a {MSU . 'f‘ ,. ‘ L g . LIBRARIES \— RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. EVIDENCE AND POSSIBLE MECHANISM FOR THE PERMANENT DECLINE IN TUBEROINFUNDIBULAR DOPAMINERGIC NEURONAL ACTIVITY AFTER CHRONIC ESTRADIOL ADMINISTRATION IN FISCHER 344 RATS By Paul Edward Gottschall A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1986 ABSTRACT EVIDENCE AND POSSIBLE MECHANISM FOR THE PERMANENT DECLINE IN TUBEROINFUNDIBULAR DOPAMINERGIC NEURONAL ACTIVITY AFTER CHRONIC ESTRADIOL ADMINISTRATION IN FISCHER 344 RATS By Paul Edward Gottschall The objective of these studies was to determine if the decline in tuberoinfundibular dopaminergic (TIDA) neuronal function observed during chronic estradiol-l7-B (EZ) administration persisted after E2 was removed. Ovariectomized (OVX) Fischer 344 rats were implanted with an Ez-containing Silastic capsule for 4 weeks. Other OVX rats received an empty or Ez-containing capsule for 4 weeks, after which the capsules were removed, and experiments were performed 4 or 26 weeks later. Anterior pituitary (AP) weight and serum prolactin was greatly increased at the end of the E2 treatment,that persisted 4 and 26 weeks after E2 was withdrawn. At the end of E2 treatment and 4 weeks after E2 was withdrawn, TIDA function, as evaluated by electrical stimulation of median eminence tissue in vitro after allowing for uptake of 3H-DA, was decreased compared to OVX controls. Also, Ez-treated rats showed a reduced serum prolactin response to drugs that act on central dapaminergic neurons, e.g., morphine, haIOperidol, and nomifensine. Therefore, the decline in TIDA neuronal function observed at the end of long-term E2 treatment persists up to 26 weeks after 32 removal. In an attempt to elucidate the mechanism by which 82 results in a "permanent" decline in TIDA function, F344 rats were given daily bromo- Paul Edward Gottschall cryptine injections in addition to a 30-day E2 treatment. Bromocryptine, a dopaminergic agonist, prevented the 82-ind-uced increases in serum prolactin and AP DNA content. TIDA neuronal release was reduced in both Ez and E2 and bromocryptine treated groups. However, by 30 days after discontinuing treatment only rats given E2 alone showed a persistent decline in TIDA function. Since animals given bromocryptine and 82 had small APs and showed recovered TIDA activity after the withdrawal period, the enlarged AP in Ez-treated rats compressed the hypothalamus and may be responsible for the permanent damage to TIDA neurons. Since permanent damage to hypothalamic neurons by an enlarged AP was speculated to be the result of E2 treatment, neurons which regulate other AP hormones may also be damaged. To evaluate this possibility, pulsatile release of prolactin, growth hormone (GH) and luteinizing hormone (LH) was evaluated in OVX control rats, chronically Ez-treated rats, and rats 120 days after chronic 82 treatment. Only the frequency of prolactin pulses, but not the frequency of GH and LH pulses, was reduced in rats 120 days after E2 treatment. This suggests selectivity in the hypothalamic damage produced by the enlarged AP. DEDICATION This thesis is dedicated to my parents William and Viola Gottschall for their enduring love, support and encouragement. Also, to the numerous peOple who have, possibly unknowingly, touched my life and renewed my spirit of hOpe in the face of adversity. ii ACKNOWLEDGEMENTS I would like to thank the members of my thesis committee, especially Dr. Keith Demarest, for their help in guiding these experiments. I also wish to thank all the present and former members of the Neuroendocrine Research Laboratory. The daily personal interaction will perhaps be my most vivid and pleasant memory of my years at Michigan State. Special acknowledement goes to Dr. William Sonntag, Dr. Dipak Sarkar, Dr. Vincent Hylka and Dr. Rodolfo Goya each of whom had important roles in the development, design and/or data analysis of these studies. Recognition should also go to ‘Kathy Quigley, Dr. Goya, Dr. Sumio Takahashi, Dr. Sonntag, Dr. Hylka and Dr. Karen Briski for their help in conducting the experiments. I am very thankful to Dr. Vicki Kingsbury for typing this thesis, manuscripts and general excellent secretarial help. Finally, I will be forever grateful to my mentor Dr. Joseph Meites, whose critical thinking and vast knowledge of endocrinology is equalled only by his patience and understanding. Dr. Meites' leadership and character contributed greatly to my scientific and personal eXperience. iii TABLE OF CONTENTS LIST OF TABLES O O O O O O O O O O O O O O O O O O O O O O 0 LIST OF FIGIJRES O O O O O O O O O O O O O O 0 O 0 O O O O 0 LIST OF ABBREVIATIONS 0 O O O O O O O O O O O O O O O O O 0 INTRODUCTION 0 O O O O O O O O O O O O O O O O O O O O O O O LITERATIJRE REVIEW 0 O O O O O O O O O O O O O O O O O O O O I. II. III. IV. Prolactin Secretion in Different Physiological States . A. B. C. D. E. F. Estrous and Menstrual Cycles . . . . . . . . . . . . Pregnancy and Pseudopregnancy . . . . . . . . . . . Lactation . . . . . . . . . . . . . . . . . . . . . Stress . . . . . . . . . . . . . . . . . . . . . Puberty and Old Age . . . . . . . . . . . . . . . . Male and Female . . . . . . . . . . . . . . . . . . Agents that Promote Prolactin Secretion . . . . . . . . A. B. C. D. Serotonin . . . . . . . . . . . . . . . . . . . . . Opiates . . . . . . . . . . . . . . . . . . . . . . Prolactin Releasing Factors . . . . . . . . . . . . Estrogen . . . . . . . . . . . . . . . . . . . . . . Agents that Inhibit Prolactin Secretion . . . . . . . . A. B. C. D. Prolactin Inhibiting Factors . . . . . . . . . . . . Acetylcholine . . . . . . . . . . . . . . . . . . . Gamma Amino Butyric Acid . . . . . . . . . . . . . . Corticosterone, Progesterone and Testosterone . . . Evidence that Dopamine is the Principal Prolactin Inhibiting Factor of the Hypothalamus . . . . . . . . . A. B. C. D. E. Criteria for Hypothalamic-Hypophysiotropic Hormones Dopamine in the Median Eminence and Hypothalamic- Hypophyseal Portal Blood . . . . . . . . . . . . . . Action of Dopamine and Dopaminergic Agonists and Antagonists . . . . . . . . . . . . . . . . . . . Variations in Portal Blood Dopamine and Physiologic- al Changes in Prolactin Secretion . . . . . . . . . Regulation of Synthesis, Release and Inactivation of Dopamine . . . . . . . . . . . . . . . . . . . . iv Page vii viii 29 3O 31 32 34 37 VI. Relation of Estrogen to Anterior Pituitary Hormone Secretion and Development of Pituitary Prolactinomas . . A. Estrogen Actions on the Anterior Pituitary . . . . . . 1. In vitro stimulation of prolactin . . . . . . . . . 2. In vivo stimulation of prolactin . . . . . . . . . 3. Interaction of estrogen and dopamine on lacto- troph mitotic activity and prolactin secretion . . B. Anatomy and PathOphysiology of Estrogen-Induced Prolactinomas . . . . . . . . . . . . . . . . . . . . Estrogen Influence on the Central Nervous System . . . . A. Distribution of Estrogen on Target Sites in the Brain B. Effect of Estrogen on Neurons and Neurotransmitter Systems . . . . . . . . . . . . . . . . . . . . . . . 1. Genomic, transcription-translation mediated effects . . . . . . . . . . . . . . . . . . . . . . 2. Effects which may Operate on neural membranes . . . C. Estradiol-induced Adult Anovulatory Syndrome . . . . . D. Estrogen and Hypothalamic Mechanisms which Regulate Prolactin Release . . . . . . . . . . . . . . . . . . l. Tuberoinfundibular dopaminergic neurons . . . . . . 2. Other neurotransmitter/neuropeptide systems affecting prolactin release . . . . . . . . . . . . MATERIALS AND METHODS O O O O O O O O O O O 0 O O O O O O O O I. II. III. IV. V. Animals, Treatments and Blood Collections . . . . . . . . Radioimmunoassay of Hormones . . . . . . . . . . . . . . Methods of Evaluating Tuberoinfundibular Dopaminergic Neuronal Activity . . . . . . . . . . . . . . . . . . . . A. In Vitro Superfusion cg Median Eminence Tissue After Accumulation of H-Dopamine . . . . . . . . . . B. Assessment of Prolactin Response After Administering Central-Acting Dopaminergic Drugs . . . . . . . . . . Catecholamine Assay . . . . . . . . . . . . . . . . . . . statistics. 0 O O O O O O O O O O O O O O O O .0 O O O O O E XPER m NTAL . O O I O O O O O O O O O O O O O O O O O O O O O I. Tuberoinfundibular Dopaminergic Neuronal Function: Effect of Chronic Estradiol Administration and Persistent Hyperprolactinemia After Removal of Chronic Estradiol . . . . . . . . . . . . . . . . . . . . . . . . A. Objectives . . . . . . . . . . . . . . . . . . . . . . B. Materials and Methods . . . . . . . . . . . . . . . . C. Results . . . . . . . . . . . . . . . . . . . . . . . D. Discussion . . . . . . . . . . . . . . . . . . . . . . 40 41 41 42 44 46 48 49 SO 51 52 52 53 54 58 6O 6O 62 62 62 65 66 67 68 68 69 71 79 II. Evidence for a Permanent Decline in Tuberoinfundibular Dopaminergic Neuronal Function After Chronic Estrogen Treatment . . . . . . . . . . . . . . . . . . . . . . . . A. Objectives . . . . . . . . . . . . . . . . . . . . . . B. Materials and Methods . . . . . . . . . . . . . . . . C. Results . . . . . . . . . . . . . . . . . . . . . . . D. Discussion . . . . . . . . . . . . . . . . . . . . . . III. Bromocryptine Prevents the Decline in Tuberoinfundibular Dopaminergic Neuronal Function After Removal of Chronic Estrogen Treatment . . . . . . . . . . . . . . . . . . . A. Objectives . . . . . . . . . . . . . . . . . . . . . . B. Materials and Methods . . . . . . . . . . . . . . . . C. Results . . . . . . . . . . . . . . . . . . . . . . . D. Discussion . . . . . . . . . . . . . . . . . . . . . . IV. Pulsatile Release Patterns of Prolactin, Luteinizing Hormone and Growth Hormone: Effect of Estradiol-Induced Anterior Pituitary Growth . . . . . . . . . . . . . . . . A. Objectives . . . . . . . . . . . . . . . . . . . . . . B. Materials and Methods . . . . . . . . . . . . . . . . C. Results . . . . . . . . . . . . . . . . . . . . . . . D. Discussion . . . . . . . . . . . . . . . . . . . . . . GENERAL DISCUSSION . . . . . . . . . . . . . . . . . . . . . . LIST OF REFERENCES . . . . . . . . . . . . . . . . . . . . . . APPENDIX I . . . . . . . . . . . . . . . . . . . . . . . . . . APPENDIX II. . . . . . . . . . . . . . . . . . . . . . . . . . CURRICULIIM VITAE O O O O O O O O O O O O O O O O O O O O O O 0 vi 83 83 83 84 93 97 97 97 99 109 112 112 112 115 122 124 131 163 169 179 LIST OF TABLES TABLE 1. Effect of acute APO, HALO or MOR injection on plasma PRL levels in F344 control rats, in rats treated with E for 4 weeks, and in rats 4 weeks after withdrawal of c ronic E2 treatment. . . . . . . . . . . . . . . . . . . . . . Effects of chronic E2 treatment and E withdrawal on AP weight and DNA content, and serum PRL and LH levels . . . . . . . . . Ability of APO to inhibit PRL secretion in OVX controls and 26 weeks after withdrawal from chronic E2 treatment . . . . . . Effect of MOR, HALO, and NOM on serum PRL in OVX controls and 26 weeks after withdrawal from chronic E2 treatment . . . . AP weight, AP DNA content and serum PRL and LH in OVX control F344 rats, in rats 26 weeks after E withdrawal, and in both groups after a 3-day E2 challenge . . . . . . . . . Effect of varying pooled plasma concentrations of AP hormones and varying sample volumes on within-assay coefficient of variation . . . . . . . . . . . . . . . . . . . Effect Of chronic E treatment and 120 days after E2 was discontinued on parameters of pulsatile prolactin secretion 0 O O O O O O O O O O O O O O O O O O O O O 0 O O O 0 Effect of chronic E2 treatment and 120 days after E2 was discontinued on parameters of pulsatile LH secretion . . . . . Effect of chronic E treatment and 120 days after E2 was discontinued on parameters of GH secretion . .-. . . . . . . . vii Page 73 78 85 87 91 114 119 120 121 LIST OF FIGURES Figure 1. 9. Stimulation-evoked release of 3H and accumulation of 3H-DA in median emience tissue in chronically E -treated rats and rats 4 weeks after withdrawal of chronic 2 treatment . . . . . Effect of acute APO, HALO and MOR on stimulation or inhibition of PRL release in chronically Ez-treated rats and in rats 4 WEEkS after E2 Withdrawal o o o o o o o o o o o o o o o o o o 0 Effect of acute MOR on stimulation of GH secretion in chronically E -treated rats and rats 4 weeks after Ez-Wi thd rawal O O O O O O O O O C O C O O O C I O O O O O O O 0 Ability of acute APO to inhibit PRL secretion in OVX controls and long (26 weeks) after withdrawal of chronic E2 treatment. . Effect of acute MOR, HALO and NOM on PRL secretion on OVX controls and long (26 weeks) after withdrawal of chronic E 2 treatment 0 O O O O O O O I O O O O O O O O O O O O O O O O Stimulation-evoked release of 3H and accumulation of 3H-DA in median eminence tissue in OVX control rats, in rats long (26 weeks) after removal of chronic E2 and after a 3-day 32 Challenge in both groups 0 O I O O O O O O O O O O O O O O 0 Effect of a chronic bromocryptine and/or E treatment on AP weight, serum PRL, AP PRL and DNA content . . . . . . . . . . . Fractional release of 3H during superfusion of median eminence after accumulation of H-DA in rats chronically treated with bromocryptine and/or E2 . . . . . . . . . . . . . . . . . . . . Stimulation-evoked release before and during infusion of 10 uM nomifensine in rats chronically treated with bromocryptine and/ or E2 0 O I O O O O O O O O O O O O O O O O O O O O O O O O O 0 viii Page 72 75 77 88 89 92 100 101 102 10. 11. 12. 13. 14. 15. 16. Median eminence 3H-DA uptake and neurointermediate lobe catecholamine content in rats chronically treated with bromocryptine and/or E2 . . . . . . . . . . . . . . . . . . . . Effect of chronic bromocryptine and/or E treatment and thereafter treatment was discontinued for 30 days before experiments were performed on AP weight, serum PRL and AP PRL and DNA content . . . . . . . . . . . . . . . . . . . . . . Fractional release of 3H during superfusion of median eminence in rats chronically treated with bromocryptine and/or E2 followed by a 30 day withdrawal period. . . . . . . . . . . . . Stimulation-evoked release before and during infusion of 10 NM nomifensine in rats chronically treated with bromocryptine and/ or E2 followed by a 30 day treatment withdrawal period. . . . . Median eminence 3H-DA uptake and neurointermediate catecholamine content in rats chronically treated with bromocryptine and/or E2 followed by a 30 day treatment withdrawal period . . . . . . Representative examples of pulsatile LH and PRL release in OVX controls, rats treated chronically with E2 and rats 120 days after withdrawal Of E2 treatment 0 O O O O O O O O O O O O O O 0 Representative examples of pulsatile GH and PRL release in OVX controls, rats treated chronically with E2 and rats 120 days after Withdrawal Of E2 treatment 0 O O O O O O O O O O O O O O 0 ix 103 105 106 107 108 117 118 LIST OF ABBREVIATIONS Word anterior pituitary apomorphine hydrochloride catechol-o—methyltransferase deoxyribonucleic acid l-dihydroxyphenylalanine dopamine dorsomedial and ventromedial hypothalamic nuclei endogenous opiate estradiol-17-B Fischer 344 follicle stimulating hormone gamma amino butyric acid gonadotropin releasing hormone growth hormone haloperidol S-hydroxytryptophan luteinizing hormone medial basal hypothalamus median eminence medial pre-optic area morphine sulfate messenger ribonucleic acid nomifensine maleate norepinephrine ovariectomized parachlorophenylalanine prolactin (in figures and tables) prolactin inhibiting factor prolactin releasing factor radioimmunoassay serotonin thyroid-stimulating hormone thyrotropin releasing hormone tuberoinfundibular dopaminergic neuron vasoactive intestinal peptide Abbreviation AP APO COMT DNA l-DOPA DA DMN-VMN EOP E 3344 FSH GABA GnRH HALO 5-HT? LH MBH MPOA MOR mRNA NOM OVX PCPA PRL PIF PRF RIA 5-HT TSH TRH TIDA VIP INTRODUCTION Anterior pituitary (AP) hormone release and synthesis are mainly regulated by chemicals (peptides) secreted from hypothalamic neurons which terminate on a portal vascular system that carries these substances directly to the AP. Prolactin secretion from the AP is controlled by a variety of stimulatory and inhibitory factors present in the hypothalamus, but the net influence on prolactin secretion is inhibitory. The net inhibition of prolactin release exerted by the hypothalamus is unique among AP hormones. Pituitary stalk section, electrical lesion of the median eminence which destroys the terminals for hypothalamic hormone release, or transplantation of the AP away from hypothalamic influence, all enhance the release of prolactin but greatly reduce the secretion of all other AP hormones (Meites et a1., 1972). Dopamine (DA), a catecholamine, has been shown to be the major substance in the hypothalamus which inhibits the tonic secretion of prolactin (Leong et a1., 1983), although DA alone does not account for the total hypothalamic prolactin inhibiting activity. Estrogen may be the most important peripheral factor involved in regulating prolactin release. Prolactin levels are higher in females than males, decline in females after ovariectomy, and estrogen replacement returns prolactin to nonrovariectomized control levels (Meites et a1., 1972). Estrogen appears to affect the release of prolactin not only by a direct action on the lactotrophs, but also by influencing hypothalamic neurotransmitter mechanisms, particularly DA (Meites, 1974). Estrogen and DA affect not only the release of prolactin, but can alter the mitotic rate of the lactotrophs as well. Estrogen administra- tion for prolonged periods in the rodent initially results in hyper- trOphy and hyperplasia of the lactotrophs, and later after one or two passages of the pituitary tissue, can form a neoplasia capable of growth in the absence of elevated estradiol levels (Furth and Clifton, 1966). The estrogen-induced AP hyperplasia can be prevented by administration of a dopaminergic agonist (Lloyd et a1., 1975). In fact, even when given alone, dopaminergic agonists can reduce, and dopaminergic antagonists stimulate AP mitotic activity (Jacobi and Lloyd, 1981; Kalberman et a1., 1980). Therefore, manipulations that chronically increase prolactin release can augment lactotroph mitoses, and those that decrease prolactin release reduce lactotroph mitotic activity (Pawlikowski, 1982). Intracellular mechanisms which are essential for the release of prolactin may also influence the regulation of mitosis in the lactotrOphs; alterations in second messengers, e.g., Ca++, the cyclic nucleotides, products of the cyclooxygenase or lipoxygenase pathways of arachadonic acid have been shown to influence the release of prolactin (Dannies and Tam, 1982). These substances, or products activated by these substances, could also activate a particular gene; the transcription-translation product may turn on DNA synthesis directly, or deactivate a mitotic regulatory repressor gene. The outcome would be the same in either case--augmented DNA synthesis. Both estrogen and DA have been shown to directly (in the case of estrogen) or indirectly (in the case of DA) alter the synthesis of prolactin mRNA levels (Mauer, 1982). Certainly, if estrogen and DA can affect gene products that regulate prolactin synthesis, then mitotic regulatory activity could also be influenced. What are the relative roles of estrogen and DA in stimulating and inhibiting mitotic activity of the lactotrophs? Estrogen can induce . pituitary prolactinomas in APs that are transplanted under a kidney capsule suggesting at least to one laboratory (Clifton and Meyer, 1956) that the hypothalamus plays a minor role in the development and growth of these tumors. However, administration of a dopaminergic antagonist concomittantly with estrogen, can significantly augment the increase in AP weight and DNA content (almost double), compared to animals treated wth estrogen alone (Kalberman et a1., 1980; Gottschall and Meites, unpublished). This indicates an important role for endogenous hypothalamic DA in the growth and possibly the develOpment of prolactinomas. Dopaminergic agonists can also prevent estrogen-induced increases in AP weight and DNA synthesis (Lloyd et a1., 1975). It is clear, then, that exogenous stimulation or blockade of DA receptors can dramatically influence the growth of estrogen-induced prolactinomas. What effect does chronic administration of estrogen have on endogenous hypothalamic dapaminergic mechanisms which control prolactin release? It is now well established that chronic treatment with estradiol decreases the activity of tuberoinfundibular dopaminergic neurons (Sarkar et al., 1983a). This decline in TIDA neuronal function after chronic estrogen treatment has been suggested to be caused by estradiol-induced degeneration of these neurons, as evidenced by (a) a decline in 3H-DA uptake into TIDA neuron terminals (Sarkar et al., 1984a), (b) increased glial reactivity in the arcuate nucleus (Drawer and Sonnenschein, 1976), and (c) distorted fluorescent fibers and the presence of autofluorescent material in the arcuate nucleus after fluorescent visualization of arcuate nucleus catecholaminergic neurons (Sarkar et a1., 1982). If chronic estrogen ‘treatment indeed causes degeneration of TIDA neurons, then reduced activity of these neurons should be sustained even when estradiol is removed. This thesis will present evidence that chronic estradiol treatment can produce a permanent reduction in TIDA neuronal activity in the Fischer 344 (F344) rat, and will attempt to elucidate the mechanism(s) by which estradiol produces this effect. Also, some evidence will be presented as to the specificity of the estradiol-induced decline in neuron function, i.e., are TIDA neurons alone permanently affected by chronic estradiol treatment? LITERATURE REVIEW I. Prolactin Secretion in Different Physiological States The initial experiments which demonstrated neural and/or hypophyseal action on reproductive and other endocrine functions were carried out by Long and Evans (1922, Evans and Long, 1922) who observed that mating of a female rat with a vasectomized male, mechanical stimulation of the cervix, or chronic injection of bovine AP tissue resulted in a prolonged period of vaginal diestrus characterized by the presence of functional corpora lutea in the ovary. That this phenomena involved AP secretion was confirmed by Smith and Engle (1927) who demonstrated that transplantation of AP tissue away from the in situ site also resulted in a "pseudopregnant" state. Astwood (1941) was the first to propose the functional existence of a third "gonadotrophin" which was able to maintain luteal function in the rat, and Evans et al. (1941) demonstrated that prolactin mediates the maintenance of the corpora lutea during pseudopregnancy. Anterior pituitary involvement in milk secretion was demonstrated by Stricker and Grfiter (1928), and prolactin, along with adrenocorticotrophic hormones were shown to be the essential requirement to initiate and maintain lactation by the classical experiments of Turner (1939) and Lyons (Lyons et a1., 1958). Following the separation of growth stimulating activity from lactogenic activity in an AP extract, a relatively pure "prolactin" was identified (Riddle, et a1., 1933) and a pigeon crop assay was developed for this preparation. A major breakthrough in "prolactinology" was the development of radioimmunoassays, initially for insulin by Yalow and Bernson (1959), and later the ‘rat prolactin radioimmunoassay was reported by Niswender et al. (1969) which for the first time allowed for quantitative measurement of physiological levels of prolactin in the serum. Prolactin was subsequently shown to be secreted in surge-like patterns in the rat on the afternoon of proestrous day during the 4-day estrous cycle, and during the first half of pregnancy (Smith et a1., 1975). During the 1960's and 1970's a wealth of evidence had substantiated the presence of hypothalamic neurotransmitters , particularly DA and serotonin (5-HT), as major modulators of prolactin secretion (Meites, 1977). Li (1972) fully characterized the amino acid sequence of rat, ovine, and human prolactin. Recently, a cloned DNA complementary to rat prolactin messenger RNA was sequenced which includes the codon for the precursor signal peptide, the amino acid sequence was determined from the complementary DNA (Cooke et a1., 1980). A. Estrous and Menstrual Cycles Intact adult female rats are spontaneous ovulators, but do not exhibit a true luteal phase. Estrous cycles are usually 4 or 5 days in length with ovulation occurring 10 or 11 hrs after a late afternoon surge of luteinizing hormone (LE), on the proestrous day of the cycle, (Everett, 1964). Contiguous with the surge of L8 is an afternoon rise in prolactin, which usually begins earlier and is maintained longer than that of LH, and is dependent on ovarian estrogen secretion (Everett, 1964). Early experiments demonstrated that AP prolactin content was higher (Reece and Leonard, 1939) and that AP prolactin release in vitro was greater, in animals killed on the day of proestrus or estrus as compared to animals killed on diestrus (Sar and Meites, 1967). Studies performed later showed a surge of prolactin in the serum, as measured by radioimmunoassay, beginning early on the afternoon of proestrus. This surge reaches a peak as the lights go off (with a 12 hr on, 12 hr off light cycle) and decreases to near basal values by the morning of estrus (Niswender et a1., 1969; Butcher et a1., 1974, Smith et a1., 1975). The surge of prolactin is dependent on a slowly rising rate of follicular estrogen secretion during diestrus II and proestrus, since injection of an antiserum to estradiol on diestrus II prevents the proestrus surge of prolactin (Neill et a1., 1971). The action of estrogen on the surge of prolactin seems to be mediated through hypothalamic mechanisms since a rostral hypothalamic cut placed immediately behind the optic chiasm blocks the proestrus rise in prolactin secretion (Neill, 1972). Studies performed in primates, including women, have usually failed to demonstrate any consistent change in the pattern of prolactin secretion throughout the menstrual cycle (Reyes et a1., 1975; McNeilly and Chard, 1974; Lenton et a1., 1982). However, these studies in women involved collecting only one blood sample per day. Reports that collected blood more frequently showed a nddcycle increase in circulating prolactin which was coincident with the LH surge (Robyn et a1., 1976), and 2-fold greater amount (Djahanbakhch et a1., 1984) than prolactin levels measured the night before the LH surge. Multiple blood sampling is especially important since prolactin has been shown to be secreted in a pulsatile fashion not only in rats (Saunders et a1., 1976), but also in primates during the mid-cycle surge of gonadotrophins. Many of these prolactin pulses are coincident with LH pulses (Backstrom et a1., 1982; Belchetz et a1., 1978). This is important because the C-terminal fragment of the precursor to gonadotrophin-releasing hormone (GnRH) has recently been isolated and shown to have potent prolactin-inhibiting activity (Nikolics et a1., 1985). It is interesting to speculate that if these two peptides, GnRH and the prolactin inhibiting C-terminal fragment, are released simultaneously from the same neurons then each pulse peak of LH should correspond to a pulse nadir of prolactin. Although this does not appear to occur during the episodic release of LH and prolactin pulses, there are a number of physiological situations when prolactin levels are high and LH levels are low and vice versa. The function of the preovulatory surge of prolactin during the menstrual cycle is unknown. During the estrous cycle of the rat, prolactin may have a role in luteolysis of corpora lutea formed during the previous estrous cycle. Regression of the corpus luteum is primarily dependent on prostaglandin production but stimuli that can alter the rate of prostaglandin synthesis can decrease the time to luteolysis (Rothchild, 1981). Injection of ergot drugs which decrease prolactin levels in the serum significantly’ increased numbers of corpora, lutea compared to saline injected controls. When exogenous prolactin was injected along with the ergot drug, luteolysis occurred similar to saline injected animals (Meites et a1., 1972). Therefore, prolactin does play a role in corpora lutea regression during the estrous cycle of the rat, although other AP factors (eg. LH) may also be important. B. Pregnancy and pseudopregnancy During the estrous cycle of the rat, progesterone is secreted from corpora lutea for about 2 days before regressing. However, mating or a variety of other cervical stimuli results in maintenance of progesterone ‘ secretion until late in gestation in the pregnant rat and for about 11-13 days in the pseudopregnant rat. There is a wealth of evidence which has identified prolactin as the stimulus which converts the corpora lutea of the estrous cycle to actively secreting corpora lutea of pregnancy (Butcher et a1., 1972; Smith et a1., 1975; Smith et a1., 1976). Prolactin, during the first half of pregnancy or during all of pseudopregnancy, is secreted in twice daily surges termed nocturnal and diurnal; the former peaks as the lights turn on, the latter as the lights go off (Smith et a1., 1975; Butcher et a1., 1972). Blockade of the prolactin surges on day 3 or later, but not on day 1 or day 2 of pseudopregnancy by administration of bromocryptine causes regression of corpora lutea. Regression can be reversed if prolactin is injected simultaneously with bromocryptine (Smith et a1., 1976). Ovariectomy the day after mating reduced the magnitude of the nocturnal and diurnal surges on day 2 of pseudopregnancy and the surges of prolactin lasted only for about 6 days (Freeman et a1., 1974; Freeman and Sterman, 1978). Steroid replacement can prolong the surges to about day 10. Prolactin surges can be induced after cervical stimulation in the long-term ovariectomized rat which demonstrates that the surges are not absolutely dependent on ovarian secretion (Smith and Neill, 19763), but maintenance of normal surges requires progesterone (Smith et a1., 1975). In the pseudopregnant state, the last prolactin nocturnal surge occurs on day 11, and on day 12 a normal proestrus surge of prolactin occurs 10 signifying a return to estrous cycles (Smith and Neill, 1976b). Termination of the diurnal and nocturnal surges of prolactin during pregnancy, which happens on day 8 and 10 of pregnancy reapectively, appears to be mediated through a rise in the secretion of rat placental lactogen by the deveIOping conceptus which occurs about ths time (Tonkowicz and Voogt, 1983). The rat placental lactogen may feedback on hypothalamic mechanisms which regulate the surges, to inhibit further prolactin secretion. Between days 9 and 12 of pregnancy, corpora lutea appear to be both LH and prolactin dependent, after which functional corpora lutea are maintained by placental hormones (Rothchild, 1981). Serum prolactin is maintained at low levels until the day before parturition, at which time there is a large surge (Linkie and Niswender, 1972). The most convincing evidence that these surges are neurally mediated endocrine reflexes, in rats is that after pelvic neurectomy, cervical stimulation does not result in pseudopregnancy and there are no prolactin pulses (Kollar, 1953). However, pelvic neurectomy does not seem to interfere with other normal hypothalamic-pituitary ovarian functions. It is this reflex which initiates the prolactin surges. The neural mechanisms which maintain the twice daily surges of prolactin during early pregnancy or pseudopregnancy are not well understood. Lesion of the medial pre-optic area (MPOA) resulted in a prolonged pseudOpregnant state and nocturnal surges but no diurnal surges. This demonstrates that the MPOA tonically inhibits the nocturnal surge of prolactin and the development of pseudopregnancy (Clemens et a1., 1976; Arita and Kawakami, 1981). Also, in rats which were cervically stimulated, electrical stimulation of the MPOA completely prevented both 11 the diurnal and noctural surge (Gunnet and Freeman, 1984). Contrary to this, is the observation that stimulation of the MPOA in pentobarbitol anesthetized female rats can induce a diurnal surge of prolactin (Gunnet and Freeman, 1984). It was suggested that these two contradictory functions of the MPOA involve two different neuronal pathways. A second important hypothalamic area involved in the regulation of the prolactin surges of pseudopregnancy is the dorso medial and ventro medial nuclei (DMN-VMN). Electrical stimulation of these areas results in both prolactin surges and a pseudopregnant state (Beach et a1., 1978; Freeman and Banks, 1980). However, like the dual action of the MPOA, lesions of the DMN-VMN in cervically stimulated females selectively abolished the diurnal surge, but the nocturnal surge remained. Therefore, both the MPOA. and the DVM-VMN apparently regulate both prolactin surges, but involve independent pathways for each surge. Studies recently carried out (Gunnett and Freeman, 1985) to investigate the interaction of these two areas suggest that the inhibitory action of the MPOA on the nocturnal surge can continue to Operate in a DVM-VMN lesioned animal. However, the stimulatory role of the MPOA in the diurnal surge, requires an intact DVM-VMN. C. Lactation The role of prolactin in the production and secretion of milk in female mammals is probably its most well known and ubiquitous function (Meites et a1., 1972; Cowie et a1., 1980). Prolactin levels increase about the time of parturition and are maintained high as long as lactation is continued by the suckling young. Prolactin is released into the circulation as a result of suckling by a typical neuroendocrine 12 reflex. However, the suckling-induced reflex release of prolactin has many characteristics which differ from the proestrus or cervical- stimulation induced prolactin surges discussed previously. Following return of young to their mothers after a period of separation, prolactin rises within 5 minutes after the initiation of suckling, reaching a peak at 30 minutes and remains at this level for at least 90 minutes (Grosvenor and Whitworth, 1974). Prolactin continues to be secreted until AP stores have been exhausted (Grosvenor et a1., 1979). In contrast to the proestrus and cervical-stimulated surges of prolactin, suckling induced prolactin release is not surge-like and does not exhibit a circadian pattern, but is tightly coupled to the stimulus of suckling and responds to a greater degree with a more intense period of suckling. Also, suckling-induced prolactin release strictly ‘requires application of the stimulus for a prolactin response to occur (Neill, 1980). At the spinal level, the central nervous system pathways involved in the suckling-induced release of prolactin appear to be common to the pathway involved in oxytocin release for milk ejection. Just central to the medial geniculate body, the prolactin pathway diverges and passes between the third ventricle and the mammilothalamic tract, to the lateral hypothalamus and up to the MPOA. At the MPOA the pathway joins with a neocortical path which descends from the orbitofrontal region. These two pathways, then pass caudally to the anterior hypothalamic area, where, at least as far as activating prolactin release by electrical stimulation is concerned, it appears to terminate. However, this may be due to changes in neurons or neurotransmitters (Tindal, 1978). The specific neurotransmitters involved in this release of prolactin, particularly between the anterior hypothalamus area and the 13 median eminence are unknown but both DA and serotonin have been implicated (Leong et a1., 1983). After parturition, a 'period of lactational. anestrus occurs, the length of which appears to be related to the intensity and frequency of suckling by the young (Van der Shoot, et a1., 1978). However, since suckling induced release of prolactin is also related to the intensity of the suckling stimulus, at least in rats, hyperprolactinemia could be involved in the lactational anestrus (Meites et a1., 1978; McNeilly, 1984). A number of mechanisms have been suggested to explain the anestrus during lactation, and 'many of these involve the ‘hyperpro— lactinemia which occurs during this time. The mechanism which has the best support is the effect of suckling and/or prolactin on the GnRH control of gonadotrOphin secretion. Immediately before parturition, there is a massive reduction in circulating steroids. When suckling begins postpartum, concentrations of follicle stimulating hormone (FSH) return to normal within a few days, but LH remains below normal diestrus levels even when estradiol is low (Taya and Greenwald, 1982). The degree of pulsatile LH secretion, which is taken to be a reflection of pul- satile GnRH secretion, is decreased in lactating rats. The greater the degree of the suckling stimulus the greater the decrease in pulsatile LH release (Fox and Smith, 1984). The ability of lactating mothers to release LH after injection of GnRH was reduced in lactating mothers compared to nonrlactating controls both in vivo (Lu et al., 1976a) and from AP tissue incubated in vitro (Lu et al., 1976b; Smith, 1985). This effect is probably due to a decrease in AP GnRH receptors which were observed to be reduced by 502 in lactating mothers compared to diestrus females (Smith, 1984). All of these results suggest a decrease in GnRH 14 secretion and demonstrate diminished ability of GnRH to release LH in lactating rats. Evidence supporting prolactin as the agent which reduces GnRH or GnRH action on the AP is that treatment of lactating mothers with ergocornine, which reduces PRL secretion, produced increases in LH release even in the presence of suckling pups (Lu et al., 1976b). Furthermore, increasing circulating prolactin by AP transplants, transplantation of a prolactin-secreting AP tumor, or injections of prolactin significantly reduced LH levels (Meites et al., 1978). In contrast to these results, Smith (1978) has shown that prolactin can decrease LH secretion in ovariectomized lactating rats only in the presence of suckling pups. Therefore, although prolactin may be a significant factor responsible for the inhibition of gonadotrophin release during postpartum lactation, it is not the sole factor, and a more direct effect of _the suckling stimulus may' also inhibit GnRH release. In addition to the potential hypothalamic effects of prolactin on GnRH during lactation, there is evidence for direct inhibition. of ovarian function by prolactin (McNeilly, 1984). In vitro, addition of prolactin (Van der Shoot'et a1., 1982) appears to inhibit the secretion of estradiol by suppressing the levels of FSH-induced aromatase within granulosa cells. A similar effect was observed in granulosa cells from lactating rats (Taya and Greenwald, 1982). However, administration of GnRH or LH can induce follicle growth and ovulation in lactating rats (Taya and Greenwald, 1982), sows (Hausler et a1., 1980), or cows (Riley et a1., 1981). Therefore, the relative contribution of the direct action of prolactin on the ovary in producing lactational anestrus remains obscure. 15 D. Stress The first indirect evidence that acute stress resulted in prolactin release from the AP was by Nicoll et al. (1960) who observed that various stressors such as restraint, injection of formaldehyde, cold or heat could induce lactation in estrogen-primed rats. This was confirmed when stress was shown to deplete AP prolactin content (Grosvenor et a1., 1965). With the development of the prolactin radioimmunoassay, it was observed that ether stress increased serum prolactin levels in female rats at all times during the estrous cycle except on the afternoon of proestrus when prolactin levels were already high (Neill, 1970). Ether and Nembutal anesthesia also caused release of prolactin (Ajika et a1., 1972). Krulich et a1. (1974) demonstrated that even mild stressors, e.g., handling of animals or transfer from room to room, could increase serum prolactin levels. In addition to stress-induced decreases in growth hormone (GH) secretion, prolactin appeared to be more susceptible to these mild stresses than other AP hormones. The release of prolactin in response to acute stress is a hypothalamic-mediated event. The effect of chronic stress on AP prolactin secretion is not as clear. Restraint stress for 2 hours a day for 20 days increased serum prolactin on day 9 and 19 when the samples were taken prior to the stress. However, there was no effect of stress on prolactin levels on day 10 and 20 when the samples were taken following the stress period .(Riegle and Meites, 1976). Moreover, in another study, chronic (42 days) cold exposure, forced exercise, or daily immobilization tended to decrease serum prolactin levels in single samples taken throughout the treatment period (Tache et a1., 1978). The functional role: of :increased circulating prolactin during acute stress is not known. 16 E. Puberty and old age Although particularly recognized for its role as a luteotropin and in mammary gland function in the female rat, prolactin appears to be important in events occurring during reproductive development. Prolactin levels gradually increase during the pre-pubertal period in the rat (Voogt et a1., 1970; Ojeda et a1., 1976), and during this time a circadian diurnal pattern is established; levels which slowly increase in the late afternoon and evening (Kimura and Kawakami, 1980). Hyper- prolactinemia, induced by administration of the dopaminergic antagonist, sulpiride or by injecting exogenous prolactin, results in precocious puberty (Clemens and Meites, 1977; Advis and Ojeda, 1978; Advis et al., 1981a). Prolactin appears to advance puberty in females by facilitating the steroidogenic response of the ovary to gonadotrOpins which are at low levels during the juvenile period; injected prolactin has been shown to elevate ovarian LH receptors (Advis et al., 1981b). As might be expected, suppression of prolactin by ergot drugs delays the onset of puberty and suppresses the steroidogenic ability of the ovary (Advis et al., 1981a). No effect was observed on LH or FSH levels in these studies, suggesting a direct action of prolactin on the ovary (McNeilly, 1984). A contrary, but interesting observation is that implantation of prolactin into the hypothalamus of pre-pubertal female rats advanced the onset of puberty, suggestive of a central action of prolactin (Clemens et a1., 1969). As can be seen, prolactin plays an important role in reproductive development, although whether its most important action is at the hypothalamic and/or ovarian level is not clear. Towards the opposite end of reproductive development, i.e., old age, prolactin secretion is radically altered as compared to the young adult 17 rat. Regularly cycling middle-aged animals (10-12 mos) exhibit an altered proestrus surge shown to be increased in Sprague-Dawley rats (Wise, 1982) but decreased in mice (Brawer and Finch, 1983) or Long-Evans rats (Gottschall and Davis, 1980). Following cessation of cycles, due to hypothalamic defects (Meites, 1982), estradiol levels are moderately elevated (ll-25 mos) and as a consequence, prolactin levels are also increased (Lu et a1., 1979). Ovariectomy’ at a young age prevents this age-related hyperprolactinemia (Lu et a1., 1979). Aging in female rats is also associated with a high incidence of prolactin-secreting pituitary microadenomas (Takahashi and Kawashima, 1983) and frank prolactin-secreting pituitary tumors (Huang et a1., 1976), which may be related to the earlier recurrent estrous cycles and to the acyclic period of moderately elevated estradiol levels (Finch et a1., 1984). This phenomenon will be discussed further in section VIc. F. Male and Female The male rat has lower circulating levels of prolactin than the female rat. This, for the most part, is due to the action of estradiol which increases prolactin release in the female. Ovariectomy decreased circulating prolactin to approximately male levels (MacLeod and Fontham, 1970), and this is surprising, in part because adult female APs contain about three times as many lactotrophs as males (Takahashi and Kawashima, 1982). Estradiol replacement in ovariectomized females returns prolactin to pre-castrate levels. The secretion of prolactin in the male rat is not surge-like at any time of the animal's life, in contrast to the female. However, neonatally castrated males can exhibit afternoon prolactin surges when given estrogen. It is thought that androgens, 18 during neonatal life, prevent the organization of a rostral hypothalamic "surge center.” In fact, the hypothalamic MPOA of the rat (Gorski et a1., 1978), as well as the human is sexually dimorphic (Swaab and Fliers, 1985). With increasing age, male rats become hyperprolactinemic but not in as great numbers or to the same extent as in females, possibly owing to the fact that female APs have a greater number of lactotrophs (Takahashi and Kawashima, 1982). II. Agents that Promote Prolactin Secretion Neuroendocrine substances which affect AP hormone secretion can generally be divided into three categories: 1) neurotransmitters/ neuromodulators which do not act on the AP, but whose activity either directly or indirectly regulates the activity of releasing hormone neurons; 2) hypothalamic hypophysial releasing or release-inhibiting hormones are the chemicals present in neurons whose terminals end on capillaries in the median eminence. The portal vasculture carries the hormones to the AP where they act directly on specific binding sites to affect AP hormone synthesis and release; and 3) substances, usually present peripherally, which can act on neurotransmitter/neuromodulator neurons, releasing/release-inhibiting neurons or directly on the AP to influence hormone release. 0f the substances discussed in Sec II and III, serotonin and the endogenous opiates belong to category #1, prolactin releasing factors and prolactin-inhibiting factors belong to category #2 and estrogen, testosterone, progesterone, and corticosterone belong to category #3. Present evidence suggests that gamma-amino butyric acid (GABA) and acetylcholine may belong to category #1 or 19 category #2. A. Serotonin (5-hydroxytryptamine; 5-HT) Convincing evidence demonstrates that S-HT stimulates the release of prolactin. Meites (1963) first reported that S-HT initiated mammary secretion in rats, and Kamberi et al. (1971) first reported that S-HT administered intracerebroventricularly (5-HT does not cross the blood brain barrier) increased serum prolactin levels. Systemic injection of tryptophan or S-hydroxytryptophan (S-HTP), synthesis precursors of SAHT, increased brain S-HT turnover and serum prolactin concentrations (Meites and Clemens, 1972; Lu and Meites, 1973; Mueller et al. 1976). Synthesis precursors of 5-HT may exert non-specific actions on other monoaminergic neurons, although the non-specific effects of tryptophan. and SAHTP appear not to involve the hypothalamic catecholaminergic neurons since catecholamine levels in the hypothalamus were not altered after S-HT precursor administration. Sub effective doses of S-HTP, when injected together with the specific S-HT reuptake inhibitor, fluoxetine, resulted in a significant rise in prolactin levels (Clemens et a1., 1977). In male rats, blockade of 5-HT synthesis by injection of parachlorophenyl- alanine (PCPA) or depletion of 5-HT by the specific serotonergic neurotoxin, 5,7 dihydroxytryptamine, significantly reduced brain S-HT and serum prolactin levels, while having no effects on brain norepinephrine or DA content (Gil-Ad et a1., 1976). S-HT does not cause release of prolactin when added to AP tissue in vitro (Birge et a1., 1970; Meites and Clemens, 1972). Injection of PCPA blocked the estrogen-induced (Caligaris and Taleisnik, 1974) or suckling-induced (Kordon et a1., 1973/74) surges of prolactin secretion. Metergoline or 20 cinanserin, both S-HT receptor antagonists, blocked the stress-induced increase in prolactin (Demarest et al., 1985a). Increases in prolactin after S-HTP administration were demonstrated after cutting all afferent input to the medial basal hypothalamus, and no effect was observed after extra hypothalamic lesions (Ohgo et a1., 1976), suggesting the importance of intrahypothalamic S-HT. However,another study showed that lesion or electrical stimulation of the mesencephalic raphe nucleus resulted in decreased and increased prolactin secretion, respectively (Advis et a1., 1979). These results taken together demonstrate that S-HT is involved in a neuronal circuit(s) mediating prolactin release. Most evidence suggests that S-HT acts through a putative prolactin-releasing factor to cause the secretion of prolactin (Clemens et a1., 1978). B. Opiates The ability of morphine to cause the release of prolactin was first suggested by Meites et a1. (1962) who demonstrated mammary' gland activation after injection of morphine in estrogen-primed rats. Isolation of the endogenous opiates (EOP; Hughes et a1., 1975; Hughes et a1., 1977) from brain tissue, and their relative high concentration in the hypothalamus, suggested that EOPs may have neuroendocrine functions. Indeed, intracerebroventricular or systemic administration of the EOP's have been shown to influence the release of a number of AP hormones, including prolactin (Dupont et a1., 1977; Bruni et a1., 1977; Van Vugt and Meites, 1980). The neuroendocrine actions of EOPs are similar to morphine, and can be blocked with the opiate receptor antagonist, naloxone. Beta-endorphin was shown to be 500 to 2000 times more potent than the enkephalins in raising serum prolactin levels in the rat (Cusan 21 et a1., 1977). It is generally agreed that morphine and the EOPs do not alter AP hormone secretion by acting directly on the AP. No effect of the EOPs on prolactin secretion was observed in AP cell culture (Rivier et a1., 1977), or on whole AP tissue (Shaar et a1., 1977), using high . concentrations of met-enkephalin. However, the EOPs, particularly beta-endorphin, have been shown to be in high concentration in the hypothalamic-hypophysial portal blood in the rat (Sarkar and Yen, 1985) and in monkeys (wardlaw et a1., 1980). The reason for the presence of beta-endorphin in the portal blood is not clear, and may represent an overflow from hypothalamic or pituitary activity. The EOPs are involved in a number of physiological stimuli which alter prolactin secretion. Naloxone in a dose-related fashion, can block the suckling-induced (Miki et a1., 1981) and stress-induced (Van Vugt et a1., 1979) release of prolactin. A single injection of naloxone can block the proestrus surge of prolactin (Ieiri et a1., 1980), and also the diurnal surges of prolactin after cervical stimulation (Sirinathsinghji and Andsley, 1985), although the involvement of EOPs in the proestrus surge of prolactin are controversial (Piva et a1., 1985). The neuronal circuits through which the EOPs act to effect prolactin release are not certain, although both dopaminergic and serotonergic systems appear to be involved (Van Vugt et a1., 1979; Arita and Porter, 1984; Koenig et a1., 1979) Administration of morphine or EOPs to rats which results in a marked rise in serum prolactin also decreases the turnover of DA in the median eminence (Deyo et a1., 1979; Van Vugt et a1., 1979), and diminishes DA levels in hypophysial portal blood (Gudelsky and Porter, 1979). These results are consistent with the belief that the EOPs increase prolactin 22 by inhibiting the activity of tuberoinfundibular dopaminergic (TIDA) neurons. However, the decrease of DA in portal blood after morphine ad- ministration may not be solely responsible for the rise in prolactin (Arita and Porter, 1984). It appears that a stimulatory factor, possibly via a serotonergic-prolactin-factor-releasing system, also takes part in the opiate-induced increase in prolactin release (Koenig et a1., 1979). C. ProlactinrReleasing Factor(s) (PRF) It has been a number of years since the postulation of a putative PRF from the prolactinrreleasing activity of hypothalamic extracts (Meites et a1., 1960). It may be that physiological increases in prolactin secretion, produced during suckling, proestrus, pregnancy etc., are at least partly due to PRF(s) secretion from the hypothalamus which act on the AP to release prolactin. Unfortunately, numerous peptides have been shown to release prolactin from AP cells in vitro. Some of these include thyrotrophin-releasing hormone (TRH; Tashjian et a1., 1971), substance P (Kato et a1., 1976), vasoactive intestinal peptide (VIP; Kato et a1., 1978), epidermal growth factor (Johnson et a1., 1980), fibrobast growth factor (Schonbrunn et a1., 1980), cholecystokinin (Malarkey et a1., 1981), angiotensin II (Steele et a1., 1981), neurotensin (Enjalbert et a1., 1982), bombesin (Westendorf and Schonbrunn, 1982), and vasOpressin (Shin, 1982). Some of these substances are present in the hypothalamus (Palkovits, 1984), and are in high concentrations in the portal blood. However, probably the most important criteria for the identification of a substance as a hypothalamic-hypophysial hormone (see Sec IV below) is the ability of the particular neuronal system hormone to alter its secretion into the 23 portal blood at times during physiological changes in secretion of the hypOphysial hormone. To date, evidence has been presented that only two of the above peptides (TRH and VIP) show alterations in release during physiological changes in prolactin release. Initially, TRH was isolated as a peptide that regulates thyroid stimulating hormone (TSH) release from the AP (Bowers et a1., 1970), but TRH was soon shown to also markedly increase prolactin secretion (Tashjian et a1., 1971). The most convincing evidence that TRH is a physiologically significant factor regulating prolactin release is that the rise in prolactin during suckling causes the release of both prolactin and TSH in the rat (Blake, 1974). TRH levels are increased in hypophysial portal blood during suckling (Fink et a1., 1981) or after mammary nerve stimulation (de Greef and Visser, 1981). Also, the amount of TRH required to release prolactin in vitro (10"9 M; Woolf and Letourneau, 1979) is in the same range of concentrations found in the portal blood (Eskay et a1., 1975). Studies have shown that suckling augments the PRL responsiveness to TRH (Leong and Neill, 1982). However, suckling does not induce a rise in circulating TSH levels in women and in many physiological states, prolactin and TSH release do not occur together, e.g., during stress, the afternoon of proestrus, cold temperature. The isolation of VIP was originally from porcine intestine, but it has been found to be widely distributed in the mammalian central nervous system, with high. concentations in the hypothalamic suprachiasmatic nucleus but only moderate levels in the median eminence (Palkovits, 1984). VIP is present in ‘rat portal blood (Said and Porter, 1979; Shimatsu et a1., 1981) and can ‘bind specifically to sites (n1 AP 24 membranes (Bataille et a1., 1979). Like TRH, VIP releases prolactin in vitro at concentrations similar to those found in portal blood (Enjalbert et a1., 1980). Passive immunization with antiserum to VIP can partially block the suckling-induced rise in prolactin, and completely prevent the stress-induced rise in prolactin (Abe et a1., 1985). Therefore, VIP may be involved in the stress-induced increase in prolactin. In summary, VIP appears to be a PRF involved in the release of PRL in certain physiological states although more work is required to confirm this. The status of TRH as a physiological releasor of prolactin remains in question. D. Estrogen The ability of chronic estrogen administration to stimulate prolactin secretion will be discussed in detail in Section V. Here, the potential for acute estrogen action to modulate the release of prolactin will be briefly covered. Estradiol was first shown to cause prolactin secretion in vivo by Turner (1939) and in vitro by Nicoll and Meites (1962). Estradiol benzoate induced prolactin release in a dose-related fashion (Chen and Meites, 1970). Acute estradiol can act directly on the AP to antagonize the inhibitory effect of DA on prolactin secretion (Lu et a1., 1971; Raymond et a1., 1978). Also, estradiol has been shown to greatly enhance prolactin responsiveness to TRH, possibly by increasing TRH binding sites on the lactotrophs (DeLean et a1., 1977), even in the presence of inhibitory amounts of DA (Raymond et a1., 1978; Labrie et a1., 1980). The effect of acute estradiol administration on VIP and other agents influencing prolactin secretion has not been investigated. Estradiol given acutely in vivo may act via the hypothalamus as well as 25 directly on the pituitary to increase prolactin secretion. One of the most studied effects of estrogen is its direct action on the AP to increase prolactin synthesis by augmenting prolactin gene transcription (Mauer, 1982). The estradiol-induced increase in prolactin mRNA occurs via a biphasic mechanism, the first phase is dependent on the conversion of the cytosolic estrogen receptor to the activated nuclear form, and is protein synthesis independent. The second phase is possibly mediated by the ability of estrogen to alter the responsiveness of the AP to another regulator of prolactin gene transcription, e.g., DA. This phase can be blocked by a protein synthesis inhibitor (Shull amd Gorski, 1985). III. Agents that Inhibit Prolactin Secretion A. Prolactin Inhibiting Factor (PIFs) The control of prolactin secretion by the hypothalamus is predominantly inhibitory, which is unique among AP hormones. The inhibitory influence of the hypothalamus on prolactin secretion was first suggested when AP transplants underneath the kidney capsule in short-term hypophysectomized female rats resulted in ‘maintenance of active corpora lutea and/or development of the mannnary gland. This indicates a continuous secretion of prolactin in the blood in the absence of hypothalamic influence. Secretion of all other AP hormones was diminished, as indicated by atrophy’ of target (glands (Everett, 1966). Prolactin was assumed to be released from the AP after elimination of hypothalamic influence, and the inhibitory factor was demonstrated to be contained within the hypothalamus. This was performed 26 by testing acid extracts of hypothalamus in vitro on AP hormone secretion, and it was found that prolactin secretion was inhibited (Talwalker et a1., 1963). The putative prolactin inhibitory substance was named prolactin inhibitory factor or PIF. Subsequently, much of the PIF activity of the hypothalamus was attributed to dopamine (DA) which will be discussed in. detail in Sec. IV. However, DA-free extracts concentrated from the mediobasal hypothalamus and the organum vasculosum of the lamina terminalis have been shown to contain PIF activity (Enjalbert et a1., 1977). Recently, the C-terminal fragment of human pre-pro GnRH has been shown to have potent PIF activity (Nikolics et a1., 1985; discussed in Sec. I). This 56 amino acid peptide may, along with DA, make up the total hypothalamic prolactin inhibition. B. Acetylcholine There is relatively minimal information regarding the action of acetylcholine on prolactin release, much of which is conflicting. Early work (Libertun and McCann, 1973, McLean and Nikitovitch-Winer, 1975) suggested a stimulatory' role for acetylcholine (n1 prolactin ‘release since very large doses of central or systemically administered atropine inhibited prolactin secretion in male and female rats. However, other reports showed that intracerebroventricular injection of acetylcholine, or the cholinergic drugs pilocarpine or physostigmine could decrease serum prolactin--an effect which could be blocked by atropine (Grandison et a1., 1974). Cholinergic agonists were also shown to block the afternoon surge of prolactin in OVX estrogen-primed rats, inhibit the rise of prolactin during restraint stress and partially block nocturnal surges of prolactin in the pseudopregnant rat (Grandison et a1., 1974, 27 Grandison and Meites, 1976, Subramanian and Gala, 1976a, Subramanian and Gala, 1976b). It was suggested that acetylcholine was affecting prolactin release through central catecholaminergic mechanisms (Grandison and Meites, 1976). More recently, cholinergic drugs have been . shown to inhibit prolactin by a direct AP action (Miikherjee et a1., 1980; de Galarreta et a1., 1981). Cholinergic receptors are present on the AP (Schaeffer and Hsueh, 1980). To date, though, the mechanism and physiological relevance of the ability of acetylcholine to affect prolactin release is unclear. C. Gamma-amino Butyric Acid (GABA) Much evidence has been accumulating in recent years indicating a role for GABA in the regulation of AP function, particularly prolactin secretion. The synthetic enzyme glutamic acid decarboxylase is present in hypothalamic tissue (Tappaz et a1., 1977) and autoradiography has localized 3I-I-GABA in different regions of the hypothalamus, including the ME (Makara et a1., 1975). Binding sites have been demonstrated for GABA on the AP (Grandison, 1981) and GABA was recently shown to be in greater concentrations in hypophysial portal compared to peripheral blood. Also, electrical stimulation of the ME results in almost an 8-fold elevation of GABA in the portal circulation (Mitchell et a1., 1983). Although early experiments suggested that GABA was effective in suppressing prolactin release when administered centrally (Mioduszewski et a1., 1976), most recently work demonstrates that GABA acts directly on the AP to inhibit prolactin secretion. Incubation of GABA with AP preparations in vitro resulted in inhibition of prolactin secretion (Enjalbert et a1., 1979; Grossman et a1., 1981), although large 28 concentrations are required relative to dopamine (Arimura and Schally, 1977). Muscimol, a GABA agonist, can reduce prolactin levels in hypophysectomized rats bearing an AP transplant under the kidney capsule, and can inhibit the rise in prolactin after administration of the DA synthesis blocker, alpha-methyl para tyrosine (Muller et a1., 1979). Interestingly, the GABA-containing dipeptide, homocarnosine, is also present in portal blood under basal prolactin conditions (Mitchell et a1., 1983) and can inhibit prolactin when incubated with AP tissue in zigrg, albeit at relatively high concentrations of 6 uM (Schally et a1., 1977). Even with respect to the evidence that GABA can suppress prolactin release in vitro, this does not rule out the possibility of a central action of GABA to inhibit prolactin. D. Corticosterone, progesterone and testosterone Adrenalectomy in the rat produces an increase in plasma prolactin (Ben David et a1., 1971), which can be prevented by administration of glucocorticoids (Chen et a1., 1976). Corticosterone or a glucocorticoid agonist can inhibit the ether or restraint stress-induced increase in prolactin in adrenalectomized (Harms et a1., 1975) or in intact male rats (Euker et a1., 1975). It appears that the influence of corticosterone on prolactin secretion is exerted at both the AP and hypothalamic levels. Incubation of AP tissue in media containing 10 ug/ml corticosterone decreases prolactin release “but does not affect other AP hormones. After adrenalectomy, medial basal hypothalamic DA turnover was not altered but a higher concentration of S-HT was found in the anterior hypothalamus (Leung et a1., 1980). Doses of fluoxetine, a 5-HT agonist, and crypoheptadine, a 5-HT antagonist, were ineffective in 29 altering prolactin release in intact rats, but fluoxetine increased and crypoheptadine suppressed serum levels of prolactin in adrenalectomized rats (Leung et a1., 1980). Therefore, the influence of the glucocorticoids, specifically corticosterone, on AP prolactin secretion is complex, with actions at both the hypothalamic and AP level. Even less is known regarding the effect of testosterone and progesterone on prolactin secretion. Progesterone is essential for maintenance of normal diurnal and nocturnal surges of prolactin which occur during pregnancy in the rat (Neill, 1980). The estrogenrinduced increase in prolactin release is partially antagonized by concurrent administration of progesterone (Chen and Meites, 1970). Castration of male rats or testosterone when administered alone does not alter or slightly lowers circulating prolactin levels (Meites, 1977). However, testosterone or the reduced metabolite, 5 -dihydrotestosterone can completely inhibit the estradiol induced increase in prolactin, under chronic treatment conditions (Brawer et a1., 1983). IV. Evidence that Dopamine is the Principal PIF of the Hypothalamus In the last 15 years an enormous amount of evidence has substantiated DA as a PIF which regulates prolactin release. In this section, the evidence which has allowed DA to meet the criteria for a hypothalamic-hypOphysiotrOpic hormone will be presented. 30 A. Criteria for Hypothalamic-HyPOphysiotrapic Hormones Certain conditions must be verified for a substance to be considered as a hypophysiotrophic hormone. Many of these criteria were established based upon the work of Geoffrey Harris (1955), who was the first to propose and substantiate a central control of AP function and Andrew Schally (Schally, 1978) and Roger Guillemin (Guillemin, 1978) were the first to isolate and characterize hypophysiotrophic hormones from the hypothalamus. These criteria are important because they aid in distinguishing true hypophysiotrOphic action from a pharmacological effect of a substance on AP hormone release. For a substance to be considered as a hypothalamic-hypophysiotrophic hormone, some of the criteria which must be verified are: l) the factor should exist in the median eminence and be present in portal blood at higher concentrations than in the systemic circulation; 2) the purified factor should be effective in altering AP hormone release in vitro at a concentration similar to that found in the portal blood; 3) the synthetic factor should act identically with the purified hypothalamic factor, and agonists and antagonists should result in appropriate hormone changes; 4) presence of binding sites for the factor should be located specifically on (in) the appropriate AP cell population; and 5) changes in portal blood levels of the factor should correspond to physiological variations in hormone secretion. In considering DA as a prolactin-inhibiting hormone, all of the above criteria have been realized. 31 B. Dopamine in the Median Eminence and Hypothalamic-Hypophyseal Portal Blood Although several nuclei containing DA have been located in the hypothalamus, it appears that only one, originating in the arcuate and periventricular nuclei is involved in the regulation of AP function. This group of perikarya is termed A12 by Fuxe et al. (1979). The A12 neurons can be divided into two groups: DA-containing neurons terminating in the pars intermedia and pars nervosa have perikarya which originate in the rostral and caudal arcuate nucleus, respectively, and are termed tuberohypophysial dopaminergic neurons; DA-containing neurons terminating in the external layer of the median eminence have perikarya diffusely distributed throughout the arcuate and periventricular nuclei, and are termed tuberoinfundibular dopaminergic (TIDA) neurons (BjBrklund et a1., 1973). The TIDA neurons appear to be intimately involved in the control of prolactin secretion. DA has not only been visualized by fluorescent anatomical methods, but also quantitated by chemical means. There are relatively high concentrations of DA in the median eminence, about 100 ng/mg protein (Rinne and Sonninen, 1968). Even though there was good evidence in the early 1970's that DA played a crucial role in regulating prolactin secretion , it wasn't until a sensitive assay was develOped to measure plasma DA (Ben-Jonathan et a1., 1977), that DA was quantitated in the portal blood. In female rats under pentobarbital anesthesia and using a radioenzymatic assay for measurement, DA was found to be in concentrations of 1-3 ng/ml in the portal blood (Ben-Jonathan et a1., 1977). Others using urethane anesthesia and a liquid chromatographic-e1ectrico-chemical detection system observed portal blood DA levels in the 5 ng/ml range (Plotsky et a1., 1978), 32 compared to peripheral levels of 0.1-0.3 ng/ml. This portal blood concentration of DA was sufficient to inhibit prolactin secretion when infused in vivo after blockade of in situ of TIDA synthesis (Gibbs and Neill, 1978). Therefore, DA meets the criteria of being in high concentrations in the median eminence and hypOphysial portal blood (greater than peripheral levels), and can inhibit prolactin release at concentrations similar to those found in the portal plasma. C. Action of D0pamine and Dopaminergic Agonists and Antagonists Pasteels (1961) and ‘Talwalker' et a1. (1961) first showed independently that acid extracts of rat hypothalamus inhibit the _i_n_ ‘yitgg secretion of prolactin from AP tissue. Subsequently, many reports indicated in vivo that dopaminergic drugs could decrease prolactin release and anti-dopaminergic drugs increase prolactin. At the time, the belief was that these dopaminergic drugs changed hypothalamic PIF activity, and therefore altered the amount of PIF released into portal blood (Ratner et a1., 1965; Nagasawa and Meites, 1970; Lu and Meites, 1972; Meites et a1., 1972). However, it was observed that TIDA neuron terminals in the median eminence are adjacent to capillaries which drain directly into the portal blood (Fuxe et a1., 1974), and that hypothalamic extracts lose their PIF activity if first incubated with a monoamine oxidase (Shaar and Clemens, 1973). This suggests that ‘DA itself could be PIF. The most convincing evidence for DA's direct action on the lactotrOph is work performed in vitro. Consistent reports showed that DA can reduce prolactin by a direct effect on the AP in vitro at concentrations of 5 x 10-7 M (Koch et a1., 1970; MacLeod and Lehmeyer, 1974), and one report observed a decrease in prolactin at 10.9 M DA 33 (Shaar and Clemens, 1974). The decrease in prolactin by DA observed in ziggg can be prevented by DA receptor blockers (MacLeod and Lehmeyer, 1974). The identification of stereospecific binding sites for DA on AP membranes (Brown et a1., 1976; Caron et a1., 1976; Calabro and MacLeod, 1978; Cronin et a1., 1978) further supports the view that DA acts directly on the lactotroph to inhibit prolactin release. These binding sites have not only been identified on AP membrane preparations, but specifically on lactotrOphs (Goldsmith et a1., 1979). The competition curves of dopaminergic agonists for 3H-dihydroergocryptine (a specific DA receptor agonist) binding sites parallels the agonists' ability to inhibit PRL release in vitro (Cronin et a1., 1978). The AP DA receptors are a pure papulation of the D2 type (binding does not increase cAMP levels) and appear to exist in an interconvertable high and low affinity form (Sibley et a1., 1982). The Ki for DA agonists binding to the receptor in the high affinity form correlate perfectly with the ICSO of the.agonists' ability to inhibit prolactin secretion in vitro (George et a1., 1985). Also, the K1 for DA of 7 nM, which displaces 3H- dihydroergocryptine from. high affinity form receptors, is well within the range of DA concentrations which have been reported in portal blood. Hence, DA binding sites located on the lactotrophs and the ability of DA agonists to inhibit prolactin secretion strengthens the hypothesis that DA is a true hypophysiotropic hormone. 34 D. Variations in TIDA Neuronal Activity, Hypophyseal Portal Blood Dopamine and Physiological Changes in Prolactin Secretion Although a number of studies have measured portal blood DA during physiological changes in prolactin secretion, e.g., proestrus, most experiments have used biochemical methods to evaluate TIDA. neuron activity. This can indicate changes in potential portal blood DA (Reymond and Porter, 1982), without the use of anesthesia. The most common method of evaluating TIDA neuron activty is based on the fact that since DA content does not appear to change with increasing or decreasing activity of the neurons (under most conditions), synthesis of DA must keep pace with release. Therefore, synthesis of DA should be a reflection of neuronal activity of dapaminergic neurons (Moore and Demarest, 1982). The two available methods to measure TIDA activity are 1) determining the rate of decline of DA after administration of the tyrosine hydroxylase inhibitor alpha-methyl-para tyrosine and is termed DA turnover, or 2) measuring accumulation of l-DOPA after inhibition of l-aromatic amino acid decarboxylase by' 3-hydroxybenzy1hydrazene (NSD 1015), which is an index of DA synthesis (Moore and Demarest, 1982). During the estrous cycle in the rat, DA levels in the hypOphysial portal blood were lower on the afternoon of proestrus as compared to estrus (Ben-Jonathan et a1., 1977), and is at least partially responsible for the afternoon proestrus surge of prolactin. This proestrus fall in portal blood DA may be due to the action of estrogen since adrenalectomized, ovariectomized rats treated with estradiol 24 hours earlier showed decreased DA in the portal blood (Cramer et a1., 1979). A slower turnover of DA, and decreased DA synthesis in the median eminence on the afternoon of proestrus as compared to estrus (Demarest 35 et a1., 1981) has also been observed which agrees with the results of DA in portal blood. No changes were observed in DA turnover during the estrous cycle by Homna and Wuttke (1980). Whether the change in TIDA neuronal activity, and therefore portal blood DA during proestrus is alone sufficient to account for the prolactin surge is not known. The diurnal and nocturnal surges of prolactin which take place during early pregnancy or after cervical stimulation in the rat appear to occur out of phase with changes in DA synthesis in the median eminence (Voogt and Carr, 1981; McKay et a1., 1982) and with the levels of DA in hypophysial portal blood (DeGreef and Neill, 1979), i.e., during the prolactin surges portal blood DA is low, in between the prolactin surges portal blood DA is high. This circadian pattern of TIDA neuronal activity appears to be independent of circulating prolactin levels (Demarest et a1., 1983), because the neuronal pattern persists even after the prolactin pattern is abolished by administered haloperidol, a DA receptor antagonist. The importance of DA in the development of these prolactin surges is questionable, however, because peripheral infusion of DA, to mimic the DA changes in portal blood which normally occur after cervical stimulation, did not result in nearly as large of a rise in prolactin compared to a normal cervical stimulation- induced prolactin surge (DeGreef and Neil, 1979). Therefore, although changes in hypophysial portal blood DA and TIDA synthesis are inversely correlated with the circadian prolactin surges of pseudopregnancy, DA is not solely responsible for these prolactin surges. Similar findings have been reported regarding hypophysial portal blood DA and the suckling- induced rise in prolactin in rats. In the rat, a decrease in portal DA by itself is not sufficient to account for the suckling-induced rise 36 in prolactin (Neill et a1., 1982). Thus, the above results imply that during physiological increases in prolactin secretion in the rat changes in DA in the portal blood, although occurring in the appropriate direction, are not of sufficient magnitude to account for the total prolactin response. This has led to the belief that even though DA seems indisputably to be PIF, its role is more important during the tonic or basal conditions of prolactin release than in physiologically stimulated prolactin release. It is interesting that in the estradiol-treated, stalk-transected monkey, DA infusion can account for most if not all of the inhibitory effect of the hypothalamus on prolactin release. Existence of other PIFs other than DA do not need to be postulated for the tonic suppression of prolactin release by the hypothalamus (Neill et a1., 1982). An important physiological mechanism demonstrating DA's role in the basal secretion of prolactin is that DA is involved in prolactin short- loop feedback. Systemic cu: intracerebroventricular injection of prolactin increases DA turnover (H8kfelt and Fuxe, 1972; Gudelsky et a1., 1976), DA synthesis (Johnston et a1., 1980), tyrosine hydroxylase activity (Nicholson et a1., 1980) and DA in the portal blood (Gudelsky and Porter, 1980). Anti-dopaminergic drugs, e.g., haloperidol, can also increase DA in the portal blood, an effect which appears to be mediated by the haloperidol-induced increase in circulating prolactin, since pre- treatment with prolactin antiserum greatly attenuates the DA response (Gudelsky and Porter, 1980). It appears, therefore, that prolactin may feed back on TIDA neurons in a positive fashion. Since DA inhibits prolactin at the AP level, this may be one mechanism by which prolactin regulates its own secretion. 37 There is good evidence that the basal secretion of prolactin progressively increases during aging in both ‘male and female rats (Meites et a1., 1984). Circulating levels of prolactin are highest in the oldest female rats which often exhibit prolactinrsecreting pituitary adenomas. It might be expected, because of the prolactin-TIDA neuronal short-loop feedback mechanism, that DA in the portal blood would be increased with age. However, the major stimulus for the age-related hyperprolactinemia is believed to be a decrease (not an increase) in TIDA neuronal activity in old rats. Both old males and females show reduced median eminence DA content (Gudelsky, 1981; Wilkes et a1., 1979), decreased DA turnover and synthesis (Simpkins et a1., 1977; Demarest et a1., 1982) and decreased DA in the portal blood (Gudelsky, 1981; Sarkar et a1., 1984a), as compared to young rats. It follows logically that prolactin-TIDA neuron short-100p feedback is impaired in old rats, as has recently been observed (Sarkar et al., 1983c). Therefore, the failure of TIDA neurons to respond to prolactin during aging may be of significance with regard to the development of hyperprolactinemia and prolactin-secreting tumors. E. Eggulation of Synthesis, Release and Inactivation of Dopamine The catecholamines, including dopamine are synthesized both in the central nervous system and the periphery from the amino acid precursor tyrosine, by a similar enzymatic sequence. Centrally, tyrosine is actively taken up in the neuron and converted to l-DOPA by tyrosine hydroxylase (Levitt et a1., 1965). Tyrosine hydroxylase catalyzes the rate-limiting step in the sequence, and requires molecular oxygen and a reduced pteridine as cofactors for its activity. l-DOPA is converted to 38 DA by the relatively non-specific ubiquitous enzyme aromatic l-amino acid decarboxylase (Carlsson et a1., 1972). The synthesis of DA takes place in the terminals of neurons as the uptake mechanism for tyrosine and both synthetic enzymes are present in synaptosomal fractions (C00per et a1., 1982). The steady-state concentration of DA in TIDA neurons is maintained relatively constant under most conditions. This is due, at least in part, to DA end-product feedback inhibition on tyrosine hydroxylase. When neuron activity increases, DA is released from the terminals and DA intraneuronal concentration temporarily falls. Along with the increase in neuronal activity, tyrosine hydroxylase appears to increase its affinity for tyrosine and the pteridine co-factor and. decrease its affinity for DA (Udenfriend et al., 1965; Costa and Neff, 1966). All of these changes in tyrosine hydroxylase increase its activity to replace the released DA. ‘With decreased neuronal activity, DA. binds to an allosteric site on tyrosine hydroxylase to decrease its activity (Cooper et a1., 1982). Catecholamines are stored within vescicles at the neuron teminals. The vescicles are synthesized in the perikarya and transported to the terminal via axonal flow (Dahlstrom et a1., 1973). The existence of two storage pools for DA has been proposed (Axelrod, 1974). There is a readily releasable pool which is closely associated with the internal synaptic membrane and is believed to consist of newly synthesized catecholamine. The other, a larger storage pool may be more distant from the synaptic. membrane and its function. is unknown. Neuron :hmpulses result in Ca++-dependent (DeRobertes and Vas Ferreira, 1957) release of newly synthesized DA (Kapin et a1., 1968). 39 Mesotelenecephalic DA neurons appear to have autoreceptors, which are activated by DA upon release of DA into the synaptic cleft. Activated autoreceptors can inhibit synthesis and possibly further release of DA from the neuron (Moore and Demarest, 1982). The regulation of the release from TIDA neurons is somewhat controversial. Evidence has been reported which suggests the lack of autoreceptors (Demarest and Moore, 1979a) or the presence of autoreceptors on TIDA neurons (Sarkar et al., 1983b). Autoreceptors on TIDA neurons were suggested as evidenced by the ability of DA and its receptor agonists to decrease the release of 3H-DA from median eminence (ME) tissue in vitro. Whether this action occurred via a receptor or by end-product feedback inhibition by the agonist after uptake into the neuron is in question. Nevertheless, 3H-DA agonist binding was demonstrated in homogenates of bovine ME tissue (Cronin et a1, 1978). Release of DA from TI neurons can be agumented by prolactin, a mechanism by which prolactin can regulate its release (Sec IV-D) (Perkins and Westfall, 1978; Demarest et a1., 1985b). Inactivation of DA, and the catecholamines in general, is thought to occur by at least two mechanisms: 1) reuptake back into the neuronal terminal, and 2) enzymatic degradation. The uptake of DA into TIDA neurons is also controversial. Sarkar et al. (1983c) showed that the uptake of 3H-DA into whole median eminence tissue was linear over time and temperature and was sodium dependent. It could be effectively blocked by the catecholamine reuptake inhibitor, nomifensine, and by non-radioactive DA. However, others have shown that 3H-DA uptake in neuronal homogenates is lower in median eminence as compared to the striatum (Demarest and Moore, 1979b). In favor of a reuptake mechanism in TIDA neurons is that nomifensine injected in vivo can increase DA 40 release into hypOphysial portal blood (Sarkar et al., 1983b) and decrease prolactin secretion (Cocchi et a1., 1979). The three major catabolic metabolites of DA in the central nervous system are dihyroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and to a much lesser extent methoxytyramine (MTA). Deamination occurs by the action of monoamine oxidase, which exists as a mitochondrial membrane bound enzyme (Nukada et a1., 1963), and requires reuptake of DA into the neuron terminal. The aldehyde is then further oxidized to the acid by aldehyde dehydrogenase. Extraneuronally, possibly of glial origin, DA can be 0-methylated by catechol-O-methyltransferase (COMT) (Alberici et a1., 1955; Axelrod, 1974). V. Relation of Estrogen to AP Hormone Secretion and Development of Pituitary Prolactinomas In many instances, the secretory function and cellular proliferation in endocrine glands are closely correlated. A. chronic increase in hormone secretion is usually accompanied by enhanced mitotic activity of that secretory cell type. With regard to prolactin secretion, there is an increase in cellular proliferation of the lactotrOphs in many cases of increased prolactin release; e.g., blockage of DA receptors (Jacobi and Lloyd, 1981; Kalberman et a1., 1980) or lesion of the MBH (Cronin et a1., 1982) which destroys TIDA neurons both of which enhance prolactin secretion and increase mitotic activity of the lactotrophs. Estrogen is a potent physiological stimulator of prolactin release (Meites, 1974) and results not only in lactotroph proliferation but also in the 41 development of prolactinomas (Furth and Clifton, 1966). In this section, the relation of physiological and pharmacological doses of estrogen to secretion and mitosis of prolactinrsecreting cells will be discussed. A. Estrogen Actions on the Anterior Pituitary Estrogen administration was first shown to enhance the pituitary prolactin content in rats and guinea pigs by Reece and Turner (1936). Since mammary gland stimulation was also observed, prolactin was assumed to be increased in the serum as well. Also, estrogen injected in vivo can enhance AP prolactin release in. vitro (Ratner et a1., 1963), suggesting estrogen can act directly on the AP to increase prolactin. After the development of radioimmunoassay, daily injections of estradiol benzoate were shown to increase serum prolactin concentrations in a dose-related fashion (Chen and Meites, 1970) although the largest pharmacological doses of estradiol did not elevate prolactin to the same extent of the lower optimal dose. Chronic estrogen treatment (at relatively high doses) can inhibit the secretion of LH, FSH, and thyrotropin TSH release from the AP, and these same doses can increase the release of prolactin and possibly GH (Meites, 1974). 1. In vitro stimulation of prolactin Estradiol, but not testosterone, progesterone, cortisol, or corticosterone, added the incubation media of AP tissue cultures increases the release of prolactin into the media (Nicoll and Meites, 1962). Estrogen also causes prolactin release in primary cultures of AP cells (Labrie et a1., 1980; West and Dannies, 1980). However, estrogen's ability to promote prolactin secretion in vitro is rather modest 42 compared to the large response observed in vivo. Two findings appear to explain this discrepancy: l) estradiol can almost completely reverse the inhibition of prolactin release by DA or DA agonists, and 2) estradiol can also sensitize the lactotrophs to the prolactin-releasing action of TRH. The DA agonist bromocryptine maximally inhibited (70%) prolactin secretion in primary AP cell cultures (Labrie et a1., 1983). However, maximal inhibition of prolactin by bromocryptine observed after preincubation with estradiol was only 202. It has been suggested that estrogen can exert an ”anti-dopaminergic”-like action. However, the potent anti-dOpaminergic effect of estrogen cannot be explained by an estradiol-induced decreased number or affinity of DA receptors (Labrie et a1., 1983), suggesting this is a post-receptor phenomenon. Furthermore, estrogens can increase the number of TRH binding sites on AP cells in culture (Gershengorn, 1979) and this action temporarily parallels estrogen's ability to sensitize the prolactin response to TRH (DeLean et a1., 1977). Although estradiol can cause a specific release of prolactin, the mechanism of release is not well understood. It is thought that under basal conditions of release in vitro, the most recent synthesized prolactin is preferentially released (Walker and Farquhar, 1980). Estradiol is known to increase the synthesis of prolacin (Mauer and Gorski, 1977; Stone et a1., 1977) at least in part by increasing transcription of prolactin 'messenger RNA (Mauer, 1982; Schull and Gorski, 1985). 2. In vivo stimulation of prolactin Painting of the skin of mice at twice weekly intervals with estrin preparations called "alpha-folliculin" or keto-hydroxy estrin and the 43 observation of mammary and pituitary tumor develOpment was the first report to establish that the carcinogenic effect of any substance was restricted to tissue remote from the site of application of the cancer-causing agent (Cramer and Horning, 1936). In this report, hypopituitarism and strain differences in the susceptibility to estrogen was also observed (Cramer and Horning, 1936; McEwen et a1., 1936). Later, it was found that the F344 rat is particularly susceptible to estrogen at the pituitary level and stilbesterol-induced pituitary adenomas up to 500 mg in weight have been observed (Dunning et a1., 1947). As is true with most experimental neoplasms induced by hormone imbalances, the estrogen-induced pituitary adenoma is initially only capable of growth in animals with the same hormone imbalance required for tumor induction (Furth et a1., 1960). However, ultimately these tumors will produce autonomous variants capable of growth in a usual endocrine environment. Normally, primary estrogen-induced tumors are dependent on elevated estradiol levels for their growth, but can become autonomous after one or two passages of the pituitary tissue (Furth and Clifton, 1966). Whether the change in estrogen sensitivity of these tumors with time is an exclusive characteristic of the autonomous cells or is just an adaptive phenomenon acquired gradually is not known (Clifton and Furth, 1961; Furth, 1969). Estrogen-induced pituitary adenomas secrete copious amounts of prolactin (Meyer and Clifton, 1956) and contain hyperplastic and hypertrophic lactotrophs (Gersten and Baker, 1970). Somatotrophin release is also moderately increased with chronic estradiol treatment (Lloyd et a1., 1972; Gottschall et a1., 1986). However, after estradiol is removed, GH, but not prolactin, returned to control levels (Gottschall et a1., 1985). Chronic estrogen 44 can decrease circulating levels of TSH, thyroxin and triiodothyronine, LH, and FSH (Nakagawa et a1., 1980). In vivo, chronic estradiol dramatically increases prolactin synthesis (Wiklund et a1., 1981) and this appears to be due to an increase in prolactin mRNA synthesis (Mauer, 1982) and turnover (De Nicola et a1., 1978) and increased DNA synthesis (De Nicola et a1., 1978). Therefore, estradiol administration appears to specifically augment prolactin release and synthesis in addition to increasing DNA synthesis and mitotic activity of the lactotrophs. 3. Interaction of estrogen and dopamine on lactotrophic mitotic activity and prolactin secretion As mentioned earlier, the proliferation of endocrine cells appears to be related to the cell's secretory response. During various states of chronic prolactin hypersecretion, there is an increase in mitotic activity of AP cells. Lactating rabbits which maintain high levels of prolactin for extended periods, exhibit increased AP mitoses (Allanson et a1., 1969). Administration of estrogens (Hunt, 1947) in a single dose (Lloyd et a1., 1972; Lloyd et a1., 1973) or maintaining high estradiol levels by implanting an estrogen-containing pellet (Kalberman et a1., 1979) increased AP mitotic activity and DNA synthesis. In each of these studies, prolactin concentrations in the serum were markedly enhanced. Furthermore, estrogen can increase DNA polymerase (Mastro and Hymer, 1973) and thymidine kinase activity (Valotaire et a1., 1975) in the AP, both indicative of enhanced DNA synthesis. Dopaminergic receptor agonists such as bromocryptine inhibit estrogen-induced prolactin secretion (Lu et a1., 1971) from normal and tumorous APs (Quadri et a1., 45 1972). By inhibiting the release of prolactin, bromocryptine can also decrease the stimulation of DNA synthesis and mitotic activity as a result of estrogen treatment (Lloyd et a1., 1975; Kalberman et a1., 1980). The DA antagonist sulpiride injected daily during estrogen treatment was able to stimulate larger APs, prolactin secretion, and DNA synthesis when compared to rats treated only with estrogen (Jahn et a1., 1982). This action of sulpiride was interesting in that acute injection of sulpiride was only effective in raising prolactin during early estrogen treatment (7 and 21 days) and was completely ineffective in increasing prolactin secretion at 45 days of estrogen treatment. Moreover, antagonists injected in vivo increased AP DNA synthesis and decreased AP prolactin content even in the absence of estrogen (Jacobi and Lloyd, 1981). Therefore, the mitotic activity of the lactotrOphs can be augmented by substances which increase prolactin secretion, i.e., estrogen and DA antagonists, and decreased DNA synthesis is observed when prolactin secretion is low, i.e., DA agonists. This close coupling of secretion and mitosis of the lactotrophs may be important when hypothalamic mechanisms controlling prolactin release do not function normally. Other factors may contribute in altering prolactin secretion and mitosis of the lactotrophs after chronic estradiol treatment. In F344 rats, estrogen-induced prolactinomas were shown to develop a direct arterial blood supply to the tumor which may dilute hypothalamic factors reaching the AP via the portal circulation, and thereby reduce their influence on prolactin secretion (Elias and Weiner, 1984). After stilbestrol treatment in F344 rats, the lactotrophs appear in two distinct pOpulations based upon gradient sedimentation which may vary in 46 their sensitivity to estrogen or hypothalamic factors (Phelps and Hymer, 1983). Also, when an AP is transplanted under the kidney capsule preceding estrogen treatment, the in situ AP exhibited greater growth and tumor development than the transplant (Welsch et a1., 1971). This would suggest a positive hypothalamic influence on lactotroph proliferation as a: result of estrogen and indeed, increased prolactin- releasing activity of hypothalamic extracts was observed after chronic estrogen treatment (Ratner and Meites, 1964; Nakagawa et a1., 1980). Chronic administration of GnRH agonists (Lamberts et a1., 1981) or GnRH antagonists (De Quijada et a1., 1983) can inhibit growth of estrogen-induced transplantable prolactin secreting 'tumors. Tumor inhibition by GnRH analogs appears to be mediated by chemical castration as evidenced by lowered estradiol levels and atrophy of the ovaries and the uterus. Ovariectomy or administration of the anti-estrogen tamoxifen can also inhibit pituitary tumor growth (Lamberts, 1984). B. Anatomy and PathOphysiology of Estroggn-Induced Prolactinomas The percentage of AP cell types in the normal adult female rat consists of about 302 lactotrophs, 302 somatotrophs, 151 gonadotrophs, 152 unidentified (or undifferentiated) chromOphobic cells with the remainder consisting of thyrotrophs, corticotrophs and unclassified cells (Takahashi and Kawashima, 1982). Estradiol treatment results in profound changes; most dramatic is a marked increase in the number of lactotrophs. Estradiol implantation into the pars distalis resulted in hypertrophied and hyperplastic lactotrophs (Gersten and Baker, 1970). Ultrastructurally, the lactotrophs contained extensive development of the rough endoplasmic reticulum arranged in concentric whorls (Shiino 47 and Rennels, 1975), an increased Golgi region volume density and a smaller number and size of secretory granules (Mc Comb et a1., 1981). All of these cellular alterations are indicative of increased synthesis and secretion of prolactin and an increased mitotic activity of the lactotrOphs. Continued estrogen treatment results in compression of the pituitary sinusoids and thickening of the sinusoid walls due to the increase in lactotroph cell number. Further estrogen administration results in a gland which is nodular in appearance with marked sinusoid compression, and occasionally open sinuses which appear as blood lakes (Clifton and Meyer, 1956). The mean weight of these Wister rat APs was 172 mg after 230 days of estrogen treatment, compared to 10 mg control AP. As mentioned earier, the F344 strain is particularly susceptible to the AP hyperplastic effect of estrogen (Dunning et a1., 1947). After only 60 days of treatment with stilbesterol, almost 602 of total AP cells were immunocytochemically identified as lactotrophs (Phelps and Hymer, 1983). This is certainly a low estimate (because many of the lactotrophs may have lacked sufficient granules to stain immunocytochemically) since the total number of cells in the AP increased 11002. When chronic estrogen treatment is discontinued, the number and size of secretory granules in the lactotrophs increased dramatically, and secondary lysozomes were present (Shiino and Rennels, 1975). The glands, although still hypertrophic, lost the nodular appearance and returned to a more normal shape (Treip, 1983). The question of whether primary estradiol-induced multiplication of lactotrOphs is a true. neoplasia or just hyperplasia has not been resolved, but the current belief is that it is a hyperplasia. Estrogen treatment produces an immediate and dramatic reduction on somatic 48 growth. This is more apparent in male than female (which exhibit a growth stasis) rats. The mechanism responsible for the estrogen-induced dimunition of growth appears to be complex. Estradiol can reduce dietary intake (Josimovich et a1., 1967) and depress levels of an insulin-like growth factor and carrier protein even in the presence of normal or elevated growth hormone levels (Drazmin et a1., 1979). In the male, estradiol may suppress anabolic steroid production by inhibiting gonadotrophin release from the AP (Attardi et a1., 1980). The induction of hyperprolactinemia after high doses of estrogen to male rats is associated with reduced circulating TSH, LH and FSH levels (Smythe et a1., 1981), although in female rats GH may be elevated (Gottschall et a1., 1986). Hyperprolactinemia also increases adrenal weight and corticosterone levels (Gottschall and Meites, unpublished). Estradiol-induced hyperprolactinemia produces marked mammary gland development (even in the absence of the adrenal), the uteri are usually hypertrophic and the ovaries are smaller than normal. In the rat, hyperprolactinemia results in a puzzling renal proximal tubular degeneration with polyuria and proteinuria (Furth and Clifton, 1966). VI. Estrogen Influence on the Central Nervous System The view that steroid hormones can influence the brain and behavior have been held for many years. The experiments of Berthold (1849), on the castration effects on sexual and aggressive behavior in the rooster, and Brown-Sequard and D'Arsonval (1891) work on the effects of testicular extracts on rejuvenation processes in the aged human, demonstrate the long~held belief that the gonads affect behavior. 49 However, it has not been until the last three decades that information regarding the mechanism(s) and site of action of steroids has been achieved. Brain implants of crystalline steroid were used to localize brain regions where steroids may be acting (Harris et a1., 1958; Kent and Liberman, 1949). Technological advances allowed for measurements of the affinity and number of steroid receptors (Jensen and Jacobson, 1962) and the receptors could be specifically localized by autoradiography (McEwen et a1., 1972). These tools made possible the identification of specific brain sites where steroids act to modify or even induce particular physiology and/or behavior. Many studies dealing with steroids throughout the sixties and seventies were concerned with steroid modulation of neuroendocrine reproduction function and behavior. This section will focus on the location of the sites and mechanism of action of steroids in the brain, and more importantly, how chronic administration of estradiol can have profound permanent actions on reproductive and other neuroendocrine processes. A. Distribution of Estrogen Target Sites in the Brain Much of the work on steroid-concentrating sites in the brain has used the rat as a model. The map of estrogen-concentrating cells in the rat brain obtained by autoradiography after injection of 3H-estradiol has revealed estrophilic perikarya in the hypothalamic hypophysiotropic area and also the corticomedial amygdala (Leiberburg and McEwen, 1977). Fewer and less intensive 3H-estradiol was found in nuclei of cells of the midbrain central gray and hippocampus (Pfaff and Keiner, 1973). There is little sex difference in 3H-estradiol uptake, i.e., short-term castrated males exhibit almost an identical pattern of uptake as do 50 short-term castrated females (McEwen, 1981). When radioactivity ‘was quantitated after extraction from nuclear fractions of brain tissue, the AP showed the greatest uptake followed by the pre-optic area, the medic-basal hypothalamus, the corticomedial amygdala, and the rest of the hypothalamus (McEwen, 1981). The percent of total perikarya containing estradiol in specific areas of the hypothalamus has also been analyzed by autoradiography (Morrell and Pfaff, 1983). The pre-Optic area, ventromedial nucleus, arcuate nucleus, and medial amygdaloid nucleus contain 24, 29, 21 and 402 respectively, estrogen-concentrating cells relative to the total number of perikarya in these areas. This uptake was shown to be saturable and an active selective process in a particular sub pOpulation of neurons. B. Effect of Estrogen on Neurons and Neurotransmitter Systems Steroids in general, and estradiol specifically, easily traverse the cell membrane because of their lipOphilic nature, and classically, were believed to bind to cytosolic receptors in specific target cells. This receptor-estrogen complex is then translocated across the nuclear membrane after ”activation" originally described as an increase in affinity of the receptor for polyanions (Higgins et al., 197; Milgrom et a1., 1973). The activated estrogen-receptor complex then interacts with the genome, possibly by altering promoter elements which control initiation of transcription, resulting in the synthesis of steroid regulated proteins (Chambon et a1., 1984). Although for many years most work. has suggested a cytosolic intracellular population, of steroid receptors, some recent evidence has postulated that this receptor, whether bound to steroid or not, actually resides in the nucleus 51 (Gorski, 1985). Recently, steroids have been demonstrated to have non-genomic mediated effects that are of very short latency (Szego, 1984). 1. Genomic, transcription-translation mediated effects There are only a few instances where steroid hormone action has been shown to result in direct genomic activation in the brain. Estradiol administration can transiently activate hypothalamic cell nuclear RNA polymerase II activity (Kelner et a1., 1980). Unlike the uterus, the brain activation lacks the sustained activation of RNA polymerase II and does not alter RNA polymerase I activity. This may be because of the absence of estrogen-induced hypertrOphy and hyperplasia in the brain. Indirect demonstrations of genomic involvement after estrogen admin- istration include the ability of the RNA synthesis inhibitor, actinomycin D, to block estrogen action on female sexual behavior (Quadagno et a1., 1971; Terkel et a1., 1973), and the preovulatory LH surge (Jackson, 1972; Kalra, 1975). The major end result of genomic activation by estrogens is the production of enzymes and other cell proteins believed to be the substances which manifest hormone action. The importance of these products is shown by their ability to 1) alter neuronal transmission, synthesis or plasticity, 2) produce stabilizing factors like nerve growth factor, 3) result in programmed cell loss, or 4) activate, de-activate or modulate pre- or post-synaptic receptor populations. There are an abundant number of examples which illustrate estrogen- induced alteration of neuronal function by transcription products. Due to the diversity of estrogen's effects, an important qualification in 52 estrogen-mediated action is that they are primary, i.e., a direct effect of estrogen. on. a particular ‘neuron, or secondary, i.e., an effect mediated by the estrogen-induced release of a hormone or neuro- transmitter (McEwen, 1981). Examples of the genomic-action of steroids will be given in Sec. VI C and D. 2. Effects which may operate on neural membranes Some actions of steroids are of short latency, too short to be acting via transcription-translation. In some instances, these short-lateny events have been reproduced on isolated synaptosomes or membranes. The best example of a short-latency action is the alteration in. diencephalon neuronal. discharge rates within. seconds after' 17-8- estradiol iontophoresis (Kelly et a1., 1977). No effect was observed after 17-a-estradiol administration. The importance of these short-latency effects as a major means of influencing neuroendocrine function has not been determined. C. Estradiol-induced Adult Anovulatory Syndrome Elevated estrogens, administered exogenously or endogenous high circulating levels result in a well-characterized anovulatory state in the female rodent that has been postulated as a model for aging (Finch et a1., 1984). A single injection of a large dose of estradiol benzoate (Brown-Grant, 1974) or estradiol valerate (Brawer et a1., 1978), or exposure to constant light (Brawer et a1., 1980) causes the cessation of regular estrous cycles in female rats and a rapid onset of persistent vaginal cornification. The ovary contains many large follicles which produce substantial amounts of estradiol and can maintain estradiol 53 levels of 30 pg/ml, the upper limit of the physiological range. This estradiol level can be sustained for up to six months or longer. The potential action of chronic estradiol administration on the hypothalamus was first suggested by observations of hypothalamic neuroanatomy after multiple monthly injections of estradiol valerate (Brawer and Sonnenschein, 1976). Histologically, the arcuate nucleus contained multiple pathological foci characterized by degenerating axons and dendrites. These foci were most apparent in the lateral regions of the nucleus. The neuronal degeneration was associated with reactive microglia containing cellular debris and reactive astrocytes that contained many pools of electron dense material and large bundles of fine filaments. The microglial response is a long-established marker for neuronal degeneration present in diseases where cell death is a well-known phenomenon (Bernheimer et a1., 1973). Interestingly, the arcuate nucleus pathology progressed with the same time course, intensity and distribution in rats given monthly injections or a single injection of estradiol valerate (Brawer and Finch, 1983). The lesion appears to be an estrogen-ovarian dependent phenomenon, since OVX prior to a single injection of estradiol or placement in constant light, does not result in the pathology (Brawer et a1., 1980). However, chronically elevated estradiol in ovariectomized rats by multiple injections can produce the arcuate nucleus lesion. D. Estrogen and Hypothalamic Mechanisms which Regulate Prolactin Release Recently, it has been shown using autoradiography that arcuate 54 nucleus neurons containing immunoreactive tyrosine hydroxylase are the major neurotransmitter class of perikarya of this nucleus that concentrate 3H-estradiol. These cell bodies are well established to belong to TIDA neurons. This important work shows that estradiol has at least the capability of directly affecting a final common pathway involved in regulating prolactin release; ie. TIDA neurons. 1. Tuberoinfundibular dapaminergic neurons A substantial amount of evidence accumulated over the last five years has demonstrated that chronic treatment of male or female rats with estradiol attenuates TIDA neuronal activity. The suggestion was apparent in an early piece of work which indicated that injection of rats with estradiol for ten. days depleted the hypothalamus of PIF (Ratner and Meites, 1964). Subsequently, the literature became quite difficult to interpret, because of the variety of estradiol-treated models, the methodological approaches used to evaluate TIDA neuronal activity, along with the complexity of action estradiol on these neurons. Sub-acute treatment of ovariectomized rats with estradiol (24 hrs) suppresses the concentration of DA in the portal blood (Cramer et a1., 1979) and may be related to the decline of portal blood DA on the day of proestrus during the estrous cycle (Ben-Jonathan et a1., 1977). Interestingly, the mechanism by 'which short-term estrogen treatment suppresses the release of DA into hypophysial portal blood may involve the action of a metabolite of estradiol. 2-hydroxyestradiol, a catechol estrogen, but not estradiol, can inhibit the activity of tyrosine hydroxylase under in vitro conditions (Lloyd and Weisz, 1978; Lloyd and 55 Ebersole, 1980; Foreman and Porter, 1980). Since synthesis of DA is directly related to it's release, inhibition of tyrosine hysroxylase by 2-hydroxyestradiol may account for the decrease of DA into portal blood after short-term estradiol. Longer treatment with estradiol (2-7 days) leads to an elevation in portal blood DA (Gudelsky et a1., 1981), increased turnover (Eikenburg et a1., 1977) and synthesis (Demarest et al,. 1984) of DA, and increased release of 3H-DA. during electrical stimulation. of ME tissue after accumulation of 3-DA (Gottschall and Meites, submitted). This augmented TIDA neuronal activity after longer estradiol treatment is a secondary action of the steroid; i.e., it is mediated via the hypothalamic and AP estradiol-induced release of prolactin. Hypophysectomy prior to estradiol administration did not result in any change in DA turnover (Eikenburg et a1., 1977). Also, methods that increase circulating levels of prolactin, i.e., injection of bovine prolactin, transplantation of an AP under the kidney capsule or injection of drugs that enhance prolactin secretion, stimulate the release of DA into the portal blood (Gudelsky and Porter, 1980) and increase the turnover of DA in the ME (Hokfelt and Fuxe, 1980; Gudelsky et a1., 1976; Gudelsky and Moore, 1977). Since estradiol exerts anti-dopaminergic effects on the lactotrOphs of the AP (Lu et a1., 1971; Raymond et a1., 1978), elevated prolactin as a result of these treatments may augment the release of DA into the portal blood in an attempt to compensated for the anti-dopaminergic action of estradiol. The first direct evidence that chronic estrogen treatment could reduce TIDA neuronal activity was that two weeks of estradiol treatment markedly decreased ME DA content and produced a substantial, but 56 non-significant, decline in mean DA turnover in the ME (Smythe and Brandstater, 1980; Dupont et a1., 1981). The magnitude of the estradiol-induced hyperprolactinemia was related to the degree of reduction in ME content of DA (Smythe and Brandstater, 1980), which raised the possibility that the loss of DA content was due to the action of prolactin and not to estradiol directly. Furthermore, it was shown by pharmacological means, that there was a loss of TIDA neuronal control of prolactin secretion in animals which were hyperprolactinemic after chronic estrogen treatment (Smythe et a1., 1982; Casaneuva. et a1., 1982). Sarkar et a1., (1982) observed an abnormal histofluorescence of dopaminergic neurons in the arcuate nucleus and ME of female rats bearing estrogen-induced prolactinomas as indicated by the presence of distorted fibers and deposits of punctate autofluorescent material. The suggestion was made that chronic hyperprolactinemia could cause degeneration of TIDA neurons, since this pathology was also seen in old hyperprolactinemic rats and in those made hyperprolactinemic by implanting a transplantable prolactinoma. Whether or not estrogen and/or prolactin is neurotoxic to TIDA neurons has not been verified or confirmed. However, there is clearly a decrease in the function of TIDA neurons after long-term estradiol treatment. Chronic estrogen can diminish DA levels in portal plasma (Sarkar et al., 1984a), decrease the synthesis and turnover of DA (Demarest et a1., 1984; Morgan et a1., 1985; Terry et a1., 1985), decrease medial basal hypothalamic tyrosine hydroxylase activity (Luine et a1., 1977), attenuate AP DA content (DiPaoLa et a1., 1985), reduce the electrically stimulated release of 3--DA from ME tissue (Gottschall et a1., 1986), and alter the serum prolactin response to central-acting dapaminergic drugs (Casaneuva et 57 a1., 1982; Willoughby et a1., 1983; Gottschall et a1., 1986). The estradiol-induced decline in TIDA neuronal function occurs at a time when monoamine oxidase activity is low, since four weeks of estradiol treatment results in. a 30% decrease in. monoamine oxidase (type A) activity in the medial basal hypothalamus (Luine et a1., 1977). The implication for the estrogen-induced loss of TIDA neuronal control of prolactin release is that the loss of the ability of prolactin to regulate it's own secretion in a negative feedback fashion may play a role in the later hypersecretion of prolactin after estrogen treatment and be involved in the development of prolactin secreting tumors. Moreover, if estradiol resulted in a loss of TIDA control of prolactin release that was permanent, it would clearly indicate a central role in the deve10pment and/or growth of prolactinomas. Microprolactinomas are relatively common in humans with large adenomas occurring less frequently' compared. to rats (Frantz, 1984). These were initially recognized by pituitary enlargement (Forbes et a1., 1954), and those without acromegalic symptoms were classified as ”functionless" tumors of the pituitary. However, elevated serum prolactin levels were demonstrated initially with bioassay (Canfield and Bates, 1965) and later by radioimmunoassay (Frantz et a1., 1972; Franks et a1., 1977) in patients bearing ”functionless" tumors. The hyperprolactinemia is responsible for symptoms of amenorrhea and galactorrhea in. many of these patients. The two most often used therapies for these tumors are treatment with a dOpaminergic agonist or surgery (Barbieri and Ryan, 1983). The observation that a majority of women with prolactin-secreting tumors developed their symptoms during or after oral contraceptive use suggests that exogenous estrogens may play 58 a role in the pathogenesis of these tumors in humans as well as in rats (Shearman and Fraser, 1977; Jaquet et a1., 1978; Schlechte et a1., 1980). However, other studies have found no correlation between the estrogen-containing oral contraceptives and the development of prolactinomas (Hulting et a1., 1983; The Pituitary Adenoma Study Group, 1983). Therefore, this question remains. There is clear evidence that exogenous estrogens and the use of oral contraceptives elevates serum prolactin levels (Hagen et a1., 1983). Interestingly, it appears that many hyperprolactinemic subjects have lost the central dopaminergic inhibition of prolactin secretion, as evidenced by an abnormal (attenuated) response pattern to central acting dopamninergic drugs. All of these same subjects can respond to a dopaminergic agonist acting at the level of the AP (Fine and Frohman, 1978; Muller et a1., 1978; Sekiya et a1., 1985). 2. Other neurotransmitter/neuropeptide systems affecting prolactin release There is sparse information regarding other neurotransmitter function after chronic estradiol treatment. Chronic estradiol adminstration in vivo increased release of TRH from the hypothalaus incubated in vitro (Franks et a1., 1984) and also decreased the content of VIP in the medial basal hypothalamus (Malletti et a1., 1982). Estrogen given for three days decreased glutamic acid decarboxylase activity in the arcuate nucleus and anterior hypothalamic areas (Wallis and Luttge, 1980). Administration of serotonergic receptor agonists, zimelidine and quipazine, in combination. with estradiol doubled AP weight compared to animals treated with estradiol alone. However, the 59 S-HT precursor, S-HTP, did not increase, or the synthesis inhibitor PCPA did not decrease the estradiol-induced increase in AP weight (Walker and Cooper, 1985). MATERIALS AND METHODS I. Animals, Treatments and Blood Collections Adult female Fischer 344 rats, an inbred strain, were used in all of the experiments. The animals weighed 160-180g each upon arrival and were housed in our animal facility for at least two weeks prior to experimentation. The rats were kept in temperature (25°C) and light (14 h light/10 h dark) controlled rooms. Ralston Purina Rat Chow (Ralston Purina Rat Chow (Ralston Purina Co., St. Louis MO) or Teklad Rat Chow (Harlan-Sprague-Dawley, Winfield IA) and tap water were provided e_d_ libitum throughout the housing and experimental periods. In each experiment, the rats were bilaterally ovariectomized prior to any treatment. At various periods after ovariectomy, animals were implanted with an empty or an estradiol-17-B (E2)-containing Silastic capsule 10 mm in length (Dow Corning, Midland MI). The E2 capsules were packed with 5-6 mg of crystalline E2 (Sigma Chemical Co., St. Louis MO). The inner diameter of the capsules was 0.078” and the outer diamter was 0.125" and the ends were sealed with Silastic Medical Grade Elastomer using a stannous octoate catalyst (Dow Corning, Midland MI). Capsules were implanted subcutaneously on the dorsal side of the animal about 5 cm posterior from the skull. The length of E2 administration and the duration of the withdrawal period after removal of E2 are stated in the Materials and Methods section of each experiment. Drugs were administered by several different routes as stated in each Materials and 60 61 Methods section. Blood was collected by decapitation, by orbital sinus puncture under light ether anesthesia or chronically from a cannula in the right atrium. Cardia cannulae were made from Silastic tubing having an inside diameter of 0.025" and an outside diameter of 0.047". The length of the tubing from the silastic pad to the bevelled tip was 25-28 mm varying according to the size of the animal at the time of surgery. The saline-filled cannula was inserted into the right atrium of ether anesthetized animals via a small incision in the right external jugular vein (after the distal end of the jugular was tied off). The cannula was secured in place by suturing above and below the stabilization pad. The free end was passed underneath the skin and exited about 1 cm posterior to the base of the skull. The cannula were flushed with heparinized saline and tied. Rats were housed individually and allowed to recover for at least 2 days. On the day of the experiment, animals were moved to a room for the blood collection, and silastic tubing extentions with syringes were attached to the cannula. After a minimum two hour adaptation period, drug injections were given and samples were withdrawn into heparinized syringes from freely moving, non-disturbed animals. The samples were immediately centrifuged, plasma separated and stored at -20°C until assay. The erythrocytes were resuspended in equal volume amounts of sterile saline and reinjected into the animal after withdrawal of the next sample. Blood collected from animals by decapitation or orbital sinus puncture was allowed to clot overnight at 4°C and serum was separated and frozen at -20°C until assayed. 62 II. Radioimmunoassay of Hormones Serum and plasma prolactin, GH and LH were measured by radioimmunoassay (RIA). These assays were performed using RIA kits from the National Pituitary Agency of the NIADDK, except for rabbit anti-rat prolactin which was provided by Dr. C.L. Chen (University of Florida, Gainesville, FL). Bound hormone was separated from free hormone by IgSORB (Enzyme Center, Boston MA). Samples were assayed in duplicate and only those volumes which gave hormone values corresponding to the linear portion of the standard curve were used. Serum and plasma hormone concentrations were expressed in terms of NIADDK rPRL-RP-B, rGH-RP-l, and rLH-RP-l. The 502 bound dose and the minimum detectable dose for the prolactin, GH and LH RIAs were 0.9 ng, 0.8 ng, 5.9 ng and 0.09 ng, 0.05 ng, and 0.33 ng/tube, respectively. The coefficients of variation for each asay will be discussed in detail in Exp. IV of the "Experimental" section. In some of the experiments AP DNA content was measured according to Burton (1956). III.Methods of Evaluating Tuberoinfundibular Dopaminergic Neuronal Activity A. In Vitro Superfusion of Median Eminence Tissue After Accumulation of 28-Dopamine After decapitation, the brain was quickly removed and placed on ice under the dissecting microscOpe. A few drops of ice cold Krebs-Henseleit buffer (see below) were placed on top of the hypothalamus, the median 63 eminence (ME) was visualized and dissected using a fine iris scissors according to the method of Cuello et a1. (1973). The length and width of the ME tissue block were approximately 1.5 and 0.6 mm, respectively, and the ME weight was < 0.3 mg. It contained a small piece of pituitary stalk. MEs were kept on ice (< 5 min) until ready for accumulation. Four rats were used for each experiment (per day), two MEs for each experimental group. Tissues of ME were placed in 10 x 75 mm glass tubes (2/tube) containing Krebs-Henseleit buffer. The tissues were pre-incubated for 5 min at 37°C under constant oxygenation. The tissues were then transferred to one ml of buffer containing 0.36 uM 3'H-DA (Amersham, Chicago IL) and 0.1 uM desipramine (Sigma Chemical Co., St. Louis MO), and incubated for 20 min. The tissues were then rinsed with buffer and transferred to a superfusion apparatus similar to that described by Aceves and Cuello (1981), except that the inner chamber was smaller and had a narrow central tunnel 2 mm in diameter and length. The tubing used was polyethylene except in the pump where Silastic was used. The dead space in the tubing between the buffer reservoir and the chamber was 500 pl, and between the chamber and fraction collector was 100 p1. Superfusion was carried out by a peristaltic pump (STA, Buchler Instruments, Ft. Lee, NJ), at a rate of 300 Ln/min. Superfusion medium "33 pre-warmed at 37°C under continuous oxygenation (952 02, 52 C02) and samples were collected at 2 min intervals using a Golden Retriever Fraction Collector (Instrument Specialty Co., Quincy MA). Electrical stimulation was used to induce release of 3H-DA from the median eminence tissue. Stimuli (bi-phasic square wave pulse for 15 sec, 6 mA magnitude 2 msec duration, at 20 hz) were applied by two silver electrodes and generated by two stimulators (Grass SD9, Grass 64 Instruments, Quincy MA), and were monitored on a calibrated oscilloscope (Type 564 storage oscilloscope, Tektronics Inc., Portland OR). At the end of the superfusion, MEs were homogenized in 0.5 ml of 0.1 N HCl. Radioactivity was determined in 400 pl aliquots of the superfusate samples and 250 pl aliquots of the homogenized tissue sample. Tritium was counted using 10 ml of aqueous counting scintillant (Amersham Corp., Arlington Heights IL)) in a Beckman LS-100 liquid scintillation counter (Beckman Instruments, Palo Alto CA). The release of tritium was expressed as a fractional rate constant (FRC) per min which was calculated by dividing the amount released and double the amount of tritium content in the tissue at the start of the respective two minute period (Jaffe and Cuello, 1980). In all experiments, a 40 or 50 min period of spontaneous release was observed before release was steady. Electrical stimulation was applied for 15 sec either 1:30 or 1:45 sec after the beginning of a fraction. This allowed for stimulation-evoked release of tritium to be observed in two fractions after stimulation. Thus, the stimulation evoked release was calculated by subtracting the radioactivity in the two fractions before stimulation (baseline area) from the two fractions after stimulation (peak area). Under these conditions, the accumulation of 3H-DA was temperature, time and Na+ dependent and reduced by unlabelled DA. The release of 3H after electrical stimulation was magnitude and frequency dependent, almost completely blocked in Ca+ free media and partially blocked in the absence of Na+ (Sarkar et a1., 1983). When TIDA neuronal ”activity” or ”function" are used in this text, it only refers to the ability of ME tissue to release radioactivity during electrecal stimulation after uptake of 3H-DA. Since this method measures only 65 release from TIDA neuronal terminals, it's relationship to in vivo action potential spike frequency is not known. 1. Materials [7,8-3H]-DA (specific activity 41, 43 or 55 Ci/mmol) was obtained from Amersham Corp. (Arlington Heights, IL). Krebs-Henseleit medium contained gelatin (0.12), ethylenediamine tetra acetic acid disodium salt (EDTA; 27 pM), ascorbic acid (130 pM), nialamide (Sigma Chemical Co., St. Louis MO; 12.5 pM), NaCl (134 mM), CaCl2 (2 mM), KCl (5 mM), I'IHZPO4 (1.25 mM), NaHCO3 (25 mM), MgSOa (1 mM) and glucose (10 mM), saturated with 52 C02, 952 02, pH 7.4. Medium was prepared fresh daily and the protocol for preparation is given in Appendix I. B. Assessment of Prolactin Response After AdministerinLCentrel-Acting Dopaminergic Drgge The ability to evaluate TIDA neuronal function by neuropharmaco- logical means is based on the observation that the "post-synaptic" biological action of TIDA neurons is a readily measurable event, i.e., the release of prolactin into the peripheral circulation. Consequently, assuming the lactotrophs have equal sensitivity to the dopaminergic inhibition of prolactin ’release, then central acting dOpaminergic or anti-dopaminergic drugs should result in release (or inhibition of release) in direct proportion to the activity of TIDA neurons. For example, if TIDA neuronal activity is higher than normal, administration of morphine which inhibits TIDA neuronal activity results in a large release of prolactin into the blood. Conversely, if TIDA neuronal activity is low, administration of nomifensine, a drug which inhibits DA neuron reuptake, will cause only a small inhibition of prolactin 66 release. The drugs used to evaluate TIDA neuronal activity by plasma prolactin responses were apomorphine hydrochloride (APO), a DA receptor agonist (Sigma Chemical Co., St. Louis MO), haloperidol (HALO), a DA receptor antagonist (McNeil Laboratories, Ft. Washington, PA), morphine sulfate (MOR), a drug which inhibits central TIDA neuron turnover (Mallenkrodt Labs, St. Louis MO), and nomifensine maleate (NOM), a catecholaminergic reuptake inhibitor (Hoechst-Roussel, Somerville NJ). IV. Catecholamine Assay The neuronal tissue in which catecholamines were measured was the neurointermediate lobe. Neurointermediate lobes were dissected and immediately sonicated in 250 pl of 0.1 N perchloric acid containing 5 mM glulathione. The homogenate was centrifuged and the supernatant stored frozen at -40°C until assay. The catecholamine assay was a modification of the radioenzymatic method of Cheng and Wooten (1980). Briefly, 50 pl samples were incubated with buffered COMT and 3 H-S-adenosyl methionine, a methyl donor (ICN Radiochemicals, Irvine CA) in a total volume of 100 ul. Both internal and external standards (2000-31.25 pg) were included in the assay. The DA and NE metabolites, methoxytryptamine and normetanephrine, respectively, were partially purified and concentrated by solvent extraction and thin layer chromatography. Amine content was determined by counting the chromatographic spots containing the 3H-labelled metabolites in the liquid scintillation counter. The complete assay protocol including procedure for partial purification of 67 liver COMT can be found in Appendix II. Neurointermediate lobe DA and norepinephrine content were expressed as ng per mg protein as measured by the method of Bradford (1976) using a Bio-Rad kit (Richmond CA). V. Statistics The data from the in vitro superfusion was evaluated with the non-parametric Mann-Whitney U analysis. Much of the data revealed a significant Fmax test for heterogeneity of variance and therefore were transformed logarithmically before parametric tests were performed. The remaining data were analyzed by analysis of variance (ANOVA) followed by Student-Newman-Keuls multiple comparison test. In all cases, a p5_0.05 was considered significant (Steel and Torrie, 1980). EXPERIMENTAL I. Tuberoinfundibular DOpaminergic Neuronal Function: Effect of Chronic Estradiol Administration and Persistent Hyperprolactinemia After Removal of Chronic Estradiol A. Objectives Long-term estrogen administration in rats produces chronic hyperprolactinemia and development of prolactin-secreting adenomas. However, the effects of chronic estrogen treatment on.‘hypothalamic dapaminergic control of prolactin release still remains unclear. There is evidence that long-term estrogen treatment can result in (a) cytopathological changes in the arcuate nucleus, including an increase in number of reactive glial cells and the appearance of axonal and dendritic degeneration (Brawer and Sonnenschein, 1976), (b) reduced neuronal catecholamine fluorescence in the ME and arcuate nucleus (Sarkar et a1., 1982), (c) depletion of DA content from the ME and reduction of DA concentration in hypophysial portal blood (Casanueva et a1., 1982; Sarkar et a1., 1984), and (d) decreased turnover of DA and an attenuated ability to release 3H-DA from ME tissue in vitro (Sarkar et a1., 1984; Demarest et a1., 1984). The decline in TIDA activity during chronic estrogen treatment may result from loss of dopaminergic neurons of the arcuate nucleus (Sarkar et a1., 1982) or loss in sensitivity of TIDA neurons to the increased levels of circulating prolactin (Demarest et a1., 1984). The former suggests that a permanent decline in TIDA function may occur, and 68 69 persist even after removal of the estrogen treatment, whereas the latter suggests the possibility that TIDA function ‘may be restored after estrogen removal. To clarify this problem, the present study was undertaken to assess TIDA neuronal activity at the end of 4 weeks of estrogen treatment, and 4» weeks after removal of chronic estrogen treatment. TIDA. neuronal activity was evaluated by two methods (a) in vitro superfusion and electrical stimulation of ME tissue after allowing for accumulation of BH-DA, and (b) testing the effectiveness of drugs that inhibit or stimulate prolactin release through dopaminergic or anti-dopaminergic mechanisms. B. Materials and Methods Animals were OVX prior to treatment and randomly divided into three groups: (a) OVX controls received (sc) an empty Silastic capsule 10 mm in length, for 4 weeks after which the capsule was removed for 4 weeks, (b) rats OVX for 4 weeks and then implanted with a Silastic capsule containing E2 for 4 weeks, and (c) OVX rats implanted with an E2 capsule for 4 weeks, followed by removal of the E2 capsule for 4 weeks. TIDA activity was estimated by determining the accumulation and release of 3H from ME tissue after allowing for accumulation of 3H-DA into the tissue as previously described in the Methods section. All experiments were performed beginning at 1100 4hr. The rats were decapitated, trunk blood was collected for RIA of LH and prolactin, and ME tissue was dissected for superfusion. After a 40 min washout period, the tissue was electrically stimulated using field stimulation. The stimulation evoked release of 3H was used as an index of TIDA activity. 70 Total uptake was expressed as cpm per pg ME protein as measured by the Bio-Rad assay. The capacity of pharmacological agents which act either on TIDA neurons or on dOpaminergic receptors on the AP to stimulate or inhibit prolactin release was assessed. The following three drugs were injected: apomorphine hydrochloride (APO) a DA receptor agonist; haloperidol (HALO), a DA receptor antagonist; and morphine sulfate (MOR), which inhibits central TIDA turnover (Deyo et a1., 1979; Gudelsky and Porter, 1979). Different animals were used for each of three experiments. The rats were implanted with an intra-atrial Silastic cannula under ether anesthesia as previously described. Two days later, the animals were adapted to the experimental room for two hours starting at 0800 hr. Blood samples were withdrawn 40 and 20 min prior to drug administration. After sc injection of APO (0.25 mg/kg, in saline) or iv injection of MOR (5 mg/kg, in saline), samples were withdrawn 15, 30, 60, and 90 min later. After sc administration of HALO (0.5 mg/kg, in 0.32 tartaric acid), blood samples were withdrawn 45, 90, 135, and 180 min later. Dosages were chosen which produce maximal effects on prolactin release in control male rats (Mueller et a1., 1976). Serum and plasma prolactin, LH, and GH were measured by RIA. The DNA assay was performed by the method of Burton (1956). Absolute values of the in vivo data were analyzed by one-way ANOVA followed by the Student-Newman-Keuls' multiple comparison test. Since basal serum prolactin levels among groups were different, data were also expressed as ng prolactin/ml plasma/estimated DNA of prolactin-cells (pg) ratio. The Wilcoxson-Mann‘Whitney analysis tested differences among 71 groups when data were expressed as a ratio. For the RIA data of Table 2, both the prolactin and LH results revealed a significant Fmax test for heterogeneity of variance. Therefore, data were transformed logarithmically before performing the ANOVA. and Student-Newman-Keuls multiple comparison test. The in vitro ME superfusion results were analyzed by the Wilcoxson-Mann-Whitney test. C. Results Figure 1 shows that at the end of 4 weeks of E treatment (E) and 2 after 4 weeks of withdrawal from E2 (WD) there was a suppression of stimulation-evoked release of 3H-DA from the ME, as compared to values in OVX controls (C). There was a trend for reduced 3H-DA accumulation after 4 weeks of E2 and after the 4 week withdrawal period which was not statistically significant. The absolute values of plasma prolactin following administration of APO, HALO and MOR to the three treatment groups are shown in Table l. APO, a DA receptor agonist, produced a significant decline in plasma prolactin values 30 and 90 minutes after injection in OVX controls, and a significant reduction in plasma prolactin levels at all time points after injection into rats treated with E2 for 4 weeks and in rats after E2 removal for 4 weeks. Injection of HALO, a DA receptor antagonist, produced an increase in circulating levels of prolactin in all groups. After administration of MOR, a drug which decreases TIDA activity, plasma prolactin was increased at 15 and 30 minutes but returned to baseline levels by 60 minutes in OVX control rats. Plasma prolactin increased only 30 minutes after MOR injection in animals at the end of 4 weeks of E2 treatment. 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MW mien one: 2E3 ANS in”: swan 3...; Susan Rug” e. 8: 3...»: £33 .23.: .232 «Hutu SEN: ASHES SEHSS u AS Wad Nu: $2: .558.“ Home; Seuss can»... as”: segue; o a: flew a dam a flu an clan flu mm” «mien g _aouusea woe .afidoo mcmaouoonlazm uo Away <29 voumlmuno ace Away u:0u:00 <2: m< flouo am macaw some new :30;a Ou—< .aCOIuwuuu Nu omcouzu mo —o:ouv:am3 swam: axes: e nuns am was 0300: c haw a cum: vouoouu new» :m .ouwu ~ouu=ou cent :m A~I\wcv n—obo— Adm slow—n co cam900m=m so: uo OA<= .omd mason mo uuouuw .~ ewpmfi 74 all time points after MOR administration in rats after E2 removal for 4 weeks. The prolactin response to pharmacological agents is also presented as the difference between the pre-injection and post-injection level of prolactin divided by an estimate of the portion of total AP DNA content which constitutes only the prolactin-secreting cells (Figure 2). Assuming blood volume and clearance of the hormone are similar among treatment groups, this ratio is an estimate which is proportional to the change in prolactin release per lactotroph in response to the drugs. This allows for evaluation of the prolactin response to the drug without the bias of differing basal plasma prolactin levels or the different number of lactotrophs among the treatment groups. The DNA attributed to the lactotrophs was calculated by first measuring total AP DNA content. For' OVX: F344 control and ‘E -treated rats, Phelps and Hymer (1983) 2 counted the number of immunocytochemically identifiable lactotrophs after separation of AP cells from red blood cells on a Ficoll-Hypaque gradient. When these authors used an E -treatment similar to ours, they 2 observed AP enlargements comparable to those reported here and 59.82 of the AP cells were identified as lactotrophs. Only 29.8% lactotrophs were identified in OVX controls. In our experiment, the rats showed a 31% decline in mean AP DNA content (539 ug to 3751J8; see Table 2) 4 weeks after the Ez-containing capsule was removed. Assuming that this decline was exclusively due to a loss of lactotrOphs, the Z loss of lactotrophs in rats after E2 withdrawal for 4 weeks would be 0.598 x 0.310 - 0.185 or an 18.5% loss of lactotrophs (thus, the Z of lactotrophs was 0.598 - 0.185 - 0.413 or 41.3%). The estimate used for the portion of AP DNA content due to the 75 0 ' TN.—_._.c I X 1\ “'9 ' b | a b p E || . 5’5 {Li ' ; . 5-2. . ~-——-L_--... . . E < 1 3: 1 5 = 5 I 2 P z O | O 1’ ' z .. g o c -4- r I: a '. a a I, a z * fi' 1 ~ In .- 3 a “- r: ‘\ < Q _ > \ao _J w o .. .. b . E k~q~~1 ----- 1E q 1 J 1 ,1\ so so so )P 0.0 140 TIME AFTER INJECTION (mln) FIGURE 2. Effects of acute administration of apomorphine (APO,left), haloperidol (HALO,center) and morphine (MOR,right) on stimulation or inhibition of prolactin (PRL) release in F344 control rats (C), in rats treated with 32 for 4 weeks (E), and in rats 4 weeks after withdrawal of chronic E2 treatment (WD). Data (X :_S.E.M. of 6-10 animals/group) are expressed as the change in plasma prolactin after drug injection (ng/ml) divided by the estimated portion of total AP DNA content which constitutes PRL-secreting cells (pg). Arrow indicates the time of drug injection. * - p S_0.05 compared to C; o - p $_0.05 compared to WD. 76 lactotrophs was 29.82 for OVX controls, 59.8% for EZ-treated rats and 41.32 for rats withdrawn from E2 for 4 weeks. The total DNA content and the estimated portion due to lactotrophs is shown in Table 1. The inhibition of prolactin per pg prolactin-cell DNA after acute APO administration (Figure 2) was significantly greater in rats at the end of E treatment (E) than in OVX controls (C) or in rats 4 weeks 2 after E removal (WD). Animals after removal of E2 (WD) showed a greater 2 prolactin inhibition per pg prolactin-cell DNA after APO than OVX controls. Blockade of DA receptors by administration of HALO resulted in similar increases in plasma prolactin in all three groups when the data were expressed per pg prolatin-cell DNA. Injection of MOR resulted in a rapid increase and then decline in prolactin release in OVX control rats (C). The prolactin rise in response to MOR was lower but more prolonged in E -treated rats (E) and in rats after withdrawal of E2 (WD). 2 Since EZ-treated animals and animals after E2 withdrawal exhibit a greatly enlarged AP, there is the possibility that the enlarged gland may impinge on blood flow between the median eminence and the AP. This could result in a smaller PRL response in the EZ-treated animals because of the limited blood flow in the portal vasculature, and reduce the passage of DA to the AP. To evaluate this possibility, the plasma GH response to acute MOR injection was also measured in the three treatment groups. Injection of 5 mg/kg MOR produced a significant increase in plasma GH (Figure 3) in animals after E removal for 4 weeks, but MOR 2 did not induce significant release of GH in control rats or in rats at the end of 4 weeks of E2 treatment. Table 2 shows that 4 weeks of E2 treatment produced greater than a 6-fold increase in AP weight and a 65-fold rise in serum prolactin as 77 500- :k 0 I\ 2400- I \ e I \ B, I \WD 5 I J.\ I 300 - I \ (D / \\ < 5, 200 - I, ° ‘J S / ’IIs .\ O. TI’I” O “ --\.\__ T E In» ’ I \"~-.. 100 P T 7 \ g “- o’ . c I \\\ N I \1’1 33.3.. 0 so so so TIME (min) FIGURE 3. Effect of morphine (MGR) on the concentration of plasma GH in OVX (C), Ez-treated (E) rats, and rats 4 weeks after withdrawal of chronic E2 treatment (WD). Arrow indicates the time of drug injection. (X i S.E.M. of 7-10 animals/group) * - p S 0.05 compared to pre-injection GH level, 0 - p i 0.05 compared to WD. 78 Table 2. Effects of E -treatment and E -withdrawal on anterior pituitary (AP) weight and DNA content, and serum PRL and LH levels. AP weight AP DNA AP DNA serum PRL serum LH (mg) (us) (us/ms) (us/m1) (us/m1) 1) ovx controlsa 1o.o:o.3 112:4 11310.4 26:4 535177 2) EZ-treated 64.514.7* 539:37" 8.110.3" 1695:208* 29:4" 3) Ez-withdrawal 30.0.3.7" 375:24" 12.3-30.4 247:26* 161:22* a X + S.E.M. of 11-12 animals/group; Group 1 was OVX for 8 weeks, Group 2 rezeived E for 4 weeks, Group 3 received E2 for 4 weeks after which E2 was withdrawn for 4 weeks * p £_0.05 compared to Group 1 79 compared to non-Ez-treated control rats. Both AP weight and serum prolactin declined by the end of 4 weeks of E withdrawal (Group 3), but 2 remained elevated when compared with OVX controls. AP DNA content increased from 112 pg in OVX controls to 539 pg after 4 weeks of E2 treatment, and remained elevated (3x) above control values 4 weeks after E2 withdrawal. When DNA content was expressed per mg AP tissue weight, Ez-treated rats showed reduced DNA content when compared with control values. Four weeks of E2 treatment significantly suppressed the LH response to OVX. This suppression of LH release persisted 4 weeks after removal of the E . 2 D. Discussion The results presented here confirm previous reports that chronic E2 treatment depresses TIDA neuronal activity (Casanueva et a1., 1982; Sarkar et a1., 1984; Demarest et a1., 1984), and indicate that after removal of long-term E2 treatment in F344 OVX rats, TIDA activity remains depressed. Attenuated TIDA neuronal activity is clearly indicated by the observation that at the end of E2 treatment or even 4 weeks after E2 removal, the electrically-stimulated release of 3H-DA from ME tissue was decreased when compared to rats not treated with E2. The plasma prolactin response to APO, HALO and MOR was expressed as the ratio of the change in plasma prolatin after injection of the drug divided by an estimated portion of total AP DNA content which is contained only in the lactotrOphs. In both Ez-treated groups, the estimates of prolactin-cell DNA are presumed to be low. Ez-treatment results in low prolactin content in the lactotrophs (per cell) and therefore it is probable that a portion of the lactotrophs were not 80 immunocytochemically identified as such. These possibly low estimates of prolactin-cell DNA are conservative in the sense that, after drug injection, they allow for greater prolactin responses in Ez-treated rats and rats' after withdrawal from E2. Decreased TIDA activity after Ez-treatment and after Ez-withdrawal is supported by the pharmacological evidence that acute administration of MOR to EZ-treated rats resulted in a slower and attenuated increase in plasma prolactin when compared with OVX control rats. Chronic E has been shown to elevate hypothalamic 2 opiate binding sites (Wilkinson et a1., 1985) which would be expected to permit a larger increase in prolactin in Ez-treated animals after MOR. However, Ez-treated rats showed a smaller rise in prolactin compared to controls which further suggests a depressed TIDA function. Depression of TIDA neurons (e.g., by MOR) which have a low basal activity, i.e., Ez-treated rats and rats after E2 withdrawal, increased prolactin to a lesser degree than rats with a higher TIDA activity, i.e., controls. After HALO injection, all groups showed similar increases in circulating prolactin levels, suggesting that a maximal blockade of DA action on the lactotrophs permitted a similar release of prolactin/ g prolactin-cell DNA in all three treatment groups. APO produced a greater inhibition of prolactin release in both Ez-treated groups as compared to controls, when the data are expressed as per lactotroph. There are potential problems in the evaluation of TIDA activity in_ 3122_ using lactotrophic responses to DA agonists and antagonists, especially in animals with enlarged AP's. A restricted blood flow in the portal vasculature or genesis of systemic arteries to the pituitary gland (Elisa and Weiner, 1984) as a result of AP growth, could decrease the concentration of DA reaching the AP lactotrophs. This should 81 effectively reduce all the releasing/release-inhibiting factor levels from the hypothalamus that regulate AP function. The combination of decreased release of DA from TIDA neurons, together with the possible limited reduction in portal blood flow and development of new vascularization to the AP, could greatly decrease the inhibitory hypothalamic influence on prolactin secretion. Thus, the reduced prolactin release in Ez-treated animals after injection of MOR could be partially the result of less DA reaching the .AP because of these physical factors and not due to diminished TIDA activity. To evaluate this possibility, the ability of MOR to increase GH was measured in controls, in Ez-treated rats and in rats after E2 withdrawal. MOR is known to increase GH by a central action on neurotransmitters, growth hormone releasing factor and somatostatin (Sonntag et a1., 1983). In the present study, MOR increased plasma GH to levels similar in control and Ez-treated animals; however a 4-fold greater increase in CH was observed in animals 4 weeks after withdrawal of Ez-treatment. If reduced portal blood flow and dilution of portal blood DA due to new vascularization were effective in reducing hypothalamic influence on the AP, the time course for the MOR-induced GH release among treatment groups should be similar to the time course of prolactin release. However, the time course for CH release was very different from prolactin, suggesting that the central nervous system mechanisms responsible for MGR-induced GH release were not significantly affected by altered portal blood flow. This interpretation should be viewed with caution, since the influence of E2 on the hypothalamic and AP mechanisms effecting GH release is not well understood at present. In summary, it is clear that TIDA neuronal function, as evaluated 82 directly by in vitro superfusion release of 3H-DA and indirectly by the plasma prolactin response to MOR, a drug which inhibits TIDA neuronal activity, is diminished in Ez-treated rats and also in rats 4 weeks after removal of the chronic E2 treatment. 83 II. Evidence for a Permanent Decline in Tuberoinfundibular Dopaminergic Neuronal Function After Chronic Estrogen Treatment A. Objectives It has been suggested that long-term estrogen exposure can cause damage to TIDA neurons in female rats (Sarkar et a1., 1982; Sarkar et a1., 1984). If estrogen does result in permanent TIDA neuronal damage, the depressive effects on TIDA neuronal function in the presence of elevated estrogen levels should remain long after removal of the estrogen. The objective of this study, therefore, was to assess TIDA neuronal activity long after removal of chronic E2 treatment. TIDA neuronal activity was evaluated by in vitro superfusion of median eminence tissue and by testing the ability of TIDA neurons to alter prolactin secretion in response to pharmacological manipulation, 26 weeks after removal of chronic estrogen treatment. B. Materials and Methods Animals were ovariectomized immediately prior to implanting (sc) an empty or E2 containing Silastic capsule The capsules were removed after 4 weeks of treatment. Experiments were performed 26 weeks after removal of the capsules. In other experiments, animals were implanted with a capsule for 4 weeks, the capsule was then removed, and 26 weeks later the rats received E for 3 days. The capacity of the following four 2 drugs to alter prolactin release was assessed in control and Ez-treated groups and used to evaluate TIDA neuron activity: 1) apomorphine 84 hydrochloride (APO), a DA receptor agonist; 2) morphine sulfate (MOR), which inhibits central TIDA turnover; 3) halOperidol (HALO), a DA receptor antagonist; and 4) nomifensine maleate (NOM), which blocks the uptake of DA into catecholaminergic neuronal terminals. Different groups of animals were used for each experiment. Blood samples were wdthdrawn by the retro-orbital technique under light ether anesthesia. After taking a pre-sample 30 min before drug injection, two blood samples were withdrawn at various times following drug adminstration via the following routes and dosages: APO, 0.25 and 0.01 mg/kg sc; HALO, 0.5 mg/kg, sc; MOR, 5 mg/kg, ip; NOM, 10 mg/kg, ip. All superfusion experiments were performed at 1100 hr. Animals were decapitated and blood collected for prolactin and LH RIA. The AP was removed, weighed, and homogenized in 2 ml 0.01 M phosphate buffered saline for later DNA. assay. The ‘ME superfusion. has been. described previously in full Methods section. One estimation of TIDA neuronal activity was the ability of the neurons to increase the radioactivity released in response to electrical stimulation termed the stimulation-evoked release. Total accumulation of 3H-DA was also used as an index of TIDA function and was expressed as cpm per pg ME protein as measured by the Bio-Rad assay. Statistical analysis was performed as in Experiment I. C. Results The ability of the dopaminergic agonist APO to inhibit prolactin secretion in OVX controls and in rats long after E2 withdrawal is shown in Table 3. Injection of 0.25 mg/kg APO reduced serum prolactin in both groups for at least 90 min. However, after 0.01 mg/kg APO, prolactin 85 Table 3. Ability of APO to inhibit PRL secretion in OVX controls and 26 weeks after withdrawal from chronic E2 treatment. OVX controls8 (n - 6) ng PRL/ml serum Pre-sample + 30 min + 90 min APO 40.8 i 8.3b 5.0 i 1.7* 13.2 i 2.1* (0.25 mg/kg) APO 38.2 :_9.3 25.8 :_9.7 34.4 :_9.7 (0.01 mg/kg) AP DNA (ug) 115 :_6 estimated PRL- 34 + 2 cell DNA (pg) 26 weeks after removal E., (n = 4) ng PRL/ml serum =- Pre-sample + 30 min + 90 min * APO 97.4 i 25.4 6.8 i 1.2" 8.7 i 1.7 (0.25 mg/kg) APO 103.4 71 19.3 41.7 i 3.8* 108.6 i 17.9 (0.01 mg/kg) AP DNA.(1g) 312 :_42 estimated PRL- 111 i_15 cell DNA (pg) 8animals were OVX and received either an empty or Ez-containing silastic capsule for 4 weeks; 26 weeks later the capsules were removed and experiments were performed. APO was injected sc and blood samples were taken at times indicated (min) b x i 5.12.11. * p S_0.0S compared to pre-injection PRL level 86 release was significantly suppressed only in the animals after E2 withdrawal and not in the controls. Table 4 shows the prolactin response after MOR, HALO or NOM administration. Injection of MOR significantly elevated serum prolactin in OVX control rats but not in animals after E2 withdrawal. HALO, a dopaminergic receptor antagonist produced a significant elevation in serum prolactin in ‘both treatment groups. Injection of NOM, a drug which blocks the re-uptake of DA in catecholaminergic neuronal terminals, significantly reduced serum prolactin in OVX controls but not in rats after Ez-withdrawal. In an attempt to compensate for the differing basal prolactin levels, the serum prolactin response to the above pharmacological agents is presented in Figures 4 and 5 as the change in serum prolactin concentration after injection of the drug, divided by an estimate of the prolactin-cell DNA content. Since basal prolactin levels are different between OVX control rats and rats after Ez-withdrawal, it is difficult to evaluate changes in serum prolactin when the drugs are acting on different numbers of lactotrophs present. Therefore, an. estimate «of prolactin-cell DNA was made, and the change in sermn prolactin was expressed as a ratio of this prolactin-cell DNA level. The AP's of OVX F344 control animals were previously reported to have 29.82 lactotrophs and F344 OVX rats treated with E2 for similar periods were reported to have 59.82 lactotrophs (Phelps and Hymer, 1983). In our experiment, rats 26 weeks after removal of E2 showed a 40.22 decline in total AP DNA content compared to animals at the end of the 4 week E treatment 2 (Gottschall, et a1., 1986). If this decline is due exclusively to a loss of lactotrophs, then the Z loss of lactotrophs would be 0.598 x 0.402 - 0.241 or a 24% loss. Therefore, the Z lactotrophs remaining in the AP 87 ”H xv “Asv cm was comuoomcm msec nemaod~Ou aolmu acmdelau woo—n ussu uoouxo .ucoluwouu no o—mauoo new n o—afih woe a~o>w~ 41m :omuuomchoun cu nonuniow ac.° H A s u—In I c n.:.u.m I <2: _aouIaea I I can __ouIsem I 2 . S. .6332... 2 . a: $5 a: n . an .6332... 2 . 2. $5 8.. ~..: n 6.3 12 u .2... 6.2 u 3...: as I... a; A...” n a... 8.... u “.2 :9. m a a u H m a a a an I <2: __ouI4ea I I oloe hogan .4003 on auuouucoo x>b usuluoouu Nu omcouzo Icon duaeuozuws nouns also: ow one a~0uu¢00 x>o cm Adlchv Adm lano- co to: use 04¢: .zox no uuouuu .e o—aah 88 3 0- 0' ( g APO = 0.25 mglkg 8 I .J I I. 2 o / a ‘ I §-o,5- -O.5- VLwo 3 I 2 \leo E ‘---——-o APO g 0.01 mglkg .l C O. 54.0- -1.0' : L 1 1 _J_ 1 1 1 1, 4 0 30 60 90 0 30 60 90 TIME (min) FIGURE 4. The ability of apomorphine (APO; 0.25 left and 0.01 mg/kg right) to inhibit prolactin (PRL) secretion in F344 OVX controls (C) and after long-withdrawal (26 weeks) from chronic E treatment (LWD). The data are expressed as the change in serum prolactin after APO injection (ng/ml) divided by an estimate of the portion of AP DNA content which constitutes PRL-secreting cells (pg). Zero time is the time of APO injection. p > 0.05 at each time point when C is compared to LWD. There were no significant differences in serum prolactin between treatment groups at either dose of APO. 89 .asu cu ooumoaou mu 0 cos: wagon mafia some up no.0.w a I « .:Ouuoon:u wan—0 no as: 05 ma we: ouoN .35 @300 wcuuouoomLS—m mousuwumcoo cows: acoucoo ao AHE\w:V :oHuoohcw mono seams c uomfiouo aspen ca owcmso osu as ooooouaxo one sumo one .Aczav assesses» w eacoucu scum Aesop: euv HuseuvsuquwcoH woumm one Auv ofiouucou x>o «emu cu acuuouoom :«uomfiouo co Auswuuuzozv ocumcomueoc no Auoucoouoaofiuou uo: owe umsu naoum Houucoo cu ooumanu mo.o v a n .ucoaumouu uo maqmuoo new m wanna com .mumu ANN + azav cutouozufisIMw ocm ANM + UV Houucoo Ca wwcoaamxu «M can Ao3gv Nw mo ~m>osou wouwm expo) em mumu ca .on mush Houucou x>o cw Amumn oonwuumv oucocwsu anyone oucu <91: mo COHumwaesuom use Amman coaoV oswmfiu masseuse cmaooe mo coHumHS um Hwowuuoofio mcfiuao tn mo ommofiou voxo>olcofiumasawuw .o mxbuHh N UJ + «m + 95.. o 03.. ,. l *- ~I||||||l||||||||||||||||||||||||||||||| 6 (cpm x103/pg ME protein) (may 8 -0 |. X) sseslsa paxone-uonelnwns 93 hyperprolactinemia in these animals did not alter stimulation-evoked release and actually decreased 3H-DA accumulation (Figure 6). D. Discussion The present study demonstrates that permanent alterations occur in TIDA neuronal function after E2 treatment for 4 weeks in F344 rats. This is supported by the failure of TIDA neurons in Ez-treated F344 rats to respond similarly to non-E2 treated animals to most stimuli used in this study. Even though there was no significant difference in the stimulation evoked release of 3H-DA from ME tissue in vitro between control rats and rats 26 weeks after E2 withdrawal, there was a 4-fold difference in serum prolactin levels between these groups. It is well established that serum prolactin levels are important in regulating TIDA activity via a short-loop feedback mechanism (Moore and Demarest, 1982). Since animals given 4 weeks of E treatment still had a 4-fold greater 2 serum prolactin concentration 26 weeks later than rats not given 82, it might be expected that the hyperprolactinemic animals would show increased TIDA activity. However, there was no measurable difference in TIDA activity in the Ez-treated and control groups, suggesting that in the 13 -treated animals, there was a failure of the TIDA neurons to 2 respond to the increased serum prolactin levels. The prolactin responses to the dapaminergic and anti-dopaminergic drugs used in the present study also demonstrate that TIDA activity was decreased after E2 removal. APO, a dopaminergic agonist, when given at a low dose (0.01 mg/kg) did not produce a significant decrease in serum prolactin in control rats, but decreased prolactin levels in rats after E2 withdrawal. This suggests that animals previously treated with E2 are 94 at least as sensitive to APO as OVX controls, and confirms previous reports of the effects of DA agonists on prolactin secretion in other chronic E2 treatment models (Smythe and Brandstater, 1980; Willoughby et a1., 1983). Injection of MOR, a drug which decreases TIDA activity, significantly raised prolactin only in OVX controls and not in the Ez-treated animals. When the change in serum prolactin was expressed as per ug estimated lactotroph DNA, OVX control rats exhibited a 2-fold greater prolactin rise than E2 treated rats. This suggests that there was a smaller MOR-induced inhibition of TIDA neurons in E2 treated animals, possibly due to reduced activity of the neurons. Both groups responded to a single injection of HALO with a significant increase in serum prolactin as compared to pre-injection levels. However, when expressed as ug prolactin-cell DNA, OVX controls exhibited a 2-fold greater increase in prolactin than E treated animals. Reduced TIDA 2 neuronal activity long after withdrawal from E2 probably accounts for this reduced response. Acute administration of NOM, a drug that inhibits DA reuptake, also resulted in a reduced prolactin response in rats long after E withdrawal. All of these data indicate that TIDA responsiveness 2 is permanently attenuated long after removal of chronic E2 treatment in F344 rats. The 3-day E2 treatment in OVX control rats doubled the evoked release of 3H-DA and increased serum prolactin from 14 i 3 ng/ml before the E2 challenge to 304 i 119 ng/ml at the end of 3 days of E2 treatment. However, 3 days of E2 administration had no significant effect on evoked 3H-DA release and decreased 3H-DA accumulation in the rats previously treated with E2 for 4 weeks. E2 also increased serum prolactin from 57 i 11 to 340 i 43 ng/ml. At least part of the action 95 of E2 in stimulating prolactin release in vivo appears to occur by antagonizing the inhibitory effect of DA at the AP level (Raymond et a1., 1978). In the control rats, E2 may have removed dopaminergic inhibition from the lactotrophs and greatly increased serum prolactin levels. In rats previously given E for 4 weeks, TIDA activity was not 2 augmented by 3 days of E2 treatment, and E2 may have been acting on the AP in the presence of low DA concentrations. E2 action on lactotrophs in the presence of little dopaminergic inhibition may account for the large increase in AP weight without a concomitant large increase in sermn prolactin levels. Old rats are known to show deficient TIDA neuronal function, and exhibit relatively small increases in prolactin secretion in response to E2 administration as compared to young rats (Shaar et a1., 1975). The mechanism(s) responsible for the decrease in 3H-DA accumulation after 3 days of E2 administration in rats previously treated with E2 for 4 weeks is unknown. It has been observed that E2 can inhibit 3H-DA accumulation in hypothalamic tissue slices when E2 was included in the incubation medium in vitro but not when injected in vivo (Endersby and Wilson, 1974). Acute E2 depresses LH secretion in OVX rats. This negative feedback effect of E on LH secretion is partly exerted at the hypothalamic 2 level. The acute E2 treatment in F344 rats treated for 4 weeks with E2 26 weeks previously, reduced LH by a smaller absolute amount than in animals not given E2 earlier. This suggests a deficiency in the negative feedback control of LH in the chronically E2 treated rats and is consistent with reports concerning the regulation of LH after different regimens of chronic E2 treatment (Finch et a1., 1984). The Ez-induced 96 deficiency in the central mechanisms controlling LH secretion is associated with an increase in glial reactivity in the arcuate nucleus and may mimic the age-related decline in hypothalamic mechanisms which control reproductive function. The permanent attenuation in TIDA neuronal function after 4 weeks of E treatment in Fischer 344 rats may not only be due to a direct action 2 of E2 or to the resultant hyperprolactinemia, but also to physical compression on the medial basal hypothalamus by the grossly enlarged pituitary that occurs at the end of Ez-treatment. 97 III. Bromocryptine Prevents the Decline in Tuberoinfundibular Dopaminergic Neuronal Function After Removal of Chronic Estrogen Treatment A. Objectives Decreased function of TIDA neurons up to 6 months after removal of prolonged (4 weeks) E2 treatment has been observed in female F344 rats. An attempt will be made to elucidate the mechanism(s) by which chronic E2 treatment in F344 rats results in apparently permanent deficiencies in TIDA neuronal function. The specific questions to be answered were: (a) when 13.2-induced AP growth and hyperprolactinemia are inhibited by simultaneous administration of a dopaminergic agonist, is TIDA neuronal activity altered?, and (b) if TIDA neuronal activity is altered when E2 is administered together with the DA agonist, is this a permanent effect or can it be reversed after treatment is discontinued? B. Materials and Methods Rats were divided into four groups as follows: (1) OVX controls implanted with an empty Silastic capsule sc for 30 days and given daily vehicle injections (2) rats given daily bromocryptine injections for 30 days (Sandoz, Hanover, NJ; 3 mg/kg, sc, 502 ethanol in saline vehicle) and implanted with an empty capsule, (3) rats given an Ez-filled capsule (10 mm in length) and daily vehicle injections, and (4) rats given bromocryptine daily together with an Ez-filled capsule. At the end of the 30-day treatment period TIDA neuronal function was evaluated i_n vitro. Other animals were treated the same as above except that 98 treatment was discontinued after 30 days, and the rats were left untreated for 30 more days before acute experiments were performed to test TIDA neuronal function. In each group, TIDA activity was measured using an in vitro superfusion technique after allowing for accumulation of 3H-DA into median eminence (ME) tissue, as previously described. The tissue was electrically stimulated for 15 sec, 50 and 80 min after the beginning of the superfusion, and the release was expressed as a stimulation-evoked release. At sixty minutes after the beginning of superfusion, nomi- fensine maleate (10 pM; Hoechst-Roussel, Somerville, NJ), was added to the medium to evaluate 3H release from the ME tissue after inhibition of DA reuptake (Gerhards et a1., 1974; Hurt et a1., 1974). Experiments were performed starting at 1100 hr. Animals were decapitated, blood collected, serum separated and frozen, and ME tissue was dissected for superfusion. The AP was separated from the neurointermediate lobe, weighed and homogenized in 2 ml of 0.01 M phosphate buffered saline (pH 7.6) and frozen for later measurement of AP prolactin and DNA content. Serum and AP prolactin were measured by RIA. The catecholamine assay was a modification of the radioenzymatic method of Cheng and Wboten (1980). Neurointermediate lobes were dissected and homogenized in 250 pl of 0.1 N HClOa containing 5 mM glutathione. The homogenate was centrifuged, and the supernatant was stored frozen at -40°C until assay. The DA and norepinephrine (NE) contents were expressed as ng cate- cholamine per mg neurointermediate lobe protein as measured by the Bio-Rad assay. AP DNA content was measured using the method of Burton (1956). The AP weight, serum and AP prolactin concentrations, and AP DNA 99 content revealed a significant Fma test for heterogeneity of variance. x The data were transformed logarithmically before performing analysis of variance and Student-Newman-Keuls multiple comparison test. Data expressed as a ratio were analyzed by the Wilcoxson-Mann-Whitney test. C. Results Thirty days of E treatment of OVX F344 rats (E) increased AP weight 2 lO-fold, serum prolactin 140-fold, AP prolactin content lS-fold and AP DNA content about 6-fold as compared to values in non-Ez-treated controls (C; Figure 7). Bromocryptine given alone for 30 days (B) reduced serum prolactin and AP prolactin content but did not alter AP weight and DNA content when compared to control values. When bromocryptine was injected daily in EZ-treated animals (BE), it completely inhibited the Ez-induced increase in serum prolactin and AP DNA content and resulted in only a modest increase in AP weight as compared to OVX controls. However, bromocryptine did not significantly alter the EZ-induced increase in AP prolactin content. Figure 8 shows the mean fractional rate constants in the superfusion experiments in the four treatment groups after allowing for accumulation of 3H-DA into ME tissue and the response to electrical stimulation in the absence and presence of 1014M nomifensine. Stimulation-evoked release of 3H in the absence of nomifensine (Open bars, Figure 9) was significantly reduced in both Ez-treated groups (E and BE) when compared to controls (C), and there was a trend for a reduced stimulation-evoked release in bromo- crypine only (B)-treated animals. The controls (C) and bromocryptine alone (B)-treated animals exhibited an increased stimulation-evoked release in the presence, as compared to the absence of 10 “M nomifensine 100 ’ * 80' AP WTHGHT m ( 9) ‘0» t P 4:. 4:1. 1.4.. D. C B E BE 2000 * SERUM PRL 1000 J J ("D/ml) 1: 1: 20 C B E BE * 200‘ * AP PRL CONTENT (pg) 100“ r- * C B E BE 800- * AP DNA CONTENT ’ (”9’ 400» p. 4:1. .121. _JL_L 11 C B E BE FIGURE 7. Effects of 30 days of vehicle injections (C), bromocryptine (B), estradiol-l7-B (E) or bromocryptine and estradiol treatment (BE) in OVX F344 rats on AP weight, serum prolactin concentration, and AP prolactin and DNA content. (X : S.E.M., n - 12). Note that estradiol-induced increases in the parameters were reduced or prevented by concommitant bromocryptine treatment. * - p < 0.05 compared to C values. 101 .ucmumcou mumu HmCOHuuwuu Ac u :v came w:u mm voucmmouo who mums .mcumcoufieo: 2: OH mo mocomoua mo oucomam us» ca osmmuu oocmcfiEo cmfiome mzu mo Aoom mfiv noduwfiseuum Hmofiuuomfio oumoavcfi maouum can mwuacue o3u huw>m wouomaaoo mum: occauomum .AucoEumouu mo maumumv now u ouswwh momv name «can x>o vmuwouu mINHIHovauumm uo\c=m mauuazuuoeoun :« <91: uo cofiumfisasoum new ucwnofiam moumm mammuu mococuem cmvaE mo :onmwumasm madman mm mo mmmonu Hmcofiuoouh .w NKDUHh .55. mi.» on as no on no vv on no ow . . . . . _ _ . q. 14 . . . . . . _ .n 44444 4 4 1 u a «noun m 4 a o a mm m ooa o a B O. a 4 no o a a l 4 0 was In. w. 2 1 oo 10— .» » - Fw wzazwbzoz Ito. \A low lNYlSNOO BLVU 1VNOIlOVU$ 102 * I h I" M. o 16 — w m ( v— uJ’E * 4 - g E 12 - 0 ol w o F *2 By W 8 h ** ** z - 9 .— 5 4* a 2 — r- .- m C B E BE FIGURE 9. Stimulation evoked release of 3H (X i_S.E.M., n - 6) before (open bars) and during infusion of 1011M nomifensine (closed bars) in bromocryptine and/or estradiol-l7- Btreated OVX F344 rats. (See Figure 7 for details of treatments.) Note that in oth estradiol-treated groups (E and BE) stimulation-evoked release of H was reduced as compared to controls and response to nomifensine was absent. ** - p < 0.05 compared to C values, * - p < 0.05 compared to evoked release in absence of NOM. 103 MEDIAN EMINENCE 3 * H“ DOPAM|~E " * T UPTAKE (cpm x 103/ pg protein) C C 3 E BE )- Neuaomrenueolnea’ 1- LOBE DOPAMINE ,. (no /rng protein) ‘ __ c a E 35 3 I- P , \ usunomrsnueolns I LOBE 2’ NOREPINEPHHINE ,.. (no/mg protein) ‘_ _L._ ____. ._ J..- C B E BE FIGURE 10. Median eminence 3H-DA uptake (X : S.E.M.,n - 6) and neurointermediate lobe DA and NE content (X + S.E.M., n -12) in bromocryptine and/or estradiol-l7-8 treated OVX F344 rats. (See Figure 7 for details of treatment.) Note that estradiol treatment alone (E) reduced neuro- intermediate lobe DA content. * . p < 0.05 compared to C values. 104 (solid bars, Figure 9). However, neither Ez-treated group (E and BE) responded significantly to nomifensine. Accumulation of 3H-DA into ME neuron terminals and neurointermediate lobe DA and NE content for each experimental group are shown in Figure 10. In Ez-treated animals (E and ' BE), ME 3H-DA accumulation was reduced when compared to controls (C). Neurointermediate lobe DA content was reduced about 502 in animals given onLy E2 (E), but not E2 and bromocryptine, as compared to OVX control values (C). Figures 11-14 show the results 30 days after withdrawal of the different treatments. Figure 11 demonstrates that 30 days after E2 withdrawal (EW), AP weight, serum prolactin levels, AP prolactin and DNA content fell markedly but remained higher than the control animals (CW). The decreases in serum and AP prolactin levels observed during treatment with bromocryptine alone were not maintained after drug removal. Bromo- cryptrtne administered during E2 treatment completely prevented the Ez-induced increases in serum prolactin, AP prolactin and DNA content, and AP weight at the end of the withdrawal period. Figure 12 shows the mean fractional rate constants during ME superfusion of the four groups after withdrawal of treatments, and Figure 13 shows the stimulation- evoked release of 3H before and after nomifensine infusion. Only in animals after withdrawal from E2 (EH) was there a reduced stimulation-evoked release (Figure 13, open bars) and no significant response to nomifensine (Figure 13, closed bars), as compared to controls. The reduction observed in the presence of combined bromocryptine and E2 treatment was not present 30 days after removal of treatment (BEN). In fact, bromocryptine and EZ-treated animals responded to NOM infusion similarly to controls at the end of the 30 day 105 40- * AP WEIGHT - m t a) 20_ cw aw 5w BEW 400 * SERUM 300 PRL :. (no/ml) IO cw aw EW DEW a: t 80 '- AP PRL CONTENT " p _:L .13. __L If]. cw 8W EW BEW 600- a: AP DNA CONTENT 400' (vs) 200' cw aw cw DEW FIGURE 11. F344 OVX rats were treated for 30 days with vehicle (CW), bromocryptine (BW), estradiol-l7-B (EW) or bromocryptine and estradiol (BEW) and thereafter treatment was discontinued for 30 days before experiments were performed. The effects shown are of the different treatments on AP weight, serum prolactin levels, AP prolactin and DNA content. (x + S.E.M.,n - 12). Note that 30 days after withdrawal of estradiol (EW), all parameters were still increa sed over c . * - . compared to CW values. ontrols p < O 05 106 .ucoumcou mums floccuuomuu Aw I my some ms» no mmucmmmuo mum mama .mcamcmmuso: 2: ea mo mocomoun no mocmmam msu cw osmmuu mucmcuso cmwcme may no Avon m3 cozmussum :ouuuomao museums.“ amouum one mouse:— oau aum>o vmuumfiaoo mum: mcoauomum .Amucosumouu mo magnumv you ~H mumwum away .3: comb x>o Aoumv omV vmum>oomu vcm vmummuu mrsutaoflomuumm uo\v=m ocuuaauooeoua ca ncentration of greater than 202 from nadir to zenith according to the Criteria of Santen and Bardin (1973). This criteria of 201 is greater than 2x the CVs listed in Table 6 for each level of prolactin, LH, and 114 Table 6. Effect of varying pooled plasma concentrations of AP hormones and varying sample volumes on within-assay coefficient of variation Hormone Sample Level Volume (in) Binding (Z) CV (Z) high prolactin 1 61-65 7.4 intermediate prolactin 10 49-51 4.8 low prolactin 30 66-69 6.9 high LH 20 25-28 6.1 intermediate LH 20 51-54 6.6 low LH 50 75-78 7.8 high GH 10 49-52 2.9 low GH 30 78-83 8.0 115 GB. The nature of pulsatile release was assessed by the following parameters (Ambrosi et a1, 1985): - the mean of all samples withdrawn from all the subjects of particular a treatment group - the coefficient of variation from the mean value for each subject - the number of secretory pulses per 3 h for each subject - the absolute and percent increment of each pulse from all the subjects of a particular treatment group. The data from each parameter were analyzed by one-way' analysis of variance followed by the Student-Newman-Keuls multiple comparison test. Data which showed a significant heterogeneity of variance were first logarithmically transformed before statistical analysis. p _<_0.05 was chosen as the level of statistical significance. C. Results The data in Figures 15 and 16 ‘were selected as representative examples of the pulsatile release of prolactin, LH, and GH in OVX controls (OVX), animals which received E for 30 days (E) and in animals 2 120 days after discontinuing the 30 day E treatment (WD). Prolactin 2 levels are presented in both figures to show the magnitude of hyperpro- lactinemia in the E -treated individuals, in addition to the pulsatile 2 release of prolactin. Tables 7-9 present the parameters used to evaluate the pulsatile pattern of prolactin, LH, and GH, respectively. The mean prolactin level and the absolute amplitude of the prolactin secretory peaks were greatest in animals at the end of E2 treatment and remained significantly above control levels 120 days after removal of E 2 (Table 7). However, the frequency and percent amplitude of the prolactin 116 pulses decreased after E2 treatment, and continued to be lower than OVX controls 120 days after E administration. The CV, for prolactin pulses, 2 an indication of total variation across the ten samples, was reduced compared to OVX controls at the end of E2 treatment and was even significantly lower 120 days after E2 treatment compared to animals during steroid administration. In contrast to the EZ-induced increase in prolactin levels, mean plasma levels of LH and the absolute amplitude of 'LH pulses were dramatically decreased as a result of E2 treatment (Table 8). After the 120-day withdrawal period, LH levels and the amplitude of the LH pulses were depressed compared to OVX controls but greater than at the end of E2 treatment. Treatment for 30 days with E2 did not significantly change the CV, or the percent amplitude of the pulses compared to OVX controls. Interestingly, in animals which were still hyperprolactinemic, 120 days after chronic E2 treatment, the CV of the LH values was significantly greater than in OVX controls. The measurable frequency of LH pulses was not altered as a result of either E -treatment regimen compared to OVX 2 animals. Treatment with E for 30 days significantly increased mean plasma GH 2 levels, 4-fold, however, after withdrawal of E2, mean GH values returned to control values (Table 9). The CV, the frequency of GH pulses, and the percent amplitude of the GH pulses were lower at the end of E2 treatment compared to control values. Although 120 days after withdrawal of treatment, the CV and the percent amplitude of the GH pulses remained lower than OVX control values, the frequency of GH pulses returned to control values. Chronic E2 treatment did not significantly affect the absolute magnitude of the GH pulses, although there was a trend toward 117 1500i ' r * 20 °. ‘3 fi 1000’” I . i i‘,‘ '0 I: i i 34 .* i ‘ 10 ‘ ' “ ‘ g to". i. \ .' V I . ’ y r 500P .: I > ’4'! I y . . t . *8 OVX o9 ovx _J l A A A | ‘ i ‘ I _L A J A A m a E so [ A ~ , «4000:: a A I \ ti 0 0'3 ' I t v - ’ 1"! 'I 1 ’ m a r ’ \‘l! “ ’1. I \V \l R’f. r - ‘I V E 40 A ’ y i “ h I ’ ‘ ’ 2000 ‘ 74% a a / \ i 5 IQ \l " P V \Pd >" U- a 43 a n4 E 427 E 3 c A L A A 4 _L 1 A A 1 _L J 1 J 1 a m -.,.-. “xi ' ‘e; " “2 wo . 250. A . . . I) I, “\c’-\ ’A A 150 ‘\ " 1‘ . \c -. V“ 413 wo a; I B 3‘. (a) 0 0 0 o o o o TWAAE FIGURE 15. Pulsatile release pattern of LH (solid lines, Open circles signify pulse peaks) and prolactin (dashed lines, open triangles signify pulse peaks) in OVX controls (OVX), rats treated with estradiol for 30 days (E), and rats which received estradiol for 30 days, estradiol withdrawn and blood drawn 120 days later (WD). Shown are representative patterns from three selected individuals from each treatment group. 118 20°F « WOO 021 OVX 429 OVX 100- r . 20 a e » . ‘ 40000 5 30° #8 s i 422 E I 9» 0'“) : «’3‘L I 5 xxx, 3: 15 lfki fd' ‘ i a i {\f r- , I 0 LJ E o o\' / I / Kl 2000N ‘5 p . _ r . > "5..., u p 4 s. ‘Q \fiévtl! ‘l\\//;*\\ '2‘EE g1 \_ , v“\l u I V '1 1 ‘ - ‘9 2'. a a a A a 1 1 J 4 a A a A 4 c ‘ 3 200' I ' I P 1300 Ms wo 423 we f 025 wo I V‘.v_.\/ 100» ° ‘ - - 150 Ifi\ aA" “: A ..... . .P\A._-.‘P ‘ " 3 1 § 2‘: s 8 TIME FIGURE 16. Pulsatile release of CH (solid lines, open circles signify pulse peaks) and prolactin (dashed lines, open triangles signify pulse peaks) in OVX controls (OVX), rats treated with estradiol for 30 days (E) and rats which received estradiol for 30 days, estradiol withdrawn and blood drawn 120 days later (WD). Shown are representative patterns from three selected individuals from each treatment group. 119 Ho>oH Hoaomnumo oncooco oo nonmasoo mc.o Hm>mH Houumoo x>o cu vmumnsou mo.o one; an. moumasoamo was some scans scum hopes: cassava“ monocuaouma "moaumuumacwsvm Howvmuumo any as mnfimumv MOM mvosumz mam mamauoumz mom .8 .8 .8 .8 88 «m ...22.:' .2.- .u a um :30 « on 14.6 « 0., so... a up :53 a pop exec on. £3 £5 .8 .8 88 o u a on c on. a one and m p.“ o u 4 on oosu « one“ «m 3.320 33 38 A3 3. 33 a a 3 p q o «.0 a «.0 N a on p m up 82:39 x>O I — .Esoc — :9 ‘ >012.er 2.59; 25:33.4 — >0 5.020:— cues. execs 320.com .coauouomm :fiuomaouo mafiummaao mo muouoEmuma co poacfiucoomwp mm: Houvmuumo nouum mzmv o- mam acoEumouu Hofivmuumo cacouno mo vacuum .5 canny 120 Hm>mH Howvmuumo uwcouzu ou vmumnaoo mo.o a u «« Hm>mH Houucoo x>o ou vmumnaoo mc.o a I « pmumasuamo mm? some guano Boom nonasc oumowvca mononucmumm “cowumuumacaavm Hoavmuumm ecu mo magnumv now vaSumz mam mandamus: mom .o: .2. .8 .8 88 «m 8.25 .2... :o m 3 .38 a as... to a Eu 3.. o u on :38 u. «a... o :3 on. 88 38 .8 .8 88 one." .oq! town.“ cm»... .23... o «morocco 88 38 .8 .8 88 q u so on m can to q a... o a on 3 q «a. o .828 .25 e _ .59. _ .3. ‘ agglszzu azééeu 82.33. 1— 3 :4 z ‘ c932 exeea :2...er .cowumuomm Amqv omoauo: wcuuwcamusa oauummaon mo muouoamuma :o poacfiucoomfiv mm: Hofipmuumm noumm mama ow. mam unmEumouu Heavmuumm cacouno mo uummum .w oHan 121 Ho>mH Hofivmuumm oficouzo ou mommasoo mo.c a I «a Hm>mH Houucoo x>o ou vmquEoo no.0 a I « voumasuamo was some £0.53 Boom pecan: mumowvcfi mononucmmnma “cofiumuumHCHEvm Hofivmuumo may no mafimuov mom va£umz mam mamwuoum: mom :8 8: .8 .8 88 am 2:25 a... .o m 3 o a no to... a as .o. m 3 to m 3 o 2.8 cu. .o.. .A.. .o. .m. .oo. .4. a .o o « so .u... « o. .u m o. .o m a! o um 2:25 88 88 .8 .8 88 c a no a. « on no a en 2 u no 4 a .8 a 22:3... .26 I _ .Exoc — an. i 3:262“. 2.505 32.33 4 >0 :o 2 $32 assoc 323.com .cofiumuomm Amov mcoauo: suaoum meummfiaa mo mumuosmumn :o pmscwucoumav mm: Howvmuumm umuum mzmv ON. mam unmeummuu Hoapmuumm owcouco mo uommwm .m «Home 122 reduced GH pulse amplitude after E2 withdrawal. Upon autopsy of rats at the end of the 30 day E2 treatment, it was observed that the large AP compressed the basal hypothalamus. Visual inspection of the hypothalamusrAP 120 days after the removal of the E2 ' treatment, revealed hypothalami that showed little or no compression of the MBH. In all Ez-treated animals the pituitary stalk remained grossly intact. D. Discussion The results of this study show that long term E2 treatment to F344 rats diminishes the frequency of prolactin pulses and that this decrease remains for at least 120 days after removal of E2. Experiment II showed that chronic E2 administered to F344 rats attenuates TIDA neuronal activity even up to 26 weeks after the E2 treatment was discontinued. The reduction. of prolactin pulses and TIDA. neuronal activity' after chronic E2 treatment suggests that TIDA neurons are responsible for the pulsatile secretion of prolactin although presently there are no data to support this claim. The negative feedback of E2 on LH secretion in OVX rats has been well characterized (Kalra and Kalra, 1983) by decreases in mean LH level and pulse amplitude, in agreement with the present results. Most previous reports on LH pulses have used short-term estradiol treatment and found that estradiol decreases (Akema et a1., 1983; Weick and Noh, 1984) LH pulse frequency. Although LH pulse frequency in this report also remains unchanged after B treatment, the diminished 'mean ‘EH 2 concentration at the end of the E2 treatment was still present 120 days after removal of E2. The fact that animals long after E2 withdrawal were 123 still hyperprolactinemic may account for the low LH levels observed, since high prolactin can decrease serum LH (Meites et a1., 1972). However, animals after withdrawal of E2 showed a CV and percent pulse amplitude of LH that was greater than in OVX controls, suggesting a functional neuronal regulatory system for LH that is qualitatively not different from OVX control rats. Chronic estradiol treatment increases mean GH levels, apparently by acting directly on the AP (Jansson et a1., 1983), although 120 days after E2 was removed, mean GH concentrations were similar to control levels. Thirty days of E2 treatment decreased the frequency of CH pulses but the frequency returned to control values after E2 was removed. The absolute amplitude of GH pulses did not decrease during or following E2 treatment, but reductions in percent amplitude and CV occurred as a result of E2 treatment when compared with controls. Therefore, the relative pulse amplitude (baseline compared to peak values) appears to diminish during and after chronic E2 treatment. Nevertheless, since no significant changes were observed in pulse frequency or absolute pulse amplitude 120 days after E was removed, the neuronal systems regulating 2 CH secretion appear to be functioning, in a qualitative fashion, not different from OVX controls. Since the relative pulse amplitude was reduced by E2, the large AP may be responsible for a quantitative change in the mechanism(s) that regulate GH pulse height. GENERAL DISCUSSION The data presented in this thesis indicate that chronic E2 treatment in F344 rats reduced the hypothalamic inhibitory control that DA exerts on prolactin secretion. Of greater significance, the diminished dopaminergic function persisted for up to 26 weeks after E2 treatment was discontinued. The apparently "permanent" decline in TIDA activity may not be a primary effect of increased circulating E2 since bromocryptine, prevented the EZ-induced AP hypertrophy and hyperprolactinemia, and thereby prevented the .apparently ”permanent" decline in TIDA activity. The final experiment showed that chronic E2 treatment decreased the pulsatile secretion of prolactin secretion and continued to do so long after E2 ‘was removed, whereas it did not significantly alter the pattern of LB or CH secretion after removal of E2. Although other studies have demonstrated reduced TIDA activity in the presence of elevated estrogen levels (Smythe and Brandstater, 1980; Casaneuva et a1., 1982; Sarkar et a1., 1982; Sarkar et al., 1983a), this is the first evidence that reduced TIDA neuronal activity is maintained long after discontinuing the E2 treatment. One group has shown reduced prolactin responsiveness to central-acting dopaminergic drugs long after a single injection of a large-dose injection of estrogen (Willoughby et a1., 1984). These authors speculated that the reason for the decreased prolactin response after the drug injection was that the mass of the 124 125 release. Even though this suggestion may be partly valid, in rats with very large APs, i.e., at the end of E2 treatment, this is not the case in the present study 4 or 26 weeks after E2 was removed, when AP size declined more nearly to that of non-Ez-treated rats. Also, the release of 3H-DA from the ME was measured directly and found to be reduced long after E2 was removed. It is interesting that other groups, using female Long-Evans rats and Fischer 344 male rats chronically treated with estrogen ‘have reported a decline in TIDA function at the end of estrogen treatment but after estrogen was removed there was a rebound increase in TIDA neuronal synthesis (Demarest et a1., 1984; Morgan et a1., 1985). The estrogen-induced AP hypertrOphy was considerably smaller than observed in the present studies. It was suggested that enhanced TIDA function after removal of estrogen may be a factor in the subsequent involution of the hyperplastic AP. Since, in the present study, there was no enhanced TIDA function in Fischer 344 female rats after discontinuing E2 treatment, the low TIDA neuronal activity may have been responsible for the persistent hyperprolactinemia and pituitary hyperplasia observed 4 and 26 weeks after removal of E2. The apparently "permanent" damaging action of E2 on TIDA neurons was demonstrated here by a lowered prolactin response to central-acting dopaminergic drugs, i.e., morphine and nomifensine, and by the absence of TIDA neuronal response to short-term E2 treatment 26 weeks after withdrawal of the E2 treatment. At 26 weeks after chronic E AP weight 2’ was still elevated about 2-fold, AP DNA content about 3-fold and serum prolactin was about 4-fold greater than OVX control levels. The failure of TIDA neurons to increase their function in the presence of the 126 hyperprolactinemia still present after removal of E2, may be responsible for the persistent and apparently permanent hypertrophy and hyperplasia of the lactotrOphs of the AP gland. Since chronic E2 treatment results in an apparent permanent decline in TIDA neuronal function, the possibility exists that E2 directly, or a secondary action of E2, is degenerating TIDA. neurons. It has been reported previously in rats that chronic estrogen treatment produces a lesion of the arcuate nucleus characterized by a hyperreactive glial response and a permanent loss of reproductive function even when AP weight and serum prolactin levels were only moderately elevated (Brawer et a1., 1983). The neurochemical nature of the neurons undergoing degeneration were not determined. Whether the length and magnitude of the E2 treatment used in the present studies was sufficient to produce a similar lesion is unknown. The present experiments have the confounding presence of a very large AP at the end of E2 treatment that physically compressed the MBH and may have mechanically damaged TIDA neurons; there was also an extreme hyperprolactinemia present at the end of E2 administration, which has been suggested to damage TIDA neurons (Sarkar et al., 1984b). When bromocryptine was administered during E2 treatment to inhibit AP growth and hyperprolactinemia, TIDA activity was still depressed when compared with control animals. When all treatment was discontinued for 30 days, animals previously treated only with E2 showed reduced TIDA function, whereas in animals treated with bromocryptine and E2 for 30 days, after which treatment was removed for 30 days, TIDA function had returned to control levels. Therefore, E2 alone can alter TIDA neuronal activity, independent of AP size and serum prolactin levels. This 127 observation is in contrast to a study which showed that shorter-term estrogen treatment in hypophysectomized rats did not alter TIDA neuronal function (Eikenburg et a1., 1977). More importantly, E2 given together with bromocryptine, resulted in normal AP weight and serum prolactin levels, but did not permanently affect TIDA neuronal function, ie. TIDA function was not different from non-E2 treated controls after removal of E2. This suggests that either the enlarged AP or hyperprolactinemia was responsible for the permanent effect of E2 on TIDA neuronal activity. Mechanical damage to TIDA neurons in animals with large APs was further indicated by a persistent decline in neurointermediate lobe catechol- amine content which is not believed to be significantly influenced (Demarest et a1., 1984; Moore and Demarest, 1982) or even increased (Barden et a1., 1982) by E2 or prolactin. These results are in agreement with a study showing that the permanent decline in TIDA neuronal function after chronic E2 is strain-specific in the Fischer rat and does not occur in the Long-Evans rat strain (Riegle et a1., 1985). Experiment IV attempted to confirm the view that physical compression of the MBH was responsible for the loss of TIDA neuronal function. Since the 'hypothalamic peptides regulating the release of other AP hormones are also secreted by neurons located in the MBH, it would be expected that if TIDA neurons are damaged by the large AP, that growth hormone releasing factor and somatostatin neurons or GnRH neurons which regulate GH and LH release, reapectively, would also be damaged. All three of these AP hormones--prolactin, CH, and LH--are normally released in an episodic manner, believed to result from the episodic nature of the releasing factor/release-inhibiting factor secretion. Only the frequency of prolactin pulses, and not the frequency of GH and LH 128 pulses, was reduced long after the removal of E2. However, the magnitude of the pulses, particularly GH pulses, was reduced long after E2 was removed. Therefore, the qualitative nature of the prolactin pulses was influenced long after E was removed, but only the magnitude of GH and 2 LH pulses were permanently affected by E2 administration. TIDA neurons may be more sensitive to the damaging effects of an enlarged AP than the peptidergic neurons which regulate GH and LH secretion. A mechanism which might account for the loss of TIDA neurons is the unique 02 requirement for neurotransmitter synthesis in catechol- aminergic neurons. The physical compression on the MBH and the large, increased blood flow to the growing AP after E2 treatment may reduce blood flow and make this area relatively hypoxic. Moderately reduced 02 levels have been shown to decrease synthesis in other catecholaminergic systems (Davis, 1977; Robin, 1980). The rate-limiting enzyme in catecholamine synthesis, tyrosine hydroxylase, requires 02 for its activity (Davis and Carlsson, 1973). Since it has been shown that E2 can reduce activity in these neurons, the combination of reduced synthesis by hypoxia and physical compression by the enlarged AP, could dramatically diminish the activity of TIDA neurons. Catecholaminergic neurons may also undergo a ”disuse atrophy", whereby axons degenerate when activity is very low. The physical compression and "disuse atrophy" may result in a permanent degeneration of TIDA neurons, which cannot regenerate when the AP returns to near normal size. Although the knowledge of the mechanism(s) by which long-term estrogen treatment influences TIDA neuronal function is beginning to emerge, it is far from complete. It was observed in these experiments that increased circulating E2 can reduce TIDA neuronal activity whether 129 or not the animal is hyperprolactinemic or has a large AP. It will be important to determine the location of estrogen action in the CNS. Does estrogen influence TIDA neurons directly or does it act on neurons in an afferent pathway that impinges on TIDA neurons? Although there is good evidence for the presence of dense estrogen-binding sites in. TIDA neurons (Sar, 1984), suggestive of a direct effect of estrogen on TIDA neurons, a recent observation indicated that administration of the opiate antagonist, naloxone, can reverse the depression on TIDA neuronal function produced by chronic estrogen-treatment (K. T. Demarest, personal communication). At least three recent studies have demonstrated the reversible action of chronic estrogen treatment on TIDA neuronal function, ie. a return of TIDA function after chronic estrogen was withdrawn from the animal, and have strongly suggested that estrogen does not exert any permanent depression on TIDA neurons (Demarest et a1., 1984; Morgan et a1., 1985a; Morgan et al., 1985b). All of these studies, used a high dose of estrogen for a period of less than 8 weeks. It would be of interest to administer estrogen at very low levels for a long period of time, similar to that which occurs in recurrent estrous cycles, and observe whether TIDA neuronal activity is depressed by the estrogen treatment. This E2 treatment regimen would be similar to the experiments of Brawer et a1. (1978; Brawer et a1., 1983) who observed only a moderate AP hypertrophy, yet animals had the characteristic arcuate nucleus lesion after the long-term E2 treatment. TIDA neuronal activity was not measured in the experiments of Brawer et a1. (1978;1983). Even after a variety of different estrogen treatments in rats and mice (Brawer and Finch, 1983; Finch et a1., 1984), glial hyperreactivity 130 was seen in the hypothalamic arcuate nucleus, indicative of neuronal degeneration. Although it has been 10 years since this very important observation. was first reported (Brawer’ and Sonnenschein, 1976), the neurochemical nature of the degenerating neurons has yet to be determined. 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APPENDICES APPENDIX I BASIC METHOD FOR UPTAKE AND RELEASE OF [3-H]-D0PAMINE FROM MEDIAN EMINENCE TISSUE 1) After decapitation, and brain removal, the brain is placed on ice under the dissecting microscOpe. A few drops of cold Krebs-Henseleit buffer (KHB) are placed on tOp of the hypothalamus and the median eminence dissected out according to Cuello (Nature 1973, 2436: 465). Median eminences are placed in ice cold KHB after dissection. Tissue and 52 CO is then pre-incubated in fresh KHB at 37°C under 952 O2 2 for 5 min. NO more than 2 ME's/tube. 2) Tissues are transferred to 1 ml of buffer containing about 2 x 10..7 M 3H-DA. This is 20 ul of Amersham 46 Ci/mmol; 295 mCi/mg [7,8-3H] dopamine (Code tRK.582). Be sure to blow Off ethanol and acetic acid first (3H-DA is stored in this), before adding 1 ml KHB. Drugs can be added to KHB at this point to effect the uptake process. Uptake is linear to 5 min. For superfusion experiments, uptake time is 20 min. 3) Following uptake, ME tissue is rinsed with fresh. KHB and very carefully placed into the superfusion chambers. (Two ME's/chamber minimum). 163 164 4) Superfusion flow rate is 300 ul/min, and dead volume should be kept 5) 6) 7) to a minimum. Spontaneous release is not stable for at least 40 min. 3H is collected in two min fractions. Physiological induced release (depolarization) can be accomplished by two methods. Electrical stimuli (biphasic square wave pulses) can be applied using chambers which contain silver electrodes and generated by two stimulators. The stimulus is monitored on a calibrated oscilliscope (see diagram). With two chambers, the stimulus can be applied to a particular chamber during the last 15 sec of a particular fraction, and then to other chamber during the first 15 sec of the next fraction. This allows for the ‘major overflow of 3H to appear in one fraction. Stimulation is usually 15 sec in duration. Based on our system, 6 mA. amplitude, 20 hz frequency and 2 msec duration gives a maximal response. A second method Of neuronal depolariztion is by infusion Of hypertonic K+. 40 mM K7 is sufficient for a good response. Be sure + to lower Na concentration accordingly to eliminate osmotic effects. When studying ionic mechanisms of 3H-DA uptake and 3H-DA release, Tris and sucrose are used to replace NaHCO3 and NaCl respectively in Na+ free KHB. For Ca++ free media, ECTA is used to replace CaClz. After superfusion, median eminences are homogenized in 0.5 ml of 0.1N HCl, centrifuged and supernatant radioactivity is quantified. Calculation of 3H efflux is performed by dividing the amount of 3H released into a fraction by the tissue content of 3H at the end Of 165 that fraction. This value is then divided by 2 to come up with an efflux fractional rate constant (EFRC)/min. EFRC - radioactivity released in fraction / radioactivity in tissue divided by 2. 166 Preparation of Krebs-Henseleit Medium for Median Eminence Chemical B-D-glucose NaHCO3 EDTA Ascorbic Acid CSF (artificial) CaCl2 Gelatin Superfusion after Uptake of [3H]-Dopamine 180.2 84.0 372.2 176.1 111.0 Final M Amount/Liter 10 mM 1.80 g 25 mM 2.10 g 27 uM 10.05 mg 130 uM 22.89 mg 200 mM (stock) 10 ml (2.948 g/100 ml) 0.1% 1 g Amount/400 ml 720 mg 840 mg 20 ml 400 mg Chemical NaCl KCl KH2P04 MgSO ' 7H 0 20 ml is then 167 Artificial CSF consists of: M_._W; Final M Amount /Liter (final) 58.4 134 mM 7.83 g 74.6 5 mM 0.37 3 136.1 1.25 mM 0.17 g 246.5 1 mM 0.25 3 added to 400ml KHB to give final molarity Amount/500 ml M in 20 ml 78.3 g 3.7 g 1.7 g 2.5 g 168 Artificial CSF which will Contain a Final [K+] of 40 mM (in final 400 ml) Chemical .ELE; Final M Amount/Liter Amount/500 m1 (final) M in 20 ml NaCl 58.4 99 mM 5.8 g 57.8 g KCl 74.6 40 mM 3.1 g 31.4 3 1012904 136.1 1.25 mM 0.17 g 1.7 3 143304 ° 7H20 246.5 1 mM 4 0.25 g 2.5 g 20 m1 is then added to 400 ml KHB to give final M TO finish solution nialamide is added to the final 400 mls to give [nialamide] of 12.5 uM nialamide - 298.6 MW = 1.5 mg/4OO mls If desipramine (DMI) is required for uptake 0.1 uM MW of HCl is 302.77 and 12 g/400 mls - 0.4 M 0.030 mg/ml - 0.1 mM DMI in saline 1) make up 0.1 mM DMI in saline 2) add 10 l of 0.1 mM DMI to 10 mls of media to give 0.1 uM DMI APPENDIX II RADIOENZYMATIC ASSAY FOR CATECHOLAMINES Principle: Catecholamines (CA), incubated with partially purified catechol-o-methyl transferase (COMT), the methyl donor S-adenosyl methionine (SAM) and Mg under suitable conditions will result in the transfer of methyl groups from SAM to the m-hydroxyl group of the CA. Dopamine will be converted to 3-methoxy-tyramine (MT), norepinephrine to normetanephrine (NNM) and epinephrine to metanephrine (MN). These three methylated CAs can then be separated by thfnrlayer chromatography (TLC). Since a labelled methyl donor is used, ( H-SAM), these three CA can be quantitated by the use of liquid scintillation counting. General Procedure 3 Samples of nervous tissue or plasma are incubated with COMT and H-SAM n an appropriate buffer with the result the conversion of CA to their H-methylated products. The products are isolated from the i cubation media by an organic extraction after elevati pH ( H-methylated products will enter orgagic phase and unreacted H-SAM will remain in aqueous phase). The H-methylated CA products are concentrated back to the aqueous phase in an acid extnfct. A.§ortion of he acetic acid is then spotted on a TLC plate and H-MT, H-NMN and HAMN separated and counted. I. Methylation of CA 1) Sample preparation: For brain tissue, either whole brain is frozen on dry ice for later dissection or tissue is dissected immediately and frozen. Tissue is then placed in appropriate amount of 0.1 N HClO with 5 mM glutathione (0811). Samples are then homogenized (or sonified), spun down at 2500 rpm (RCB-Z) and the supernatant used for the assay. Be sure to save aliquot of homogenate for protein assay. Recommended volume of 0.1 N HCIOA with GSH is as follows: Tissue 0.1 N HClOL w/ GSH whole hypothalamus 4 ml preoptic area / anterior hypothalamus (POA/AHA) 1 m1 median eminence 0.5 ml 169 170 2) Standard preparation: All standards are prepared with 0.1 N HClO with GSH. Internal standards (standard plus tissue homogenate), rather than external standards are used because recovery is low and there is significant inhibition of the reaction by the sample (especially plasma). Standards are prepared such that a concentration of DA and NE are run with the same sample. Standard Curve for CA Assay For nervous tissue samples, only DA and NE are required for the standards. For plasma, use DA, NE and E. Standards run from 1 ng to 31.25 pg. Easiest for nervous tissue to prepare each standard amount for DA and NE per 5 ul, separately, and combine to give amount per 10 ul. e.g., weighed 42.3 ug DA we want 1 ng/S ul 8 200ng/ml and q.s. with 10 m1 H010 with GSH [5 42,300 ng DA/lO ml I 4230 ng DA/ml - 423 ng DA/100 ul if we take 100 pl, we have 423 ng DA/"x" . 200 ng DA/ml, therefore x - 2.11 ml q.s. 100 ul to 2.11 ml and this is equal to 1 ng/S (a, dilute 1:1 to get 500 pg/S “1, 250 pg/S pl, 125 pg/S 61, 62.5 pg/S 61 and 31.25 ps/ 51:1 Do the same for NE and combine DA and NE 1:1 to give standards amount/1011 3) Prepare buffer for incubation: for 19 samples 2 M Tris (pH 9.1) 500 pl 0.25 M EGTA (pH 9.1) 100 1n 1 M M’gCl2 150 11 0.25 M GSH 100 11 H0 150 m 2 4) Prepare reaction mix: buffer for incubation 3H-SAM (a) COMT (b) (a) 3H-SAM from 200 pl of 180 p1 H O (b) isolation of COMT from rat liver is described 171 for 19 samples ICN comes as 1 mCi/ml H-SAM from the bottle is diluted to 200 pl with 400 pl 200 pl (20 C1) 200 p1 S) Pipet assay: Use 13 x 100 mm cultures tubes and be sure to add each component directly to bottom of tube A. for nervous tissue: 0.1 M 110104 w/ GSH CA standard sample homogenate reaction mix B. for plasma 0.1 N 8010‘ w/ GSH dialyzed plasma (a) CA standard sample plasma reaction mix Reagent Blank 60 pl 40 pl 10 pl 50 pl 40 pl Internal Standard 10 50 40 10 50 40 p1 p1 pl pl pl p1 Sui—mas 10 pl 50 pl 40 pl 10 pl 50 pl 40 p1 (a) Dialyzed plasma was prepared by dialyzing plasma against two changes of saline for 24 hrs. Theoretically, it contains no CA. 6) Incubate, well-parafilmed tubes in Dubonoff shaking incubator at 37°C for 75 min. II. Extraction of 3H-methylated CA 7) Place all tubes on ice; mixture: add to each tube 55 pl of the following 172 for 19 tubes 0.45 M borate buffer (pH 10.5) (a) 1 ml "carrier" (b) 0.1 m1 (a) may require heating to go into solution (b) "carrier" is mixture of nonrradioactive MT, MN and NMN, each 10 mg/ml in 0.01 N HCl. This reduces loss of tritiated MT, MN and NMN during processing and to allow visualization after TLC. 8) Add to each tube 2 m1 of toluene: iso-amyl-alcohol (3:2); (24 ml 16 ml for 19 tubes) 9) Vortex l min, centrifuge 2500-3000 rpm, 5 min III. Back extraction 10) Transfer organic phase into 15 ml/conical centrifuge tubes which contain 40 1 0.1 N acetic acid in the bottom tip (pasteur pipets work well for transfer). Important to completely exclude the aqueous phase since this contains a large amount of unreacted H-SAM. ll) Vortex l min, centrifuge 2500-3000 rpm, 5 min IV. TLC 12) Carefully discard organic phase using suction 13) Spot 25 1 of the acetic acid extract onto Whatman silica gel plates with preabsorption area. After complete dryness, develop for about 3 hours in a solvent system composed of: Ratio for l chamber tertiary amyl alcohol 10 40 ml toluene 4 16 ml methylamine (401) 5 20 ml 14) Remove plate, allow to dry under hood, and place under UV lamp for a minimum of 4 hours, preferably overnight. - if TLC plates without pre-absorption area are used, spot one-half of sample at a time and allow for complete dryness between them 15) 173 the developing system requires saturation, e.g., use tape on the chamber lid or vacuum grease with a heavy object on top under the above conditions, Rf is as follows: MT I 0.67, MN I 0.53, NMN I 0.47. There is no cross-over between MT and NMN so if only DA and/or NE are present in the sample (e.g., brain tissue) no correction is needed. With other combinations of CA products, there is cross-over, and the percent cross over should be determined using the standards and the results corrected accordingly. Liquid scintillation (LS) counting When the carrier spots are clearly visible, scrape the spots into LS vials. 16) Add to each vial 1 m1 of mixture of ethyl acetate: glacial acetic 17) VI. For 1. acid: H20 (3:3:1) After 30 min, add 10 ml of ACS (Amersham), shake, and count. Calculations nervous tissue: Since DA and NE come as HCl, the weight due to the 1101 must be subtracted off the standard amounts; e.g., DA MW I 153.18; DA HCl MW I 189.64 19.22 is due to HCl 1000 pg DA HCl I 808 pg DA 500 pg DA HCl I 404 pg DA 250 pg DA HCl I 202 pg DA 125 pg DA HCl I 101 pg DA 62.5 pg DA HCl I 50.5 pg DA 31.25 pg DA HCl I 25.25 pg DA NE MW 169.18 17.72 is due to HCl NEHCl MW 205.64 1000 pg NE HCl I 823 pg NE 500 pg NE HCl I 411.5 pg NE 250 pg NE 801 I 205.7 pg NE 125 pg NE HCl I 102.9 pg NE 62.5 pg NE HCl I 51.4 pg NE 31.25 pg NE HCl I 25.7 pg NE Plot standard curve after subtracting cpms of reagent blank and cpms of sample used for the standard from each standard point Calculate best straight line using linear regression analysis. Be sure that 0,0 is included as a standard value. Subtract reagent blank cpms from each sample and calculate pg from regression equation 174 Multiply by tissue dilution factor to get total CA in whole tissue Can express per tissue protein, or wet weight or even DNA same as above for plasma except CA usually expressed as pg/ml plasma minimum detectable dose is taken to be 2x the cpms of the reagent blank and is usually 25-35 pg/tube. Reagents and Materials for CA Assay 2.. 0.1 N HClO plus 5 mM GSH CA stds D HCl NE HCl sample for internal standard reaction mixture 2 M Tris-buffer (pH 9.1) 0.25 M EGTA (pH 9.1) 1 M MgCl 0.25 M G H H 0 3H-sXM (ICN) 1 mCi/1.0 ml in H 2 carrier mixture 3 methoxytyramine, normetanephrine, metanephrine each at 10 mg/ml in 0.01 N HCl 0.45 M borate buffer (pH 10.5) toluene:isoamy1alcohol 0.1 N acetic acid solvent system for TLC tertiary amyl alcohol:toluene:methylamine (402) 304/Eton (9:1), 14 c1/mm61 Tris HCl 158 g/mole x 2 moles/l I 316 g/l x 0.050 1 I 15.8 g in 50 ml Tris base 121.1 g/mole x 2 moles/l I 242.2 g/l x 0.50 1 = 12.12 g in 50 ml pH 2M Tris base with 2M Tris HCl to pH 9.1 50 ml 0.25 M EGTA 380.4 g/mole x 0.25 mole/l x 0.050 1 I 4.75 g in 50 ml pH to 9.1 w/ NaOH 175 1 M MgCl we have MgCl2 ° 6H 0 MW 203.3 2 2 203.3 g/mole x l mole/l x 0.05 1 I 101.g in 50 ml 0.25 M GSH 307.3 g/mole x 0.25 mole/l x 0.01 1 I 0.76 g in 10 ml 0.45 M Borate (pH 11) NaBO4 ° 10 H20 381.37 g/mole x 0.45 mole/l x 0.05 l I 8.58 g in 50 ml pH to 10.5 w/ NaOH and heat until it goes in 0.01 N HCl we have 2 N HCl we want 50 ml 0.25 ml of 2 N HCl in 49.75 m1 weigh 30 mg each of 3-MT, NM & M and add 3 ml 0.01 N HCl. Store in brown bottle. we want 0.1 N HClO4 plus 5 mM glutathione MW HClO4 I 100.46 100.46 g/mole x 0.1 mole/l x 0.5 l I 5.023 g HClO4 in 500 ml we have 702 HClO 70 g/100 ml I 5.023/"x" ; we need 7.17 ml 8010 4 4 in 500 m1 H20 MW GSH I 307.3 g/mole 307.3 g/mole x 0.005 mole/l x 0.5 1 I 0.768 g or 768 mg 176 Procedure for COMT Purification 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11) 12) Everything performed at 4°C; 50 g of rat liver is homogenized in 200 ml 0.154 M KCl. Centrifuge for 30 min at 28,000 x g Filter supernatant through cotton gauze and titrate to pH 5 with 1.0 M acetic acid Centrifuge at 12,000 x g for 20 min Save supernatant and measure out 150 ml Add 26 g of ammonium sulfate (0-301), stir and centrifuge at 12,000 x g for 20 min. Save supernatant Add 17 g of ammonium sulfate to supernatant (30-502). Stir, centrifuge at 12,000 x g for 20 min and save pellet Add 25 ml of lmM phosphate buffer (pH 7.0) to pellet, suspend it, and dialyze against two changes of 2 liters of phosphate buffer (pH 7.0) containing dithiothreitol at 0.1 mM Centrifuge dialyzed solution at 12,000 x g for 30 min to remove precipitate Titrate supernatant to pH 8.1 with 2M Tris buffer (pH 8.2) Add sufficient dithiothreitol and pargyline to make final concentrations of 5 mM and 0.1 mM respectively Aliquot (500 ul/tube), ended with 32 ml enzyme) Reagents Needed for COMT Purification 400 ml KCl 0.154 M 1 M acetic acid ammonium sulfate 26 g and 17 g 25 m1 lmM phosphate buffer 3 liter lmM phosphate buffer with 0.1 mM dithiothreitol dithiothreitol and pargyline were added to final solution to make final concentrations of 5 mM and 0.1 mM respectively 1... 177 To make up 1 mM or 0.001 M phosphate buffer pKa2 of P04 I 7.2 7.0 - 7.2 - log [HP05:31_ ; x - [HPO4--], y - [H2P04-] [1121304 1 Ratio I 0.631 since we want 4 1 0.01 M x 4 l I 0.004 moles x.- 0.631 also x + y I 0.004 y 0.631 y + y I 0.004 x I 0.631 1.631 y I 0.004 y I .0024 moles x I 0016 moles 0.0016 mole [MPOA-2] dibasic x 141.96 g/mole I 0.2271 g 0.0024 mole [82P04-] monobasic x 138.01 g/mole = 0.3312 g 0.154 moles KCl/l x 0.4 l I 0.0616 moles 74.55 g KCl/mole x 0.0616 moles I 4.59 g in 400 m1 100 ml 1M AcA 60.05 g/mole 60.05 g/liter 6.00 g/100 m1 3 0 2 6.00 ml/100 m1 H20 4 1 P04 buffer 0.1 mM DTT 154.25 g/mole x 0.0001 moles/1 - 0.01542 g/l x 4 1 - 0.0617 g 178 30 mls enzyme pargyline MW 195.7 dithiothreitol MW 154.2 0.005 M dithiothreitol x 0.03 1 I 0.00015 moles 154.2 g/mole x 0.00015 mole I 0.02313 g or 23.1 mg 0.001 M pargyline x 0.03 l I 0.000003 moles 195.7 g/mole x 0.000003 I 0.00058 g or 0.58 mg CURRICULUM VITAE Name: Paul Edward Gottschall Birth Date: June 29, 1956 Birth Place: Dayton, Ohio USA Nationality: American Present Address: Department of Physiology 242 Giltner Hall Michigan State University East Lansing, MI 48824 Future Address: Tulane University Medical Center Laboratory for Molecular Neuroendocrinology and Diabetes School of Medicine Herbert Research Center Belle Chasse, LA 70037 Position Held: Research/Teaching Assistant Department of Physiology Michigan State University East Lansing, MI 48824 Education: Wright State University, B.S. 1978, Dayton Ohio Wright State University, M.S. 1980, Dayton Ohio Major Research: Effect of chronic estrogen treat- ment on hypothalamic dopaminergic control of prolactin secretion. 179 Original Articles Gottschall PE, Quigley KQ, Goya RG, and Meites J. Pulsatile patterns of plasma prolactin, luteinizing hormone and growth hormone during and long after chronic estradiol treatment. (in preparation) Gottschall PE and Meites J. Bromocryptine prevents the decline in tuberoinfundibular dopaminergic neuronal function after removal of chronic estrogen treatment in Fischer 344 rats. (submitted 1985) Gottschall PE and Meites J. Evidence for a permanent decline in tuberoinfundibular dOpaminergic neuronal function after chronic estrogen treatment is terminated in Fischer 344 rats. (submitted 1985) Sonntag, W.E., Gottschall, P.E., and Meites, J. Increased secretion of somatostatin-28 from hypothalamic neurons of aged rats ig_ vitro (submitted, 1985). Gottschall, P.E., Sarkar, D.K., Hylka, V.W., and Meites, J. Persistence of low ‘hypothalamic dopaminergic activity' after removal of chronic estrogen treatment Proc Soc Exp Biol Med 181: ???, 1986. Sarkar, D.K., Gottschall, P.E., and Mbites, J. Inhibition of tuberoinfundibular dopaminergic neuronal function after chronic hyperprolactinemia in rats. Endocrinology 115: 1269-1274, 1984. Sarkar, D.K., Gottschall, P.E., Xie, Q.W., and Meites, J. Reduced tuberoinfundibular dopaminergic neuronal function in rats with in situ prolactin secreting pituitary tumors. Neuroendocrinology 38: 498-503, 1984. Sarkar, D.K., Gottschall, P.E., Meites, J., H rn, A., Dow, R.C., Fink, G., and Cuello, A.C. Uptake and release of HIdopamine by the median eminence: evidence for pre-synaptic dopaminergic receptors and for dopaminergic feedback inhibition. Neuroscience 10: 821-830, 1983. Sarkar, D.K., Gottschall, P.E., and Meites, J. Damage to hypothalamic dopaminergic neurons is associated with development of prolactin-secreting pituitary tumors. Science 218: 684-686, 1982. Sonntag, W.E., Forman, L.J., Miki, N., Trapp, J.M., Gottschall, P.E., and Meites, J. L-DOPA restores amplitude of growth hormone pulses in old male rats to that observed in young male rats. Neuroendocrinology 34: 163-168, 1982. 180 Chapters $2_Books Hylka, V.W., Briski, K.P., Gottschall, P.E., Sonntag, W.E., and Meites, J. Hypothalamic-hypophysiotropic releasing and release-inhibiting hormones: Agonists and antagonists. In: Pharmacologic Methodologies for the Study of the Neuroendocrine System, R.W. Steger and A. Johns (eds.), CRC Press, Boca Raton, Florida (1985). Meites, J., Sonntag, W.E., Forman, L.J., Sarkar, D.K., Gottschall, P.E., and Hylka, V.W. Growth hormone and prolactin secretion in aging rats. In: Altered Endocrine States During Aging, V.J. Cristofalo, G.T. Baker III, R.C. Adelman, and J. Roberts (eds.), Alan R. Liss, New York, 1984. Sarkar, D.K., Gottschall, P.E., and Meites, J. Relation of the neuroendocrine system to development of prolactin-secreting pituitary tumors. In: Neuroendocrinology of Aging, J. Meites (ed.), Plenum Press, New York, 1983. Papers Presented Sonntag, W.E., Gottschall, P.E., and Meites, J. Increased secretion of somatostatin-28 from 'hypothalamic neurons of aged rats .ig_ vitro in response to 55 mM postassium. 67th Annual Meeting of the Endocrine Society, 1985, Baltimore, MD. Gottschall, P.E., Sar r, D.K., Quigley, K., and Meites, J. Prolactin (PRL) stimulation of ( )Idopamine (DA) release from rat median eminence tissue: depressant effect of chronic estrogen treatment. 13th Annual Meeting of the Society for Neuroscience, 1983, Boston, MA. Sarkar, D.K., Gottschall, P.E., Xie, Q.W., and Meites, J. Hyperprolac- tinemia can be destructive of tuberoinfundibular dOpaminergic neurons. 65th Annual Meeting of The Endocrine Society, 1983, San Antonio, TX. Gottschall, P.E., Sarkar, D.K., and Meites, J. Further evidence that loss of tuberoinfundibular dopaminergic (TIDA) function is involved in estrogen (E2)-induced pituitary tumor growth. Annual Meeting of The Federation of American Societies for Experimental Biology, 1983, Chicago, IL. Sarkar, D.K., Gottschall, P.E., and Meites, J. Evidence for degeneration of tuberoinfundibular dopaminergic neurons in aging rats with prolactin-secreting pituitary tumors. 12th Annual Meeting of the Society for Neuroscience, 1982, Minneapolis, MN. Sonntag, W.E., Forman, L.J., Miki, N., Gottschall, P.E., Trapp, J., and Meites, J. Restoration of normal pulsatile release of growth hormone in old male rats by L-DOPA. 63rd Annual Meeting of The Endocrine Society, 1981, Cincinnati, OH. 181