. .. -. v-- "“‘m r." A .a-" "U L I B R A R ' Michigan 8 tate University This is to certify that the thesis entitled RELATION OF BRAIN MONOAMINE METABOLISM T0 SECRETION 0F GONADOTROPINS AND PROLACTIN presented by James William Simpkins has been accepted towards fulfillment of the requirements for Ph.D. degree in Physiology Date 7/23/27 7 0-7639 RELATION OF BRAIN MONOAMINE METABOLISM TO SECRETION OF GONADOTROPINS AND PROLACTIN By James William Simpkins A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1977 ABSTRACT RELATION OF BRAIN MONOAMINE METABOLISM TO SECRETION OF GONADOTROPINS AND PROLACTIN By James William Simpkins 1. Treatment of ovariectomized, estrogen-primed rats with 500 ug progesterone (P) per kg body weight resulted in a subsequent LH and prolactin surge. Serum LH concentration increased significantly by 4 h and was elevated dramatically by 6 and 9 after P administration. Serum prolactin levels were elevated by 2 h, peaked at 4 h and remained elevated at 6 and 9 h after P treatment. A 2-fold increase in anterior hypothalamic norepinephrine (NE) turnover was observed by 4 h after P treatment which returned to control (Pre-P) levels by 6 and 9 h after P administration. Anterior hypothalamic (AH) dopamine (DA) turnover decreased significantly by 6 and 9 h after P treatment. No significant difference in catecholamine concentration or turnover was observed in posterior hypo- thalamic fragments at any of the sampling times. It is con- cluded that the increase in AH~NE turnover and the decrease in AH-DA turnover may partially mediate the P induced surges of LH and prolactin. low . an. James William Simpkins 2. The tyrosine hydroxylase inhibitor, alpha-methyl-para- tyrosine (ampt), completely inhibited the P-induced LH surge in ovariectomized, estrogen-primed rats when administered at the time of P treatment. Alpha—mpt, when injected 1 h before decapitation, was able to block partially LH release at 4,6 and 9 h after P administration, but was ineffective at 2 h and increased LH secretion at 0 h after P treatment. This treatment resulted in elevated prolactin at 0 and 2 h but not at 4 and 6 h after P administration. Since NE turnover in- creases and DA turnover decreases after P treatment, these observations suggest that the ampt blockade of NE synthesis results in blockade of the LH surge while its blockade of DA synthesis increases serum prolactin. 3. Sustained administration of the DA agonist, piribedil, blocked the P-induced prolactin increase in ovariectomized, estrogen—primed animals but was ineffective in altering peak levels of serum LH. These findings indicate that the decrease in AH-DA turnover is not essential for mediating the P—induced LH surge, but may indicate that a decreased DA turnover is necessary for mediating prolactin release by P administration to ovariectomized, estrogen-primed rats. 4. Small doses of 6—hydroxydopamine 6-OH—DA implanted into the suprachiasmatic nucleus (SCN) 24 h before P treatment to ovariectomized, estrogen-primed rats, depleted anterior hypothalamus NE by 83% and blocked the subsequent LH surge, whereas median eminence (ME) 6-OH-DA implants decreased ME-NE of RC 5011 A U W la tenin James William Simpkins by 57%, but were ineffective in altering LH secretion. Neither treatment significantly altered DA concentration. Implants of 6-OH-DA into ME caused a slight decrease in serum LH concentration at the time of P treatment (0900 h). It is concluded that rostral hypothalamic noradrenergic synap- ses mediate the P-induced LH surge in this experimental model, whereas ME noradrenergic synapses are not involved in this process. 5. Orchidectomy in rats rapidly increased circulating levels of LH (6 h) which remained elevated through 48 h. A 2-fold increase in hypothalamic NE turnover occurred by 6 h and returned to sham castrate levels by 48 h post-castration although serum LH levels continued to be elevated. Hypothala- mic DA turnover appeared to increase by 6 h and was elevated at 48 h post-castration. Medial basal hypothalamic (MBH) implantation of the neurotoxin, 6—hydroxydopamine (6-0H-DA), 24 h before castration was effective in partially inhibiting the post-castration LH increase. These studies indicate that the transient increase in NE turnover observed at 6 h may be involved in hastening the post-castration LH increase, although the long term response of LH to castration may be independent of noradrenergic input. The increased DA turnover which follows castration may be in part responsible for the decrease or lack of response of prolactin secretion to castration. 6. The steady state concentration of DA, NE and sero- tonin (SHT) was determined and turnover estimated in several James William Simpkins brain regions of young (3-4 mo) and old (21 mo) male Wistar rats. In old male rats, MBH-DA concentration and turnover were significantly lower than in young males. In the remain- ing hypothalamus, DA concentration was slightly lower in old than young rats although DA turnover was not different in the 2 groups. DA concentration and turnover in olfactory tuber- cles were the same in both age groups. The steady state con- centration of NE in the MBH and remaining hypothalamus and hypothalamic NE turnover were significantly lower in old than young male rats. Since DA inhibits prolactin secretion and NE stimulates LH and FSH secretion, agesrelated alterations in central CA‘s may account for the observed increase in serum prolactin and decrease in serum LH and FSH in old male rats. In both brain and hypothalamus, steady-state concentra- tions of SHT were the same in young and old rats, but by 30 min after monoamine oxidase inhibition with pargyline, hypo- thalamic but not brain SHT increased more in old than in young male rats. S-Hydroxyindoleacetic acid (SHIAA) concentration was 25% higher in brains of old than young males but decreased less in response to treatment with pargyline. These results may indicate a greater turnover of SHT in the hypothalamus in old than young male rats and a deficiency in the clearance of SHIAA from the brains of old male rats. Since SHT has been implicated in stimulating prolactin and inhibiting LH and FSH secretion in rats, the apparent increase in hypothalamic SHT turnover may be involved in elevation of prolactin and depres- sion LH and FSH in old male rats. Dedication This thesis is dedicated to my wife, Janet, and my children, Christopher and Gretchen, for joining me in this venture. Their sacrifices en route to this thesis have been so much greater than mine. ii ACKNOWLEDGEMENTS The author wishes to thank the faculty, staff and students of the Department of Physiology, Michigan State University for their contribution to my education. My thanks is also extended to the following individuals who assisted in various aspects of this thesis work: Charles Hodson, Henry Huang, Warren Chen, Juan Advis, William Mallard, Greg Mueller and Claire Twothy. I also wish to express my deepest appreciation to Professor Joseph Meites for his unwavering dedication to my education. His knowl- edge of endocrinology and his zeal for research has made my tenure in the Neuroendocrine Research Laboratory a very pleasurable and productive experience. iii 'l" m. TABLE OF CONTENTS LIST OF TABLES ...................................... LIST OF FIGURES ..................................... INTRODUCTION. LITERATURE REVIEW ................................... I. Hypothalamic Control of Anterior Pituitary Function ................................... A. B. C. D. Classical Observation ................. Portal Vessels and Neurosecretion ..... Hypophysiotropic Hormones ............. Hypothalamic Anatomy ....... .... ...... . II. Monoaminergic Pathways in the Hypothalamus.. A. B. C. Noradrenergic Pathways. ............... Dopaminergic Pathways ................. Serotonergic Pathways ................. III. Catecholamine and Serotonin Metabolism ...... IV. Hypothalamic Control of Prolactin Secretion. A. B C D. E F G Prolactin Levels and Physiological States. .............................. Inhibitory Influence of the Hypothal- amus on Prolactin Secretion ........... . Stimulatory Influence of the Hypothal- amus on Prolactin Secretion ........... Influence of Steroids on Prolactin Secretion ............................. . Dopaminergic Effects on Prolactin Secretion ............................. . Noradrenergic Effects on Prolactin Secretion ............................ Serotonergic Effects on Prolactin Secretion ............................. iv Page viii ix V. Hypothalamic Control of Luteinizing Hormone Secretion .................................... A. Feedback of Gonadal Steroids on LH Secretion .............................. . Noradrenergic Effects on Luteinizing Hormone Secretion ..... . ............ .... . Dopaminergic Effects on Luteinizing Hormone Secretion ...................... Serotonergic Effects on Luteinizing Hormone Secretion ...................... Inhibitory Effects of Prolactin on LH Secretion .................... ..... ..... macaw MATERIALS AND METHODS ................................ I. II. III. Animals, Treatments and Blood Collection ..... Radioimmunoassay of Serum Hormones ........... Assay of Dopamine and Norepinephrine in Brain Tissue ..... . .......... .. ................. A. Isolation and Preparation of Brain Tissue. ............................ B. Radioenzymatic Assay of D0pamine (DA) and Norepinephrine (NE) ....... ... ...... EXPERIMENTAL ......................................... I. Effects of Progesterone on Steady State Con- centration and Turnover of Dopamine (DA) and Norepinephrine (NE) in Anterior and Posterior Portions of the Hypothalamus, and on Serum Luteinizing Hormone and Prolactin Levels in Ovariectomized, Estrogen-Primed Rats. ...... A. Objectives ............................ B. Materials and Methods .................. C. Results ................................ D. Discussion ................ ............. Further Study on the Effect of Progesterone on Dopamine Turnover in Ovariectomized- Estrogen- -Primed Rats ........... ....... ....... A. Objectives ........................... B. Materials and Methods ............ . ..... C. Results ................................ D. Discussion ............................. V Page 47 47 54 58 61 63 66 66 67 68 68 70 72 II. III. IV. VI. Effects of Alpha-Methyl—Para-Tyrosine on Serum LH and Prolactin Surges Induced by Progesterone Treatment of Ovariectomized, Estrogen-Primed Rats ......................... A. Objectives ................. . ........... B. Materials and Methods ................. . C. Results ................................ D. Discussion .............. . .............. Effects of Multiple Piribedil Injection on Serum LH and Prolactin Surges Induced by Progesterone in Ovariectomized, Estrogen- Primed Rats ................. ...... ........... A. Objectives ............................. B. Materials and Methods. ................. C. Results ................................ D. Discussion ................... . ......... Effect of Implants of 6-Hydroxydopamine in the Suprachiasmatic Nucleus (SCN) and Median Eminence (MB) on Anterior Hypothalamic and Median Eminence Dopamine and Norepinephrine Concentration, and on Serum Luteinizing Hormone and Prolactin Surges Induced by Progesterone in Ovariectomized, Estrogen- Primed Rats .......... ...... .................. A. Objectives ............................. B. Materials and Methods .................. C. Results ................................ D. Discussion ............................. . Measurement of D0pamine and Norepinephrine Turnover and Serum Luteinizing Hormone After Short Term Castration in Male Rats... ..... ... A. Objectives ................... . ......... B. Materials and Methods .................. C. Results ................................ D. Discussion ............................. Effects of Medial Basal Hypothalamic Implants of 6-Hydroxyd0pamine on the Post-Castration Increase in Serum Luteinizing Hormone ........ A. Objectives ............................. B. Materials and Methods .................. C. Results ................................ D. Discussion ............................. vi Page 93 95 96 96 101 101 102 103 106 108 108 109 111 111 Page VII. Measurement of Concentration and Turnover of Brain Dopamine, Norepinephrine and Serotonin and Serum Luteinizing Hormone, Follicle Stimulating Hormone and Prolactin in Young and Old Male Rats ............................ 112 A. Objectives.... ......... . ............... 112 B. Materials and Methods ....... . .......... 114 Study 1 ............................. 114 Study 2 ............................. 115 C. Results ................................ 116 D. Discussion ............................. 123 GENERAL DISCUSSION ................................... 128 BIBLIOGRAPHY ......................................... 134 APPENDICES A. COYLE AND HENRY CATECHOLAMINE ASSAY PROCEDURE 175 B. BEN-JONATHEN AND PORTER CATECHOLAMINE ASSAY PROCEDURES...... ..... . ...... ..... ............ 177 C. RESEARCH PUBLICATIONS .......... . ............. 179 CURRICULUM VITAE ..................................... 182 vii LIST OF TABLES TABLE Page 1. Effects of Alpha-Methyl-Para-Tyrosine (ampt) on Serum Luteinizing Hormone (LH) at Various Times After Progesterone Administration to Ovariectom- ized, Estrogen-Primed Rats...................... 87 2. Effects of Alpha-Methyl-Para-Tyrosine (ampt) on Serum Prolactin (PRL) at Various Times After Progesterone Administration to Ovariectomized, Estrogen-Primed Rats ........ .................... 88 3. Effects of Suprachiasmatic Nucleus (SCN) and Median Eminence (ME) Implants of 6-Hydroxydopa- mine on Serum Luteinizing Hormone (LH) in Ovariectomized, Estrogen-Primed Rats ...... ...... 99 4. Average Body Weights and Weights of Brain Tissue Used for Catecholamine (Study 1) and Serotonin (Study 2) Determinations ......... ... .......... .. 116 5. Basal Serum Hormone Levels in 3-4 and 21 Month Old Male Rats.. ...... . .......................... 123 viii FIGURE 1. LIST OF FIGURES Effects of progesterone administration to ovari- ectomized, estrogen-primed rats on steady state concentration and alpha-methyl-para-tyrosine induced depletion of anterior hypothalamic catecholamines and on serum luteinizing hormone (LH) and prolactin (PRL) ..... .................. Effect of progesterone administration to ovari- ectomized, estrogen-primed rats on steady state concentration and alpha-methyl-para-tyrosine induced depletion of posterior hypothalamic catecholamines ................ ...... ........... Long term effects of progesterone administra- tion to ovariectomized, estrogen—primed rats on alpha-methyl-para-tyrosine induced depletion of anterior hypothalamic d0pamine and on serum luteinizing hormone (LH) and prolactin (PRL)... Long term effects of alpha—methyl-para-tyrosine on progesterone induced LH secretion in ovari- ectomized, estrogen-primed rats. ............... . Effect of sustained administration of piribedil mesylate (ET-495) on the progesterone induced luteinizing hormone (LH) and prolactin (PRL) secretion in ovariectomized, estrogen-primed rats..... ............................. ......... . Effects of suprachiasmatic nucleus (SCN) and median eminence (ME) implants of 6-hydroxy- d0pamine (6-OH-DA) on anterior hypothalamic and median eminence concentration of norepinephrine and dOpamine ...... . ............................ . Effect of suprachiasmatic nucleus (SCN) and median eminence (ME) implants of 6-OH-DA or cocoa butter (CB) on progesterone induced luteinizing hormone (LH) secretion in ovariec- tomized, estrogen-primed rats........... ....... ix Page 76 77 82 86 92 97 98 FIGURE Page 8. Effect of orchidectomy on concentration and alpha—methyl-para—tyrosine induced depletion of hypothalamic norepinephrine and dOpamine and serum LH concentration ......................... 105 9. Effect of bilateral ventromedial nuclei (VMN) implants of 6-hydroxyd0pamine (6-OH-DA) on post-castration rise in serum luteinizing hormone (LH) ................................... 110 10. Medial basal hypothalamic (MBH) d0pamine and norepinephrine concentration and levels 1 h after alpha-methyl~para—tyrosine treatment in 3-4 and 21 month old male rats ................. 118 11. Remaining hypothalamus dopamine and norepineph— rine concentration and levels 1 h after alpha- methyl—para-tyrosine treatment in 3-4 and 21 month old male rats ............................ 119 12. Olfactory tubercle dopamine concentration and levels 1 h after alpha~methy1-para-tyrosine treatment in 3-4 and 21 month old male rats.... 120 13. Hypothalamic serotonin (SHT) concentration and levels 30 min after treatment with pargyline in 3-4 and 21 month old male rats .............. 121 14. Brain serotonin (SHT) and S—hydroxyindoleacetic acid (SHIAA) concentration and levels 30 min after treatment with pargyline in 3-4 and 21 month old male rats ............................ 122 3'5 '51 r'f I'D n) INTRODUCTION The central nervous system (CNS) exerts a profound influ- ence on the endocrine system through its regulation of pituitary gland function. The synthesis and secretion of anterior pituitary (AP) hormones is regulated by neurohormones produced in and released from neurosecretory cells of the hypothalamus. These neurons act as neuroendocrine trans— ducers, receiving input from the autonomic nervous system and providing endocrine output through the release of neurohor- mones. The input to neurosecretory cells has been extensively studied using pharmacological agents which alter the metabol- ism and/or synaptic transmission of putative central neuro- transmitters. These studies have been valuable in demonstrat- ing a possible role for several putative neurotransmitters in the control of AP function, but have several limitations. First, all drugs have effects other than those for which they are administered. For catecholaminergic and serotonergic drugs, these effects can be exerted on other autonomic inputs affecting AP hormone secretion. Thus the prototype noradren- ergic agonist, clonidine may effect both the central sero- tonergic and the histaminergic systems, and the serotonergic antagonist, methysergide, is a partial d0pamine (DA) agonist. The catecholamine precursor, L-dihydroxyphenylalanine (L-dOpa), can be taken up and decarboxylated to DA in d0pa- minergic, noradrenergic, and serotonergic neurons. Second, the ability of a drug which alters CNS function to induce changes in AP hormone secretion does not, in itself, demon- strate a physiological involvement of that system in hormone secretion. Thus, pharmacologic effects do not always have physiological significance. An alternative approach to the evaluation of the role of putative neurotransmitters in AP hormone secretion is to determine if changes in brain metabolism or transmission of neurotransmitters occur during states of altered AP hormone secretion. Correlations between the metabolism of central neurotransmitters and the rate of AP hormone secretion pro- vide additional support (though not definite proof) for the involvement of a neurotransmitter in the regulation of hormone secretion. Initial attempts at measuring catechol- amine (CA) turnover during states of altered hormone secre- tion have been made primarily in the laboratories of Fuxé, Wurtman, and Donoso. The work in this thesis was conducted to assess further the role of hypothalamic noradrenergic, d0pamineragic and serotonergic systems in the regulation of AP function by simultaneously estimating their turnover and AP hormone secretory rates during a variety of experimental states . Pharmacological studies indicate that drugs which modify central noradrenergic activity affect the rate of secretion of luteinizing hormone (LH), but there is controversy as to the role of central noradrenergic systems in controlling prolactin secretion. Similarly there is general agreement that the tuberoinfundibular dopamine (DA) system inhibits prolactin secretien, but there is no agreement as to the role of DA in the control of LH release. This thesis was in part devoted to an evaluation of alterations in both DA and NE turnover during conditions in which LH and prolactin secretory rates increase simultaneously, diverge in Opposite directions, or respond selectively to the stimulus. Comparison of re- sults among these three experimental models has provided fur- ther evidence on the role of both CA's in the secretion of LH and prolactin. Endocrine glands are part of a homeostatic system, responding to a variety of enteroceptive and exteroceptive stimuli. The AP, like other endocrine glands exhibits both acute and chronic responses to changes in the environment. In general, previous studies attempting to relate CA metabol- ism with endocrine states have employed chronic stimuli. If the CNS is involved in mediating acute changes in AP hor- mone secretion, one should be able to demonstrate alteration in CA turnover which precedes or accompanies changes in hor- mone secretion. A part of this thesis is devoted to determining the time course of the response of the central CA systems to acute endocrine alterations. Rostral areas of the hypothalamus are believed to be involved in mediating gonadal steroid feedback which results in an increase in secretion of LH. This positive feedback of estrogen and progesterone occurs on the day of proestrus of the estrous cycle in rats, on the day preceding ovulation in human subjects, and prior to the onset of cyclic reproduc- tive function in both animal models and human subjects. In rodents, disruption of rostral (but not caudal) hypothalamic nuclei can block the surge in serum LH on proestrus and LH surges normally induced by administration of gonadal steroids. It would appear then that afferents terminating in the ros- tral hypothalamus or passing through this area en route to the medial basal hypothalamus (MBH) mediate the steroid induced release of LH. Part of this thesis is devoted to determining whether the rostral hypothalamus functions is a center for integrating afferent information or simply is a hypothalamic area through which nerve tracts pass to reach the MBH. Finally, senescence in rodents and humans results in a variety of endocrine deficiencies. In rodents, gonadal and thyroid functions decrease as an apparent result of a de- creased ability of the AP to secrete hormones in response to appropriate stimuli. Cyclicity in female and fertility in male rats decreases with age. Old animals are less respon- sive to several types of stimuli which release LH and follicle stimulating hormone (FSH) and are more responsive to some stimuli which release prolactin. Similarly, old rats are less responsive to stimuli which normally release TSH. Thus expo- sure to low ambient temperature releases less TSH in old than in young male rats. To assess the possible involvement of CA's in the decreased responsiveness of the AP to physio- logical stimuli, basal serum concentrations of AP hormones and concentrations and turnover of central biogenic amines are compared in young and old male rats.. ini LITERATURE REVIEW I. Hypothalamic Control of Anterior Pituitary Function A. Classical Observations The existence of the pituitary located deep within the cranium has been known for some 2,000 years. The pituitary, because of its central location in the brain case, has been claimed to have many supernatural functions by ancient anatomists (Harris, 1972). This structure was considered to be part of the brain until 1838 when Rathke demonstrated that a portion (anterior pituitary) developed from a non-neural stomite. The pioneering work of Cushing and colleagues in the first decade of this century demonstrated the importance of the anterior pituitary gland in the maintenance of normal life and in the genesis of several diseases (Crowe, Cushing and Homans, 1910; Cushing, 1909). The discovery that the pituitary gland contained substances which stimulate body growth (Evans and Long, 1921, 1922), growth and maturation of the ovaries (Smith, 1926a; Zondek and Ashheim, 1926), initiation of milk production (Stricker and Grueter, 1928; Evans and Simpson, 1929), restoration of atrophic thyroids (Allen, 1919; Smith and Smith, 1922; Anderson and Collip, 1932) and restoration of atrophic adrenals (Allen, 1922; Smith, 1926b) left no doubt that the anterior pituitary gland 6 r‘f Af—Ji secretes hormones which exert dramatic effects on physio- logical processes. Early indication for a role of the CNS in regulation of AP function came from diencephalon lesions and subsequent atrophy of endocrine glands. Ascher (1912) reported that anterior hypothalamic lesions caused gonadal atrophy in dogs. These observations were later extended to other species (Camus and Roussy, 1920; Dey, 1943). Hypothalamic lesions were subsequently shown to block stress-induced adrenal hypertrophy (Ganong and Hume, 1954) and produce thyroid atrOphy (Cahane and Cahane, 1936). Electrical stimulation of the hypothalamus has been shown to induce ovulation in rabbits (Haterius, 1937; Harris, 1948a), and increase thyroid (Harris and Woods, 1958) and adrenal activity (deGroot and Harris, 1950). These effects were not observed with elec- trical stimulation of the AP (Markee g; 31., 1946) indicat- ing that the hypothalamus exerts a profound influence on hormone secretion from the AP. Additional evidence for the influence of the hypothala- mus on the normal adenohypophysial function were obtained by interruption of the hypothalamo-hypOphysial connection. Dott (1923) and Mahoney and Sheehan (1936) first observed that pituitary stalk transection caused atrophy of both gonads and thyroid glands. Stalk transection was later shown to interfere with normal adrenal responses (Fortier gt a1,, 1957; ill. at at .H\ r, g Lazolo and DeWied, 1966). Transplantation of the in sigg pituitary to the anterior chamber of the eye or under the renal capsule has been shown to cause a variety of metabolic changes including atrophy of the gonads, adrenals and thyroid glands (Harris, 1948, 1955). However, ectopic pituitaries were able to maintain corpora lutea (Everett, 1954, 1956) and mammary gland (Meites, 1967) function due to the increase in PRL release from ectopic pituitaries (Meites gt 21-» 1961; Pasteel, 1961). These early experiments demonstrated both a stimulatory and an inhibitory influence of the hypothalamus on release of AP hormones. B. Portal Vessels and Neurosecretion Popa and Fielding (1930) first observed that the blood vessels supplying the anterior pituitary gland were part of a portal system connecting the sinusoids of the AP with a capillary plexus in the median eminence. They proposed blood flow from the pituitary to the hypothalamus. Houssay et 31. (1935) followed the direction of blood flow in this portal system of toads and correctly proposed a hypothalamus to pituitary blood flow. One year later, Wislocki and King (1936) observed the movement of dyes from the hypothalamus to the pituitary after a systemic injection, providing strong evidence that the anterior pituitary received blood after its circulation through the medial basal hypothalamus. The capillary plexus of the superior hypophyseal artery appears to give rise to the long portal vessel which travels along the lateral and anterior aspects of the infundibulum (Netter, 1965). In mammals, 70-90% of the blood supplied to the anterior pituitary is via these long portal vessels (Adams gt 31., 1963; Porter gt 31., 1967). In most species, the remaining blood supply to the AP is believed to come from a plexus at the distal end of the infundibulum and travel via short vessels, deep within the infundibulum to the pituitary (Netter, 1965). In mammals, except the rabbit, 100% of the blood supply appears to reach the anterior pitui— tary via the hypophysial portal system (Harris, 1947; Goldman and Sapirstein, 1962). Thus, anatomical evidence for a link between the nervous and endocrine systems is well documented. The possibility that nervous tissue was capable of secreting endocrine active substances into the hypophysial portal system (Haterius, 1937; Hensey, 1937) or into the general circulation (Scharrer and Scharrer, 1940) was pro- posed in the late 1930's and early 1940's. Within the next decade, neurosecretion had been demonstrated and the pathways for oxytocin and vaSOpressin synthesis, transport and secre- tion elucidated (Bargman and Scharrer, 1951; Scharrer, 1952; Scharrer and Scharrer, 1954). This pioneering work demon- strated that the paraventricular and supraoptic nuclei of the hypothalamus contained cell bodies of nerves which synthesized 10 these two hormones. Axons from these nuclei traveled ventro- caudally to the nervous tissue of the pars nervalis (posterior pituitary) where oxytocin and vasopressin are stored prior to release into the circulatory system. The "chemotransmitter hypothesis" was proposed and p0pu- 1arized by G. H. Harris (1948b) based upon the anatomical relationship between the hypothalamic, hypOphysial portal circulation, and the anterior pituitary and the previously proposed concept of neurosecretion. Harris proposed that nervous activity in the hypothalamus induced the release of humors from neurosecretory cells into the intercellular space which bathe the capillary plexus of the median eminence. These humors were proposed to be transported to the anterior pituitary gland via the hypOphyseal portal vessels where they were pr0posed to act on AP cells to alter the release of hormones. This hypothesis has withstood 35 years of experi- mental testing and today is a basic premise of the science of neuroendocrinology. C. Hypophysiotropic Hormones The first demonstration of a hypothalamic factor capable of altering the release of an anterior pituitary hormone was provided by Saffran and Schally (1955) and Guillemin and Rosenberg (1955). Using a hypothalamus—pituitary coincuba- tion system, the former demonstrated that the addition of norepinephrine to the media increased the release of 11 adrenocorticotrOpin (ACTH), an effect which was not observed in the absence of hypothalamic tissue in the media. Subse- quent to these observations several groups demonstrated the existence of hypothalamic factors capable of altering the release of AP hormones 13_31333. In the late 1950's and early 1960's hypothalamic releasing activity was demonstrated for TSH (Shibusawa 33 31., 1956), LH (McCann 33 31., 1960), PRL (Meites 33 31., 1960), FSH (Igarshi and McCann, 1964; Mittler and Meites, 1964) and GH (Deuben and Meites, 1964). Hypothalamic release, inhibiting activity has been demon- strated for PRL (Talwalker 33 31., 1961, 1963; Pasteels, 1961) and GH (Krulich 33 31., 1968). Attempts at purification and identification of hypothala- mic releasing and release inhibiting hormones have been only partially successful. Corticotropin-releasing factor, the first hypothalamic releasing factor, is yet to be identified although pepsin sensitive CRF material has been demonstrated and partially purified (Royce and Sayers, 1958) and a pr0posed l3-amino acid peptide has been published (Schally and Bowers, 1964). Likewise proposed structures for GRF (Schally 33 31., 1973) and PIF (Schally 33_31,, 1976) have been reported, but as yet unconfirmed. Thyrotropin-releasing hormone was the first hypothalamic releasing factor purified and synthesized (Burger 33 31., 1969; Folkers 33 31., 1969; Boler 33 31., 1969). This 12 tripeptide is identical in all species tested, has been shown to release TSH from all species tested, and has been identified in hypophysial portal blood. Thus, TRH appears to be a true hypothalamic hormone. Although releasing factors were originally believed to be specific for one anterior pituitary hormone (and so named), recent studies have shown that TRH releases PRL as well as TSH (Jacobs 33 31,, 1971; Muller 33 31., 1973; Convey 33 31., 1973). Luteinizing hormone releasing hormone (LHRH) was iso- lated, its amino acid sequence determined, and synthesized in the laboratories of Andrew Schally (Matsuo 33 31., 1971a; Matsuo, 1971b). This decapeptide has been shown to be effective in releasing LH and FSH (Schally 33 31., 1971), leading to the common use of the term "gonadotropin releas— ing hormone" (GnRH) and follicle stimulating hormone releas- ing hormone (FSH-RH). Since the synthesis of the native LHRH, about 75 analogues have been synthesized (Saffran, 1974), most of which decrease its LH releasing activity. Growth hormone-release inhibiting hormone (GIF or soma- tostatin) was first isolated from ovine hypothalami, identi- fied and synthesized by Guillemin's group (Brazeau 33 31., 1973). Although somatostatin is effective in inhibiting GH release, like the other identified hypothalamic hormones, it is not specific for GH. Somatostatin inhibits TRH induced TSH and prolactin release (Vale 33 31., 1974; Chen 13 and Meites, 1975). In addition, somatostatin has been local- ized in the alimentary tract (Mortimer 33 31., 1974) and pancreas (Alberti 33 31,, 1973) and may be important in the regulation of both insulin and glucagon release (Gerich 33 31., 1974). Somatostatin administration has also been shown to exert an antidiabetic effect in both rats and primates (Mortimer 33 31., 1974). D. Hypothalamic Anatomy The hypothalamus is an anatomically diverse structure containing clusters of cell bodies intermixed with a variety of afferent and efferent pathways (Jenkins, 1972). The hypo- thalamus extends rostrally to the sulcus limitans and caudal- ly to the mammillary bodies at the dorsal border. The hypo— thalamic sulci separate the hypothalamus from the thalmus. The ventral border is free from nervous connections, except for the extension of the supraOpticohypophysial tract to the neurohypOphysis. The lateral borders of the hypothalamus are formed in part by the internal capsule. In rats, this area of the diencephalon constitutes about 1/100th of the weight of the whole brain. In general, cell bodies distribute themselves in three major gray regions in the hypothalamus. The anterior and intermediate gray areas of the hypothalamus appear to be essential for central mediation of anterior pituitary hor- mone release; whereas, the posterior gray area appears to be 14 relatively unimportant in this function. Except for the arcuate nucleus and median eminence, hypothalamic nuclei are located bilaterally on either side of the third ventricle. The anterior hypothalamic area includes the preOptic area (POA) located rostral to the Optic chiasm; the suprachias- matic nucleus (SCN) which lays immediately dorasl to the optic chiasm; the anterior hypothalamic nucleus (AHN) which is located dorsolateral to the SCN; the paraventricular nucleus (PVN) which extends rostrally from the POA and dorso- caudally along the wall of the third ventricle; and a rather diffuse supraoptic nucleus (SON). The integrity of the POA- AHN-SCN complex is vital for the cyclic release of luteiniz- ing hormone in rodents (Halasz and Pupp, 1965; Gorski, 1966) but may be less important in premates (Krey 33 31., 1975). The intermediate group of nuclei include the lateral hypothalamic nucleus (LHN), the ventromedial nucleus (VMN), the dorsomedial nucleus (DMN), the arcuate nucleus (AN), and the median eminence (MB). In addition to their well docu- mented role in food intake (Mayer, 1953; Mayer and Arees, 1968) the LHN and VMN have been reported to influence the secretion of growth hormone (Martin et al., 1975). The arcuate nucleus is located in the mediobasal region of the tuber cinereum and is continuous with the median eminence. This nucleus has been shown to contain cell bodies of the tuberoinfundibular DA system which extends to the external 15 layers of the median eminence and nerve terminals containing both norepinephrine and serotonin (Fuxé and H6kfelt, 1966; Ungersted, 1971). In addition to neurons of the arcuate nucleus, specialized ependymal cells at the base of the third ventricle extend through the arcuate nucleus to form foot pads on the vessels of the hypophyseal plexes (Bleier, 1971; Mitchell, 1975). These tanocytes have been pr0posed to be an alternative route for cerebrospinal fluid born amines and releasing hormones to reach the anterior pituitary gland (Zimmerman 33_31., 1974). The very location of the median eminence at the neuro- vascular junction suggests its importance in the control of anterior pituitary physiology. The densely packed nerve terminals of the median eminence surround the capillary plexus of the hypophyseal portal system (Harris, 1948) and contain high concentrations of DA, LHRH, TRH, and somatostatin (Brownstein 33 31., 1975). The hypothalamus receives afferents primarily from two brain regions: the brainstem reticular formation and the limbic system (Nauta and Haymaker, 1969). From the brain stem, afferents reach the hypothalamus via the mammillary peduncle to terminate in the mammillary bodies and lateral hypothalamus; the dorsal longitudinal fasiculus which extends from the para-aquaductal gray of the midbrain to the posteri- or-hypothalamic area; and the medial forebrain bundle which 16 also originates in the para-aquaductal gray area of the brainstem. Afferents from the limbic system to the hypothalamus include the fornix, the medial forebrain bundle and the stria terminalis. The fornix follows a course from the hippocampus cranially running ventral to the corpus callosum. At the level of the anterior commissure the fornix divides into two columns and extends caudally to the mammillary bodies of the hypothalamus. The stria terminalis originates in the amygdala and terminates in the anterior gray of the hypothalamus and perhaps other areas (Nauta, 1958). The descending portion of the medial forebrain bundle originates in the olfactory tubercle and septal regions and descends to the preopticohypothalamic region and medial hypothalamus. Although much evidence has been presented for the above mentioned afferents to the hypothalamus, other pathways to the hypothalamus have been proposed and some evidence pre- sented. The inferior thalamic peduncle appears to extend from the medial thalamic region to the preoptic nucleus (Ingram, 1940; Nauta, 1962). In addition, evidence for afferents reaching from the globus pallidus, the cortex (Ward and McCullock, 1947), and the retina (R155 33 31., 1963) has been reported. CUR. 17 II. Monoaminergjc Pathways in the _Hypothalamus A. Noradrenergic Pathways The first evidence that norepinephrine (NE) might be involved as a neurotransmitter in the central nervous system came from the biochemical observation of NE in various brain regions (Amin 33 31., 1954; Vogt, 1954). The Falck-Hillarp histofluorescence technique provided the first demonstration of the cellular localization of NE (Carlsson 33 31., 1962) in nerve terminals and cell bodies (Dahlstrom and Fuxé, 1964, 1965; Fuxé, 1965a, 1965b). Because of the relatively low concentration of monoamines in axons, physical or chemical lesions of pathways is required to concentrate monoamines in axons to the extent needed for histofluorescence detection (Ungerstedt, 1971). These techniques demonstrated that nor— adrenergic cell bodies were located in the locus ceruleus (Loizou, 1969) and midbrain reticular areas, with axons entering the ascending medial forebrain bundle. Projections from the medial forebrain bundle provide the noradrenergic component to the cerebellum, cerebrum, lower brain stem, mesencephalon and diencephalon (Loizou, 1969; Ungerstedt, 1971). The ventral portion of the medial forebrain bundle innervates the entire hypothalamus (Ungerstedt, 1971). Of particular interest to the control of AP function is the observation of noradrenergic projections from the medial 18 forebrain bundle to the anterior hypothalamic nuclei (POA, SON, and PVN) and the intermedial nuclei (AN and internal layers of the median eminence). The recent development of a micropunch technique (Palkovits, 1973) and sensitive radioenzymatic assays for monoamines (Coyle and Henry, 1973; Cuello 33 31., 1973; Moore and Phillipson, 1975; Ben—Jonathan and Porter, 1976) has permitted a systematic evaluation of the monoamine con- tent of discrete hypothalamic areas. Although NE is rather evenly distributed in the hypothalamus, highest concentra- tions are found in the anterior hypothalamic nuclei and the arcuate nucleus (Palkovits 33 31., 1974). In general, NE concentration in hypothalamic nuclei correlates well with the activity of d0pamine-B-hydroxylase, the enzyme that hydroxylates DA to NE (Saavedra 33 31., 1974a). Several lines of evidence indicate that the noradrener— gic component of the hypothalamus originates from cell bodies located in extra-hypothalamic brain areas. Histo- fluorescence studies have been unable to detect any nor- adrenergic cell bodies in the hypothalamus (Understedt, 1971). Lesions in the midbrain tegmentum (Anden 33 31,, 1966a), locus ceruleus (Loizou, 1969) and medial forebrain bundle (Kabayashi 33 31., 1974) result in a varying decrease in NE concentration in the hypothalamus. Hypothalamic deafferentation results in a total loss of d0pamine-B-hydrox- lyase activity from several hypothalamic nuclei (Brownstein 19 33 31., 1976) and a decrease in NE content. These studies suggest that the noradrenergic terminals in the hypothalamus originate from other brain areas. However, all noradren- ergic input to the hypothalamus may not come from cell bodies in the locus ceruleus since lesions in this area do not completely eliminate NE in the hypothalamus (Forzon, 1969; Ungerstedt, 1971). Some of the NE remaining in the hypo- thalamus following midbrain or medial forebrain bundle lesions may be present in neuroglial cells (Iversen, 1974) and thus is unresponsive to removal of noradrenergic input. B. D3paminergic Pathways There is an abundance of evidence for three distance DA systems in the brains of laboratory animals. The nigro- striatal DA system appears to have cell bodies in the A9 cell group of the zona compacta of the midbrain tegmentum and extends rostrally to provide DA terminals to the globus pallidus, the caudate nucleus and the putamen (Anden 33 31., 1964, 1965, 1966c; H6kfelt and Ungerstedt, 1969). This dopa- minergic system appears to be involved in the normal control of extra-pyramidal upper motor neurons, and a deficiency in this system has been demonstrated in Parkinson's disease (Hornykiewicz, 1963). A second dopaminergic system appears to originate just dorsal to the interpeduncular nucleus of the midbrain (A10 cell group) and pass rostrally to innervate the olfactory tubercles and the nucleus accumbens (Anden 33 31,, 1966b; Ungerstedt, 1971). Neither of these two 20 dopamine systems appear to send branches to the hypothalamus as indicated by the inability of hypothalamic deafferentation to decrease DA content (Weiner 33 31., 1972a). The tuberoinfundibular DA system appears to originate from a tightly packed band of cell bodies in the arcuate nucleus and the lateral periventricular nucleus (Fuxé, 1963; Fuxé and kufelt, 1966). Axons from these A12 cell bodies pass ventral to terminate in the external layer of the median eminence. This system appears to be the only d0paminergic input to the median eminence. Dopaminergic cell bodies have been observed in the suprachiasmatic nucleus (Fidbrink 33 31., 1974). Further, Bjorklund 33 31. (1975) has presented evidence for a second hypothalamic DA system with cell bodies in the rostral peri- ventricular mucleus (A14 cell group) and axons extending to the medial preoptic area and to the suprachiasmatic nucleus. Palkovits 33 31. (1976) have presented evidence for decreased DA content in the medial basal hypothalamus and the presence of degenerative hypothalamic axons after lesions placed in the substancia nigra. However, this study has been ques- tioned since total deafferentation of the hypothalamus does not decrease hypothalamic DA content (Weiner 33 31., 1972a). The bulk of evidence indicates that DA in the hypothalamus originates from intrahypothalamic systems. 21 C. Serotonergic Pathways The relatively weak fluorescence of SHT produced by histofluorometric techniques have made it more difficult to determine the distribution of serotonin than catecholamines in mammalian brains. Lesions and subsequent biochemical analysis have been used to partially map central serotonergic pathways. Yellow fluorescent axons have been shown to emerge from a narrow band of cells in the mid portion of the lower pons and upper medulla, the raphe nuclei (Dahlstrom and Fuxé, 1964; Ungerstedt, 1971). These axons ascend as part of the medial forebrain bundle and appear to provide serotonergic nerve terminals to most if not all of the brain (Kuhar 33 31., 1972). Lesions of the medial and/or dorsal raphe nuclei and medial forebrain bundle have been shown to decrease SHT concentration and tryptOphan hydroxylase activity in fore- brain areas (Kuhar 33 31., 1972; Ungerstadt, 1971; Herr and Rothe, 1972) including areas of the hypothalamus (Saavedra 33 31., 1974b). Electrical stimulation of the raphe nuclei increases serotonin turnover in the forebrain (Sheard and Aghajanian, 1968) and the hypothalamus (Advis, Simpkins, Bennett and Meites, unpublished). In the hypothalamus, serotonin can consistently be visualized in the suprachiasmatic nucleus (SCN) by histo- fluorescence techniques. This is consistent with the observa— tion that enzymatically determined SHT and tryptOphan liv- Sui 22 hydroxylase are high in this nucleus (Saavedra 33 31., 1974b). Serotonin and tryptOphan hydroxylase appear also to be highly concentrated in the periventricular nucleus, arcuate nucleus and median eminence, and rather evenly distributed in the remaining hypothalamic nuclei. III. Catecholamine and Serotonin Metabolism The biosynthesis of catecholamines and serotonin is mediated by enzymes produced in nerve cell bodies and de- livered to presynaptic nerve terminals by axonal transport (McClure, 1972; Ochs, 1972). The neutral amino acid L-tyro- sine is actively pumped into catecholaminergic neurons (Iversen, 1971) and hydroxylated to the catechol, dihydroxy- phenylalanine (DOPA) by the rate limiting enzyme in cate- cholamine synthesis, tyrosine hydroxylase (Levitt 33 31., 1965, 1967). Since brain concentrations of tyrosine are sufficiently high under most conditions to saturate this enzyme, changes in circulating levels of tyrosine do not appear to influence catecholamine synthesis (Nagatsu 33 31., 1964; Copper 33 31., 1971). Rather intraneuronal concentra- tion of dopamine exerts an inhibitory influence on tyrosine hydroxylase activity by competing with the cofactor, tetra- hydrOpteridine, for binding to this soluble enzyme (Udenfriend 33 31., 1965; Costa and Neff, 1966; Alousi and Weiner, 1966; Spector 33 31., 1969). Thus, activation of catecholaminergic 23 neurons, which decreases intracellular d0pamine concentra- tion, activates tyrosine hydroxylase; whereas, reduced activ- ity in catecholaminergic neurons increase d0pamine stores and depresses tyrosine hydroxylase activity. The decarboxylation of DOPA to dopamine is catalyzed by the enzyme L-aromatic amino acid decarboxylase, an enzyme which is common not only to catecholaminergic and seroton- ergic neurons but to other tissues which metabolize neutral amino acids (Goldstein 33 31., 1974). In the mammalian brain and in tissue receiving sympathetic innervation, DOPA concentrations are extremely low, a result of the high activ- ity of this soluble enzyme (Km = 4 x 10"4 M; Goldstein 33 31., 1974). In adrenergic neurons, d0pamine-B-hydroxylase (DBH), which hydroxylates dopamine to norepinephrine is present (Kaufman, 1966; Fuxé 33 31., 1971). This enzyme appears to be localized on membranes of storage granules (Thomas 33 31., 1973) and has a high'copper content (Friedman and Kaufman, 1965; Blumberg 33 31., 1965), a characteristic which makes it sensitive to chelating agents (Goldstein, 1966; Sulser and Saunders-Bush, 1971). DBH has been used as a specific, sensitive marker for noradrenergic neurons (Geffen 33_31,, 1969; Hartman and Udenfriend, 1970; Hartman, 1973). Phenylethanolamine-N-methyltransferase (PNMT), a solu- ble enzyme which catalyzes the N-methylation of 24 norepinephrine to epinephrine is found in high concentra- tions in the adrenal medulla (Axelrod, 1962) and in rather low concentrations in the mammalian brain (Axelrod, 1962; McGeer and McGeer, 1964). The presence of this enzyme in low concentrations in the brain suggest a role for epinephrine in central nervous system physiology which, however, has as yet not been elucidated. Biological inactivation of catecholamines can occur either by enzymation degradation of the neurotransmitter to an inactive form or by the "recapture" of the catecholamine in storage granules of presynaptic nerve terminals. Enzymatic breakdown of catecholamines occurs via the action of monoamine oxidase (MAO) and/or catecholamine-O-methyl- transferase (COMT). MAO deaminates dopamine and norephineph- rine to their respective aldehydes, 3,4-dehydroxyphenylact- aldehyde and 3,4-dehydroxyphenyl-g1ycolaldehyde. COMT catalyzes the 0-methy1ation of dopamine to 3-methoxytyramine and norepinephrine to normetanephrine (Axelrod and Tomchik, 1958; Axelrod 33 31., 1959). Although high concentrations of both of these enzymes have been observed in mammalian brain, their role in inactivating catecholamines released into the synaptic cleft appears to be very minor, as indi- cated by the observation that treatment with either MAO or COMT inhibitors does not greatly potentiate the effect of 25 sympathetic stimulation (Pletscher, 1973). A major means of inactivation of catecholamines appears to be through an active reuptake into presynaptic vessels for both norepinephrine (Glowinski 33 31., 1965; Glowinski and Axelrod, 1966) and d0pamine (Coyle and Snyder, 1969; Horn 33 31., 1971). It appears that a large proportion (50-90%) of NE released into the synaptic cleft can be inactivated by this method (Langer, 1970; Haggendal, 1970). The apparent physiological importance of reuptake in terminat- ing catecholamine action is indicated by the observation that inhibition of reuptake by cocaine, phenoxybenzamine, or imipramine caused a marked potentiation of the effects of adrenergic stimulation (Iversen, 1972b). Considerable evidence indicates that both the secretion and turnover of neurotransmitters are closely coupled to electrical activity in catecholamine neurons. Electrical stimulation of peripheral adrenergic neurons results in increased tyrosine hydroxylase activity (Sedvall 33 31., 1968; Dairman 33 31., 1968) and norepinephrine turnover (Alousi and Weiner, 1966; Gordon 33 31., 1966; Roth 33 31., 1966; Sedvall and KOpin, 1967; Sedvall 33 31., 1968; Weiner and Rabadjuja, 1968). Similarly electrical stimulation of the locus ceruleus or its projections results in an increase in norepinephrine turnover in the cerebral cortex and hippo- campus (Arbuthnott 33 31., 1970; Korf 33 31., 1973a) whereas 26 lesions of the locus ceruleus decrease norepinephrine turn- over in the cerebral cortex (Korf 33 31., 1973b). Stimula- tion of the medulla oblongata increases norepinephrine turnover in the spinal cord (Dahlstrom 33 31., 1965); whereas, spinal transection decreases norepinephrine turn- over caudal to the lesion (Anden 33 31., 1966d, 1969). A similar relationship between nervous activity and turn- over appears to exist in both d0paminergic and serotonergic neurons. Stimulation of the zona compacta of the substancia nigra results in an increase in dopamine turnover in the striatum (VonVoigtlander and Moore, 1971), an effect which appears to be frequency dependent (Roth 33 31., 1973). Stimulation of the raphe nuclei results in a frequency- dependent increase in serotonin synthesis (Shields and Eccleston, 1973) and accumulation of 5-hydroxyindole acetic acid in the forebrain (Sheard and Aghajanian, 1968). Raphé nucleus lesion decreases forebrain serotonin turnover (Herr and Roth, 1972). Each of the methods available for estimating monoamine turnover has advantages and disadvantages as indicated by the critical review of Costa (1970). Estimation of catecholamine turnover by measuring the depletion of norepinephrine speci- fic activity after intraventricular infusion of labeled norepinephrine was introduced by Glowinski's group (Mith and Glowinski, 1963; Glowinski 33 31., 1965). In this method HON [Ihi 1963 ity adre nore from synt Glow Pitf; 27 norepinephrine is transported into noradrenergic neurons by a reuptake mechanism and mixes with endogenous norepinephrine (Whitby 33 31., 1961; Hertting and Axelrod, 1961; Iversen, 1963). The rate of decrease in specific norepinephrine activ— ity is presumed to reflect its rate of replacement in nor- adrenergic neurons by newly synthesized norepinephrine. The depletion of specific norepinephrine activity over the first 8 h after injection exhibits an exponential decrease (Glowinski 33 31., 1965) and yields values for whole brain norepinephrine turnover which are not significantly different from those obtained using the radiolabeled precursors or the synthesis inhibition method to estimate turnover (Iversen and Glowinski, 1966). However, this method has several inherent pitfalls. First, since norepinephrine can be taken up into both dopaminergic and serotonergic neurons (Green 33 31., 1969) the depletion of labeled norepinephrine may reflect activity in these neurons as well as in noradrenergic neurons. Second, because of the low specific activity of available norepinephrine, turnover can be measured in only large pieces of tissue. And finally, since long periods of time (8 h) are required for the radiolabeled norepinephrine depletion, this method is unacceptable for estimation of norepinephrine turn- over in response to acute stimuli. The measurement of the acute rate of appearance of labeled catecholamines following systemic injection of 28 catecholamine precursors (Zigmond and Wurtman, 1970; Zschaeck and Wurtman, 1972) has several advantages over the previous technique. The trace doses of labeled precursors used can be easily administered and do not appear to influ- ence endogenous amine concentrations (Zigmond and Wurtman, 1970; Hyyppa 33 31., 1973). Further, the rate of production of catecholamines can be determined over a very short time interval (less than 10 min.), making this technique accept- able for acute studies. Since this technique measures acutely the accumulation of labeled catecholamines, synthesis rather than turnover is measured. The major disadvantages of this technique are that catecholamine precursors can be taken up by non-catecholaminergic cells. Thus, one can not be certain where the conversion of precursors to d0pamine and norepinephrine is occurring. This technique also requires the use of larger pieces of tissue and can not be employed for measuring catecholamine synthesis in very localized areas of the brain. Two methods have been introduced utilizing synthesis inhibitors to estimate catecholamine turnover. One method used extensively by Carlsson's group (Carlsson33 31., 1972a, 1972b) utilizes the inhibition of aromaticHLaamino acid decarboxylase (AAAD) to measure the rate of accumulation of L~dopa. This method has the advantage of allowing acute measurement of catecholamine (30 min.) turnover and the (“f U? an,- 3161‘- 29 simultaneous measurement of both catecholamine and serotonin metabolism since AAAD blocks both systems. However, since DOPA is an intermediate in both dopamine and norepinephrine biosynthesis, one cannot distinguish between the two systems. Further, the relative lack of sensitivity of assays for dOpa require the use of large pieces of tissue (Kehr 33 31., 1972). A final method for measuring catecholamine turnover utilizes the rate of d0pamine and norepinephrine depletion following a-methyl-para-tyrosine (ampt) induced inhibition of tyrosine hydroxylase (Brodie 33 31., 1966; Costa and Neff, 1966). Catecholamine depletion following ampt treatment is exponential (Brodie 33 31., 1966) and yields estimates of turnover rates consistent with those obtained using other techniques (Carlsson 33 31., 1972a; Costa, 1970). This method has the advantages of convenience and applicability to acute studies. Further, the recent development of sensitive radio- enzymatic assays for both dopamine and norepinephrine (Coyle and Henry, 1973; Cuello 33 31., 1973; Moore and Phillipson, 1975; Ben Johnathen and Porter, 1976) allows its use in measuring catecholamine turnover in small pieces of brain tissue. However, this method has several drawbacks. First, the intraperitoneal administration of ampt does not cause an instantaneous inhibition of catecholamine synthesis (Westfall and Osada, 1969) and thus the rate of catecholamine depletion may vary with time after ampt administration. In addition, 3O ampt may have an effect on catecholaminergic neurons other than just tyrosine hydroxylase inhibition which could affect the estimation of catecholamine turnover. Even with these shortcomings, this appears to be the best method available for measuring catecholamine turnover small brain tissue and is the method employed in the research in this thesis. IV. fiypothalamic Control of Prolactin Secretion A. Prolactin Levels and P3ysiological States A role for prolactin has been demonstrated or implicated in no less than 82 different physiological phenomena in vertebrates (Nicoll and Burn, 1972). In mammals, prolactin has been clearly demonstrated to stimulate mammary gland growth (Talwalker and Meites, 1961: Meites, 1965), initiate and maintain lactation (Stricker and Grueter, 1928; Turner and Gardner, 1931), and mammary tumorogenesis and growth (Liebelt and Liebelt, 1961; Boot 33 31., 1962). In rats pro- lactin and LH appear to be luteotrOphic (Gospodarowicz and Legault-Demare, 1963). In males, prolactin has been shown to stimulate prostate and seminal vesicle growth (Antliff 33 31., 1960). The influence of prolactin on the physiological events enumerated above appears to be mediated in part by increased secretion of prolactin from the anterior pituitary gland. In both male and female rats and in humans, serum levels of 31 prolactin increase during prepubertal development (Meites and Turner, 1948; Minaguchi 33 31., 1968; Voogt 33 31., 1970; Ojeda and McCann, 1975; Dohler and Wuttke, 1975), and seminal vesicle-prostate growth in males. Further, the suckling stimulus, which is necessary to maintain milk pro- duction, is a powerful stimulator of prolactin secretion (Sar and Meites, 1969; Amenomori 33 31., 1970; Reece and Turner, 1937; Grosvenor and Turner, 1958). Increased pro- lactin release from the anterior pituitary gland occurs in response to increasing serum titers of estrogen during the estrous cycle in rats (Clark and Meites, unpublished; Neill 33 31., 1971; Chen and Meites, 1970) and appears to be in- volved in corpora lutea formation and maintenance in rodents (Meites, 1968). Diurnal and nocturnal surges of prolactin release occur during pseudopregnancy and pregnancy in the female rat (Neill, 1975) and may be important to the mainte- nance of luteal function and progesterone secretion (Everett, 1954). B. Inhibitory Influence of the flypgthalamus on Prolactin Secretion The mammalian hypothalamus normally exerts an inhibitory influence on synthesis and release of prolactin from the anterior pituitary gland. Everett (1954) first observed that ectOpic relocation of the anterior pituitary resulted in long- term maintenance of functional corpora lutea and 32 pseudopregnancy. Implantation of l, 2 or 4 APs underneath the kidney capsule increased circulating prolactin prOpor- tional to the number of APs present (Chen 33 31., 1970). Lesions in the median eminence, which disrupt the hypothal- amo-hypophyseal connections initiate lactation in the rabbit (Haun and Sawyer, 1960) and rat (DeVoe 33_31., 1966), to induce pseudopregnancy in rats (Flerko and Bordos, 1959) and to cause a greater than 10-fold increase in circulating pro- lactin levels (Welsch 33 31., 1971). Talwalker 33 31. (1961) and Pasteels (1961) first observed prolactin-release-inhibit- ing activity in hypothalamic extracts from rats. Subsequently, these observations were extended to other species (Talwalker 33 31., 1963; Schally 33_31., 1965). These acid extracts of the hypothalamus were effective in inhibiting prolactin release both 13 3133 and 13_y1333_in all species tested. Kamberi 33 31, (1971a) demonstrated that hypothalamic ex- tracts infused into portal vessels suppressed serum prolactin in a dose-responsive manner. Cerebrocortical extracts had no such effect. C. Stimulato3y Influence of the Hypothalamus on Prolactin Sécretion The mammalian hypothalamus appears to contain a prolac- tin-releasing factor (PRF) as well as PIF. Injection of acid extracts of rat hypothalamus into estrogen-primed rats initi- ates milk secretion (Meites 33 31., 1960; Minhkinsky 33 31., 33 1968). Nicoll 33 31. (1970) later provided evidence for a hypothalamic PRF 13_31333. Incubation of neutralized hypo- thalamic extracts with anterior pituitaries appear first to inhibit and later to stimulate prolactin secretion. Methanol extracts of both rat and porcine stalk-median eminence stimu- late prolactin release in a dose-response manner (Valverde 33 31., 1972). The chromatographically separated PRF frac- tion of the methanol extraction was reported to be free of TRH, oxytocin, and vasopressin activity. Recently, Mitnick 33 31. (1973) reported PRF synthesis and release by rat hypo- thalami incubated 13,31333. In avian species, the hypothalamus appears to stimulate rather than to inhibit prolactin secretion. 13_y1333_incuba- tion of pigeon pituitaries decreases (Nicoll and Meites, 1962) and addition of hypothalamic extracts increased release of prolactin (Kragt and Meites, 1965). Subsequently, PRF activity was found in several avian species (Meites, 1967; Nicoll, 1965; Gourdji and Tixier-Vidal, 1966, and Chen 33 31., 1968). The possibility that thyrotrOpin-releasing hormone (TRH) is the active component in hypothalami responsible for PRF activity is based upon the observation that TRH stimulates release of prolactin in the rat, cow and human (Mueller (D ("P n) H ., 1973; Vale 33 al., 1973; Convey 33 31., 1973; Snyder 33 al., 1973; Noel 33_al., 1974). This TRH-induced release 34 of prolactin appears to be through a direct action upon the pituitary rather than via a hypothalamic mechanism since the effect is observed 13_31333 using normal pituitaries (Mueller 33 31., 1973) or pituitary tumor cells (Tashjian 33 31., 1971) and 13 3133 in animals bearing median eminence lesions (Porteus and Melven, 1974). The physiological significance of TRH in normal regula- tion of prolactin secretion has been questioned in that prolactin and TSH secretion rates appear to be independently controlled in many conditions. Low ambient temperature, which has been reported to stimulate hypothalamic TRH synthe- sis (Reichlin 33 31., 1972; Hefco 33 31., 1975) and release (Montoya 33 31., 1975), increased TSH and decreased prolactin release (Mueller 33 31,, 1974; Chen and Meites, 1975; and Reichlin 33 31., 1972). Stress caused a rapid increase in prolactin and a decrease in TSH secretion (Brown-Grant 33 31., 1954; and Nicoll 33 31., 1960). Suckling, which is a well known stimulator of prolactin secretion, has been reported not to affect TSH release rate (Sar and Meites, 1969; Gautvik 33 31., 1974). These results indicate that during many conditions, TSH and prolactin secretion are independently controlled and that TRH does not appear to be the physio- logical PRF. However, Koch 33 31. (1977) has observed that intraperitoneal injection of TRH antibodies decreases circu- lating levels of both TSH and PRL in estrogen-primed rats. 35 This suggests a role for TRH in estrogen stimulation of pro- lactin secretion. Further, since somatostatin has been reported to block TRH induced TSH release without effecting TSH stimulation of PRL release (Vale 33 31., 1974), a differ- ential control of TSH and PRL secretion by TRH is possible. D. Influence of Steroids on Prolactin Secretion Reece and Turner (1936, 1937) and later Meites and Turner (1942, 1948) were first to present evidence that estro- gen increased both synthesis and release of prolactin in male and female rats, male guinea pigs, and rabbits. These initial observations were later confirmed by Chen 33 31. (1970) using radioimmunoassay to measure both serum and pituitary prolac- tin, and by MacLeod (1975) using labeled leucine incorpora— tion into de novo synthesized prolactin. In ovariectomized female rats, all doses of estradiol were effective in increas— ing serum prolactin, although low doses were more effective than higher doses. In the rat and rabbit, estrogen is con- .siderably more effective in females than in males (Meites and Turner, 1948), although no sex difference has been found in human subjects (Frantz 33 31., 1972). Increased serum titers of estrogen are associated with an increase in prolactin secretion during several physio- logical states. The increased prolactin secretion which pre- cedes the onset of puberty coincides with an increase in serum estrogen levels (Brown-Grant 33 31., 1970; Naftolin 36 _3 31., 1972; Dohler and Wuttke, 1974, 1976) and can be blocked by ovariectomy (Simpkins and Meites, unpublished). During proestrus of the estrous cycle increased titers of serum estrogen are followed by a prolactin surge (Meites and Clemens, 1972; Meites 33 31., 1972). This proestrous pro- lactin surge can be blocked by ovariectomy on the morning of proestrus or by administration of an antiserum to estrogen (Neill 33 31., 1971). During diestrus of the estrous cycle and during most of pregnancy, serum levels of both estrogen and prolactin are low (Meites 33 31., 1972; Yoshinaga 33 31., 1969). Estrogen can affect PRL secretion by both a direct action on the anterior pituitary or indirectly through the central nervous system. Administration of estrogen to hypo- physectomized rats bearing ectOpic pituitary graphs increases serum PRL levels (Chen 33_31., 1970). A similar effect of estrogen on PRL secretion was observed in animals implanted with pituitary tumors (Mizuno 33 31., 1964). These studies, which suggested a direct effect of estrogen on the anterior pituitary, did not eliminate the possibility that estrogen increased PRF or decreased PIF release into the circulatory system. Definitive evidence for a direct action of estrogen on pituitary PRL release was provided by the observation of increased PRL release induced by estrogen in 3 h (Lu 33 31., 1971) and 3 day (Nicoll and Meites, 1962) incubations with 37 pituitary cell cultures. Further evidence for a direct action Of estrogen is the ability Of the anterior pituitary ‘to concentrate labeled estrogen (Vertes 33 31., 1973), per- Ihaps indicating the presence of estrogen receptors. Evidence for a hypothalamic mediation of the estrogen stimulation Of PRL secretion are indirect. Estradiol ben- zoate (EB) implants in the median eminence (but not other brain areas) tripled serum PRL levels in 25 days (Nagasawa I33_31,, 1969). However, this study did not eliminate the jpossibility that EB was delivered to the AP through the hypo- jphyseal portal system and exerted its effect directly. ()ther studies have shown that steroids implanted in the hypo— ‘thalamus reach the AP (Bogdanove, 1964). Experimental alteration Of plasma estradiol concentra— tions modify metabolism of hypothalamic neurons. Estrogen administration to female rats has been reported to decrease hypothalamic PIF activity as measured by a bioassay system (Ratner and Meites, 1964). Ovariectomy has been reported to decrease and subsequent estrogen treatment to increase median eminence (Fuxé 33 31., 1969) and anterior hypothalamic DA turnover (Huang 33 31., 1977). Since dopaminergic neurons appear to exert an inhibitory influence on PRL secretion, these changes in d0pamine metabolism are Opposite to the expected effects Of ovariectomy and estrogen replacement on serum PRL levels. Further estrogen binding has been reported 38 in several areas of the hypothalamus and limbic system (Stump 33 31., 1977). Although evidence exists that estrogen can affect the metabolism Of nervous tissue, there is no direct evidence that the estrogen induced increase in PRL secretion is centrally mediated. Although estrogen is the primary steroid stimulating jprolactin release, other circulating steroids may influence the release Of PRL from the AP. 13 3133, large doses of either progesterone or testosterone have been reported to linduce only small increases in serum PRL levels (Meites, 1959). Since both Of these steroids are ineffective in altering PRL release rate 13 31333_(Nicoll and Meites, 1964), ‘the Observed effects 13_3133_may indicate a conversion to estrogen. Progesterone has been reported to inhibit partially ‘the estrogen—induced prolactin release in ovariectomized rats (Chen and Meites, 1970). High concentrations Of cortisol have been reported to inhibit PRL secretion both 13 3133 and 13_31333 (Nicoll and Meites, 1964). However, the high doses of this steroid needed to inhibit PRL secretion make its physiological significance questionable since such levels of glucocorticoids are never achieved normally. Evidence has been presented that thyroid hormone can stimulate PRL release both 13 3133_(Meites 33 31., 1963) and 13 31333 (Meites and Nicoll, 1965) at relatively low doses. Since changes in circulating levels Of thyroid hormones occur 39 only very slowly and do not generally coincide with the circulating PRL levels, thyroid hormones are probably not directly involved in the regulation of prolactin secretion. Rather thyroid hormones probably have a permissive function in the synthesis and release Of PRL as well as other AP hor- mones . E. Dopaminergjc Effects on Prolactin Secretion The inhibitory influence of the mammalian hypothalamus on prolactin secretion appears to be mediated through central catecholaminergic systems. Early evidence indicating an in- hibitory influence Of catecholaminergic systems on prolactin secretion included the Observation that reserpine, which depletes storage pools of monoamines (Holzbauer and Vogt, 1956; Sheppard and Zimmerman, 1960), stimulates lactation in human subjects (Sulman and Winnik, 1956; Rabinowitz and Freedman, 1961) and experimental animals (Meites, 1957). In later experiments, serum prolactin, as measured by radio- immunoassay, has been shown to be elevated following reser- pine treatment (Lu 33 31., 1970). Other pharmacological agents which deplete central catecholamine stores cause a similar increase in serum prolactin. A single injection of alpha-methyl-para-tyrosine, alpha—methyl-meta—tyrosine, M-dOpa and 3-Odotyrosine increase serum prolactin as measured by radioimmunoassay (Lu 33 31., 1970; Lu and Meites, 1971; Smythe 33_31., 1974). L-dOpa, which is readily taken up into 40 both dopaminergic and noradrenergic neurons, and which in- creases central levels Of both DA and NE, has been reported to inhibit postpartum lactation in rats (Mizuno 33_31., .1964) and to lower serum prolactin levels in rats (Lu and ldeites, 1972) and humans (Turkington, 1972). The antipsychotic drugs which appear to exert their behavioral effect by blocking DA receptors (Clemens, 1975) are among the most powerful stimulators Of prolactin secre- tion. ChlorOpromazine, perphenazine, and haloperidol, ‘which are believed to block both d0paminergic and noradren- ergic receptors all cause a large increase in circulating levels Of prolactin (Lu 33_31., 1970; Ben-David 33 31., 1971; Dickerman 33 31., 1972). Pimozide, a specific DA re— ceptor blocker (Janssen 33_31., 1968) has been reported to induce a rapid increase in prolactin secretion (Clemens 33 31,, 1974). Sulpiride, which is believed to block DA receptor, has also been reported to increase serum prolactin (Clemens 33 31., 1974; MacLeod 33 31., 1977; Pass and Meites, 1977). Apomorphine, a specific DA agonist (Anden 33 31., 1967; Ferrini and Miragolin, 1972; Lal 33 31., 1972), has been shown to inhibit prolactin secretion 13 3133 (Smalstig and Clemens, 1974; MacLeOd and Lehmeyer, 1974; Mueller 33 31. 9 1976 and 13 vitro (Smalstig 33 31., 1974) in rats and 13 vivo in humans (Martin 33_31., 1974). Piribedil (ET 495), another 41 specific DA receptor agonist (Corrodi 33 31., 1973) has been ‘reported to reduce prolactin secretion in rats (Nicoll _e_3 a_1_., 1970; Nagasawa and Meites, 1970; Wuttke 33 31., 1972; bdalven and Hoge, 1971; Shaar and Clemens, 1972) and in laumans (Besser 33 31., 1972; del Pozo 33_31,, 1972, 1974). 'These DA agonists interfere with the ability Of haloperidol zand pimozide to increase prolactin secretion (Smalstig and (Zlemens, 1974; Mueller 33_31., 1976) indicating that the ef- :fects Of these drugs on prolactin secretion occurs at DA 'receptors. There is little question that a hypothalamic dopaminer— gic system exerts a tonic inhibitory influence on prolactin secretion, however, there is still controversy as to whether this effect is mediated through a hypothalamic PIF or by DA acting directly on the anterior pituitary. Infusion of DA or its agonist, apomorphine, into the third ventricle or Systemic injection Of L-dopa have been reported to elevate hypothalamic and portal blood concentration Of PIF and de- crease serum prolactin (Kamberi 33_31,, 1973; Lu and Meites, 1972; Ojeda 33 31., 1974). In addition, systemic injection 0f ergot alkaloids (Wuttke, Cassell and Meites, 1971), mono— amine oxidase inhibitors (Lu and Meites, 1971) and prolactin itself (Chen 33_31., 1967; Clemens and Meites, 1968) have been reported tO increase hypothalamic PIP activity. In con~ trast, the suckling stimulus, systemic reserpine injection, estrogen administration and stress, all of which increase 42 serum prolactin levels, decrease hypothalamic PIF activity (Mittler and Meites, 1967). The bioassay used in these studies to determine PIF activity could not distinguish be- tween this and other hypothalamic factors which can inhibit jprolactin secretion by a direct action on the anterior pituitary. The possibility that DA could act directly on the anterior pituitary in mediating the inhibitory influence Of the hypothalamus on prolactin secretion was suggested ini- tially by the Observation that doses Of DA (but not NE or E) below those found in the medial basal hypothalamus can in- .hibit prolactin secretion 13_31333_(C1emens, 1975). The observation Of tuberoinfundibular dopaminergic nerve endings in the area Of the capillary plexus of the hypophyseal portal vessels (Ajeka and HOkfelt, 1975) and the apparent presence of DA in portal blood (Fuxé 33 31., 1974; Neill 33 31., 1977) indicates that DA could directly affect the secretion of prolactin. However, no correlation between hypOphyseal portal blood DA concentration and circulating levels Of pro- lactin have yet been determined. Shaar and Clemens (1974) reported that removal of cate- cholamines from hypothalamic extracts by incubation with monoamine oxidase or by binding catecholamines to aluminum oxide removed PIF activity as measured by the ability Of the extract to inhibit prolactin release 13_31333. .Addition of the aluminum oxide bound catecholamines to the extract 43 restored PIF activity. Further PIF activity was reported to be resistant tO pepsin treatment. The hypothalamus contains factors other than DA which can inhibit prolactin release as demonstrated by the ability of a single rat hypothalamic equivalent (which contains 20-30 ng Of DA) to decrease serum prolactin levels shortly after 13 3133 injection (Watson 33 31., 1971) and its ability to prevent the suckling and stress induced decrease in pituitary prolactin. Lu 33_31, (1970) demonstrated that intra- venous administration Of 5-10 ug Of DA was ineffective in inhibiting prolactin release, and Blake (1976) has reported that infusion of at least 80 ug DA per hour are required to inhibit prolactin secretion in rats. Clearly, the hypothal- amus is much more effective in inhibiting prolactin secretion than DA alone. In addition, pretreatment Of hypothalamic- anterior pituitary coincubation systems with either pimozide (Vale 33_31., 1973) or haloperidol (Ojeda 33_31,, 1974) did not prevent hypothalamic inhibition Of prolactin secretion. Several laboratories have recently reported evidence for non- catecholamine hypothalamic substances with PIF activity (Takahara 33 31., 1974; Dular 33 31., 1974; Greibrokk 33 31., 1974; Schally 33 31., 1977), and Kordon (personal communica- tion) has demonstrated PIF activity in a catecholamine-free synaptosome preparation from the rat median eminence. These results indicate that hypothalamic d0pamine can inhibit pro- lactin secretion, but other factors are probably also involved. 44 F. Noradrenergic Effects on Prolactin Secretion The influence of the noradrenergic system on prolactin secretion is less clear than hypothalamic d0paminergic effects for two reasons. First, noradrenergic input to the prolactin release mechanism appears to be minor. Thus, removal of nor- adrenergic afferents to the hypothalamus results in only slight changes in serum prolactin levels (Weiner and Ganong, 1972). Second, few drugs are selective for noradrenergic neurons. The a-adrenergic receptor blocker, phentolamine, is able to partially block the inhibitory effects of d0pamine on prolactin secretion 13_31333_(MacLeOd and Lehmeyer, 1974), suggesting that phentolamine is a partial DA antagonist. Clonidine, a reputed a-adrenergic agonist (Anden 33 31., 1970) may exert its central effect by acting at pre- rather than postsynaptic receptors, thus decreasing central nor— adrenergic activity (Dollery and Reid, 1973). Clonidine has also been reported to stimulate central histamine receptors. The most common drugs used to block dopamine B-hydroxylase (DBH), the enzyme which hydroxylates DA to NE, acts by chelating the COpper ions in DBH (Morgan and Cogan, 1975). Certainly other COpper and iron requiring enzymes are also affected. Thus, it is not surprising that conflicting data concerning the role Of NE in regulation of prolactin secre- tion have appeared. ‘ 13 31333, high doses Of NE incubated with anterior pituitaries has been shown to inhibit prolactin secretion 4S (Koch 33_31., 1970; Shaar and Clemens, 1974), however, high doses Of NE injected intraventricularly or intravenously are without effect on PRL secretion (Kamberi 33 31., 1971a; Lu 33_31., 1970). Administration of L-dihydroxyphenylserine (L-DOPS), a noradrenergic precursor has been reported to increase prolactin secretion (Donoso 33 31., 1971) and Disul- fram, a DBH inhibitor appears to decrease prolactin secre- tion (Meites and Clemens, 1972). Administration Of the a-agonist, clonidine decreases prolactin in male rats (Mueller 33 31., unpublished) and blocks the proestrous prolactin surge in female rats (Clemens, unpublished). In ovariectom- ized, estrogen primed rats, clonidine can stimulate prolactin secretion (Lawson and Gala, 1975). Alpha adrenergic receptor blockers have been reported to partially inhibit (Du Ruisseau 33 31., 1977) and to have nO effect (Meltzer 33 31., 1976) on the stress-induced rise in prolactin. Clearly, the role of the central noradrenergic system in the control Of prolac- tin has not been elucidated. G. Serotonergic Effects on Prolactin Secretion The central serotonergic system appears to exert a stimulatory influence on serum prolactin secretion. Systemic injection Of serotonin (5 mg daily) has been shown tO stimu- late mammary growth and lactation in rats and rabbits (Meites 33 31., 1959; Meites 33_31., 1960). Lu 33 31. (1970) was unable to show any effect Of systemically administered 46 serotonin on prolactin levels 60 min. after injection; whereas, Lawson and Gala (1975) reported a transient increase in serum prolactin 30 min. after serotonin injections. The discrepancy in the above two reports appears to be due to the timing of blood sampling. Kamberi 33 31. (1971a) re- ported that serotonin injection into the third ventricle of rats causes a prompt rise in serum prolactin. Administration Of the tryptOphan hydroxylase inhibitor, p-chlorOphenylalanine systemically (Caligaris and Taleisnik, 1974; Advis 33_31., unpublished) or into the third ventricle (Caligaris and Taleisnik, 1974) is able to decrease serum prolactin levels in both male and ovariectomized, estrogen primed female rats. Administration Of the serotonin precursor, S-hydroxytrypto- phan (5 HTP) partially reversed the PCPA induced prolactin decrease (Caligaris and Taleisnik, 1974), lowers serum pro- lactin in ovariectomized estrogen primed rats (Lu and Meites, 1973; Chen and Meites, 1975) and blocks suckling-induced prolactin increase (Kordon 33 31., 1974b). The neutral amino acid precursor, L-tryptophan, has been shown to elevate serum prolactin and tO enhance stress-induced prolactin re- lease (Mueller 33_31,, 1976). Enhancement of postsynaptic stimulation Of serotonergic receptors by blockade Of pre- synaptic reuptake with Lilly 110140 (fluoxitine) stimulates prolactin secretion when administered with low doses Of.5HTP (Clemens 33 31., 1977). Blockade of serotonergic receptors 47 with methylsergide (Gallo 33 31., 1975) decreases prolactin secretion in lactating and in ovariectomized rats; whereas, quipazine, a serotonin receptor agonist (Hong and Pardo, 1966; Rodriguez 33 31., 1973) stimulates prolactin secretion (Clemens, 1975; Clemens 33 31., 1977). Electrolytic or radiofrequency lesions of the raphe nuclei have been shown tO impair lactation (Barophy and Harvey, 1975) and decrease serum prolactin in male rats (Advis 33 31., unpublished); whereas, electrical stimulation Of the dorsal raphe increases prolactin secretion (Advis 33 31., unpublished). These changes in prolactin secretion are thought to be mediated via changes in the turnover rate of serotonin since raphe lesions decrease (Kuhar 33 31., 1972; Herr and Roth, 1972) and raphé stimulation increases serotonin turnover (Sheard and Aghajanian, 1968). V. Hypothalamic Control Of Luteinizi3g Hormone Secretion A. Feedback of Gonadal Steroids on LH Secretion Luteinizing hormone (LH) secretion appears to be regu- lated primarily by circulating levels of estrogens and pro- gesterone in the female and testosterone in the male (Martin 33 31., 1977) through feedback mechanisms acting directly on the anterior pituitary or mediated through the hypothalamus. In female mammals, which show cyclic release Of LH, both 48 stimulatory and inhibitory gonadal steroid feedback mechan- isms are well documented (McCann, 1974); whereas, in males inhibitory, but not stimulatory feedback appear to be present (Bloch _£._l-: 1974; Turner, 1973). Teleologically, the existence Of a dual effect of gonadal steroids in females allows for the rhythmic surges in LH, FSH, and prolactin which are necessary for the maintenance of ovarian cyclicity. In males, in which the function of LH and FSH is to stimulate continuously testosterone secretion, spermatogenesis and spermiogenesis, a tonic rather than a cyclic control of LH secretion is necessary. This sex difference in adult LH secretory action is determined by the steroid environment of the hypothalamus during a very short postnatal period in the rat (Coy, 1970). Ovariectomy and orchidectomy causes an increase in serum LH which persists indefinitely (Gay and Midgley, 1969; Yamamoto 33 31., 1970; Blackwell and Amoss, 1971), an effect which can be inhibited by the administration Of estrogen or progesterone (Smith and Davidson, 1974; Chowers and McCann, 1967; Ramirez 33 31,, 1964). Medial basal hypothalamic (MBH) implants Of gonadal steroids appear to be more effective in inhibiting LH secretion than intrapituitary implants (Davidson 33 31., 1969; Davidson, 1969; Sawyer and Hilliard, 1972), suggesting that the MBH is an important hypothalamic site for inhibitory steroid feedback. However, Bogdanove 49 (1964) has proposed that MBH implants result in a more efficient delivery of steroids to the pituitary than intra— pituitary implants, i.e., the "implantation paradox." In male rats, we have demonstrated that 3H-testosterone im- planted into the MBH causes a decrease in LH secretion before substantial label reaches the pituitary gland (Turner and Simpkins, unpublished) suggesting that the MBH can medi- ate the inhibitory feedback Of steroids on LH secretion. The administration of estrogen and progesterone, alone or in combination can stimulate LH surges in both female rats and humans. Administration of estrogen in both 4- and 5-day cycling rats during diestrus has been shown to advance the time of ovulation (Everett, 1948; Brown-Grant, 1969; Weick 33 31., 1971). The LH surge which occurs on proestrus Of the estrous cycle and that induced by estrogen adminis- tration can be blocked by the administration of antibodies against estrogen (Ferin 33 31,, 1969, 1974; Knobil 33 31., 1974). Estrogen administration tO long-term ovariectomized rats causes a daily LH surge (Legan and Karsch, 1975). The ability Of progesterone to induce LH surges appears to depend upon the existing estrogen environment. Thus, pro- gesterone can stimulate LH surges if administered on the third day of diestrus in 5-day cycling rats or on proestrus in 4-day cycling rats (Everett, 1948; Zerlmaker, 1966; Brown- Grant and Naftolin, 1972). Administration Of progesterone 50 in early diestrus inhibits LH secretion (Everett, 1948) and interferes with LHRH induced LH release (Martin 33_31., 1974). Estrogen and progesterone appear to interact synergis- tically tO stimulate LH surges. Progesterone administration tO estrogen primed, long term ovariectomized rats, stimulate an LH surge which is similar to the preovulatory LH surge both in magnitude and timing (Caligaris 33 31., 1971). The similarity Of the response to this sequence Of steroid treat- ments to that which occurs on proestrus is indicated by the Observation Of both an FSH and a prolactin surge accompanying the LH surge (Caligaris 33 31., 1974). The possible role of a progesterone-estrogen interaction to induce the preovula- tory LH surge has been questioned because of conflicting reports regarding levels Of progesterone on the morning of proestrous (Schwartz, 1969; Everett, 1964; Redman, 1968; Hori 33 31,, 1968; Goldman 33 31., 1969; Piacsek 33 31., 1971; and Barraclough 33 31., 1971). However, recent studies utilizing frequent blood sampling times have demonstrated daily morning progesterone peaks during the estrous cycle (Mann and Barraclough, 1973) and a large progesterone surge on the morning Of proestrous (Kalra and Kalra, 1974). Thus, a synergistic interaction between progesterone and estrogen may normally occur during the estrous cycle. Lisk and Reuter (1977) have recently reported that progesterone enhanced 51 binding of 3H-estradiol to both preoptico-anterior hypo- thalamic and median eminence-arcuate tissue in the rat hypothalamus. The locus of the stimulatory feedback of gonadal ster- oids on LH secretion has not yet been conclusively estab- lished, although direct anterior pituitary and hypothalamic mediated stimulatory feedback have been described. Removal of afferents to the MBH by deafferentations which exclude the preoptico-anterior hypothalamic area blocks ovulation (Hillarp, 1949; Halasz and Gorski, 1967; Halasz, 1972) and gonadotropin secretion (Palka 33 31., 1969; B1ake.33 31., 1972; Weiner 33 31., 1972b). Hypothalamic islands which in- clude the preoptico—anterior hypothalamus in the islands do not interfere with cyclic release Of LH (Halasz, 1972). Electrical lesions Of the preOptic anterior hypothalamus (Barraclough, 1966) or the suprachiasmatic nucleus (Schneider and McCann, 1969a; Mess 33 31., 1966) has a similar effect on LH secretion. Deafferentation and anterior hypothalamic lesions also block the steroid induced LH surge (Taleisnik 33 31., 1970; Bishop 33 31,, 1972). Electrical stimulation of preOptic-anterior hypothalamic areas, as well as the arcuate-median eminence area stimulates ovulation (Sawyer, 1975), LH secretion (Clemens 33 31., 1971; Cramer and Barraclough, 1973) and increase LHRH levels in portal blood (EsRay 33 31., 1977). In primates, the role Of the 52 preOptic-anterior hypothalamus in mediating positive steroid feedback has been questioned by Krey 33 31. (1975) based upon the Observation Of normal cyclicity and normal response to estrogen after medial basal hypothalamic deafferentation. In addition to the well known presence of LHRH contain- ing cell bodies and nerve terminals in the median eminence- arcuate nucleus in the mouse (Zimmerman 33 31., 1974) and rat (Barry and Dubois, 1973; Baker 33 31., 1974; Kordon 33_31,, 1974), LHRH perikarya have been demonstrated in the preoptic nuclei of the guinea pig (Leonardelli 33 31., 1973; Barry and Dubois, 1973), the cat and dog (Barry and Dubois, 1975) and the rat (Baker 33 31., 1975). These rostral LHRH containing cells may send axons to the medial basal hypothala- mus (MBH). Deafferentation in male and female rats has been shown to cause a depletion Of MBH-LHRH concentration and an increase in preOptic-anterior hypothalamic LHRH (Kalra, 1976; Kalra 33 31., 1977). In addition to the localization Of LHRH containing cell bodies in the rostral aspects of the hypo- thalamus, there appears tO be a large serotonergic and nor- adrenergic input to rostral hypothalamic nuclei (Ungerstadt, 1971; Brownstein 33 31., 1976). Pharmacological studies indi- cate that both of these amines may be important neurotrans- mitters in mediating stimulatory steroid feedback (Kalra and McCann, 1972; Coen and MacKinnon, 1976). 53 An alternative site Of the pOsitive feedback of gonadal steroid is directly on gonadotropin producing cells of the anterior pituitary gland. Arimura and Schally (1971) first Observed that 24 or 48 hours exposure to estrogen increased LHRH induced release of LH 13 3133. Later studies have since shown that estrogen can potentiate LHRH induced LH release from the anterior pituitary and from pituitary cell suspensions 13 31333 (Labrie 33 31., 1976), and in animals with MBH deafferentation (Greeley 33 31., 1975). Cyclic variations in pituitary responsiveness Of LH secretion to LHRH stimulation have been Observed (Gordon and Reichlin, 1974; Aiyer 33 31., 1974a; Martin 33 31., 1974; Zeballos and McCann, 1975). The most sensitive stage of the estrous cycle for LHRH-induced LH release is the afternoon Of proestrus, when circulating estrogen titers are high. In addition, estrogen appears to increase the self-priming action Of LHRH on LHRH induced LH secretion (Aiyer 33 31., 1974b; Greeley 33 31., 1975). Consistent with these reports are the Observa- tions that specific binding Of 125 I-LHRH to anterior pitui- tary membranes is highest on proestrous (Kyringza 33 31., 1975) indicating that estrogen may increase the synthesis of plasma membrane LHRH receptors. Progesterone appears to have nO effect on LHRH induced LH secretion; however, large doses can interfere with estrogen potentiation Of LHRH induced LH secretion (Aiyer 33 31., 1974a; Greley 33 31,, 1975). 54 B. Noradrenergic Effects on Luteinizing Hormone Secretion Several pharmacological approaches have been employed to determine the role of catecholamines in the secretion of gonadotropins. These include: a) administration of CA de- pleting drugs, b) administration Of receptor agonists and antagonists, and c) ventricular infusion of monoamines. Alpha—methyl-p-tyrosine (ampt) which depletes central CA stores by competitively inhibiting the activity Of tyrosine hydroxylase (Spector 33 31., 1965; Corrodi and Hansen, 1965), the rate limiting enzymes in catecholamine synthesis, has been shown to block ovulation (Brown, 1967; Kordon and Glowinski, 1969), the proestrous LH surge (Kalra and McCann, 1973), the release Of LH in response to castration (Ojeda and McCann, 1973), and stimulatory gonadal steroid feedback Of LH secretion (Kalra 33 31., 1972). Reserpine, which depletes catecholamine storage vessels (Dahlstrom 33 31,, 1965) is able to block ovulation (Brown, 1967) and prevents LH secre- tion in response to administration of Pregnant Mare Serum (PMS) (Barraclough and Sawyer, 1957). 6—HydroxydOpamine, which selectively destroys catecholaminergic nerve terminals and axons (Thoenen and Tranzer, 1968; Uretsky and Iverson, 1970) blocks LH release on the afternoon of proestrous and in response tO steroid administration. This antigonadotropic effect occurs whether infusion is into the third ventricle (Kalra 33 31., 1974) or into the ventral noradrenergic tract (Martinovic and McCann, 1977). 55 High doses of alpha-adrenergic receptor blocking drugs have been reported to block ovulation in the rabbit (Kordon and Glowinski, 1969), to decrease LH levels in castrated females (Schneider and McCann, 1969b), and to block estrogen induced LH release (Schneider and McCann, 1970a). The selec- tivity Of high doses of a-adrenergic receptor blockers has recently been questioned on the basis of the Observation that these drugs are able to block the reported DA-induced increase in LHRH secretion into the hypOphysial system (Kamberi, 1976) and increased LH secretion (Kamberi, 1970c; Kalra and McCann, 1973). Intraventricular infusion of norepinephrine induces ovulation (Sawyer 33 31., 1949; Rubinstein and Sawyer, 1970; Sawyer, 1975; Tima and Flerko, 1975), and increases serum LH in the rat and rabbit (Sawyer, 1975; Krieg and Sawyer, 1976) and increases LHRH release into the hypophyseal portal circu- lation in female rats (Portal 33 31., 1976). 13_31333 incu- bation Of NE in a hypothalamo-pituitary incubation system stimulates LH secretion (McCann and Moss, 1976), an effect which appears to be mediated through an a-adrenergic mechan- ism (Godde and Schwilling, 1976). In a series of detailed experiments Kalra and McCann (1972) undertook an analysis Of the relative importance of d0paminergic and noradrenergic systems in mediating the stimulatory feedback Of estrogen in ovariectomised and 56 progesterone in ovariectomized estrogen primed rats on LH secretion. Administration Of the dopamine-B-hydroxylase inhibitors, diethyldithiocarbarmate (DDC) or l-pheny1-3- (2-thiazolyl)thiourea (U-l4,624), which are believed to depress central NE level while not affecting or increasing central DA levels (Goldstein and Nakajima, 1967; Johnson 33 31., 1970) blocked the progesterone induced LH surge in ovariectomized, estrogen-primed rats. Administration Of L-dOpa to DBH blocked rats was ineffective; whereas, dihy- droxyphenylserine (DOPS) was effective in reestablishing the LH surge. Since in DBH blocked animals L-dOpa should selec- tively increase DA concentration; whereas, DOPS should selectively increase NE, these studies suggest that proges- terone induced LH surge is noradrenergic, but not dopamin- ergic mediated. Ojeda and McCann (1973) demonstrated that both ampt or DDC are able to block the post-castration rise in serum LH in male rats. However, administration Of neither L-DOPA or DOPS was successful in restoring the post-castration LH rise. Thus, the involvement of CA5 in the post—castration LH rise in male rats is uncertain. In 4-day cycling rats, systemic administration or third ventricle infusion Of either Ompt or PIA-63, a dopamine-B- hydroxylase inhibitor, on the second day Of diestrus blocked ovulation and reduced proestrous LH levels (Terasawa 33 31., 57 1975). Administration Of L-DOPA or DOPS reversed the ampt block in about half Of the animals tested. These studies indicate that a central noradrenergic mechanism may be in- volved in stimulating LH secretion in several different conditions. Increases in NE turnover have been described during states Of increased LH secretion. Ovariectomy increases anterior hypothalamic concentration Of NE (Donoso 33 31., 1967), increases rate of depletion of 3H-NE from whole brains at 6 and 21 (but not 2) days post-castration (Anton- Tay and Wurtman, 1968), and triples turnover by 2 weeks post-castration as estimated by measuring the rate Of cate- cholamine depletion following ampt treatment (COppOla, 1969). 3H—tyrosine also increases after The synthesis of NE from ovariectomy (Anton-Tay 33 31., 1970; Bapna 33 31., 1971). Hypothalamic tyrosine hydroxylase activity increases 2-3 fold from 4 to at least 60 days post-ovariectomy in female rats, an effect which can be blocked by progesterone, but not estrogen, replacement (Beattie 33 31., 1972; Beattie and Soyka, 1973). Changes in hypothalamic concentration and metabolism of norepinephrine occur during states Of stimulatory steroid feedback. On proestrus, anterior hypothalamic (Stefano and Donoso, 1967; COppola, 1969) and median eminence (Selmanoff 33 31., 1976) norepinephrine concentration and whole brain 58 norepinephrine turnover (Zachaeck and Wurtman, 1973) increase. During the first estrous cycle, hypothalamic norepinephrine turnover increases during early proestrus, then decreases in late proestrus and estrus (Advis 33 31., 1977). A similar transient increase in norepinephrine turnover was Observed in ovariectomized, estrogen-primed rats in response to progester- one administration (Simpkins, Huang, Advis and Meites, unpub- lished). In general, these studies indicate that a central noradrenergic mechanism is involved in stimulating LH secre- tion during several conditions. C. Dopaminergic Effects on Luteinizing Hormone Secretion There is no general agreement concerning the role of central dopaminergic mechanisms in the regulation Of LH secretion. DOpamine infusion into the third ventricle has been reported to increase LH and FSH secretion (Kamberi 33 31., 1970b, 1971c; Schneider and McCann, 1970b,c), portal vessel LRF activity (Kamberi, 1969) and plasma LRF activity in hypOphysectomized rats (Schneider and McCann, 1970b). In contrast, Cramer and Porter (1973) Observed no effect Of third ventricle infusion of DA on plasma LH in intact or estrogen treated rats. Sawyer's group Observed that third ventricle DA infusion does not effect LH but is able to block the LH increase stimulated by third ventricle NE in- fusion (Sawyer 33 31., 1974; Krieg and Sawyer, 1976). 59 In an attempt to explain these differences, McCann and Moss (1976) suggested that the steroid environment of the animal is important in modulating the effect Of intraventricular DA on LH secretion. During the estrous cycle, intraventricu- lar DA stimulates LH secretion on the second day of diestrus and on proestrus, when estrogen titers are high, but not on estrus or the first day of diestrus (Schneider and McCann, 1970a). Infusion Of DA into the third ventricle stimulated LH secretion in ovariectomized, estrogen-progesterone primed animals, but failed tO effect LH secretion in ovariectomized animals (Vijayan and McCann, 1977). Furthermore, DA stimu- lates the 13 31333_re1ease of LHRH from medial basal hypO- thalami of ovariectomized, estrogen-primed rats, but not from ovariectomized rats (Rotsztejn 33 31., 1976). In addition to a possible role Of gonadal steroids in modulating the DA effect, it is possible that DA is taken up by noradrenergic neurons and converted tO norepinephrine before it affects LH secretion. The O-adrenergic receptor blockers inhibit the reported DA induced LH release both 13 31333 (Kamberi 33 31,, 1970c; Schneider and McCann, 1969b) and 13 3133 (Schneider and McCann, 1970a) which indicates that DA may act through a adrenergic system or be converted to NE before interaction with the noradrenergic receptor. Studies utilizing systemic administration of dopamine receptor stimulators or blockers have produced equally 60 conflicting data. The neuroleptic haloperidol (Janssen, 1967) has been reported to block ovulation in the rat (Boris 33 31., 1970) and decrease serum levels Of LH and FSH and hypothalamic LHRH activity when administered on early proestrous (Dickerman 33 31., 1974). In contrast, the dopamine receptor blocker, d-butaclanol (Voith and Herr, 1975; Lippman 33 31., 1975; Lippman and Pugsley, 1975) ap- pear tO have no effect on pulsatile LH release in ovari- ectomized rats (Drouva and Gallo, 1977) and heloperidol has no effect on basal LH levels in male rats (Mueller 33 31., 1976). Pimozide administration has been reported to have no significant effect on the post-castration LH rise (Ojeda and McCann, 1973) or on pulsatile LH release in ovariectomized rats (Drouva and Gallo, 1976). Dopamine receptor stimulation with apomorphine and CB-154 blocks pulsatile LH secretion (Drouva and Gallo, 1976), but apomorphine has no effect on basal levels Of LH in male rats (Mueller 33 31., 1976). Piribedil has been reported to decrease basal LH levels in male rats (Mueller 33 31., 1976), but has no effect on the post-castration LH rise in rats (Grandison, Hodson and Meites, unpublished). Fuxé and co-workers have provided evidence that the tuberoinfundibular DA system may be involved in inhibiting LH secretion. Castration decreases and administration Of estrogen increases tuberoinfundibular DA turnover as 61 measured by histofluorescence (Fuxé 33 31., 1969). Tubero- infundibular DA turnover decreases on proestrous and early estrus of the rat estrous cycle (Ahren 33 31., 1971). Similarly, d0pamine turnover in the whole hypothalamus has been reported tO decrease on late proestrous of the first estrous cycle in rats (Advis 33 31., 1977). During lactation in the rat, serum LH levels are low and tuberoinfundibular DA metabolism is high (Fuxé 33 31., 1967; Ben-Jonathan and Porter, 1976). D. Serotonergic Effects on Luteinizing Hormone Secretion Evidence for both a stimulatory and an inhibitory role of serotonin on LH secretion have been presented. Intra- ventricular infusion Of serotonin has been reported to sup- press LH and FSH secretion (Kamberi, 1970b; Schneider and McCann, 1970c) and inhibit the proestrus surge of LH and FSH (Kamberi, 1973). Systemic administration of 5 HTP, a sero- tonin precursor, blocks ovulation and the proestrous surge of LH and FSH (Kamberi, 1973). Electrical stimulation of the raphe nuclei increases hypothalamic 5 HT turnover and inhibits ovulation (Carrer and Taleisnik, 1970, 1972); whereas, administration Of PCPA on the morning Of proestrus facili- tates ovulation (Kordon and Glowinski, 1972). However, other studies suggest a stimulatory role for 5 HT in LH and FSH release. Coen and MacKinnon (1976) were able to block the 62 evening surges Of LH in ovariectomized, estrogen-primed rats with p-chlorOphenylalanine (PCPA), p-chloroamphetamine (PCA) and 5,6-dihydroxytryptamine (5,6 DHT), all Of which deplete central serotonin levels. Infusion Of 5,6-DHT into medial and dorsal raphe nuclei, which causes complete disappearance of 5—HT terminals in the suprachiasmatic nuclei 10 days after administration (Daly 33 31,, 1973), blocks estrogen induced LH surges. Administration Of 5-HTP in PCPA depleted, ovari- ectomized, estrogen primed rats causes a reappearance of the evening LH surge (Hery 33 31., 1976). Further, the timing of serotonin depletion by PCPA appears to be important, since administration just before the LH surge was ineffective;' whereas, administration earlier causes blockade of the LH surge (Hery 33 31., 1975). Kordon's group has suggested two functionally distinct serotonergic systems influence LH secretion. An inhibitory system is indicated by the Observation that median eminence- arcuate area is the only hypothalamic area able to mediate the inhibitory effect Of implanted serotonin on LH secretion (Kordon, 1969). A stimulatory system is indicated by the observation of a large serotonergic input to the suprachias- matic nucleus (Fuxé, 1965; Ungerstadt, 1971; Brownstein 33 31., 1975), an area whose integrity is vital to cyclic release of LH (Clemens 33 31., 1973), and the Observation that serotonergic depleting drugs can block cyclic LH surges 63 but have little effect on basal LH secretion (Coen and MacKinnon, 1976; Hery 33_31,, 1975, 1976). E. Inhibitory Effects of Prolactin on LH Secretion In many physiological, pathological and experimental states an inverse relationship between serum prolactin and gonadotropin exists. During postpartum lactation in rats, serum prolactin is high; whereas, serum LH and FSH are low (Meites, 1966). Ovariectomy in rats results in an increase in gonadotropins and a decrease in prolactin secretion (Meites and Clemens, 1972). In humans, a similar relation- ship between serum prolactin and LH appears to exist during normal lactation and states Of inappropriate lactation (Martin 33 31., 1977). Forbes-Albright and Chiari~Frommel syndromes are conditions in which hyperprolactinemia is associated with infertility. In these conditions, the high circulating levels of pro- lactin are believed to be responsible for the low serum LH. Suppression of serum prolactin in both postpartum rats (Lu _3 _1., 1976) and in humans (Seri _3__1., 1974) with the DA agonist CB-154 (Z-bromo—ergocryptine) results in an increased gonadotropin secretion. In humans, L-DOPA or CB-154 induced decreases in prolactin and results in resumption Of menstrual cycles in Forbes-Albright (Turkington, 1974) and Chiari- Frommel (Seri and Seri, 1974) patients. The resumption of 64 reproductive function is believed to be due to a decrease in circulating prolactin since CB-154 has never been shown to stimulate LH secretion (Fuxé 33 31., 1976). Induction Of hyperprolactinemia by exogenous administration Of ovine pro- lactin (Gudelsky 33 31., 1977) and by implantation of anterior pituitary glands beneath the kidney capsule or subcutaneous implant Of pituitary tumor tissue (Grandison 33 31., 1977) decreased LH secretion in both castrated males and females. The prolactin-induced inhibition of gonadotropin secre- tion appears to be centrally mediated. Systemic injections of prolactin and subcutaneous pituitary tumor implantation increase tuberoinfundibular as well as anterior hypothalamic DA turnover (HOkfelt and Fuxé, 1972; Gudelsky 33 31., 1977; Hodson 33_31., unpublished). These treatments do not effect other central d0paminergic systems and have no effect on norepinephrine turnover. Since the inhibitory effect Of prolactin on gonadotropin occurs in castrated animals, the prolactin effect does not appear to be secondary to its effect on the gonads. Muralidhar 33 31. (1977) reported a decreased responsiveness Of the pituitary to a single injec- tion of LHRH in rats treated with prolactin. However, using multiple LHRH injections, Hodson 33 31. (1977) have been unable to demonstrate any effect Of prolactin on LHRH induced LH release. The explanation of the difference between these two studies is at present uncertain. However, it is possible 65 that prolactin could effect pituitary sensitivity by decreas- ing the release Of LHRH. This could decrease the priming of the pituitary which could result in a decreased responsive- ness Of the pituitary to a single LHRH injection. Multiple LHRH injections would mask this effect. MATERIALS AND METHODS I. AnimalsL Treatments and Blood Collection Mature male and female rats used in these studies were obtained from four sources (Spartan Research Animals, Haslett, MI; Charles Rivers, Madison, WI; Blue-Spruce Farms, Altamont, N.Y.; and Harlan Industries, Indianapolis, IN). Animals were housed in light (14 h on, 10 h Off) and temperature (25°31°C) controlled rooms and provided with Purina Rat Chow (Ralston Purina CO., St. Louis, MO) and tap water 33 libitum. Animals were orchidectomized or ovariectomized within 3—5 min while under deep ether anesthesia. Cannulae were implanted in ether anesthetized rats with the aid Of a Kopf Stereotaxic Instru- ment using coordinates described in the rat brain atlas of Skinner (1971). Correct placement Of annulae was confirmed by macroscopic Observation following decapitation of the animals. Piribedil mesylate (provided by Dr. M. Derome«Tremblay, Les Laboratories Servier, Neuilly sur Seine, France), alpha- methyl-para-tyrosine methyl ester HCl (Regis Chemical CO., Morton Grove, IL) and pargyline hydrochloride (Sigma Chemical CO., St. Louis, M0) were dissolved in 0.89% NaCl just before use. Estradiol benzoate (EB) and progesterone (P) (Nutri- tional Biochemicals Corp., Cleveland, OH) were dissolved in 66 67 corn Oil. 6-HydroxydOpamine hydrobromide (6-OH—DA) (Regis Chemical CO., Morton Grove, IL) for intrahypothalamic implantation, was tapped into 26-gauge stainless steel wire cannulae. The combined weights Of 50 cannulae determined before and after tapping the drug indicates that 1-2 mg of 6-OH-DA were administered per implant. Blood samples were taken by decapitation or by orbital sinus cannulation under light ether anesthesia except in Experiment V in which sampling was done via chronically im- planted jugular cannulae in unanesthetized, unrestrained animals. Blood samples were stored at 4°31°C for 24 h to allow clot formation and serum was separated and stored at -20°C until assayed for hormone concentration. 11. Radioimmunoassay Of Serum Hormones Serum concentrations of luteinizing hormones (LH), pro- lactin, and thyroid stimulating hormone (TSH) were deter- mined using standard double antibody ratioimmunoassay pro- cedures. Serum prolactin was assayed using the method of Niswender 33 31. (1969), while serum LH and TSH were deter— mined by the method described in the NIAMDD-kits. Hormone concentrations are expressed in terms of the standard refer- ence preparations NIAMDD-rat prolactin-RP-l, NIAMDD rat LH—RP-l, and NIAMDD-rat TSH-RP-l. All serum samples were 68 assayed in duplicate or triplicate. Samples from individual experiments were all tested in the same assay to avoid inter- assay variability. Hormone concentration was determined only from serum volumes which resulted in hormone values on the linear portion Of the standard curve. 111. Assay of Dopamine and Norep1nephrine in Brain Tissues A. Isolation and Preparation of Brain Tissue Immediately after decapitation, brains were removed from the cranium and laid dorsal side down (to avoid deformation of the hypothalamus) in a pOOl of ice cold 0.89% NaCl. The hypothalamus was excised using the following landmarks: anterior, immediately rostral to the Optic chiasm; posterior, middle Of the mammilary bodies; lateral, the lateral hypothal- amic sulci; dorsal, 2-3 mm dorsal to the ventral hypothalamic surface. In the same experiments (see Experimental) the anterior and posterior portions Of the hypothalamus were separated by making a frontal cut just caudal to the infundi- bular stalk. Such a procedure produced anterior hypothalami (AH) weighting 22.8:0.4 mg and posterior hypothalami (PH) weighting 11.0:0.4 mg (N=64). The whole hypothalamus or hypothalamic fragments were immediately frozen on dry ice until assayed for catecholamine concentration. The median eminence (ME; Experiment IV) was dissected from the remaining hypothalamus using fine iris scissors. 69 Cuts were made at the posterior border Of the infundibular stalk and along the lateral aspects Of the tuber cinereum at an angle of about 20° from the ventral hypothalamic sur- face. The piece of tissue produced by this procedure corre- sponded roughly to the superficial basal hypothalamic layer described by Kavanagh and Weize (1973) and contained 1831 pg protein (N=l4) as assayed by the micro-method of Lowry 33 31. (1951). Medial basal hypothalamus (MBH; Experiment VII) was dissected by making cuts similar to those for the ME except at a 45° angle from the ventral hypothalamic surface. This dissection corresponded to the intermediate basal hypothalamic layer described by Kavanagh and Weize (1973) and the tissue contained 3414 pg proteins (N=32). The ME was homogenized in 25 ul and the MBH in 40 ul of 0.4 N perchloric acid (PCA) containing 10 mg percent EDTA. Whole hypothalami as well as anterior and posterior hypothal- amic portions were hemogenized at a concentration Of 1 mg tissue per 10 ul 0.4N PCA (plus 10 mg percent EDTA). Tissues were homogenized using microhomogenizers (Micrometric Instru- ments Inc., Cleveland, OH) and centrifuged (microcentrifuge, Coleman Inst., Oak Brook, IL) to separate the particulate portion from the supernatant. DOpamine (DA) and norepinephrine (NE) were assayed in 10 ul of supernatant by the method described below. 70 B. Radioenzymatic Assay of DOpamine (D31 andINOrepinephrine (N31? Tissue DA and NE were assayed by the method of Coyle and Henry (1973) or by a modification Of the method Of Ben- Jonathan and Porter (1976). Both assays utilized 10 ul of tissue supernatant or standard DA and NE (Sigma Chemical CO., St. Louis, MO) incubated in the presence Of buffered cate- cholamine-O-methyl transferase (COMT) and the methyl donor 3H-S—adenosyl methionine (New England Nuclear, Boston, MA). COMT was partially purified from rat liver by the method of leodijevic 33 31. (1970). The procedure for extraction and separation of 3-0- methoxynormetanephrine and 3-0-methoxytyramine (the o-methyl- ated metabolites Of NE and DA) by the method of Coyle and Henry (1973) is described in Appendix A. Briefly, after side chain cleavage of normethanephrine with sodium meta- periodate (Mallinckrodt Chemical CO., St. Louis, M0), the NE and DA metabolites were separated utilizing a series of acid- case and organic-aqueous phase extractions. As a result of incomplete oxidation of normetanephrine, 4.8% Of the counts in the DA fraction were from tissue or standard norepineph- rine. Both standard and tissue counts were corrected for this contamination before DA and NE content were calculated. The Ben-Jonathan and Porter method (1976) utilizes solvent extraction and thin layer chromatography to separate normethanephrine and methoxytyramine. This procedure 71 simplified the cumbersome Coyle and Henry method and improved the assay sensitivity (see Appendix B). Amine content Of samples were determined after separation by counting chroma- tographic spots containing the 3H-labeled metabolites in class scintillation vials containing 10 ml of Scintiverse (Fisher Scientific Products, Livonia, MI). Samples were counted in a New England Nuclear, Mark II, scintillation (201111128 r . EXPERIMENTAL I. Effects Of Pr3gesterone on Steady State Concentration and Turnover Of Dopamine (DA) and Norepjnephrine (NE; in Anterior anleosterior POrtions Of the Hypo- thalamus, andIOn Serum Luteifiizinngormone and ProlactinILevels in Ovariectomized, Estrogeninimed’Rats A. Objectives Several experimental approaches indicate that central noradrenergic and dopaminergic systems influence the secre- tion Of anterior pituitary hormones (Wurtman, 1971; de Wied and de Jong, 1974; Meites 33 31., 1976). The anterior hypothalamus is Of particular importance in mediating the stimulating influence of gonadal steroids on the secretion of gonadotropins. Destruction Of anterior hypothalamic nuclei or surgical separation Of the rostral hypothalamus from the MBH can block ovulation (Hillarp, 1949; Halasz and Gorski, 1967; Halsaz, 1972), the proestrus surge Of LH (Palka 33,31,, 1969; Blake, 1972), and LH release normally seen in response to progesterone (P) administration to ovari- ectomized, estrogen-primed rats. The present study was undertaken to determine if alterations in anterior or pos- terior hypothalamic DA and NE metabolism occur after P treat- ment Of ovariectomized, estrogen—primed rats. 72 73 B. Materials and Methods Female Long—Evans rats, weighing 200.255 g, were bi- laterally ovariectomized and 10 days later received a single s.c. injection of estradiol benzoate (BB; 100 ug/kg body weight). Three days after the EB injection, animals received a single s.c. injection Of P (500 ug/kg body weight) at 0900 hrs. This treatment has been shown to induce a surge in both LH (Caligaris 33_31,, 1971) and prolactin (Caligaris 33 31., 1974). Animals were killed by decapitation 0, 2, 4 and 6 h after the P administration. DA and NE turnover were esti- mated by a modification Of the non-steady state method described by Brodie 33 31. (1966). At the time of sacrifice, animals were randomly placed in two groups. One group Of the animals received an i.p. injection Of 250 mg alpha-methyl« para-tyrosine per kg body weight 1 h before decapitation. The other group Of animals received vehicle (0.89% NaCl). At the time Of decapitation, trunk blood was collected for determination of serum LH and prolactin concentration. Anterior and posterior hypothalamic fragments were dissected as described in general "Materials and Methods." Anterior hypothalamus (AH) and posterior hypothalamus (PH) weighed 22.8 3 0.4 and 11.0 3 0.4 mg, respectively (N=64). The significance of differences among groups was deter- mined by ANOVA and Student-Newman—Keuls tests (Sokal and Rohlf, 1969). Two-way ANOVA was used to test the significance 74 Of interaction between factors (time after P treatment and drug treatment). The level of significance chosen was P<:0.05. C. Results The upper panel Of Figure 1 shows that the steady state concentration of anterior hypothalamic NE does not change significantly between 0 and 4 h after progesterone adminis- tration. However, a significant decrease (P‘<0.05) in NE concentration occurs between 4 and 6 h post-progesterone. NE turnover, as estimated by the increase in NE depletion after exposure to ampt, increased 2-fold (P‘<0.05) at 4 h after P administration. By 6 h post-progesterone turnover was not different from 0900 h controls. No significant alteration in the steady state concentra- tion of anterior hypothalamic DA occurred at any Of the times tested. However, a progressive decrease beginning at 4 and becoming significant (P<<0.05) at 6 h, in DA turnover occurred. Serum LH levels increased slightly by 4 h and were in- creased dramatically by 6 h after P administration. Serum prolactin levels were elevated by 2 h, peaked at 4 h and remained elevated at 6 h after P administration. NO change in posterior hypothalamic NE or DA concentra- tion or turnover was Observed in this study (Figure 2). 75 Figure 1. Effects of progesterone administration to ovari- ectomized, estrogen-primed rats on steady state concentration and alpha-methy1-para-tyrosine induced depletion Of anterior hypothalamic cate- cholamines and on serum luteinizing hormone (LH) and prolactin (PRL). The solid bars indicate steady state concentration and hatched bars indicate amine concentration 1 h after i.p. injection Of ampt (N=6-8). The numbers above the sets of bars indicate percentage depletion of amines induced by ampt. The star above the solid bar indicates a significant differ— ence in amine concentration (P< 0.05) between that group and the 0 h group. Stars above the numbers indicate a signifi- cant interaction between ampt treatment and time after pro- gesterone as analyzed by a Two Way ANOVA (P< 0.05). LH and prolactin represented in the lower panel are from sera of non-drug treated, control animals. Stars above the points indicate a significant increase above 0 h values (P<<0.05). The verticle line in all 3 panels represents 1 standard error of the means (SEM). _.__- __..__... ..__._._ ...—.v ._.._ 76 2_2 ~ 3 41 A Herman 2100 15.. NE (IE/6|) an 3|. i ii. _45_ 1. “mm" 5.0 I,“ 3.0 (IS/BI) 7.0 500 s 55'". [N.PIl Pll (IS/ll) 300 m I ' :I“=::::::;I---—-. I 2 4 3 Hours arm metsmm Figure 1 2: _l_|_ 24 s % mumu l mo ' mo N I 800 (IE/GI) 808 400 200 52 u _50__ AL % warm I» DA 400 (Ii/BI) 30. _.... -w- --_— ”— . 2 4 6 ""8 “TE! PIIGESIEIOIE Figure 2. Effect Of progesterone administration to ovari- ectomized, estrogen-primed rats on steady state concentration and alpha-methyl—para—tyrosine induced depletion Of posterior hypothalamus. The solid bars indicate steady state concentration and the hatched bars indicate amine concentration 1 h after i.p. injection of ampt (N=6-8). Verticle lines indicate 1 SEM. The number above the sets of bars indicates percentage depletion induced by ampt. 78 D. Discussion The observation that anterior, but not posterior hypo- thalamic catecholamine metabolism is altered in response to a stimulatory regimen of gonadal steroids is consistent with the hypothesis that anterior hypothalamic areas regulate the stimulatory influence of gonadal steroids on LH secretion (Gorski, 1966). Earlier Observations indicate that estrogen or a combination Of estrogen and progesterone implanted in the anterior hypothalamus stimulate LH secretion (McCann, 1974). Anterior hypothalamic destruction blocks ovulation and the LH surge normally occurring on proestrus and follow- ing steroid administration (Hillarp, 1949; Mess 33 31., 1966; Halasz, 1972; Weiner 33_31., 1972). However, steroid implants or lesions in the posterior hypothalamus have little or no effect on LH secretion (McCann, 1974). The doubling of NE turnover by 4 h after the P injec- tion is similar in magnitude to that Observed in other condi- tions of increased LH secretion. Anton-Tay and Wurtman (1968) Observed a doubling in the rate Of depletion Of whole 3H-NE at 2 weeks post-castration, whereas Coppola brain (1969) observed a doubling of total catecholamine turnover in anterior hypothalamus 2 weeks post-castration. Tyrosine hydroxylase activity has been reported to increase 2-3 fold from 4 to 60 days post-ovariectomy in rats (Beattie 33 31., 1972). The present study indicates that an increase in NE 79 turnover can occur quickly (4 h) in response to stimulatory steroid feedback, while the increased NE turnover reduced by ovariectomy requires days to develop (Anton-Tay and Wurtman, 1968). The long latency in response to ovariectomy in females may be due to the long half-life Of gonadal steroids. The increased turnover Of anterior hypothalamic NE occurs at the onset of the LH surge and at the time of peak serum prolactin. The Observation that the dopamine B-hydroxylase inhibitor, diethyldithiocarbarnate (DDC) can block the P induced LH surge in ovariectomized, estrogen-primed rats sug- gests a causative relationship between the increased NE turn- over and the onset Of the LH surge. On the afternoon of proestrus in the rat, when a steroid induced LH surge normal- ly occurs (Ferin 33 31., 1969, 1974; Kalra and Kalra, 1974) hypothalamic (Advis, Simpkins, Chen and Meites, unpublished Observation) as well as whole brain (Zachaeck and Wurtman, 1973) NE turnover appears to increase. A causative relationship between the increase in NE turn- over and the prolactin surge seems unlikely since a signifi- cant increase in serum prolactin is Observed before a significant elevation in anterior hypothalamic NE turnover occurs. Other studies using pharmacological agents which alter central NE metabolism have not demonstrated a consis- tent affect On prolactin secretion (Meites 33 31., 1976). 80 The decreased DA turnover which appears to begin at 4 h and which is significant at 6 h after P administration is associated with both the rise in serum LH and peak in serum prolactin levels. The decreased DA turnover may cause the elevation in serum prolactin since in all mammalian species yet tested, a central d0paminergic mechanism appears to inhibit prolactin secretion (Meites 33 31., 1972). However, pharmacological Observations have produced contradictory data as tO the role Of DA in controlling LH secretion (McCann and Moss, 1975; Meites 33 31,, 1976). Thus, subsequent studies have been conducted to determine the role of both DA and NE in mediating the P induced LH and prolactin surges in ovari- ectomized rats. Further Study on the Effect Of Progesterone on DOpamine Turnover inIOVariectomized- Estr3gen-Primed Rats A. Objectives The effect of P injection on NB turnover appeared to be transient whereas its influence on DA metabolism persisted through 6 h. Since the peak in serum LH leVels occurs after 8 to 10 h, in response to P administration into ovariectom- ized, estrogen primed rats, it was Of interest to measure DA and NE turnover in the anterior hypothalamus at these times. 81 B. Materials and Methods Treatment was the same as in the previous experiment except that animals were killed at the time of and 9 h after P administration. TO estimate catecholamine turnover, rats received an injection of ampt (250 mg/kg body weight) at either 90, 45 or 0 min before decapitation. This treatment allowed the depletion curves of DA and NE to be evaluated. The significance of differences between lepes of the deple- tion curves was evaluated by ANOVA and Student-Newman Keuls test (Sokal and ROlfh, 1969). C. Results Figure 3 shows the effect of ampt on anterior hypotha- lamic d0pamine (AH-DA) at 0 and 9 h after P administration to ovariectomized, estrogen-primed rats. Nine h of P exposure resulted in a decrease in DA depletion at 45 and 90 min after ampt treatment. There was no significant differ- ence in the rate Of NE depletion between 0 and 9 h after P administration. All vehicle treated control animals showed elevated levels Of LH (> 2,000 ng/ml) and high circulating levels Of prolactin at 9 h after P treatment. D. Discussion The results presented here indicate that the decreased DA turnover which begins between 4 and 6 h after progesterone administration continues through at least 9 h and that the 82 400 I DA my ~ (us/cl) zoo . l TIME AFTER aMPT (nfinutes) Hours Post Progesterone 0 9 PRL 105 383 (us/ml) 139 :47 l La 81 >2,ooo (us/n1) 18 I Figure 3. Long term effects Of progesterone administration to ovariectomized, estrogen-primed rats-on alpha— methyl-para-tyrosine induced depletion Of anterior hypothalamic dopamine and on serum luteinizing hormone (LH) and prolectin (PRL). Upper panel shows the effect of progesterone treatment on anterior hypothalamic DA concentration of rats decapitated at 0, 45, or 90 min after i.p. injection of 250 mg ampt/kg body weight. Solid bars represent animals killed at 9 h after progesterone treatment and hatched bars represent animals killed at 0 h after progesterone treatment. Verticle lines represent SEM. The lower table shows serum LH and prolactin (PRL) concentration in control (non-drug treated) rats at 0 and 9 h after progesterone administration. 83 increase in NE turnover observed at 4 h post-progesterone was indeed transient, since no significant difference in NE turn- over was Observed between 0 and 9 h. These data indicate that the P-induced increase in serum prolactin is more close- 1y associated with the decreased DA turnover than the increased NE turnover since NE turnover returned to normal whereas prolactin levels remained elevated. Similarly, the changes in serum LH correlated better with the decreased DA than the increased NE turnover. Two hypotheses may explain the role of DA and NE in the genesis Of the P-induced LH surge in ovariectomized, estrogen- primed rats. First, the increased NE turnover observed at 4 h after P treatment may be sufficient to initiate a series Of events (i.e., increased LHRH synthesis and release) lead— ing to the LH surge. Consistent with this hypothesis are the observations that the P—induced LH surge can be eliminated by the interruption of NE metabolism (Kalra and McCann, 1972) or by blockade of a-adrenergic receptors (Kalra 33 31., 1974). Second, the decreased DA turnover may cause or at leaSt permit the P-induced LH surge. This hypothesis is supported by the Observation that third ventricle infusion Of DA can block the LH surge induced by subsequent third ven- tricle infusion Of NE (Sawyer 33 31,, 1974; Krieg and Sawyer, 1976). In order to distinguish between these two hypotheses, selective alteration in either DA or NE metabolism is required. 84 11. Effects Of Alpha-Methyl-Para-1yrosine on Serum LH and Prolactin Surges Inducedby Progesterone Treatment of Ovariectomiied, EEtrogen-Primed Rats A. Objectives Alpha-methyl-p-tyrosine (ampt), which depletes central catecholamine stores by competitively inhibiting tyrosine hydroxylase (Spector 33 31., 1965; Corrodi and Hansen, 1965), the rate limiting enzyme in catecholamine synthesis, has been shown to block ovulation (Brown, 1967; Kordon and Glowin- ski, 1967), the proestrus LH surge (Kalra and McCann, 1973), the release Of LH in response tO castration (Ojeda and McCann, 1973) and stimulatory steroid feedback on LH secretion (Kalra 33 31., 1972). In view of our Observation that NE turnover increases while DA turnover decreases in response to P, the ability Of ampt to block LH secretion may be due to its effect on noradrenergic neurons. If the decrease in DA turnover were critical tO the mediation Of the P-induced LH surge, a further decrease in DA turnover with ampt should enhance rather than inhibit LH secretion. The Objective of this set of experiments was to characterize further the effects of ampt on P-induced LH secretion in ovariectomized estrogen-primed rats. B. Materials and Methods Female Long Evans rats (Blue Spruce Farms, Altamont, N.Y.) weighing 200-250 g were subjected to surgical and 85 steroid treatment as described in Experiment I. In one experiment, ampt (250 mg/kg body weight) was injected i.p. at the time of the 0900 h P administration. Blood samples were Obtained via orbital sinus cannulation at 0, 7, and 9 h after P treatment. In a second experiment, 250 mg ampt/kg body weight was administered i.p. to P treated ovariectomized, estrogen-primed rats at 45, 60 or 90 min before decapitation. Animals were killed at 0, 2, 4, 6, or 9 h after P treatment. C. Results Administration of 250 mg in Ompt/kg body weight at the time of progesterone administration completely blocked the P-induced LH surge (Figure 4). Administration Of ampt was ineffective in decreasing serum LH when administered 1 h before the 0 and 2 h blood samples (Table 1). Treatment with ampt 1 h before the 4 and 6 h blood samples was effective in decreasing serum LH by 4638 and 84:3%, respectively. At 9 h after P treatment, 45 min Of ampt exposure was ineffective in decreasing serum LH, but 90 min exposure to ampt decreased serum LH by 69:13%. Treatment with ampt 45, 60, or 90 min before the 0 h blood sampling caused a slight but significant increase in serum LH. Exposure to ampt for 60 min increased serum prolactin in animals sampled at 0 and 2, but not 4 and 6 h after P administration (Table 2). 86 5mm Lu (pg/ml) HOURS AFTER FROG Figure 4. Long term effects Of alpha-methyl~paravtyrosine on progesterone induced LH secretion in ovari- ectomized, estrogen-primed rats. A single i.p. injection of 250 mg ampt/kg body weight (solid line) or saline, its vehicle (dashed line) was administered at the time Of the progesterone treatment. Each point repre- sents the means Of 6 to 8 determinations and the verticle bar represents 1 SEM. Stars above the points indicate that the mean is significantly different from the means of the 0 h sample (P‘<0.05). 87 Table 1. Effects Of Alpha-Methyl-Para-Tyrosine (ampt) on Serum Luteinizing Hormone (LH) at Various Times After Progesterone Administration to Ovariectom- ized, Estrogen-Primed Rats Min After Hours After Progpsterone ampt 0 2 4 6 ISerum LH 0+ 38 3 3 46 3 6 85 3 9 773 3 189 60 52 3 6* 42 3 6 46 3 7* 125 3 23* Min After Hours After Progesterone ampt 0 Serum LH Off 81 3 8 2979 3 1332 45 143 3 21* 2282 3 267 90 177 3 33* 984 3 401* t These groups received 0.89% NaCl at 60 min before decapi- tation. it These groups received 0.89% NaCl at 45 min before decapi- tation. * Indicates a significant difference when compared with control (0) group (P<:0.05). 88 Table 2. Effects of Alpha-Methyl-Para—Tyrosine (ampt on Serum Prolactin (PRL) at Various Times After Progesterone Administration to Ovariectomized Estrogen-Primed Rats Hours After Progesterone Min After 0 2_’ 34* 6 ampt Serum PREP 0t 31 3 2 152 3 29 685 3 107 349 3 55 60 586 3 48* 665 3 47* 623 3 43 378 3 32 t These groups received 0.89% NaCl at 60 min before decapi- tation. * Indicates a significant difference when compared with con- trol (0) group (P<:0.05). D. Discussion The Observation that ampt injected at the time of P administration can completely inhibit the progesterone in- duced LH surge is consistent with its ability to block ovula- tion (Brown, 1967; Kordon and Glowinski, 1973) and the proestrous LH surge (Kalra and McCann, 1973). Kalra 33_31. (1972) observed that DDC is as effective as ampt in blocking the progesterone induced LH surge in ovariectomized, estrogen primed rats. This suggests that ampt inhibits the LH surge by interfering with NE rather than DA synthesis. The Observation that acute exposure to ampt can decrease LH levels at 4, 6 and 9 h after P administration suggests that NE synthesis is required to maintain sustained LH 89 secretion. Since ampt inhibits LH release at the time of increased NE turnover (4 h) and after NE turnover has re- turned to normal (6 and 9 h), it is possible that some aspect Of NE metabolism and transmission other than increased turnover is maintaining the high secretory rate Of LH. However, it is also possible that the increased NE turnover seen at 4 h is needed to initiate the LH surge and that a lower turnover rate is required to sustain high LH secretion. The explanation for the increase in serum LH in response to ampt at 0 h is not clear. The high DA turnover at 0 h may inhibit LH secretion at this time and the ampt induced decrease in DA synthesis may release this inhibition. In ovariectomized rats, estrogen administration has been shown tO stimulate DA turnover (Fuxé 33 31., 1969, Gudelsky 33 31., 1977) and DA has been reported to inhibit LH secretion (Sawyer 33 31., 1974; Mueller 33 31., 1976). The Observation that acute exposure to ampt stimulates prolactin release when DA turnover is high, but has no effect on prolactin release when DA turnover is low, are in accord with the suggested (Experimental 1) close relationship between DA turnover and prolactin levels in response to P administra- tion. 90 111. Effects Of Multiple Piribedil Injection on Serum LH and Prolactin Sfirg3s Induced' bngrogesterone in Ovariectomized, EstrogeniPrimed Rats A. Objectives The Observation that DA turnover decreases in response to P treatment of ovariectomized, estrogen—primed rats (Experiment 1) suggests that decreased DA activity may be important in mediating the LH and prolactin surges. Since the role Of central dopaminergic systems in the control Of LH is unclear and reports on this subject are contradictory (see section V.C. Of Literature Review), it was of interest to determine the importance of the decreased DA turnover in the steroid-induced LH surge. Therefore, in the present study LH and prolactin levels were determined following P administration to ovariectomized, estrogen primed rats treated with the DA agonist, piribedil (Corrodi 33 31., 1973; Miller and Iversen, 1973). B. Materials and Methods Female Sprague-Dawley rats (Spartan Farms, Haslett, MI), weighing 200-250 g were ovariectomized and subjected to steroid treatment as described in Experimental IB. Animals received a single i.p. injection of piribedil mesylate (1 mg/kg body weight) or 0.89% NaCl (its vehicle) at 0, 3 and 6 h after P injection. This dose of piribedil is the minimally effective dose for decreasing serum LH in intact 91 male rats (Mueller 33 1., 1976). Blood samples (1 ml) were Obtained via orbital sinus cannulation at 0, 7, and 9 h after P treatment. The significance of differences among means was tested by ANOVA and Student-Neuman Keuls test (Sokal and Rohlf, 1969). C. Results Piribedil administration from 0 through 6 h after P treatment had no effect on peak LH concentration (7 h), whereas serum LH concentration was significantly higher in piribedil treated than in control animals at 9 h after P treatment. Serum prolactin levels were significantly lower in piribedil than in saline treated rats at 7 h after P, but did not differ from controls by 9 h after P treatment. D. Discussion These data indicated that sustained DA receptor stimu- lation does not block the P induced LH surge in ovariectom- ized, estrogen-primed rats, and suggests that the decreased DA turnover Observed following P administration is not essential in mediating the steroid induced LH surge. DA receptor blockade with pimozide (Ojeda and McCann, 1973) or DA receptor stimulation with piribedil (Grandison, Hodson and Meites, unpublished Observation) has previously been shown tO be ineffective in altering the post-castration in- crease in serum LH in male rats. Figure 5. 92 2.0 E \ a O I ‘3 L5 Mnufll : all 2 s ' fine a 1.0 III a 300 liriluil SERUM PRL (no/ml) N 8 HOURS AFTER PIOGESTEIOIE Effect of sustained administration of piribedil mesylate (ET-495) on the progesterone induced luteinizing hormone (LH) and prolactin (PRL) secretion in ovariectomized, estrogen-primed rats. Rats were injected i.p. with 1 mg peribedil mesylate/kg body weight or 0.89% NaCl, its vehicle, at 0, 3 and 6 h after progesterone administration. Each point represents the means Of 8 determinations and the verticle lines indicate 1 SEM. Stars indicate significant difference between means Of drug and saline treated animals at a particular sampling time. 93 The ability Of piribedil to block the P-induced increase in serum prolactin at 7 h after P treatment is consistent with the findings of several laboratories (see section IV-E of Literature Review). These investigators demonstrated the inhibitory influence Of DA on prolactin secretion. The present study, together with the previous study (Experimentl) suggests that a decrease in DA turnover may be necessary to mediate the P-induced increase in serum prolactin. The increased rate Of prolactin secretion (rebound effect) following termination Of piribedil treatment Observed in this study, was previously reported in experiments utiliz- ing DA infusion in humans (LeBlanc 33 31., 1976). A similar rebound effect cannot account for the less rapid rate Of decrease in serum LH between 7 and 9 h after P treatment in the piribedil as compared with the saline-treated group since piribedil does not appear to inhibit the LH surge. IV. Effect Of Implants of 6-Hydroxyd3pamine in the Suprachiasmatic Nucleus (SCN) and—Median Eminence (MB) on AnteriOr Hypothalamic and Median EminencefDopamifie andINOrepinephrine Concentration, and on Serum Luteinizing Hormone and Prolactin Surges Induced 33 Pr3gesterone in Ovariectomized, Estrogen-Primed Rats A. Objectives The integrity Of rostral areas Of the hypothalamus ap- pears tO be critical in mediating the stimulatory effect Of 94 steroids on LH secretion in female rats. Rostral deafferen- tiation (Halasz, 1969) and electrolytic lesions of anterior hypothalamic nuclei (Mess 33 31., 1966) inhibited whereas electrical stimulation (Clemens 33 31., 1971) and anterior hypothalamic estrogen implantation (Davidson, 1969) stimu- lated LH secretion. The Observation that NE turnover in- creases in the anterior (but not the posterior) hypothalamus prior to the LH surge induced by progesterone in ovariectom- ized, estrogen-primed rats (Experiment I) suggests that nor- adrenergic nerve terminals in the anterior hypothalamus may mediate positive steroid feedback. Since the rostral hypothalamus contains both noradren- ergic nerve tracts en route to the median eminence (ME) and noradrenergic nerve terminals (Loizou, 1969; Ungerstadt, 1971), physical destruction of the anterior hypothalamus could alter LH secretion by disrupting synapses, nerve tracts or both. The purpose Of the present investigation was to selectively disrupt anterior hypothalamic and ME noradrenergic nerve terminals with the neurotoxin, 6-hydroxydopamine (6-OH- DA; Thoenen and Tranzer, 1968), and determine its effect on P-induced LH release in ovariectomized, estrogen-primed rats. To determine the specificity and magnitude of the 6-OH-DA effect, DA and NE concentrations in the anterior hypothalamus and median eminence were determined in both drug and sham- implanted animals. 95 B. Materials and Methods Female Sprague-Dawley rats (Spartan Farms, Haslett, MI) weighing 200-250 g, received surgical and steroid treatment as described in Experiment 1. Preliminary Observations indi- cated that intrahypothalamic implantation Of crystalline 6-OH-DA selectively depleted NE for 24 h after implantation. Thus SCN and ME implants were made 24 h before the P injec— tion. 6-OH-DA was tapped into 26 gauge tubing previously packed with cocoa butter (CB). Cannulae for control animals contained CB alone. Blood samples were taken under light ether anesthesia by orbital sinus cannulation 0, 7, and 9 h after the 0900 h P injection. Immediately after the last bleeding, animals were killed by decapitation and brains were quickly removed from the cranium. The area immediately over the Optic chiasm was dissected in the SCN implanted animals by cutting rostral and caudal to the Optic chiasm and lateral- ly at the hypothalamic sulci. The cube of tissue produced by cutting at a depth of 2-3 mm was frozen for later catecholamine assay. In ME implanted animals, the external layer of the ME was dissected as described in general Material and Methods section. DA and NE were assayed by the method described in Appendix B and are expressed as ng/g wet weight for the anterior hypothalamus and ng/mg protein for ME. 96 C. Results The effect of 6-OH-DA implants into the SCN on anterior hypothalamic DA and NE are shown in the top 2 panels of Figure 6. SCN implants Of 6-OH-DA decreased NE concentration by 83% (P‘<0.05), DA concentration by 24% (not significant) and significantly reduced the P induced LH surges when com— pared with sham-implanted ovariectomized, estrogen-primed rats (Figure 7). However, a slight, but significant increase in LH was Observed at 7 and 9 h in 6-OH-DA implanted animals. Median eminence (ME) 6-OH-DA implants decreased NE by 57% (P< 0.05) and DA by 11% in the ME (Figure 6). This treatment had no effect on LH levels at 7 and 9 h after P administra- tion (Figure 7) when compared with sham-implanted animals. ME implants of 6-OH—DA significantly decreased LH levels when measured at 0900 h (before P administration); whereas, SCN implants had no effect (Table 3). D. Discussion The present studies indicate that anterior hypothalamic noradrenergic nerve terminals are important in mediating the stimulatory effect of P on the release of LH in ovariectom- ized, estrogen-primed rats; whereas, ME noradrenergic nerve terminals may be unimportant in this regard. These data are consistent with the Observation that 6—OH-DA injected into the third ventricle (Kalra, 1975) or infused into the ventral noradrenergic tract (Martinovic and McCann, 1977), can block 97 2000 0 A 1500 300 lG/Bl IG/GI woo 200 III M 3. 06/" mrm I0/I0 Film! 0 4| 4 2| Figure 6. Effects of suprachiasmatic nucleus (SCN) and median eminence (ME) implants Of 6-hydroxydOpa— mine (6-OH-DA) on anterior hypothalamic and median eminence concentration of norepinephrine and dopamine. Upper panels indicate anterior hypothalamus (AH) and lower panels indicate median eminence (ME) norepinephrine and d0pamine concentration. Solid bars indicate amine levels in cocoa butter implanted and hatched bars indicate levels 33 h after 6-OH-DA implantation (N=6-8). Verticle lines represent 1 SEM and stars above verticle line indicate a significant (P<:0.05) decrease in amine concentration. 98 SCI IIPLAIIS 3mm 2000 I000 SE00. ll 3 (IS/ll) IE IIPLAITS 0 7 "003$ AFTER PIOSESTEIOIE Figure 7. Effect Of suprachiasmatic nucleus (SCN) and median eminence (ME) implants Of 6-OH—DA or cocoa butter (CB) on progesterone induced luteinizing hormone (LH) secretion in ovariectom- ized, estrogen-primed rats. Rats were implanted with 6-OH-DA in the SCN (upper panel) or ME (lower panel) or CB 24 h before progesterone administra~ tion. Each point represents the means of 6-8 determinations and the verticle line indicates 1 SEM. Stars above the point indicate a significant difference between means Of 6-OH-DA and CB implanted animals at a particular sampling time. 99 Table 3. Effects Of Implants of 6-Hydroxydopamine in Supra- chiasmatic Nucleus (SCN) and Median Eminence (ME) on Serum Luteinizing Hormone (LH) in Ovariectom- ized, Estrogen-Primed Rats Type of 93_ 6-OH-DA Implant Serum LH ME 109 3 17 72 3 10* SCN 127 3 25 149 3 20 + Implants were made 24 h before blood sampling. it Values represent serum LH concentration at 900 h (before progesterone administration). * Indicates a significant difference when compared to cocoa butter (CB) implanted animals (P 0.05). LH secretion induced by gonadal steroids. The observation that both noradrenergic nerve terminals (Loizou, 1969; Ungerstedt, 1971) and cell bodies Of neurons containing luteinizing hormone-releasing hormone (LHRH) are in rostral hypothalamic nuclei Of rats (Baker 33 31., 1975), provide anatomical support for the present Observation indi- cating the importance Of anterior hypothalamic noradrenergic synapses in the mediation of P induced LH secretion. Destruction Of noradrenergic nerve terminals in the anterior hypothalamus by local application of 6—OH-DA may interfere with NE stimulation Of LHRH containing cells in the anterior hypothalamus. Since ME noradrenergic nerve terminals appear not to be involved in mediation Of P induced LH secretion, 100 the effect of anterior hypothalamic 6-OH-DA implants is not mediated by a destruction of noradrenergic nerve tracts enroute to the ME. These data are supported by the Observa- tion of Kalra and McCann (1973) that blockade Of NE synthesis decreases LH release in response to anterior hypothalamic, but not ME, electrical stimulation. However, the present study does not eliminate the possibility that noradrenergic nerve terminals in the anterior hypothalamus synapse with non-LHRH containing neurons which transmit impulses to LHRH containing cells in the arcuate-median eminence area. The Observation that 6-OH-DA implants in the ME (but not SCN) decreased LH levels before P administration, may indicate that noradrenergic nerve terminals tonically stimu- late LH secretion. This Observation is consistent with the hypothesis that the medial basal hypothalamus exerts a tonic stimulatory influence on LH secretion (Gorski, 1966). However, since the present eXperiment was not designed to test this point, further studies using different experimental models will be needed. The ability Of SCN 6-OH-DA implants to block P induced LH release without significantly affecting anterior hypothal- amic DA concentration indicates the relative lack Of import- ance of anterior hypothalamic DA synaptic terminals in mediating LH release. However, this study does not eliminate the involvement Of DA in the events leading to the LH surge. 101 DA and DA agonists have been shown to stimulate, inhibit and tO have no effect on LH secretion (see section V-C of Literature Review) which may indicate a dopaminergic modula- tion Of the LH releasing system. Selective depletion of DA nerve terminals in specific areas of the hypothalamus may help clarify the role Of this putative neurotransmitter in LH release. In light of our previous demonstration that anterior hypothalamic NE turnover increased in response to P treat- ment Of ovariectomized, estrogen—primed rats (Experiment I) and the present Observation that disruption Of anterior hypothalamic noradrenergic synapses blocked the P-induced LH surge, it appears that the noradrenergic component Of the anterior hypothalamus is responsible for mediating most, if not all, Of the P stimulation of LH secretion in ovariectom- ized, estrogen-primed rats. V. Measurement of Dopamine and Norepinephrine Turnover and Serum LuteiniEing Hormone AfterTShort Term castration in MalélRats A. Objectives The experimental manipulation used in Experiments I-IV resulted in a surge in both serum LH and prolactin. The LH surges appear to be mediated by an increase in NE turnover; whereas, the prolactin surge is probably mediated by the de- creased DA turnover. If alterations in catecholamine 102 metabolism are a necessary requirement for the central medi- ation of LH and prolactin secretion, endocrine manipulations which result in selective changes in secretion of either LH or prolactin should result in selective alterations in DA and NE metabolism. Castration results in a slow rise in serum LH in females (Gay and Midgley, 1969) and a rapid increase in serum LH in males (Gay and Midgley, 1969; Yamamoto 33 31., 1970; Ojeda and McCann, 1973; Schwartz and Justo, 1977); whereas, serum prolactin levels decrease in both male and female rats follow- ing castration (Meites 33 31., 1972). This rapid selective increase in serum LH makes the acutely castrated male rat a suitable model in which to measure catecholamine metabolism for the purpose of comparison with changes Observed during stimulatory steroid feedback in female rats (Experiment I). B. Materials and Methods Male Sprague¥Daw1ey rats (Spartan Farms, Haslett, MI), weighing 225-250 g, were maintained in climate—controlled rooms for at least 4 days before the onset Of experimenta- tion. Three groups of 16 rats were orchidectomized at 48, 24, or 6 h before decapitation. One group of 16 rats was sham~operated 6 h before decapitation to eliminate the pos- sible influence Of surgical stress on catecholamine metabol- ism and serum LH levels. In sham—Operated animals, the testes were externalized, then replaced into the scrotum. 103 Both operations required the same length of time to complete (3-5 min). TO estimate catecholamine turnover, each group of rats received an i.p. injection of either ampt or 0.89% NaCl (its vehicle) 1 h before decapitation (see Materials and Methods, Section I). At the time of decapitation (1500 to 1700 h) blood was collected for assay of serum LH and the hypothalamus was dissected. Hypothalamic catecholamines were assayed by the method Of Ben-Jonathan and Porter (1976; Appendix B) and are expressed as ng DA or NE per g wet weight. The significance Of difference among group means was deter- mined by ANOVA and Student-Neuman Keuls test (Sokal and Rohlf, 1969). C. Results Serum LH increased from 7 3 3 ng/ml serum in sham castrates to 53 3 14 ng/ml serum by 6 h post—castration (Figure 8). By 24 and 48 h post-castration, serum LH levels were greater than 400 ng/ml. Hypothalamic NE concentration did not change at 6 or 24 h, but was slightly elevated by 48 h post-castration. Hypothalamic NE turnover doubled within 6 h after castration (P< 0.05), then returned to sham castration levels by 48 h. A large increase in hypothalamic DA concentration occurred by 6 h post-castration (P< 0.05) and returned to sham castrate levels by 24 and 48 h. DA turnover as estimated, by the percent depletion Of the 104 Figure 8. Effect of orchidectomy on concentration and alpha-methyl—para-tyrosine induced depletion of hypothalamic norepinephrine and d0pamine and serum LH concentration. The solid bars indicate steady state concentration and hatch- ed bars indicate amine concentration 1 h after i.p. injection of ampt (N=8). The numbers above the sets of bars indicate percentage depletion of amines induced by ampt. The stars above the solid bars indicate a significant difference (P< 0.05) in amine concentration between that group and the sham castrated group. Stars above the numbers indicate a significant interaction between time after castration and ampt treatment as analyzed by Two Way ANOVA (P< 0.05). Luteinizing hormone (LH) concentrations (lower panel) are from sera of non-drug treated animals. Stars above the points in- dicate a significant increase (P<:0.05) when compared to sham castrated control rats. The verticle line in all 3 panels represents 1 SEM. It (IE/El) u (Ia/cu) SEMI. ll (IE/ll.) 105 m. 13. Q 13. 15. mo m no no no 500 m SIG 400 / 7/ 3m 6 2 4 4 I nouns arm casmmu Figure 8 106 monoamine induced by ampt, doubled by 6 h (P‘<0.0S) and was elevated at 24 (non-significant) and 48 h (P< 0.05) post- castration. D. Discussion The rapid doubling in NE turnover, and rise in serum LH concentration, following orchidectomy in male rats is in marked contrast to the very slow changes in both parameters which occurs in female rats following ovariectomy. A 2-3 fold increase in NE turnover occurs by 6 (but not 2) days after ovariectomy in whole brain (Anton-Tay and Wurtman, 1968) and hypothalamus (Coppola, 1969) in rats. This long latent period correlates with the relatively long (3-4 days) post-castration period needed for a significant increase in serum LH in female rats (Gay and Midgley, 1969; Yamamoto 3; _l., 1970). The transient nature of this increase in NE metabolism in the males following orchidectomy also is in contrast to the long term increase in whole brain and hypo- thalamic NE turnover seen in ovariectomized rats (Anton-Tay and Wurtman, 1968; Coppola, 1969). The LH secretion rate remains high in both male and female rats indefinitely even though NB turnover returns to precastration levels by 48 h in males and remains elevated in females. The noradrenergic system appears to be important in the acute post-castration rise in serum LH. Alpha-mpt can block the post-castration LH rise if given by 2 h after castration; 107 and umpt and the d0pamine-B-hydroxylase inhibitor, DDC, are equally effective in decreasing serum LH when administered 18 h after castration (Ojeda and McCann, 1973). Similarly, treatment with the alpha adrenergic receptor blocker, phenoxybenzamine, but not the B-receptor blocker, prone- thalol, was as effective as ampt or DDC in decreasing serum LH when administered 18 h post-castration. The effects of orchidectomy on DA turnover at 6 h post- castration is difficult to assess in view of the large increase in steady state concentration of DA at that sampling time. The increased hypothalamic DA concentration may indi- cate an increase in DA synthesis or a decrease in DA release. The large decrease in DA following treatment with ampt and the increased steady state concentration, suggests that DA synthesis and release is accelerated at 6 h. At 24 h post- castration, DA concentration and depletion are slightly, but not significantly elevated. The increased depletion of DA without a change in steady state concentration indicated an increased DA turnover at 48 h post-castration. This increased DA turnover in the hypothalamus appears to persist at least 14 days after castration in male hypothalamus (Hodson, Simpkins and Meites, unpublished). Hypothalamic d0paminergic systems do not appear to play a role in the post-castration rise in serum LH. Treatment with the DA receptor blocker, pimozide (Janssen gt al., 1968), 108 or the DA receptor agonist, piribedil (Corrodi gt al., 1973; Miller and Iverson, 1973), had no effect on serum LH after castration (Ojeda and McCann, 1973; Grandison and Hodson, unpublished). Further blockade of NE synthesis is as effec— tive as blockade of both NE and DA synthesis in decreasing the post-castration rise in LH (Ojeda and McCann, 1973). The depressed pituitary and serum prolactin observed after castration in both male and female rats (Meites 33 al., 1972), may be due to the increased DA turnover which appears to follow castration. DOpaminergic agonists have been shown to inhibit both release and synthesis of prolactin (Meites, 1972; MacLeod, 1976). The differential response of prolac- tin to gonadal steroids in female rats (Experiment I), and orchidectomy in male rats, may be due to the observed de- crease in DA turnover in the former study and the apparent increase in DA turnover in the present study since NE turn- over increased transiently in both studies. VI. Effects of Medial Basal Hypothalamic Implants of 6-Hydroxydopamine on the Post-Castration Increase in Serum Luteiniiing Hormone A. Objectives The transient increase in NE turnover which follows castration in male rats appears to be primarily responsible for mediation of the acute post—castration LH increase. However, it is not clear if the transient increase in NE 109 turnover is able to stimulate sustained LH secretion. It is possible that the increased NE turnover is required to initiate LH secretion in response to castration, since a single administration of 250 mg ampt/kg body weight can block LH secretion through 24 h post-castration (Ojeda and McCann, 1973). However, it is not known whether blockade of NE synthesis can decrease serum LH when administered after long term castration. A second possible mode of noradren- ergic involvement is that the increase in LH secretion can occur independently of noradrenergic input, but the increase in NE turnover hastens the post-castration LH surge. Preliminary observations and Experiment IV indicate that a 24-h exposure to 6-OH-DA implants can depress median eminence NE concentration without significantly affecting DA concentration. The present study was conducted to determine the affect of medial basal hypothalamus NE depletion with 6-OH-DA on both acute and chronic LH secretion. B. Materials and Methods Male Sprague-Dawley rats (Spartan Research Animals, Haslett, M1) were implanted bilaterally with permanent cannu- lae into the ventromedial nuclei. After 1 wk recovery, chronic jugular cannulae were implanted to allow blood sampl- ing from unrestrained animals. Three days later an "inner" cannula containing 6-OH-DA or no drug (sham) was inserted into the permanent cannulae at 0900 h. One day later, SERUI [H 100 (lfimfl) Figure 9. 110 t 1300 SHAH "MI NW ‘ Hill-DA mm “M "W 14 HOURS DAYS TIIE AFTER CASTIIIIOI Effect of bilateral ventromedial nuclei (VMN) implants of 6—hydroxyd0pamine (6-OH-DA) of post- castration rise in serum luteinizing hormone (LH). 6-Hydroxydopamine (6-OH-DA) or empty cannulae (sham) were implanted bilaterally into the ventromedial nuclei (VMN) 24 h before orchidectomy and blood samples were made via chronic jugular cannulae. Each point indicates the means of 7-8 determinations and the verticle bars represent 1 SEM. Stars above points indicate a significant difference (P‘<0.05) between the drug and sham implanted group at a particular sampling time. 111 animals were castrated and blood samples were taken 6, 12, 24 h and 7 and 14 days following castration. C. Results MBH implants of 6-OH-DA blocked 67:12 and 69:8% of the post-castration rise in serum LH at 6 and 12 h after castra- tion. By 24 h 48:58 inhibition of LH release was observed and persisted through 2 wks post-castration. D. Discussion The inability of 6-OH-DA to completely block the post- castration LH increase on the day of and 7 and 14 days after castration may indicate that the LH increase is in part independent of noradrenergic mediation. Halasz 33 11' (1972) reported that the long term post-castration rise in LH occurs in deafferentated rats and Mitchell and Kalra (1977) recently demonstrated that the depletion of MBH-LHRH which normally follows castration still occurs in deafferentated male rats. Since long term deafferentation destroys nor- adrenergic nerve terminals in the hypothalamus (Weiner gt al., 1972a; Halasz gt al., 1972), and since d0paminergic and serotonergic drugs do not appear to alter the post-castration LH increase (Ojeda and McCann, 1973), these data may indi- cate that LHRH neurons themselves are sensitive to circulat- ing levels of testosterone. Thus ME lesions, which destroy LHRH neurons, are much more effective in blocking the 112 post-castration LH increase than is deafferentation on 6-OH-DA treatment (Davidson and Sawyer, 1961; Turner and Simpkins, 1977). An alternative explanation for these data is that 6-OH-DA does not completely destroy the noradrenergic input to the MBH and thus a noradrenergic mediated LH increase can still occur. We have observed that by 2 wks after bilateral VMN implants of 6-OH-DA, MBH-NE is depleted by about 70% (Mallard, Simpkins and Meites, unpublished observations). The remaining NE is probably confined to glial cells and adrenergic nerve terminals of the hypothalamic vasculariza- tion (Brownstein 33 al., 1976) and thus is not involved in the regulation of LH secretion. However, Uretsky 3: a1. (1971) observed that following intraventricular administra- tion of 6-OH-DA, NE turnover increases in surviving noradren- ergic neurons. Thus, it is possible that some noradrenergic nerve terminals remain functional following 6-OH-DA implan- tation and mediate part of the post-castration LH increase. VII. Measurement of Concentration and Turnover of Brain Dopamine,’Norepifiephrine and Serotonin and Serum Luteinizing Hormone, Follicle Stimulating Hormone and Prolactin in Young and Old Male Rats A. Objectives Aging female rats pass through a series of abnormal re- productive states characterized by irregular estrous cycles, 113 constant estrus, irregular pseudOpregnancies and anestrus (Aschheim, 1961; Clemens gt gt., 1969; Clemens and Meites, 1971; Huang and Meites, 1975). In general, old female rats show less capacity to secrete LH and FSH and a higher level of prolactin secretion under a variety of experimental condi- tions. After ovariectomy or estrogen-progesterone treatment of ovariectomized rats, old females release less LH and FSH than young females (Shaar gt gt., 1975; Watkins gt_gt,, 1975; Huang EE.§l-: 1976; Meites and Huang, 1976). Aging male rats also show a gradual decline in fertility (Adams, 1972) and have lower serum LH, FSH and testosterone (Shaar gt gt., 1975; Bruni gt gt., 1976) and higher prolactin (Bruni gt_gt., 1976) than young males. In response to cas- tration or ether stress, old male rats release less LH and FSH than young males (Huang gt gt., 1976; Riegle and Meites, 1976). Thyroid function in old male rats appears to be depressed as indicated by lower circulating T4, lower thyroid weight and accumulation of colloidal material in the thyroid. Old male rats release less TSH and T4 in response to low ambient temperature, and exhibit a less pronounced decrease in serum TSH in response to stress (Simpkins gt_gt., 1977; Huang, Chen and Meites, unpublished). The influence of the autonomic nervous system on the control of anterior pituitary function in young animals is now well established (see section I-V of Literature Review). 114 The first experimental evidence for a role of the autonomic nervous system in the decreased endocrine function of aging was provided by the demonstration that drugs which stimu- late the catecholamine system or inhibit serotonin metabol- ism could initiate regular estrous cycles in old constant estrous and regular cycling rats (Clemens gt gt., 1971; Quadri gt gt., 1973). In aging male mice, NE and DA metab- olism appears to be depressed in several brain regions (Finch, 1973). The present study was undertaken to determine if central catechol- and indoleamine metabolism changed in aging male rats and to assess the importance of these changes in terms of their possible influence on anterior pituitary func- tion. B. Materials and Methods Study 1 Male Wistar rats 3-4 and 21 mo old (Harlan Industries, Indianapolis, IN) were maintained in climate controlled rooms (see general Materials and Methods) and given Purina Rat Chow and water ad libitum supplemented with whole wheat bread and oranges for at least 2 wks prior to experimentation. To measure basal hormone levels and concentration and turnover of catecholamines, animals received 250 mg ampt/kg body weight or 0.89% NaCl 1 h before decapitation. At the time of decapi- tation (0800-0930 h) trunk blood was collected and brains were quickly removed and immersed in ice cold saline. 115 The MBH was dissected as previously described. The MBH used in this study corresponded roughly to the intermediate basal hypothalamic layer described by Kavanagh and Weisz (1973). The remaining hypothalamus and olfactory tubercles were also dissected and immediately frozen on dry ice. DA and NE were assayed by the method of Coyle and Henry (1973); see Appendix A) and are expressed as ng/mg protein for MBH ng/g wet weight for remaining hypothalamus and olfac- tory tubercles. Statistical analysis of data was done by ANOVA and Student-Neuman-Keuls tests. A significant inter- action between age and ampt treatment was taken as evidence for a change in turnover rate. The level of significance chosen was P< 0.05. Study 2 In a second study, 3—4 and 21 mo old male Wistar rats (Harlan Industries, Indianapolis, IN) were injected i.p. with 75 mg pargyline HCl/kg body weight or its vehicle 0.89% NaCl. After 30 min, animals were killed by decapitation and trunk blood was collected. Brains were quickly removed from the cranium immersed in ice cold saline, and the hypothalamus was dissected (as described in general Materials and Methods). The cerebellum was separated from the rest of the brain and discarded. Brain tissues were frozen until assayed for serotonin (SHT) and S-hydroxyindoleacetic acid (SHIAA). 116 Brain and hypothalamic 5HT and brain SHIAA were assayed according to the method of Curzon and Green (1970) as modi- fied by Hyyppa gtht. (1973). The recovery of brain and hypothalamic SHT was 100% and of brain SHIAA was 43%. The assay for SHIAA was not sensitive enough to measure hypo— thalamic levels. The tissue concentration of SHT and SHIAA are expressed as ng/g wet weight. The weights of tissues and the body weights of animals used in both studies are shown in Table 4. Table 4. Average Body Weights and Weights of Brain Tissue Used for Catecholamine (Study 1) and Serotonin (Study 2) Determinations Study 1 Study 2 Young Old Young Old MBH ( g protein) 36.1:4.6 32.6:4.1 ---* --- Hypothalamus (mg) 40 :2 44 :2 45:1 Olfactory Tubercles + l 15 +1 —-- --- (mg) 14 _. ._ Brain (g) --- -—- 1.71:0.02 1.87:0.07 Body weight (g) 391 :9 906 :48 405 :5 834 :47 a Dashes indicate that the tissue was not assayed. C. Results DOpamine concentration in the MBH was significantly lower in old than in young males (53:8 vs 90 :9 ng/mg protein, 117 respectively, Figure 10). One h after ampt treatment, DA decreased by 23:4% in old rats and 37:6% in young rats (P< 0.05), suggesting a greater turnover of DA in the young rats. In the remaining hypothalamus, DA content was signifi- cantly lower in old than young males. After treatment with ampt, the percentage of DA depletion was the same in both old and young rats (Figure 11). In the olfactory tubercles, DA concentration before and depletion after ampt treatment were the same in young and old rats (Figure 12). Norepinephrine concentration in the MBH was signifi- cantly lower in old than in young male rats (Figure 10). Treatment with ampt had no significant effect on NB content in either age group. In the remaining hypothalamus (Figure 11) NE concentration was 33% lower in old than in young males. Treatment with ampt resulted in a NE decrease of 24:8% in old rats and 48:3% in young rats (P‘<0.05), suggesting a greater turnover of NE in the young rats. Hypothalamic and brain concentrations of SHT were the same in young and old males. By 30 min after treatment with 75 mg pargyline/kg (Figure 13), hypothalamic SHT rose 34:5% in old and 20:3% in young males (P 0.05). The increase in brain SHT after pargyline treatment was the same in both young and old males (Figure 14). Brain SHIAA levels were 25% higher in old than in young animals (P<:0.05). After mono— amine oxidase inhibition with pargyline, the decrease in SHIAA was Significantly greater in young than in old rats. ng DA, NE/mg Protein 118 Medial Basal Hypothalamus DA NE ! Saline a 0 mp? Young Old Figure 10. Medial basal hypothalamus (MBH) dopamine and norepinephrine concentration and levels 1 h after alpha-methyl-para-tyrosine treatment in 3-4 and 21 month old male rats. Rats received a single i.p. injection of 250 mg ampt/kg body weight, or saline, 1 h before decapitation. Solid bars indi- cate steady state concentration and hatched bars indicate levels 1 h after ampt treatment for 6—8 determinations. Verticle lines indicate 1 SEM. 119 no NE/g Wot Weight .aowuwamamxo pow 0H opzmfim mom .muwn mama vac cocoa Hm cam e-m a“ unoEumouu enamoyxp-mhmm -Hxauoe-m:mfim uopmw : H mHo>oH can :ofiumuuaooqou ocflhamonfimouo: can oafiEmmow mssmHmnpomxa maficwmaom 1.0 0.3.; 3.0 0:30» COO Ar r $9"? 000.“. T 80.". l u3Eo_o;.on>I .HH mesmfim u 68 a 0 h 5 . M u M O 62. m. m. 63 ug DA/g We! Weight Figure 12. 120 Ofactory Tubercle DA Young Old Olfactory tubercle dopamine concentration and levels 1 h after alpha-methyl-para-tyrosine in 3-4 and 21 month old male rats. See Figure 10 for explanation. 121 Hypothalamus S‘HT 1600] \\"I Porgylino A \\\\\\\\\\\\\\\\\\\\\\\\\\V— ng SHT/g Wot Weight Young Old Figure 13. Hypothalamic serotonin (SHT) concentration and levels 30 min after treatment with pargyline in 3-4 and 21 month old male rats. Rats received a single i.p. injection of 75 mg pargyline HCl/kg body weight or saline, 30 min before decapitation. Solid bars indicate SHT steady state concentration and hatched bars indicate levels 1 h after pargyline treatment for 7-10 determinations. Verticle lines indicate 1 SEM. 122 .GOfiumcmHaxo How ma madman mom .mpmh mHms vac canoe am can v-m :fl mafiaxmumm spa: uquumoHu Hmumm awe om mHo>oH use :ofiumhucooqoo m<