Brown, 8. Boss. J. Rivier, R. Burgus. N. Ling. and R. Guillemin: Somatostatin. Recent Progr. Hormone Rog, 31:365-397. 1975. Valverde. R. C.. V. Chieffo. and S. Reichlin: Prolactin-releasing factor in porcine and rat hypothalamic tissue. Endocrinology 91:982- 993. 1972. Vogt, M.: The concentration of sympathin in different parts of the central nervous system under normal conditions and after the administration of drugs. J. Physiol. (London) 123:451-481. 1954. Voogt, J. L. and J. Meites: Effects of an implant of prolactin in median eminence of pseudopregnant rats on serum and pituitary LH, FSH and prolactin. Endocrinology 88:286-292. 1971. Voogt. J. L. and J. Meites: Suppression of proestrous and suckling- induced increase in serum prolactin by hypothalamic implant of prolactin. Proc. Soc. Exp: Biol. Med. 142:1056-1058. 1973. Voogt, J. L. and W. F. Ganong: Io_vitro evidence against the anterior pituitary as a site of negative feedback of prolactin. Proc. Soc. Exper. Biol. Med. 147:795-797. 1974. Watson, J. T., L. Krulich. and S. M. McCann: Effect of crude rat hypo- thalamic extract on serum gonadotropin and prolactin levels in normal and orchidectomized male rats. Endocrinology 89:1412- 1418. 1971. Weiner, N.: A critical assessment of methods for the determination of monoamine synthesis turnover rates jo_vivo. Io; Neuropsycho- pharmacology of Monoamines and Their Rogulatory Enzymes, edited by E. Usdlin. pp. 143-159, Raven Press. New York, 1974. 168 Weiss. 8. L. and G. K. Aghajanian: Activation of brain serotonin metab- olism by heat: role of midbrain raphe neurones. Brain Res. 26:37-48. 1971. Welsch, C. W.. M. D. Squiers. E. Cassell, C. L. Chen, and J. Meites: Median eminence lesions and serum prolactin: influence of ovariectomy and ergocornine. Am. J. Physiol. 221:1714-1717. 1971. Wettemann. R. P. and H. A. Tucker: Relationship of ambient temperature to serum prolactin in heifers. Proc. Soc. Exper. Biol. Med. 146:908-911. 1974. White. W. F.. M. T. Hedlund. G. F. Weber. R. H. Rippel, E. S. Johnson. and J. F. Wilber: The pineal gland: a supplemental source of hypothalamic-releasing hormones. Endocrinology 94:1422-1426, 1974. Williams. R. H., H. Jaffe. and C. Kemp: Effect of severe stress upon thyroid function. Amer. J. Physiol. 159:291-297. 1949. Wilson. C. A.: Hypothalamic amines and the release of gonadotropins and other anterior pituitary hormones. Advances in Drog Research 8:119-205. 1974. Winters. A. J., R. L. Eskay. and J. C. Porter: Concentration and distribution of TRH and LRH in the human fetal brain. J. Clin. Endocrin. Metab. 39:960-963. 1974. Wislocki. G. B. and L. S. King: The permeability of the hypophysis and hypothalamus to vital dyes. with a study of the hypophyseal vascular supply. Am. J. Anat. 58:421-472. 1936. Wong. 0. T., F. P. Bymaster. J. S. Horng, and B. B. Molloy: 3-(p-tri- f1uoromethylphenoxy)-N-methyl-3-phenylpropy1amine (Lilly 110140) a specific inhibitor of serotonin uptake into synatosomes of rat brain. Fed. Proc. 33:255. 1974. Wurtman, R. J.: Brain monoamines and endocrine function. Neurosci. Res. Symp. Summ. 6:171-297. 1972. Wuttke. W. and J. Meites: Luteolytic role of prolactin during the estrous cycle of the rat. Proc. Soc. Exp. Biol. Med. 137:988- 991. 1971. Wuttke, W.. E. Cassell, and J. Meites: Effects of ergocornine on serum prolactin and LH, and on hypothalamic content of PIF and LRF. Endocrinology 88:737-741. 1971. 169 Yamada. Y.: Effects of iontophoretically-applied prolactin on unit activity on the rat brain. Neuroendocrinology 18:263-271. 1975. Yen. S. S. C.. B. L. Lasley. C. F. Wang. H. Leblanc. and T. M. Siler: The operating characteristics of the hypothalamic-pituitary system during the menstrual cycle and observations of biological action of somatostatin. Recent Progr. Hormone Res. 31:321-357. 1975. Yuwiler, A.: Conversion of O- and L-tryptophan to brain serotonin and 5-hydroxyindoleacetic acid and to blood serotonin. J. Neurochem. 20:1099-1109. 1973. Zimmerman, E. A., K. C. Hsu. M. Ferin. and G. P. Kozlowski: Localization of gonadotropin-releasing hormone (Gn-RH) in the hypothalamus of the mouse by immunoperoxidase technique. Endocrinology_95: 1-8, 1974. Zschaeck. L. L. and R. J. Wurtman: Brain 3H-catechol synthesis and the vaginal estrous cycle. Neuroendocrinology 11:144-149. 1973. APPENDICES APPENDIX A Serotonin and 5-HIAA Assaprrocedures Reagents: Acid Butanol 1 Liter butanol plus 0.85 ml concen- trated hydrochloric acid (HCl) 1% Cysteine 0.1 gm L-cysteine (Sigma Chemical Comp.) dissolved in 10 ml 0.1 N HCl OPT solution 4.0 mg O-phthaldehyde (A grade, Calbio- chem) dissolved in 100 m1 concentrated HCl 0.5 M Phosphate Buffer Make separate 0.5 M solutions mono and dibasic Na phosphate Monobasic = 6.0 gm HaHzPO -H 0 in 100 4 2 ml H20 Dibasic = 7.1 gm NazHOP in 100 m1 H 0 4 2 Titrate dibasic with monobasic to pH 7.5 Stock serotonin standard Dissolve 43.97 mg serotonin creatinine sulfate (Sigma Chemical Comp.) in 100 m1 of 0.1 N HCl (20 mg free base/100 ml), store at 4°C for up to three weeks Stock 5-HIAA Standard Dissolve 20 mg 5-HIAA (Sigma Chemical Comp.) in 100 ml of 0.1 N HCl and store at 4°C for up to three weeks Procedure for Brain Samples: 1. Homogenize brain (about 1.45 gm tissue) in 7.0 m1 acid butanol, 2. Centrifuge at 15,000 RPM (27.000 x G) for 15 min. at 4°C. (supernatant volume = 7.75 ml fOr volume correction) 170 10. 11. 12. 171 . Transfer 3.0 ml of supernatant to a glass screw cap test tube contain- ing 2.5 m1 of 0.1 N HCl and 6.0 ml heptane. . Shake for 10 min. . Centrifuge at maximum speed in an International Clinical Centrifuge (table top model) for 5 min. . Transfer 5.0 ml or organic phase (containing 5-HIAA) to a glass screw test tube containing 1.5 ml of 0.5 M phosphate buffer pH 7.5. Continue at step 8. . Draw off remaining organic phase by suction and transfer 1.0 m1 aqueous phase (containing serotonin) to a glass screw cap test tube containing 100 ul of 1% cysteine. Continue at step 10. . Shake for 10 min. and centrifuge for 5 min. . Draw off organic phase and transfer 1.0 ml aqueous phase (containing S-HIAA) to a glass screw cap test tube containing 100 ul of 1% cysteine. Add 2.0 m1 OPT solution to serotonin and 5-HIAA fractions. mix and cap. Heat in 100°C water bath for 10 min. Cool to room temperature and read sample fluorescence at 355 nm excitation and 470 nm emission. Procedure for Hypothalamus Samples: 1. #wN Homogenize hypothalamus in 2.0 ml acid butanol. Rinse with 2.0 ml acid butanol and combine with initial homogenate. Centrifuge 10,000 RPM (12.350 x G) for 15 min at 4°C. . Transfer 3.0 ml supernatant to a glass screw cap test tube and pro- ceed at step 4 in the procedure for brain samples. Procedure for Standards: 1. Separately dilute lOO ul aliquots of serotonin and 5-HIAA stock standards 1 to 100 with 0.1 N HCl (100 ul Stb. + 9.9 ml 0.1 N HCl). Final concentration of working standards is 2 ng/ul. 172 . Place 100 ul of both serotonin and 5-HIAA standards together in 9.8 ml 0.1 N HCl to make a "combined" working standard. . To determine % recovery for brain extraction procedure, place in duplicate: 0 ul. 100 ul and 200 ul of combined working standard (0 ng/200 ng and 400 ng) in glass screw cap test tubes containing 3.0 ml acid butanol. 6.0 ml heotane and 2.5 ml. 2.4 ml and 2.3 ml 0.1 N HCl respectively. Begin at step 4 in the procedure for brain samples. . To determine % recovery for hypothalamus extraction procedure. place in duplicate: 0 ul. 50 ul and 100 ul working serotonin standard (0 ng, 100 ng and 200 ng) in glass screw cap test tubes containing 3.0 m1 acid butanol, 6.0 m1 heptane and 1.50 ml. 1.45 ml and 1.40 ml 0.1 N HCl respectively. Begin at step 4 in the procedure for brain samples. (Note. hypothalamic levels of 5-HIAA are below the limits of detection by this assay.) . Serotonin standard curves can be run at 0 ng. 50 ng. 100 ng and 200 ng in duplicate. Place 0 ul. 25 ul, 50 ul and 100 ul of working standard in glass screw cap test tubes containing 0.1 m1 1% cysteine. and 1.0 m1, 0.975 ml. 0.950 ml and 0.900 ml 0.1 N HCl. Begin at step 10 in the procedure for brain samples. . 5-HIAA standard curves are made up similar to those for serotonin with the exception that the glass screw cap tubes contain 0.5 M phos- phate buffer pH 7.5 in place of 0.1 N HCl. APPENDIX B Tryptophan Assoy Procedure Reagents: 1O -4 N HC1 75% Trichloroacetic Acid (TCA) 10’4 N HC1/75% TCA solution 23 ml 10‘4 HC1 + 3 ml 75% TCA HCHO solution 1.0 ml 37% Formaldehyde + 19.5 ml H20 HCHO/FeCl3 48.6 mg FeCl3 in 100 ml HCHO solution 0.1 N NH40H 6.75 ml NH4OH diluted up to 1 liter with H20 Stock Tryptophan Standard 20 mg of L-tryptophan (Sigma Chemical Comp.) dissolved in 100 ml 0.1 N NH OH (200 ng/ul). Store at 4°C for up td three weeks. Working Tryptophan Standard Dilute 1.0 ul Stock 9.9 ml 0.1 N NH 0H (2 ng/ul). 4 Procedure for Samples: 1. Chm-b0.) Homogenize tissue in 5 to 20 volumes of 10"4 volume based on the tissue weight.) HC1. (Use a convenient . Transfer 300 ul of homogenate to a plastic centrifuge tube containing 2.0 ml 10-4 N HC1. . Add 300 ul 75% TCA and vortex. Centrifuge at 10,000 RPM (12,350 x G) for 15 min at 4°C. Transfer 2.0 m1 supernatant to a glass screw cap test tube. . Add 200 ul HCHO/FeCl3 reagent and vortex. 173 174 7. Cap and heat at 100°C for 60 min. 8. Cool toroom temperature and read sample fluorescence at 371 nm excitation, 443 nm emission. Procedure for Standards: 1. To determine % recovery for extraction procedure place in duplicate: O u . 5 U1 and 15 ul stock standard (0. 1 ug and 3 ug) in a volume of 10' HCl comparable to the volumes used for tissue homogenization. Begin at step two in the procedure for samples. 2. Tryptophan standard curves can be run at 0 no. 50 ng. 100 ng and 200 ng. Place 0 ul. 25 ul, 50 ul and 100 ul of working standard in glass screw cap test tubes containing 2.0 ml. 1.975 ml. 1.950 ml and 1.900 ml of 10'4 HC1/75% TCA solution respectively. Begin at step 6 in the procedure for samples. CURRICULUM VITAE NAME: Gregory Paul Mueller DATE OF BIRTH: December 22. 1948 PLACE OF BIRTH: Madison, Wisconsin. USA NATIONALITY: American SEX: Male MARITAL STATUS: Single PRESENT ADDRESS: Department of Physiology Michigan State University East Lansing. Michigan 48824 PRESENT PHONE: (517) 353-6358 FUTURE ADDRESS: Department of Nutrition and Food Science Laboratory of Neuroendocrine Regulation Massachusetts Institute of Technology Cambridge. Massachusetts 02139 EDUCATION: University of Montana 1971 B.A. Zoology Michigan State University 1976 Ph.D. Physiology POSITIONS HELD: Research Assistant, Department of Physiology. Michigan State University. East Lansing, Michigan, September 1971 to present Teaching Assistant, Department of Physiology, Michigan State University. East Lansing, Michigan, September 1973 to December 1973; September 1974 to December 1974 AWARDS: Postdoctoral Fellowship Award for two years (April 1977 to April 1979). from the National Institute of Health, Bethesda. Maryland Sigma Xi Graduate Student Award for Meritorious Research in Physiology (May 1976) 175 176 Curriculum Vitae Gregory Paul Mueller Page 2 TALKS PRESENTED AT SCIENTIFIC MEETINGS: Meeting Date Topic 58th Annual FASEB Meeting 1974 Effects of estrogen. T4 and TRH Atlantic City. New Jersey on Serum Prolactin and TSH in Rats 60th Annual FASEB Meeting 1976 Effect of L-tryptophan and Anaheim. California Restraint Stress on Serotonin Turnover and TSH and Prolactin (PRL) Release in Rats RESEARCH PUBLICATIONS: 1. Dibbet. J. A., J. F. Bruni, G. P. Mueller. H. J. Chen, and J. Meites: Ip_vivo and jp_vitro stimulation of prolactin secretion by synthetic TRH in rats. Endocrinology gggA-l39. 1973 (Abstract). . Mueller. G. P., H. J. Chen. and J. Meites: In vivo stimulation of of prolactin release in the rat by synthetic-TRH. Proc. Soc. Exp. Biol. Med. 144:613-765. 1973. . Chen. H. J., G. P. Mueller, and J. Meites: Effects of L-dopa and somatostatin on the suckling-induced release of prolactin and GH. Endo. Res. Comm. 15283-291. 1974. . Mueller, G. P., H. T. Chen, J. A. Dibbet. H. J. Chen. and J. Meites: Effects of warm and cold temperatures on the release of TSH. GH and prolactin in the rat. Proc. Soc. Exp. Biol. Med. 147:698-700. 1974. . Mueller. G. P., H. J. Chen. and J. W. Thomas: Effects of estrogen. T4. and TRH on serum prolactin and TSH in rats. Fed. Proc., Fed. Amer. Soc. Exp. Biol. 33:197. 1974 (Abstract and presentation). . Jacoby. J. H., G. Mueller. and R. J. Wurtman: Thyroid state and brain monoamine metabolism. Endocrinologyhgzgl332-1335. 1975. . Mueller, G. P., C. P. Twohy. H. T. Chen, and J. P. Advis: Effects of L-tryptophan and restraint stress on serotonin turnover and TSH and prolactin (PRL) release in rats. Fed. Proc.. Fed. Amer. Soc. Exp. Biol.. 1976 (Abstract and presentation). . Mueller. G. P., C. P. Twohy. H. T. Chen, J. P. Advis, and J. Meites: Effects of L-tryptophan and restraint stress on hypothalamic and brain serotonin turnover and pituitary TSH and prolactin release in rats. Life Sci. 183715-724. 1976. 177 Curriculum Vitae Gregory Paul Mueller Page 3 9. Mueller. G. P., J. Simpkins. J. Meites. and K. E. Moore: Differential 10. effects of dopamine agonists and haloperidol on release of prolactin, thyroid stimulating hormone. growth hormone and luteinizing hormone in rats. Neuroendocrinology. 1976 (in press) Simpkins, J. S.. G. P. Mueller. H. H. Huang. and J. Meites: Changes in the metabolism of dopamine. norepinephrine and serotonin. and rela- tion to gonadotropin secretion in aging male rats (Abstract. American Physiological Society. Fall Meetings. 1976). "Illlllllllllllll'lllls