MSU LIBRARIES .—_——. RETURNING MATERIALS: P1ace in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. ANTERIOR HYPOTHALAMIC AFFERENT REGULATION OF TUBEROINFUNDIBULAR DOPAMINERGIC NEURONAL ACTIVITY By Anne Catherine Barton A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pharmacology and Toxicology 1988 ABSTRACT Anterior Hypothalamic Afferent Regulation of Tuberoinfundibular Dopaminergic Neuronal Activity by Anne Catherine Barton The role of afferent connections: 1) in maintaining basal activity of tuberoinfundibular dopaminergic (TIDA) neurons, 2) in the stimulatory action of prolactin on these neurons, and 3) in the inhibitory action of restraint stress was examined. Neuronal activity was estimated by measuring the rates of synthesis and turnover, and the metabolism of DA in the terminals of TIDA neurons in the median eminence (ME). Retrochiasmatic deafferentation decreased the rates of synthesis and turnover, and the metabolism of DA in the ME of gonadally-intact female rats, whereas a knife cut 1 mm rostral to and bilateral cuts of the retrochiasmatic area had no effect. Thus, afferents originating in the caudal region of the anterior hypothalamus, in particular, the suprachiasmatic and/or paraventricular nucleus, mediate a stimulatory influence on TIDA neurons in the intact female rat. In contrast, these afferents do not regulate basal THDA neuronal activity in male rats since retrochiasmatic deafferentation did not alter ‘ the rates of DA synthesis or turnover in the ME of male rats. Despite a decrease in basal activity following retrochiasmatic deafferenta- tion in the female rat, manipulations which increased prolactin levels increased TIDA neuronal activity while those which decreased prolactin levels were associated with a further decrease in 'I'IDA neuronal activity. These findings Anne Catherine Barton together with the identification of prolactin binding sites in the ME provides evidence for a direct action of prolactin on the terminals of TIDA neurons. The deafferentation-induced decrease in TIDA neuronal activity in intact female rats may operate via a neuronal mechanism which also functions to decrease TIDA neuronal activity following ovariectomy. Both retrochiasmatic deafferentation and ovariectomy decreased DA synthesis in the ME yet both procedures together produced no further decrease than either procedure alone. This neuronal mechanism which stimulates TIDA neurons in female rats is not operational in either intact or orchidectomized male rats, possibly due to the effects of neonatal androgen exposure. Inhibitory afferents which decrease TIDA neuronal activity and increase circulating prolactin concentrations during restraint stress also originate rostral to the mediobasal hypothalamus. Retrochiasmatic deafferentation blocked the stress-induced decrease in TIDA neuronal activity and attenuated the stress- induced increase in the secretion of prolactin. Thus, both stimulatory and inhibitory signals to the TIDA neurons originate in or pass through the anterior hypothalamus. To my husband, Ken, for his love and encouragement in supporting my career as a scientist, and to our daughter Lauren who has brought new meaning to my life and has nurtured my career as a mother. ii ACKNOWLEDGEMENTS I wish to express my sincere gratitude to Kenneth E. Moore whose confidence in me was instrumental in directing my graduate career. I greatly appreciate his commitment to my training, his tolerance of my second career and his encouragement to pursue collaborations both within and outside of the university. His leadership and guidance have been inspirational to the my scientific development. I would like to thank Keith Demarest who sparked my interest and enthusiasm for the field of neuropharmacology/neuroendocrinology. In addition, I gratefully acknowledge the collaborative efforts and helpful discussions with Drs. Keith Lookingland, Robert Lahti, Monty Piercey and Glenn Hatton. Along with our chairman emeritus, Dr. Theodore Brody, you formed a willing and helpful committee. I wish to thank all those individuals who provided technical support, especially Sue Stahl and Jill Stegman. Their dedication and friendship helped to ease many burdens. I extend a special thanks to Marty Burns for the preparation of manuscripts and to Diane Hummel for her timely and flawless assistance in the preparation of this thesis. Finally, I gratefully acknowledge the love and support given me by my parents, Frank and Claire Gredler. Their faith in me and their interest in my education have been inspirational and invaluable. iii TABLE OF CONTENTS LIST OF FIGURES LIST OF TABLES LIST OF ABBREVIATIONS INTRODUCTION A. Anatomy of Dopaminergic Neuronal Systems B. Biochemistry of Dopamine Neurons C. Methods for Estimating Tuberoinfundibular Dopaminergic Neuronal Activity 1. Rate of dopamine synthesis 2. a-Methyltyrosine induced decline of dopamine 3. Release of dopamine 4. Concentration of dopamine metabolites D. Regulation of Tuberoinfundibular Dopaminergic Neuronal Activity 1. Prolactin Z. Gonadal steroids 3. Afferent neuronal influences STATEMENT OF PURPOSE MATERIALS AND METHODS A. B. C. ”P Animals Drugs Surgical Procedures 1. Anesthesia Z. Deafferentation i. Knife construction ii. Deafferentation procedures 3. Electrolytic lesions 4. Intrac erebroventricular cannulation 5. Castration Restraint Stress Tissue Dissection iv Page vii xi «N 11 12 13 13 14 15 15 19 21 23 24 Z4 Z4 25 25 25 25 26 31 33 38 38 38 TABLE OF CONTENTS (Continued) 5” firm K. RESULTS A. Histology Measurement of Monoamines 1. 2. Radioenzymatic analyses High performance liquid chromatography with electro- chemical detection Biochemical Estimates of Neuronal Activity Radioimmunoassay Autoradiographic Analysis of Prolactin Binding Sites 1. 2. 3. Animals and tissue preparation Receptor labeling Autoradiography and data analysis Statistical Analyses Role of Afferent Neuronal Input in the Regulation of Basal Tuberoinfundibular Dopaminergic Neuronal Activity in the Female Rat 1. 2. Effect of deafferentation of the mediobasal hypothala- mus on basal tuberoinfundibular dopaminergic neuronal activity Effect of electrolytic lesions of anterior hypothalamic nuclei on basal tuberoinfundibular dopaminergic neuron- a1 activity Afferent Regulation of Tuberoinfundibular Dopaminergic Neuronal Activity in the Female Rat: Role of Prolactin l. 2. 3. Effect of retrochiasmatic deafferentation on the delayed activation of tuberoinfundibular dopaminergic neurons by prolactin Effect of retrochiasmatic deafferentation on the tonic activation of tuberoinfundibular dopaminergic neurons by prolactin Identification of prolactin binding sites in rat brain Role of Afferent Neuronal Input in the Regulation of Tubero- infundibular Dapaminergic Neuronal Activity in the Male Rat 1. Effect of complete and retrochiasmatic deafferentation of the mediobasal hypothalamus on basal tuberoinfundi- bular dopaminergic neuronal activity Effect of retrochiasmatic deafferentation on the pro- lactin-induced activation of tuberoinfundibular dopami- nergic neurons Effect of retrochiasmatic deafferentation on tubero- infundibular dopaminergic neuronal activity in cas- trated female and male rats Page 39 40 4O 42 43 43 44 44 44 45 46 47 47 47 55 58 S8 60 63 66 66 70 70 TABLE OF CONTENTS (Continued) Page D. Inhibitory Afferent Regulation of Tuberoinfundibular Dop- aminergic Neuronal Activity in the Female Rat 73 1. Effect of complete deafferentation of the mediobasal hypothalamus on the stress-induced changes in tubero- infundibular dopaminergic neuronal activity and pitui- tary prolactin secretion 73 2. Effect of retrochiasmatic deafferentation on the stress-induced changes in tuberoinfundibular dapami- nergic neuronal activity and pituitary prolactin secre- tion 76 DISCUSSION 83 A. Afferent Neuronal Regulation of Basal Tuberoinfundibular Dopaminergic Neuronal Activity in the Female Rat: Anterior Hypothalamic Origin 83 B. Stimulatory Afferent Input Does Not Mediate the Action of Prolactin on Tuberoinfundibular Dopaminergic Neurons 87 1. Delayed and tonic activation of tuberoinfundibular dop- aminergic neurons in the absence of afferent input from the anterior hypothalamus 87 2. Site of action of prolactin 89 C. Sexual difference in the Afferent Neuronal Regulation of Tuberoinfundibular Dapaminergic Neuronal Activity: Influ— ence of the Gonadal Steroids 92 D. Inhibitory Afferent Neuronal Regulation of Tuberoinfundibu- lar Dopaminergic Neuronal Activity: Anterior Hypothalamic Origin 94 SUMMARY AND CONCLUSIONS 98 BIBLIOGRAPHY 100 o "1 10 11 12 LIST OF FIGU RES Schematic of a sagittal section of the rat brain depicting the location of NE (Al-A7) and DA (A8-A16) perikarya Schematic of a midsagittal view of the rat hypothalamus indicating the location of the tuberoinfundibular and tuberohypOphysial dopaminergic neurons Schematic of a (a) nigrostriatal and (b) tuberoinfundibular DA neuron Schematic of sagittal and ventral views of the rat brain illustrating the complete and retrochiasmatic deafferenta- tion of the mediobasal hypothalamus Schematic of the topography of the lateral retrochiasmatic area showing cell groups which project axons through it to the median eminence The effect of retrochiasmatic deafferentation on the estrous cycle of the female rat The effect of increasing current intensity on the size of the electrolytic lesions produced with NE-300 electrodes Frontal brain sections illustrating a sham and electrolytic lesion of the suprachiasmatic nucleus Frontal brain sections illustrating a sham and electrolytic lesion of the anterior hypothalamic area Frontal brain sections illustrating a sham and electrolytic lesion of the paraventricular nucleus The effect of complete and retrochiasmatic deafferentation of the mediobasal hypothalamus on DOPA accumulation in the median eminence and neurointermediate lobe of the pituitary of female rats The effect of retrochiasmatic deafferentation on DA and NE content and turnover in the median eminence of female rats vii 27 30 32 34 35 36 37 48 49 LIST Figure 13 14 15 16 17 18 19 20 21 22 23 24 OF FIGURES (Continued) A time course for the effect of retrochiasmatic deafferen- tation on DOPAC and DA concentrations in the median eminence of female rats A time course for the effect of retrochiasmatic deafferen- tation on serum prolactin concentrations in female rats The effect of knife cuts made at the retrochiasmatic region and at 0.5 mm increments rostral to this region on DOPA accumulation in the median eminence of female rats The effect of retrochiasmatic deafferentation and deaffer- entation of the lateral retrochiasmatic area on DOPA accu- mulation in the median eminence of female rats The effect of electrolytic lesions of the suprachiasmatic nucleus, anterior hypothalamic area and paraventricular nucleus on DOPA accumulation in the median eminence of female rats The effect of retrochiasmatic deafferentation on the pro- lactin-induced increase in DOPA accumulation in the median eminence of female rats The effect of retrochiasmatic deafferentation on the halo- peridol-induced increase in DOPA accumulation in the median eminence of female rats The effect of retrochiasmatic deafferentation on the bromocriptine-induced decrease and the prolactin-induced increase in DOPA accumulation in the median eminence of female rats Autoradiographs of consecutive frontal sections of rat brain taken 0.3 mm posterior to bregma Autoradiographs of consecutive frontal sections of rat brain taken 2. 8 mm posterior to bregma The effect of complete and retrochiasmatic deafferentation of the mediobasal hypothalamus on DOPA accumulation in the median eminence of female and male rats The effect of retrochiasmatic deafferentation on DA con- tent and turnover in the median eminence of female and male rats viii 52 53 54 56 57 59 61 62 64 65 68 69 LIST OF FIGURES (Continued) Figure Page 25 The effect of retrochiasmatic deafferentation on the pro- lactin-induced increase in DOPA accumulation in the median eminence of female and male rats 72 26 The effect of retrochiasmatic deafferentation on DOPA accumulation in the median eminence of intact and ovariec- tomized female rats 74 27 The effect of retrochiasmatic deafferentation on DOPA accumulation in the median eminence of intact and orchi- dectomized male rats 75 28 The effect of complete deafferentation of the mediobasal hypothalamus on the stress-induced decrease in DOPA accu- mulation in the median eminence of female rats 77 29 The effect of complete deafferentation of the mediobasal hypothalamus on the stress—induced decrease in DOPAC concentrations in the median eminence of female rats 78 30 The effect of retrochiasmatic deafferentation on the stress- induced decrease in DOPA accumulation in the median eminence of female rats 81 ix Table LIST OF TABLES The effect of complete deafferentation of the mediobasal hypothalamus on NE and DA content in the median emi- nence of female rats The effect of retrochiasmatic deafferentation on DOPAC and DA concentrations in the median eminence and serum prolactin concentrations of female rats Specific binding of 125I-ovine prolactin in various regions of the female rat brain The effect of retrochiasmatic deafferentation on NE con- tent and turnover in the median eminence of male rats The effect of complete deafferentation of the mediobasal hypothalamus on the stress-induced increase in plasma pro- lactin concentrations The effect of retrochiasmatic deafferentation on the stress- induced increase in plasma prolactin concentrations 28 50 67 71 79 82 AHA AR BROMO CA COMT DA .DMN DOPA DOPAC EPI GBL HALOPER HPLC IH ICV MAO MBH GMT NE NIL NSD 1015 OC OT ORCHID X OVX PEN PH LIST OF ABBREVIATIONS anterior hypothalamic area arcuate nucleus bromocriptine anterior commissure catechol-O-methyl transferase dopamine dorsomedial nucleus 3,4-dihydroxyphenylalanine 3,4-dihydroxyphenylacetic acid epinephrine y-butyrolactone haloperidol high performance liquid chromatography incertohypothalamic intracerebroventricular monoamine oxidase complete deafferentation of the mediobasal hypothalamus a-methyltyrosine norepinephrine neurointermediate lobe of the pituitary 3-hydroxybenzy1hydrazine optic chiasm optic tract orchidectomized ovariectomized periventricular nucleus posterior hypothalamic nucleus xi LIST OF ABBREVIATIONS (Continued) POA PVN RC RC AL rPRL SCN SON THDA preoptic area paraventricular nucleus retrochiasmatic deafferentation of the mediobasal hypothalamus deafferentation of the lateral retrochiasmatic area rat prolactin suprachiasmatic nucleus supraoptic nucleus tuberohypophysi a1 tuberohypophysial dopaminergic tuberoinfundibular tuberoinfundibular dopaminergic ventromedial nucleus .0 v11 INTRODUCTION Neuroanatomists have identified a number of distinct dOpamine (DA)- containing neuronal systems within the mammalian brain. Although, most of what is known about DA neurons has been learned from studies on the nigrostriatal system, different DA neurons appear to have evolved distinctive properties that are suited to the unique requirements of their specialized functions (Moore and Wuerthele, 1979; Moore, 1987a). This is most apparent when the neurochemical properties and the responses to pharmacological and endocrinological manipula- tions of the different DA neurons are compared. For example, the tuberoinfundi- bular dopaminergic (TIDA) neurons which are located in the mediobasal hypothala- mus differ from the major ascending nigrostriatal DA neurons in that the activity of TIDA neurons: (1) is modulated by prolactin, (2) is markedly different in males and females, (3) exhibits characteristic cyclical patterns during pregnancy, and (4) is inhibited by suckling and stressful manipulations (for review, see Moore and Demarest, 1982; Moore, 1987a; 1987b). Early efforts to characterize TIDA neurons focused on the ability of hormones to alter the activity of these neurons (Ho'kfelt and Fuxe, 1972; Gudelsky §_t_ 11., 1976; Eikenburg §_t_ 31., 1977; Annunziato g 31:, 1978). TIDA neurons were considered to be part of a hormonal-neuronal feedback system controlling prolactin release independent of afferent neuronal influences. Results of more recent studies in pregnant, suckling and restraint stressed rats (McKay g g, 1982: Demarest g_t__al., 1983a; 1983b; 1985a) reveal that the TIDA neurons are not only influenced by hormones but they are also regulated by inhibitory afferent neuronal systems. The emphasis of this disserta- tion research has been to characterize the regulation of TIDA neuronal activity via afferent neuronal mechanisms. A. Anatomy of Dopaminergic Neuronal Systems The paraformaldehyde procedure of Falck-Hillarp has been used extensively to examine catecholamine-containing neuronal systems within the mammalian brain. Although this method has been extremely valuable, it requires freeze- drying, a procedure which compromises the histological quality of the tissue. Furthermore, the isoquinolone fluorescent reaction product degrades rapidly upon exposure to light causing the fluorescent label to diminish over time. The more sensitive glyoxylic acid histofluorescent technique on cryostat sections has replaced this earlier method (for review, see Bjorklund and Lindvall, 1978; Moore and Bloom, 1978; Lindvall and ij'rklund, 1983). Because of the similarities in the spectral characteristics of the catecholamines, it was difficult to distinquish DA, norepinephrine (NE) and epinephrine (EPI) neurons unless these histofluorescent studies were done in conjunction with pharmacological manipulations (i.e. inhibi- tion of DA-B-hydroxylase) or selective lesions. Thus, the use of immunocyto- chemical procedures to localize catecholamine-synthesizing enzymes has made it possible to identify DA, NE and EPI containing neurons (H8kfelt _e_t_ 2.1., 1974; Swanson and Hartman, 1975; Pickel gt a}, 1975). Prior to the advent of immunohistological techniques, Dahlstriim and Fuxe (1964) devised a numbering system to delineate various groups of catecholamine- containing neurons (Figure 1). The more caudal cells (Al-A7) in the pons medulla represent noradrenergic neurons while the rostral cells, extending from the mesencephalon (AS-A10) to the olfactory bulb (A16), are exclusively dopaminer- gic. .. Cortex ._.Ai6' '-" CC Corobollum e O Figure 1. Schematic of a sagittal section of the rat brain depicting the location of NE (Al-A7) and DA (A8-A16) perikarya. DA perikarya located in the pars compacta of the substantia nigra (A8-A9) and in the ventromedial mesencephalon (A10) give rise to the major ascending DA neuronal system, classified as the mesotelencephalic system (ij'rklund and Lindvall, 1978). These neurons are more commonly referred to as nigrostriatal, mesolimbic and mesocortical neurons which project to the caudate/putamen (striatum), nucleus accumbens and olfactory tubercle, and to various regions of the cerebral cortex, respectively. DA cell bodies have also been identified in more rostral regions of the brain, in the olfactory bulb (A16, Halasz g gl., 1977) and in four separate regions of the hypothalamus (All-A14; Lindvall gt gl_., 1984). Within the hypothalamus, DA neurons have been divided into two major groups, those comprising the incerto- hypothalamic (IH) DA system and those originally referred to as the tuberohypo- physial system (Ungerstedt, 1971). Perikarya of the IHDA system are found in the caudal hypothalamus and zona incerta (groups All and A13; Fuxe g 2g” 1969b; Bjiirklund and Nobin, 1973) and in the rostral periventricular hypothalamus (group A14; ij'rkltmd and Nobin, 1973). Cell bodies of the tuberohypophysial system are located in the mediobasal hypothalamus, in the arcuate and periventricular nucleus (A12). These neurons have been further subdivided on the basis of their anatomical distribution (Figure 2): tuberoinfundibular (TI) DA neurons project to the median eminence, and tuberohypophysial (TH) DA project to the neural and intermediate lobes of the pituitary. Carlsson gt_ a_l. (1962) and Fuxe (1964) made the initial observation of a massive supply of catecholamine fibers to the median eminence of various mammalian species. Later studies in the rat and mouse provided evidence that part of this innervation originated in the arcuate nucleus, constituting a TIDA system (Fuxe and H6kfelt, 1966; Lichtensteiger and Langemann 1966). Through the use of microspectrofluorometric analysis and stereotaxic lesions, axons of the I o .. . O - IO. . .'o ' . o o . . I I I . .0 . . . . . . o 9 O o ' o o ., 0's o .o O " . «a. ' o ' ' - o - a. ‘ o o ' _, . ' '0 .‘ ' ' I i . . . a a cl. _ ' . u . ‘ on u . ' . . I. can'. on_ n - a ‘ ..-.'. . u o u . .-.c. . I. o .0 '.'. a. o. . n . Q .I O . I . a I O . . .. . I I on U’. I. I o ....o , ". o. .n .- . u.. o. 0 0’ e on u . . .00 . .0 .g. 3”; .1 j I , ' /l/// //////////////(/,///l"2 ttttt ("II/”)7 (II/[l [fill/I I , . . [III] I Hm ’ ”ll/110mm;mum/mm(”annual/Ml” ’ ‘ I I Figure 2. Schematic of a midsagittal view of the rat hypothalamus indicating the location of the tuberoinfundibular and tuberohyp0physial dopaminergic neurons. AP = anterior pituitary; DA = dopamine; NL = neural lobe; IL = intermediate lobe; THDA = tuberohypOphysial dopamine neurons; TIDA = tuberoinfundibular dop- amine neurons. TIDA neurons were found to arise from the arcuate and ventral periventricular nuclei, project ventrally and then turn medially into the median eminence (ij'rklund gt gt., 1970, 1973a, 1973b; Jonsson gt gt., 1972; Ajika and Hkaelt, 1973). In the median eminence, they make an exceedingly dense plexus of very fine varicosities that is predominantly in the external zone, but is present in the internal and ubependymal zones as well (Fuxe, 1964; Fuxe gt a_l., 1969b; ij'rklund gt g” 1970, 1973b, Jonsson gt _a_l_., 1972; Rethelyi and Halasz, 1970; Ajika and H6kfelt, 1973, 1975). The entire dopaminergic input to the median eminence appears to originate in the mediobasal hypothalamus, but there is an additional noradrenergic innervation which originates in the lower brainstem (Fuxe, 1964; 1965; ij‘rklund gt a_l., 1973a). The noradrenergic neurons terminate in the internal and subepen- dymal layers and some have been shown to innervate the external layer of the median eminence as well (ijrklund gt gt, 1970; Cuello gt a_l., 1973; L8fstr8m gt a_l., 1976a). For example, following complete deafferentation of the mediobasal hypothalamus, a significant decrease in catecholamine fluorescence and a large number of degenerating axons were observed in the internal and subependymal zones of the median eminence with a slight decrease in catecholamine fluor- escence to the medial region of the external layer. Although Kizer gt gt. (1976) found a significant (40%) decrease in DA content in the median eminence after large lesions of DA cells of the ventral tegmental area, this is not in agreement with a number of other investigators who, using histofluorescent (Jonsson gt a_l., 1972; L3fstrb'm g gl_., 1976a) and biochemical techniques (Weiner gt at, 1972; Brownstein gt 3.1., 1976b; Gallardo gt gt, 1976; Gudelsky gt 11., 1978; Barton gt a_l., 1988) found no change in DA innervation and DA content in the median eminence after complete hypothalamic deafferentation. Overall, catecholamine- containing fibers constitute about 33% of all boutons in the lateral part of the external layer of the median eminence and 13% in the medial part (Ajika and H6kfelt 1973). The DA terminals constitute about 10-20% of the total catechol- aminergic innervation in the subependymal layer, about 50-7 5% in the medial part of the external layer, and more than 80% in the lateral external layer. The anatomically-related THDA neurons also have their cell bodies in the arcuate nucleus but in the more rostral aspects of this nucleus (Figure 2; BjBrkltmd gt gl., 1973b). The axons of the THDA neurons project through the median eminence and infundibular stalk to terminate in the neural and inter- mediate lobes of the pituitary (Fuxe, 1964; Bj6rklund gt gl_., 1970, 1973b; Smith and Fink, 1975). Conversely, the TIDA neuronal projections connect the rostral to caudal extent of the arcuate nucleus with a corresponding part of the median eminence. The DA terminals in the median eminence do not form true synaptic connections with other tissue elements but show sites of close contact with non- aminergic neurons and ependymal cells, as well as with the precapillary space of the hypophysial portal vessels (Hfikfelt, 1973; Ajika and Hkaelt, 1973). These anatomical arrangements have been taken to support the idea that DA released from the TIDA nerve terminals in the median eminence can act on neighboring peptidergic axon terminals or be released into the portal blood. For example, DA released from nerve terminals in the medial external zone of the median eminence is transported via the hypophysial portal blood to the anterior pituitary where it activates receptors on lactotrophs to inhibit the release of prolactin (MacLeod, 1976). B. Biochemistry of Dopamine Neurons Early or pioneering information on the neurochemical events occurring in DA nerve terminals has been obtained from studies on nigrostriatal DA neuro‘ns ' - 18 (Moore and Wuerthele, 1979). Tyrosine is transported into DA nerve termina where it is converted to 3,4-dihydroxyphenylalanine (DOPA) by tyrosine hydroxyl- ase, the enzyme that regulates this rate-limiting step in DA synthesis (Figure 3). This enzyme is regulated, in part, by an end product inhibitory process such that decreases in intraneuronal DA concentrations result in increased DA synthesis and vice versa. By this mechanism, concentrations of DA within the nerve terminal remain fairly constant despite alterations in the amount of transmitter released. DOPA, which is synthesized from tyrosine, is decarboxylated by L-aromatic amino acid decarboxylase to form DA. The newly synthesized DA can be stored in synaptic vesicles and be released in response to the arrival of nerve action potentials. DA which is released from nigrostriatal nerve terminals can activate receptors on dendrites or cell bodies of postsynaptic neurons. In addition, DA in the synaptic cleft can activate presynaptic or autoreceptors (Carlsson, 1975). Activation of these receptors results in the inhibition of the synthesis and release of DA (Roth 3 a_l., 1975; Christiansen and Squires, 1974; Nowycky and Roth, 1978). The actions of DA at pre- and post-synaptic receptors is terminated when DA is removed from the synaptic cleft by a high affinity active uptake mechanism which transports the amine back into the presynaptic nerve terminal (Snyder and Coyle, 1969). Once inside the nerve terminal, DA can be repackaged into vesicles and be re-released in response to appropriate stimuli or it can be oxidatively deaminated to 3,4-dihydroxyphenylacetic acid (DOPAC) by mitochondrial mono- amine oxidase (MAO). Although many of the synthetic and metabolic processes are similar to nigrostriatal DA neurons, TIDA neurons differ in several important respects (see Figure 3). Synthesis of DA in TIDA neurons is regulated by end product inhibition just as in nigrostriatal neurons. This is evidenced by the fact that inhibitors of MAO increase DA concentrations and decrease the rate of DA synthesis in the HVA com/ DOPAC TYROSINE f" —--DOPA ——DA 7 “ l TYROSINE oopAc TYROSINE —‘-:L- DA] POSTSYNAPTIC CELL BODY OR DENDRITE DA] ANTERIOR PITUITARY Figure 3. Schematic of a (a) nigrostriatal and (b) tuberoinfundibular DA neuron. COMT = catechol-O-methyl transferase; DA = dopamine; DOPA = 3,4-dihydroxy- phenylalanine; DOPAC = 3,4-dihydroxyphenylacetic acid; HVA = homovanillic acid; MAO = monoamine oxidase; 3MT = 3-methoxytyramine. 10 terminals of these neurons (Demarest and Moore, 1981b). Conversely, reserpine reduces DA concentrations and, thereby, increases DA synthesis in the same brain regions (Demarest and Moore, 1979a). Most TIDA neurons do not form classical synapses (Ajika and HESkfelt, 1973) but terminate adjacent to perivascular spaces in the external layer of the median eminence. DA released from these neurons enters the blood and is carried by the hypophysial portal veins to the anterior pituitary. The concentration of DOPAC, when compared to that of DA, is much lower in the median eminence than it is in other brain regions that contain DA nerve terminals (Fekete Q 11., 1979; Umezu and Moore, 1979; Lookingland gt 11., 1987b). This may be due, in part, to the fact that following release from the TI neuron, DA is quickly transported away from the terminal by the blood, thereby reducing the amount of amine that can be recaptured. In addition, the DA uptake system in terminals of TIDA neurons has a lower affinity for DA than do nigrostriatal DA terminals (Demarest and Moore, 1979b; Annunziato gt a_l., 1980; George and vanLoon, 1982). This is probably the reason that TIDA neurons are less sensitive than other catecholaminergic neurons to the neurotoxic actions of 6-hydroxydopamine (Jonsson gt 11., 1972; Cuello gt 11., 1974; Smith gt 11., 1974; Demarest gt 11_., 1980). Finally, TIDA neurons differ from nigrostriatal neurons in that they lack autoreceptors (Demarest and Moore, 1979a). Although an isolated report by Sarkar and coworkers (1983) suggests that TIDA neurons may regulate their activity via autoreceptor activation, several investigators (Fuxe and Ho'k- felt, 1974; Fuxe gt 11., 1975a; 1975b; Gudelsky and Moore, 1976; Gudelsky and Moore, 1977; Demarest and Moore, 1979a) have demonstrated that TIDA neurons do not respond acutely to DA agonists or antagonists. This has been confirmed with direct experimentation with drugs (baclofen and y-butyrolactone, GBL) which decrease impulse flow in DA neurons. As a result, DA release ceases, the activation of autoreceptors staps and the synthesis of DA is no longer inhibited. 11 Thus, these drugs cause the synthesis and the concentration of DA in the terminals of nigrostriatal neurons to increase. Baclofen and GBL decrease neuronal firing on TIDA neurons as evidenced by a decrease in DA turnover (Demarest and Moore, 1979a) and DOPAC concentrations (Lookingland gt a_l., 1987a) in the median eminence. The rate of synthesis and the concentration of DA in the terminals of TIDA neurons, however, does not increase (Gudelsky and Moore, 1976; Moore and Gudelsky, 1977; Moore gt 11., 1978; Demarest and Moore, 1979a; Moore and Demarest, 1980). Thus, it appears that TIDA neurons lack autoreceptor mechanisms for regulating DA synthesis. C. Methods for Estimating TIDA Neuronal Activity Electrophysiological techniques have been used extensively to record acti- vity on the mesotelencephalic DA neurons, the perikarya of which are tightly packed in discrete nuclei in the ventral midbrain. The diffuse distribution of THDA perikarya among non-DA neurons and the anatomically related THDA neurons has made it difficult to employ electrophysiological techniques to examine TIDA neuronal activity. Thus, investigators have relied on biochemical techniques to estimate activity of TIDA neurons. Neurochemical estimates are based on coupled relationships among DA synthesis, release and metabolism. For example, except when impulse traffic ceases, the concentration of DA in the terminal regions of neurons does not change. Thus, DA synthesis must keep pace with DA which is released. Little of the released DA is recaptured and subsequently metabolized to DOPAC in the terminals of the TIDA neurons (Lookingland gt 11., 1987a, 1987b). The amount of unreleased DA that is metabolized, however, is proportional to the amount of DA synthesized, and procedures that increase or decrease DA synthesis produce corresponding change?» . 's in DOPAC concentrations in the median eminence. Thus, changes in synthe51 ’ 12 release and metabolism of DA are related so that a change in any one of the processes can be used to estimate changes in the activity of TIDA neurons. 1. Rate of dopamine synthesis Since the synthesis of DA is regulated at the step catalyzed by tyrosine hydroxylase, estimates of TIDA neuronal activity have been based on the activity of this enzyme. Measurements of tyrosine hydroxylase activity have been made i_n gtt_r_o_ in response to a variety of endocrinological manipulations (Kizer gt 11., 1974; Kizer gt 11., 1978), but because of the limitations in sensitivity of the assay, these early studies required pooling of median eminences from two or more animals. The effects of similar endocrinological manipulations on the activity of tyrosine hydroxylase have also been examined i_n gitg (Wang and Porter, 1986). Changes in the quantity (Porter, 1986) of enzyme, however, make it difficult to interpret changes in tyrosine hydroxylase activity. The method first introduced by Carlsson (1972) to estimate the activity of tyrosine hydroxylase i_n 11:32 by measuring the rate of accumulation of DOPA has been used extensively. This procedure involves measuring the rate of accumulation of DOPA following systemic administration of 3-hydroxybenzy1hydrazine (NSD 1015), an inhibitor of L-aromatic amino acid decarboxylase. In the absence of a decarboxylase inhibitor, the concentration of DOPA in the terminal region of TIDA neurons is essentially zero, but following the administration of NSD 1015, the concentration of DOPA in the median eminence increases linearly with time at a rate that is proportional to the activity of these neurons (Demarest and Moore, 1980; Gunnet gt_11., 1987). One shortcoming of the DOPA accumulation technique is that follow- ing inhibition of the decarboxylase enzyme, DOPA accumulates in both NE and DA neurons. The accumulation of DOPA in the median eminence, however, generally reflects changes in TIDA neuronal activity since the density and activity 13 of DA neurons in this region greatly exceed those of NE neurons (Kizer gt 11., 1976; Demarest g 11., 1979). 2. G-Methyltyrosine-induced decline of dopamine The advantage of the G-methyltyrosine (GMT) method is that it permits the concurrent estimation of DA and NE neuronal activities in the same brain regions. Following the administration of GMT, an inhibitor of the rate- limiting enzyme tyrosine hydroxylase, the concentrations of all catecholamines in the brain fall in an exponential manner at a rate that is prOportional to the activity of catecholaminergic neurons. Fuxe and collaborators conducted the initial studies, using semiquantitative (Fuxe gt 11., 1969b) and subsequently more quantitative microfluorometric techniques (L'dfstriim gt a_l., 1976b) to estimate the decline of DA in the median eminence after GMT administration. A problem with the use of enzyme inhibitors such as NSD 1015 and GMT for estimating TIDA neuronal activity is that by blocking DA synthesis and, thus the amount of DA released into the hypophysial blood, these procedures also increase prolactin secretion. This interferes with the concurrent measurement of TIDA neuronal activity and circulating prolactin concentrations and precludes the use of these techniques for certain neuroendocrine studies. In addition, these drugs require a minimal period of time for their action and this limits the determination of acute effects of some manipulations on TIDA neurons. 3. Release of dopamine The release of DA from the rat median eminence has been measured i2 gitrg (Perkins and Westfall, 1978; Wilkes and Yen, 1980; Annunziato gt a_l., 1981; Forman and Porter, 1981) and 11 _v_i_gg using voltammetric electrodes implanted into the median eminence (Plotsky and Neill, 1982) and by quantifying DA concentrations in the hypophysial portal blood (Ben-Jonathan gt 11., 1977; Gibbs and Neill, 1978). This latter procedure has an advantage of providing a direct 14 measure of DA release from TIDA neurons and thus an index of impulse traffic of these neurons. One disadvantage of this technique is that it must be carried out in surgically-stressed, anesthetized animals which may alter TIDA neuronal activity (Pilotte gt11., 1980). Although small amounts of DA are found within the cells of the anterior pituitary gland, this tissue has neither DA nerve terminals nor the capacity to synthesize this amine (Saavedra fl 1., 1975; Dahlster and Fuxe, 1966). Thus, it has been suggested that the concentration of DA in the anterior pituitary represents released DA from TIDA neurons which has been transported via the blood and internalized in prolactin-secreting cells of the anterior pituitary (Nansel gt 11., 1979; Gudelsky gt a_l., 1980). Subsequent studies, however, have revealed that changes in TIDA neuronal activity do not correlate with the anterior pituitary DA content, indicating that the latter measure is not a reliable index of impulse traffic on TIDA neurons (Demarest gt 11., 1984a; 1984c). 4. Concentration of dopamine metabolites Results from early studies (Fekete gt 1.1., 1979; Umezu and Moore, 1979) suggested that DOPAC concentrations in the median eminence could not serve as an index of TIDA neuronal activity since measurable changes in DOPAC concentrations were not observed in response to various stimuli known to influence DA synthesis and turnover. Using more sensitive methods for detecting picogram quantities of amines and their metabolites, Lookingland gt 11. (1987a; 1987b) have demonstrated that DOPAC concentrations in the median eminence increase and decrease in response to the appropriate pharmacological and physiological stimuli. An advantage of this technique for estimating TIDA neuronal activity is that it does not require drug pretreatments and thereby allows acute neurochemical estimates of activity to be made. This procedure also permits the concurrent measurement of hormone concentrations. One drawback 15 is that DOPAC concentrations may vary independent of the activity of TIDA neurons if MAO activity or DA stores are interfered with. Thus, results of key experiments are generally confirmed using more than one neurochemical tech- nique for estimating TIDA neuronal activity. D. Regulation of Tuberoinfundibular Dopaminergic Neuronal Activity 1. Prolactin TIDA neurons exhibit a prominent inhibitory action on prolactin release from the anterior pituitary. In turn, prolactin plays a role in a short-100p feedback mechanism controlling its own release by activating TIDA neurons. Systemic injections of rat or ovine prolactin increase the GMT-induced decline of DA in the median eminence, but not in other DA-rich regions of the brain. This effect was first observed histochemically (H3kfelt and Fuxe, 1972) and subse- quently confirmed biochemically (Gudelsky gt 11., 1976; Selmanoff, 1981). There is a characteristic delay of 12 to 24 hours before this effect is observed. For example, repeated systemic injections of prolactin increase the GMT-induced decline of DA in the median eminence at 26 hours but not at 10 hours after the start of the injections. Similarly, injections of ovine or rat prolactin into the cerebroventricular system increase the GMT-induced decline of DA (Annunziato and Moore, 1978), the accumulation of DOPA (Johnston gt 11., 1980) and the activity of tyrosine hydroxylase (Nicholson gt 11., 1980) in the median eminence. The concentration of DA in the hypophysial portal blood is also increased following prolactin administration (Gudelsky and Porter, 1980). These changes, however, were not seen until at least 12 to 16 hours after the injections of prolactin, at a time when the hormone had probably disappeared from the cerebroventricular system (Login and MacLeod, 1977). Thus, prolactin appears to 16 trigger a neuronal event which ultimately leads to a delayed activation of the TIDA neurons. A number of endocrinological and pharmacological manipulations that result in sustained increases in serum prolactin concentrations also cause a delayed activation of TIDA neurons. Transplantation of anterior pituitaries under the renal capsule elevates serum prolactin and thus results in an increase in the GMT-induced decline of DA and the activity of tyrosine hydroxylase in the median eminence-arcuate nucleus (Olson gt 1.1., 1972; Hohn and Wuttke, 1978; Krieger and Wuttke, 1980; Morgan and Herbert, 1980; Nicholson gt 1.1., 1980). Rats with prolactin secreting tumors release increased amounts of DA into pituitary stalk blood (Cramer gt a_l., 1979a). The DA receptor antagonist, haloperidol, increases serum prolactin concentrations by blocking the inhibitory action of DA on lactotrophs. This, in turn, stimulates TIDA neuronal activity 12 to 30 hours after injection (Gudelsky g g, 1976; Gudelsky and Moore, 1977) and after 24 hours produces an enhanced release of DA into the hypOphysial portal blood (Gudelsky and Porter, 1980). The mechanism of the delayed activation of TIDA neurons by prolactin is not well understood, but studies with cycloheximide reveal that this inhibitor of protein synthesis prevents the ability of prolactin administered into the lateral ventricle to increase DA synthesis in the median eminence (Johnston gt a_l., 1980). This has prompted the suggestion that prolactin may induce the synthesis of a protein, possibly tyrosine hydroxylase, in the somas of the TIDA neurons and the latent period is due to the time required for this newly synthesized enzyme to be transported down TIDA axons to their terminals in the median eminence. Consistent with this proposal is the finding that prolactin increases maximal activity of tyrosine hydroxylase (Nicholson 3 11., 1980). Gonzalez and Porter (1988), have later fomd no change in the quantity of tyrosine hydroxylase in the 17 median eminence in response to hyperprolactinemia. Thus, the mechanism of the prolactin-induced activation of TIDA neurons remains to be elucidated. The actions of prolactin on the TIDA neurons consist of two separate components. Results from i3 y_1y_o_ experiments (as described above) suggest that prolactin stimulates TIDA neurons in a sluggish manner. On the other hand, i_n_ !i_tgg experiments demonstrate that prolactin causes a prompt increase in DA synthesis and release from tissue fragments of mediobasal hypothalamus (Forman and Porter, 1981; Perkins and Westfall, 1978). The conflicting results appears to relate to the fact that with i3 _v_i_vg, but not with i_rt 2111-3, experiments TIDA neurons are constantly exposed to endogenous prolactin which may tonically stimulate them and thereby mask the acute effects of the exogenous hormone. The problem is exacerbated by the fact that pharmacological manipulations needed to make biochemical measurements of TIDA neuronal activity inhibit DA synthesis and thereby remove the tonic inhibitory control of this amine on prolactin secretion. Thus, i_n_ 3132 rates of DA synthesis and turnover are actually values obtained in the presence of high circulating concentrations of prolactin. This confounding variable can be avoided by pretreating animals with a DA agonist (ie. bromocriptine; Demarest gt 11., 1984d). Bromocriptine does not influence the activity of TIDA neurons directly (Demarest and Moore, 1979a) but activates DA receptors on lactotrophs thereby reducing the secretion of prolactin. As a result of reduced circulating levels of prolactin, there is a reduction in TIDA neuronal activity (Demarest gt 11., 1984d). Systemic and icv administration of exogenous prolactin to a bromocriptine-pretreated rat increases TIDA neuronal activity as early as 4 hours later. Thus, there is both a rapid "tonic" component and a delayed "induction" component to the prolactin-induced activation of TIDA neurons (Demarest gt 11., 1984d; 1986). 18 There are marked sexual differences in the basal rate of activity of TIDA neurons and in the responses of these neurons to prolactin. Basal activity refers to that level of TIDA neuronal activity observed in a gonadally-intact, unmanipulated female or male rat. The higher basal rate of activity of TIDA neurons in female rats is evidenced by a higher rate of DA synthesis and turnover in the median eminence (Demarest and Moore, 1981a; Gunnet gt a_l., 1986). Since there is no difference in the steady state concentrations of DA in the median eminence of male and female rats, the differences in rates of synthesis and turnover of DA in this region are not a consequence of a greater density of DA neurons, but rather to increased TIDA neuronal activity in the female. This is consistent with the report that DA content in portal blood is several fold higher in the female than in the male rat (Ben-Jonathan gt 11., 1977; Gudelsky and Porter, 1981). The greater basal activity of TIDA neurons in the female is due, in part, to a greater sensitivity of these neurons to the stimulatory action of prolactin (Demarest and Moore, 1981a). For example, TIDA neuronal activity is reduced if serum levels of prolactin are reduced by hypophysectomy or by pretreatment with bromocriptine and this effect is much more pronounced in the female rat (Demarest and Moore, 1981a; Demarest g a_l., 1984d). In fact, bromocriptine reduces the rate of DA synthesis in the median eminence of female rats to values equivalent to those in male rats and this effect can be prevented by concurrent injections of prolactin. Dose response curves to icv-administered prolactin reveal that the magnitude of the stimulation of TIDA neurons is greater and the dose of prolactin needed to produce this stimulation is lower in the female (Demarest and Moore, 1981a). Thus, the higher level of basal activity or normal resting tone of TIDA neurons in the female rat results, in part, from a greater sensitivity to and, consequently, a continuous or "tonic" activation by prolactin. 19 2. Gonadal steroids The sexual difference in TTDA neuronal activity also appears to be due, in part, to both androgen-induced changes in neuronal differentiation during development and to differences in the hormonal environment in the adult. For example, the higher rate of DA synthesis in the median eminence of the adult female can be reduced to values similar to those in an adult male if the female is administered testosterone on day 5 after birth (Demarest gt a_l., 1981). Con- versely, higher female-like values for DA synthesis are observed in adult males in which androgenization of the brain is prevented by castration on day 1 after birth (Demarest gt 9, 1981). Thus, testosterone secreted during the neonatal period alters the basal rate of activity of TIDA neurons. In addition, the adult gonadal steroid hormonal milieu of both sexes affect TTDA neuronal activity. In female rats, the TIDA neurons are influenced by estrogen such that removal of the ovaries results in a decrease in DA turnover in the median eminence (Gunnet g_t 11., 1986). A stimulatory effect of estrogen is in agreement with a number of published reports. Repeated systemic injections of estrogen to adult female and male rats increase the GMT-induced decline in catecholamine histofluorescence (Lb'fstrb'm gt 11., 1977) and increase the rate of DA synthesis and turnover in the median eminence (Fuxe g 11., 1969a; Wiesel gt 11., 1978; Eikenburg gt a_l., 1977; Demarest and Moore, 1980; Crowley, 1982). Acute estrogen treatment also enhances DA release into the hypophysial portal blood (Gudelsky gt 11., 1981). Since the former effects were not observed in hypophysectomized rats (Eikenburg gt Q” 1977; Demarest and Moore, 1980), it has been postulated that the estrogen- induced activation of TIDA neurons is not due to a direct action of this hormone, but rather to its ability to increase circulating concentrations of prolactin. A complex pattern of effects on TIDA neurons is observed if rats are exposed to estrogens for longer periods (more than 2 weeks). These include a 20 decrease in the concentration, synthesis and turnover of DA in the median eminence and a reduced responsiveness of TTDA neurons to prolactin (Casaneuva gt a_l., 1982; Demarest gt 11., 1984b; Terry gt 11., 1985). These effects of estrogens may be due to a direct effect of the hormone or to enlargement and possible tumor development in the anterior pituitary which can compress TIDA neurons (Riegle gt a_l., 1985). Most of the effects of chronic estrogen treatment on TIDA neurons are reversible (Demarest gt 2., 1984b) but there are some reports that long term exposure to this hormone damages TIDA neurons (Sarkar gt 11., 1982). Removal of the source of estrogen is believed to be responsible for the decrease in TIDA neuronal activity following ovariectomy. Although the source of progesterone is also removed, this gonadal steroid has no effect on catechol- amine turnover in the median eminence (Fuxe gt 11., 1969a). Although Cramer gt _a_l. (1979b) reported an enhanced release of DA into pituitary stalk blood of ovariectomized-adrenalectomized rats administered progesterone, Wang and Por- ter (1986) reported a decrease in the quantitiy of tyrosine hydroxylase in the median eminence following progesterone pretreatment. In contrast to the ovariectomy-induced decrease in TIDA neuronal activity, castration in the male rat increases the rate of DA synthesis and turnover in the median eminence (Kizer gt 11., 1978; Gunnet e_t_ 11., 1986). Thus, TIDA neurons appear to be tonically inhibited by testosterone. This inhibitory influence of testosterone is supported by the observation that this hormone inhibits the activity of tyrosine hydroxylase (Brawer gt 11., 1986; Nakahara gt 11., 1976). In addition, the enhanced rate of DA turnover in the median eminence of orchidectomized rats can be reversed by testosterone replacement (Gunnet gt 11., 1986). The ability of testosterone to reduce TIDA neuronal activity is indepen- dent of any changes in prolactin secretion (Nazian and Mahesh, 1979)- Thus, the 21 hormonal environment to which the TIDA neurons are exposed in neonatal and adult life influence and, in part, are responsible for a sexual difference in the basal rate of activity of these neurons. 3. Afferent neuronal influences Inhibitory afferent systems decrease TIDA neuronal activity during physiological states that occur exclusively in the female. For example, during the first half of pregnancy in the rat, there are daily nocturnal and diurnal surges of prolactin (Butcher _e_t a_l., 1972) which are important for the initiation and maintenance of luteal function (Smith gt 11., 1975). TIDA neurons exhibit a semi- circadian pattern of activity during early pregnancy which occurs out of phase with the surges of prolactin (McKay g a_l., 1982). Thus, the increased serum prolactin concentrations occur at times when TTDA neuronal activity is low. A similar pattern of TIDA neuronal activity and serum prolactin levels is seen in rats made pseudopregnant by electrical stimulation of the uterine cervix (Free- man fi 11., 1974; Voogt and Carr, 1981; McKay g a_l., 1982). During lactation, increases in serum prolactin concentrations in response to suckling are due, in part, to a reduced release of DA into the hypOphysial portal blood (Ben-Jonathan gt 11., 1977). Suckling causes an immediate reduction in the rate of synthesis and turnover of DA in the median eminence (Demarest gt 11., 1983a; Selmanoff and Wise, 1981) and this decrease may facilitate the surge of prolactin. Various types of stressful manipulations are also potent stimulators of prolactin secretion in both female and male rats (Ajika _e_t 11., 1972; Krulich g a_l., 1974; Riegle and Meites, 1976). The increase in serum prolactin induced by acute restraint stress in the female rat is associated with a prompt reduction in the rates of synthesis and turnover of DA in the median eminence (Demarest _e_t a_l., 1985a). Restraint stress has been reported to decrease the GMT-induced decline in DA fluorescence (Fuxe gt 11., 1983) and have no effect on DA synthesis in the 22 median eminence of male rats (Demarest gt a_l., 1985b). This latter effect has been suggested to result from the inhibitory influence of testosterone in the adult male, since a stress-induced decrease in DA synthesis in the median eminence was observed in male rats following castration (Demarest g 11., 1985b). Thus, inhibitory neuronal mechanisms decrease TIDA neuronal activity during preg- nancy, pseudopregnancy, suckling and restraint stress in the female rat. The decrease in TIDA neuronal activity may contribute to the increase in prolactin secretion associated with these physiological states. Several investigators have studied the importance of neural connec- tions to the mediobasal hypothalamus in mediating the prolactin response to various stimuli. Deafferentation of neuronal afferents from anterior hypothala- mus to mediobasal hypothalamus was found to significantly attenuate the increase in prolactin produced by restraint stress (Mioduszewski and Critchlow, 1981; Kawakami and Higuchi, 1981; Kaler gt 11., 1984) ether stress (Mioduszewski and Critchlow, 1981) heat stress (Siegel gt 11., 1979) and pseudopregnancy induced by cervical stimulation (Freeman gt a_l., 1974). Blockade of these prolactin surges by deafferentation suggests that afferent neuronal pathways from the anterior hypothalamus may project to and inhibit TIDA neuronal activity and thereby increase prolactin secretion. STATEMENT OF PURPOSE Little is known about stimulatory afferent projections to TIDA neurons and whether afferent input mediates the prolactin-induced activation of these neurons. Hypothalamic receptors for prolactin have been identified in the rabbit (DiCarlo and Muccioli, 1981) but not in the rat hypothalamus (Dube gt 91., 1980; Walsh gt 11., 1978; DiCarlo gt 11., 1983). It is, therefore, uncertain whether prolactin alters TTDA neuronal activity directly or via afferent input originating within or outside of the hypothalamus. In addition, the role of afferent neurons in the regulation of basal TIDA neuronal activity has yet to be determined. Complete deafferentation of the mediobasal hypothalamus does not alter DA content (Weiner gt 11., 1972) or turnover (Gudelsky gt 11., 1978) in the median eminence of male rats. Since there are major. sex differences in the basal rate of activity on TIDA neurons it would be of interest to determine the effects of deafferentation in the female rat. Thus, the objective of the present studies was to examine the role of afferent neuronal input to the TIDA neurons: (1) in maintaining basal activity on these neurons, (2) in mediating the stimulatory action of prolactin, and (3) in mediating the inhibitory influence of restraint stress. The role of afferent neuronal regulation of basal TIDA neuronal activity was investigated in intact and castrated rats to determine the influence of the gonadal steroids. The origin of afferent input to the TIDA neurons was investigated through the use of deafferentation and lesion techniques. 9‘! MATERIALS AND METHODS A. Animals Female and male Long Evans rats were obtained from Charles River Breeding Labs (Wilmington, MA) and Blue Spruce Farms, Inc. (Altamont, NY) weighing 200-250 gm. Animals were housed 4 per cage in a temperature- (21 i 1°C) and light-controlled (lights on from 7:00 am to 7:00 pm) room with food and tap water available 11 libitum. Animals used in restraint stress paradigms were housed 2 per cage 3 days prior to experimentation. This eliminates a "stress effect” which occurs frequently when animals are removed sequentially from their cages and can be demonstrated by an increase in circulating prolactin concentra- tions in the third and fourth animal decapitated. B. Drugs Rat prolactin (kindly supplied by Dr. S. Raiti of the National Hormone and Pituitary Program, University of Maryland, through a contract program of NIDDK) was dissolved in 0.9% saline and administered by intracerebroventricular (icv) injection. The DA antagonist, ha10peridol (McNeil Laboratories, Fort Washington, PA), was dissolved in 0.3% tartaric acid while the DA agonist, bromocriptine (Sandoz Pharmaceuticals, E. Hanover, NJ), was dissolved in a few drops of glacial acetic acid and brought to volume with 0.1% ascorbic acid. The aromatic L-amino acid decarboxylase inhibitor, NSD 1015 (3-hydroxybenzylhydra- zine; Sigma Chemical Co., St. Louis, MO) was dissolved in 0.9% saline. 7A. 25 _D,L-alpha-methyl tyrosine methyl ester dihydrochloride (GMT; Sigma Chemical Co., St. Louis, MO) was brought to volume with distilled water just prior to use. C. Surgical procedures 1. Anesthesia Surgical procedures (deafferentations, electrolytic lesions, electrical stimulation and icv cannulation), performed with the aid of a rat stereotaxic instrument (David Kopf Instruments, Tujunga, CA), were carried out in animals anesthetized with ketamine (VetalarR; Parke Davis, Morris Plains, NJ) and xylazine (RompumR; Mobax Corp., Shawnee, KS). Ketamine (100 mg/ml) and xylazine (100 mg/ml) were diluted in distilled water to 88 mg/ml and 20 mg/ml respectively. On the day of surgery, equal parts of the two solutions were mixed and injected at a dose of 44 mg/kg ketamine/10 mg/kg xylazine. This anesthesia was chosen as it does not interfere with the estrous cycle, an effect reported to occur with barbiturate anesthesia (Kaneko, 1980). Ovariectomies and orchidectomies were performed under ether ane- sthesia. Animals were initially placed in a chamber saturated with ether vapors. During the subsequent removal of the gonads, anesthesia was maintained with an ether nose cone. 2. Deafferentation i. Knife construction Complete and retrochiasmatic deafferentations of the medio- basal hypothalamus and anterior hypothalamic deafferentations were performed using a modified Halasz knife (height, 2.0 mm; radius, 1.5 mm; Halasz and Pupp, 1965). The knife was constructed from stainless steel wire (SWX-023, diameter, 0.023 inches, Small parts Inc., Miami, Fla.). A 5 cm piece of wire was ground at the tip to form a double-edged blade, the uniformity of which was examined under 26 a dissecting microscope. The 3.5 mm knife edge was then bent to the above dimensions. The shaft of the knife was placed into a length of 20 gauge stainless steel tubing (HTX-20, outer diameter, 0.035 inches, wall diameter, 0.0045 inches). The remaining wire not encased in the tubing was bent to form a handle and placed into a stereotaxic micromanipulator. Bilateral deafferentation of the retrochiasmatic area was per- formed using two knives constructed from SWX-023 stainless steel wire, a modified method of Palkovits (1982). Straight double edged blades were ground from two lengths of wire which were secured together in a parallel arrangement so that their tips were 1.8 mm apart. 9 ii. Deafferentation procedures Animals were placed in a stereotaxic frame with the incisor bar set 8 mm below the intraaural line and prepared for deafferentation by exposing the skull. The approximate anatomical locations of the complete and retrochias- matic deafferentation of the mediobasal hypothalamus are illustrated in Figure 4. For complete deafferentation, the modified Halasz knife was lowered at the midline, 1.5 mm posterior to bregma, with the vertical blade in the rostral position. The knife tip was lowered to the base of the brain to the caudal border of the optic chiasm. The knife was turned 90° to the left, then the knife was moved posterior 2 mm and rotated 180°. The knife was then moved anterior 2 mm and rotated 90° back to the point of origin. This procedure was repeated twice and the knife was withdrawn in the same manner as it was lowered. The effectiveness of the complete deafferentation was determined postmortem by measuring NE concentrations in the median eminence (Table l). Noradrenergic neurons in the brain stem provide 50 to 60% of the innervation to the hypothala- mus (Dahlstrb'm and Fuxe, 1964; Fuxe 1964; 1965) while approximately 40% is intrinsic and remains in the median eminence after complete deafferentation. 27 , '\ fi- /“ r” ‘ i ‘ l \\‘ \ \PVN‘ (\ \PH : ' \ POA \ ‘ -‘-’ \ DMN‘?’ g ‘s \ \ \ AHA ’-~ .~-- )a‘ VMN 3 I"‘ ‘- ‘\~:::‘ oc SCN , a; / MEDIAN EMINENCE Figure 4. Schematic of sagittal and ventral views of the rat brain illustrating the complete and retrochiasmatic deafferentation of the mediobasal hypothalamus. The bold solid line represents the site of retrochiasmatic deafferentation, while the bold solid line and the bold broken line represent the complete deafferenta- tion. AHA = anterior hypothalamic area; AR = arcuate nucleus; CA = anterior commissure; DMN = dorsomedial nucleus; CC = optic chiasm; PH = posterior hypothalamic nucleus; POA = preoptic area; PVN = paraventricular nucleus; SCN = suprachiasmatic nucleus; VMN = ventromedial nucleus. 28 TABLE 1 The Effect of Complete Deafferentation of the Mediobasal Hypothalamus (MBH) on NE and DA Content in the Median Eminence of Female Rats ng/mg protein NE DA Sham 19.5 i 1.9 110 14.8 MBH 6.3 :1.0* 109 i 5.5 Values represent the means i 1 SE of 4-8 determinations. *Value significantly different from respective sham control rats, p < 0.05. 29 Thus, norepinephrine concentrations are significantly reduced in the median eminence while DA concentrations remain unaltered after complete deafferen- tation of the mediobasal hypothalamus. This observation is consistent with previous reports (Weiner g 11., 1972; Brownstein gt 11., 1976b; Gudelsky gt a_l., 1978). Retrochiasmatic deafferentation was produced by lowering the knife (as with complete deafferentation) to the base of the brain until resistance was met at the ventral surface of the skull. The knife was turned 90° to the left and right two times to produce a semicircular cut and the knife was then removed in the same manner as it was lowered (Halasz and Gorski, 1967). Anterior hypothalamic deafferentations were performed similar to retrochiasmatic deafferentation. Rather than a semicircular cut made 1.5 mm posterior to bregma, anterior hypothalamic deafferentations were made 1.0 and 0.5 mm posterior to bregma, at bregma, and 0.5 and 1.0 mm anterior to bregma (a total of 5 different anterior hypothalamic knife cuts). The most anterior cut was made at the rostral border of the preoptic area, the most anterior part of the hypothalamus. Bilateral deafferentation of the lateral retrochiasmatic area, the anatomical location of which is illustrated in Figure 5, was performed as previously described (Palkovits, 1982) with some minor modifications. The bilateral knife assembly was centered over the midline with the tip of each knife approximately 900 um lateral to the midline. The knives were lowered, 1.5 mm posterior to bregma, to the ventral surface of the brain where they were manipulated 1.0 mm anterior and 1.0 mm posterior and withdrawn in the same manner as they were lowered. Sham operations consisted simply of lowering the respective knife 5 mm through the midline without rotation. Animals were allowed to 30 Figure 5. Schematic of the topography of the lateral retrochiasmatic area showing cell groups which project axons through it to the median eminence. CA = anterior commissure; ME = median eminence; MFB = medial forebrain bundle; PEN = periventricular nucleus; POA = preoptic area; PVN — paraventricular nucleus (Modified from Palkovits, 1982). p4 h< C0 ph Fig Sire Witl Stee Mec‘ C00: 0.8I 31 recover for 7 days prior to experimentation except in the case of a time course experiment where the animals were allowed to recover from 1 day up to 3 weeks after surgery. The anatomical location of the deafferentations were verified postmortem on all frozen sections of brains analyzed neurochemically. Initially, anatomical confirmation of the retrochiasmatic deafferentation was made on horizontal fixed sections of brains stained with cresyl violet. Verification of knife cut placement along with an observation that retrochiasmatic deafferentation induced a state of persistent vaginal estrus formed the inclusion criteria for acceptance of an animal into a study. The pattern of persistent vaginal cornification following retrochiasmatic deafferentation had been demonstrated previously (Halasz and Gorski, 1967; Brownstein gt a_l., 1976a) and is depicted in Figure 6. Only those deafferentated females displaying at least 4 days of persistent vaginal estrus were used in these experiments. Following surgery, the skull was repaired with bone wax and the scalp was closed with stainless steel wound clips. All animals received a 0.1 ml subcutaneous injection of CombioticR (200, 000 U procaine penicillin and dihydro- streptomycin per ml; Pfizer, Inc., New York, NY). 3. Electrolytic lesions Electrolytic lesions were made in rats placed in a stereotaxic frame with the incisor bar positioned 3 mm below the intraaural line. NE-300 stainless steel electrodes (0.25 mm tip diameter and 0.5 mm exposed length; Rhodes Medical, Woodland Hills, CA) were bilaterally placed in the suprachiasmatic nucleus (SCN), anterior hypothalamic area (AHA) or paraventricular nucleus (PVN). Using the atlas of K3nig and Klippel (1963) as a reference, the electrode coordinates were approximately C 0.4 mm, L 0.2 mm, V 9.4 mm for the SCN, C 0.8 mm, L 0.75 mm, V 8.9 mm for the AHA, and C 1.4 mm, L 0.4 mm, V 8.3 mm for the PVN. The abbreviations C, L and V represent the caudal, lateral and 32 '00 P SHAM D olssmus 23 PROES‘I’RUS 7° ' I esraus ‘ T .— _ _ '/ a / 25- g; - w o .. J7” 21' x - j. I 4 6 7 <1 m. .J IOOr- RC LESION - .< j. o 'n- _ .— ~2 ° so F1 - _ 1 25 KC 7 / /’ r/’. '// O _ .j ,/ a 2 3 4 5 3 DAYS AFTER SURGERY Figure 6. The effect of retrochiasmatic deafferentation (RC) on the estrous cycle of female rats. Columns represent the percent of total rats (32 sham or 32 RC) whose vaginal cytology were indicative of a given stage (diestrus, proestrus or estrus) of the estrous cycle at 1 to 7 days following sham or retrochiasmatic deaf ferent ation. 33 ventral placement of the electrodes with respect to bregma and the skull. An anodal DC current of 300-500 11A intensity was supplied for 15 seconds to the electrodes by a Grass constant current unit (Grass Instrument Co., Ann Arbor, MI) which was driven by a Grass SD-9 stimulator. An initial experiment was done to determine the size of the lesion versus the amount of applied current in the PVN (Figure 7). Lesion size was measured through the use of a micrometer while viewing the brain section under a dissecting microscope. Thus, the current intensity varied depending upon the size of the region to be lesioned, i.e 300 11A for SCN, 500 11A for AHA and 400 11A for PVN. Sham lesions were made by lowering the electrodes along the same coordinates without passing current. Anatomical confirmation of lesion placement was made on frontal sections of fixed brains stained with thionin. A representative sham and electrolytic lesion of the SCN, AHA and PVN are shown in Figures 8, 9 and 10 respectively. 4. Intracerebroventricular cannulation ICV cannulae were implanted in rats placed in a stereotaxic frame with the incisor bar set 5 mm above the intraaural line (Pellegrino and Cushman, 1968). A cannula guide consisting of a 10 mm length of stainless steel tubing (HTX-23, outer diameter 0.025 inches, wall diameter 0.006 inches, Small Parts Inc., Miami, FL) was inserted into the lateral ventricle (C 0.0, L 1.5, V 3.2 mm with respect to bregma and dura) 7 days prior to experimentation. The guide was affixed to the skull with stainless steel screws and dental cement (Repair Material, Dentsply, York, PA) and occluded with a stylet. Hormones were infused at a rate of 1 111 per minute into the lateral ventricle by replacing the stylet with a 30 gauge stainless steel injector connected with polyethylene tubing to a Hamilton syringe. 34 2.4- 2.0: ’8‘ LG- 5 o: m I- LZ‘ Lu 2 S c: 2 0.8‘ O a m _J 0.4“ 0 I T i r j 0 0.4 0.8 |.2 l.6 2.0 CURRENT INTENSITY (mA) Figure 7. The effect of increasing current intensity on the size of electrolytic lesions produced with NE-300 electrodes. Values represent the means of lesion diameters (3-4 determinations) of the PVN following 0.1, 0.3, 1 and 2 mA of current applied for 15 sec. 35 Figure 8. Frontal brain sections illustrating a sham (A) and electrolytic lesion (B) of the suprachiasmatic nucleus (SCN). SON = supraoptic nucleus; V = third ventricle. 36 Figure 9. Frontal brain sections illustrating a sham (A) and electrolytic lesion (B) of the anterior hypothalamic area (AHA). OT = optic tract; V = third ventricle. 37 Figure 10. Frontal brain sections illustrating a sham (A) and electrolytic lesion (B) of the paraventricular nucleus (PVN). OT = optic tract; V = third ventricle. 38 5. Castration Rats were anesthetized with ether and ovariectomized or orchidecto- mized 2 weeks prior to experimentation (1 week prior to deafferentation). A small incision was made through the abdominal wall to externalize and remove the ovaries. The muscle wall was then sutured and the incision closed with stainless steel wound clips. For removal of the testes, an incision was made in the surrounding skin and pressure was applied to the abdomen to externalize the gonads. Blood flow to the testes was tied off, the gonads were removed and the incision was closed with wound clips. D. Restraint Stress The stress regimen consisted of 30 minutes of restraint in which rats were lightly anesthetized with diethyl ether and then immobilized in a supine position (Demarest gt a_l., 1985a). In studies where the effect of stress on DOPA accumulation was determined, NSD 1015 was administered immediately prior to the initiation of the 30-minute period of stress. E. Tissue Dissection Following the appropriate treatments, rats were decapitated and their brains were quickly removed and placed on a cold plate for dissection of the posterior pituitary and median eminence (Demarest and Moore, 1979a) or rapidly frozen over dry ice and processed using a modified method of Palkovits (1973). Consecutive 600 um frontal sections beginning at about A9220 um (K3nig and Klippel, 1963) were prepared from the frozen brains and thaw mounted on a glass slide. Frozen sections were viewed through a stereodissecting microscope and selected brain regions were removed using various sizes of stainless steel tubing 39 by a modification (Lookingland and Moore, 1984) of the method originally described by Palkovits (1973). Once dissected, the tissue samples were homogenized in polyethylene microsample tubes containing various volumes of cold 0.2 N perchloric acid with 10 mg% EGTA for radioenzymatic analysis. Samples were then centrifuged and the supernatants stored at -20°C for later assay. Tissue samples were placed in 0.1M phosphate-citrate buffer (pH 2.7) with 0.1 mM EDTA, 0.035% sodium octylsulfate and 20% methanol for analysis by high performance liquid chromato- graphy (HPLC) with electrochemical detection. Samples were then sonicated for 3 seconds (Sonicator Cell Disruptor, Heat Systems-Ultrasonics, Plainview, NY), centrifuged and the supernatants stored at -20°C. The remaining protein pellets were dissolved in 1 N sodium hydroxide and the content was determined by the method of Lowry 3311. (1951). F. Histology In the initial studies involving retrochiasmatic deafferentation and in studies employing electrolytic lesion techniques brains were preserved in a 4% formalin solution buffered in 0.1 M phosphate (pH 7.4). Fixed tissue was sectioned (50 um) on a freezing microtome and mounted to gelatin-coated slides. Sections were stained with either cresyl violet (cresyl escht violett, Chroma Gesellschaagt, Schmid and Co.) or thionin (Fischer Scientific Co., Fairlawn, NJ). The anatomical location of the knife cuts and electrolytic lesions was confirmed by examining the tissue under a microscope. 40 G. Measurement of Monoamines 1. Radioenzymatic analyses Ten microliter aliquots of tissue supernatants were assayed for DA and NE (Umezu and Moore, 1979) and DOPA (Demarest and Moore, 1980) via radioenzymatic assay (data in Figures 11, 12, 15, 23 and 24 and Table 4) . In this procedure, DA, NE and DOPA in standards and tissue extracts were incubated in the presence of partially purified catechol-o-methyltransferase and S-adenosyl-L- [methyl-3H] methionine ([3H] SAM) resulting in the formation of radioactive o- methylated 3-methoxytyramine, normetanephrine and 3-methoxytyrosine respec- tively. The first two of these products were separated by organic extraction and thin layer chromatography and the later by cation/anion exchange chromato- graphy. The contents of o-methylated catecholamines in tissue extracts were calculated directly from standards after subtracting blank values. Ten microliters of perchloric acid extracts of brain tissue or standards (0.25-8 ng DA and NE, 0.125-4 ng DOPA) were added to 5 ml conical tubes on ice. Twenty-five microliters of a freshly prepared incubation mix were then added to each tube. This mixture (for 30 samples) contained: 50 111 20 mM EGTA-sodium (pH 7.2); 125 111 catechol-o-methyltransferase prepared as described previously (Moore and Phillipson, 1975); 75 111 [3H] SAM (250 uCi/ml, New England Nuclear Corp, Boston, MA); 50 111 o-benzylhydroxylamine and pargyline (18 mg/ml and 8 mg/ml in 10% mercaptoethanol); 325 111 1M Tris base (pH 10.2) and 75 111 of distilled water. This mixture was incubated at 37°C for 30 minutes. For separation of DA and NE 30 111 of a solution consisting of 10 volumes of 0.45 M borate buffer and 1 volume of methoxyamine carrier (2.5 mg of each of the following compounds per ml of 0.025% sodium metabisulfite: 3- methoxytyramine HCl, normetanephrine HCl and metanephrine HCl; Sigma Chemical Co., St. Louis, MO) was added. Twenty 111 of 1.5% tetraphenyl boron 41 and 500 111 of toluene-iSOpentyl alcohol (3:2) was then added in quick succession. The tubes were centrifuged and 475 1110f the organic phase was transferred to 5 ml conical tubes containing 250 111 of 0.45M borate buffer (pH 8.0). After vortexing and centrifugation, 400 111 of the organic phase was transferred to 5 ml conical tubes containing 40 111 of 0.1 N HCl. After vortexing and centrifugation, the organic phase was aspirated and the acid phase washed with 250 111 of ethylacetate saturated with water. After aspiration of the ethylacetate, 25 1110f the acid phase was spotted on thin layer chromatography plates (LK6D, Linear K, Whatman, Clifton, NJ). The chromatographs were developed with methylamine- ethanol-chloroform (5:18:40) and visualized after spraying with Folin reagent diluted 1:1 with water. The 3-methoxytyramine and normetanephrine spots were scraped into scintillation vials and extracted with 0.5 ml of 3 M ammonium hydroxide. After the addition of 3 ml of Safety Solve (Beckman, Palo Alto, CA), radioactivity was determined in a scintillation counter. For separation of DOPA, after incubation, enzymatic activity was quenched by the addition of 3 ml of 0.5 N acetic acid containing 10 ug/ml 3- methoxytyrosine. The samples were then passed over cation exchange columns (15 x 5 mm, AG 50W—X4, H+, 200-400 mesh) which were prepared with 10 ml 0.2 M ammonium acetate (pH 6.5) and 5 ml of distilled water. The columns were then washed with 5 ml 0.1 M citrate buffer and 0.5 ml 0.2 M ammonium acetate (pH 4.5). Radioactive 3-methoxytyrosine was eluted with 3 ml 0.2M ammonium acetate (pH 4.5) into 12 x 75 glass test tubes. The samples were adjusted to pH 10-10.5 by the addition of 250 111 of 5 N sodium hydroxide and passed over a 30 x 5 mm anion exchange column (AG 1-X2, 200-400 mesh, OH- form) prepared by passing 5 ml 2 N sodium hydroxide, 5 ml distilled water, 5 ml 10% sodium chloride and 5 ml distilled water. After addition of the sample, the columns were washed with 5 ml distilled water, 5 ml 0.45 M borate buffer (pH 10.0) and 1 ml 0.45 M 42 borate buffer (pH 7.0). The radioactive 3-methoxytyrosine was eluted with 5 ml 0.45 M borate buffer (pH 7.0) directly into scintillation vials or into 13 x 100 glass test tubes. In some experiments, the 3-methoxytyrosine was further purified by adsorption onto paraffin-treated activated charcoal. Fifty microliters of a charcoal slurry, prepared by stirring 2.5 g treated charcoal in 5 ml water were added to each column eluate and vortexed. After centrifugation, the aqueous supernatant was aspirated and the remaining charcoal washed with 2.5 ml of 0.5% acetic acid. After centrifugation and aspiration, 3-methoxytyrosine was eluted from the charcoal with 1 ml of 5% phenol which was then transferred to a scintillation vial. Fifteen ml of Safety Solve was added and tritium was analyzed by liquid scintillation spectrometry. 2. High performance liquid chromatography Fifty microliters of tissue supernatant was analyzed for DA, NE, DOPA and DOPAC (data in Figures 13, 14, 16-20, 25-30, and Tables 1 and 2) by injecting the sample onto a C18 reverse-phase analytical column (Bioanalytical Systems, Inc., West Lafayette, Ind.) which was protected by a precolumn cartridge filter (10 11m spheres, 4.6 mm internal diameter x 3 cm; Bioanalytical Systems). The HPLC column was coupled to an electrochemical detector (LC4A; Bioanalyti- cal Systems) equipped with a TL-S glassy carbon electrode set at a potential of +0.75 V relative to an Ag/AgCl reference electrode. Depending on the age of the column, the concentrations of the mobile phase constituents and the pH of the mobile phase were altered slightly to attain maximal separation of the compounds of intere t while minimizing the total retention time (Chapin gt 11., 1986). All separations were performed at a flow rate of approximately 0.8 ml/min and a pressure of 2500 psi. The amount of compounds in each sample was determined by comparing peak heights with those obtained from standards as reported by a 43 Hewlett Packard Integrator (Model 3390A). The limit of sensitivity was approxi- mately 20 pg for the compounds measured. H. Biochemical Estimates of Neuronal Activity TIDA neuronal activity was estimated from measurements of the concentra- tions of DOPAC (Lookingland gt 11., 1987), the major metabolite of DA, and from the rates of synthesis and turnover of DA in the median eminence which contains the terminals of these neurons. The E 1132 rate of DA synthesis was estimated by measuring DOPA accumulation 30 minutes after the administration of the decarboxylase inhibitor NSD 1015 (100 mg/kg, i.p.; Demarest and Moore, 1980). Turnover rates of DA and NE were determined by linear regression analysis of the decline of DA and NE 30 and 60 minutes following GMT administration (an inhibitor of tyrosine hydroxylase, 250 mg/kg free base, i.p.; Lookingland and Moore, 1984). I. Radioimmunoassay Prolactin concentrations were measured in the serum or plasma using reagents and procedures in the rat prolactin radioimmunoassay kit supplied through the NIDDK Rat Pituitary Hormone Distribution Program by Drs. S. Raiti and A.F. Parlow. These reagents include an antiserum to rat prolactin produced in rabbits, a rat prolactin reference preparation to serve as a standard and a rat 1251 (Amersham, Arlington Heights, IL) prolactin antigen which was labelled with using the lactoperoxidase technique (Chard, 1982). Rat serum or plasma samples were incubated at room temperature with 24-hour periods between the addition of hormone antibody, iodinated hormone and precipitating second antibody (goat anti-rabbit gamma globulin; Arnel Products, New York, NY). Twenty-four hours after addition of second antibody, the mixture was centrifuged and the precipitate 44 counted in a Micromedic gamma counter (Model 4/200). The confidence limits of the assay were 1 to 12 ng/ml prolactin in 50 111 of plasma or serum. The intra- and inter-assay coefficients of variation were approximately 10% and 17%, respectively. J. Autoradiographic Analysis of Prolactin Binding Sites 1. Animals and tissue preparation Female Sprague Dawley rats obtained from Charles River Breeding Labs weighing 200-225 gm were housed as previously described. Daily vaginal cytology was monitored to confirm the stage of the cycle and only those females displaying a vaginal smear consistent with the second day of diestrus were used in the experiments. Animals were anesthetized with ketamine/xylazine (44 mg/kg/ 10 mg/kg, i.p.) and then perfused through the aorta with equal parts of phosphate- buffered saline and 0.32 M sucrose (pH 7.4). The brains were rapidly removed, frozen in liquid nitrogen for 10-15 seconds and stored at -70°C. The tissues were then sectioned (10 um thickness) using a Leitz 1720 digital motorized cryostat at -16°C and thaw-mounted on supercleaned glass slides. Six serial sections of brain were taken at various levels, 500 um apart, from 0.3 mm to 3.8 mm posterior to bregma according to the atlas of Paxinos and Watson (1982). Tissues were stored at -70°C Lmtil assay. 2. Receptor labeling Slide mounted tissue sections were brought to room temperature and humidified for a period of 15 minutes. The incubation procedure was carried out as previously described (Dube gt 11., 1980; DiCarlo and Muccioli, 1981) with some 125 modifications. The incubation contained 0.4 nM I-ovine prolactin (custom prepared by New England Nuclear using a chloramine-T iodination procedure, 45 specific activity 98 11Ci/ 11g). Ovine prolactin was chosen as the ligand since it is less sensitive to oxidative damage during iodination and is more potent at inhibiting binding of homologous hormone to its receptors than is rat prolactin 125I-ovine prolactin was chosen (Barkey fl a_l., 1981). A concentration of 0.4 nM since it represents the KD for hypothalamic prolactin receptors in rabbit (DiCarlo and Muccioli, 1981) and would thereby permit binding to the more relevant, high- affinity prolactin receptor. One experiment was run with a concentration of labeled prolactin of 0.04 nM in an attempt to decrease the amount of nonspecific binding and enhance specific binding. Iodinated prolactin was buffered in 25 mM Tris-H01 (pH 7.4) containing 10 mM MgC12, 0.1% bovine serum albumin, aprotinin (100 Kl'U/ml) and 0.1 mM bacitracin. To correct for nonspecific binding adjacent sections were incubated with iodinated prolactin in buffer containing 11M concen- trations of unlabeled prolactin. Tissue sections were preincubated in tissue buffer for 15 minutes followed by a 60 minute incubation period. After incubation, tissue sections were rinsed with 25 mM Tris-HCl buffer, dipped in deionized water, and dried rapidly under a stream of cold, dry air. 3. Autoradiography and data analysis The dried, labeled, slide-mounted sections were exposed to 3H- Ultrafilm (LKB Instruments, Gaithersburg, MD), and after 2-3 days of exposure at 4°C the autoradiographs were developed. Optical density readings were converted to dpms and subsequently to fmols using standard computer programs in the Amersham RAS computerized image analysis system. This system was initially calibrated with an 125 I microscale (Amersham, Arlington Heights, IL) cut to the same thickness (10 11111) as the tissue sections. The dpm concentrations which were generated for each standard were fit with a third order polynomial curve. Those brain regions containing bilateral structures were sampled and the binding quantified from both the left and right hemispheres in a minimum of 3 total 46 binding and 3 nonspecific binding sections. Midline brain regions (median eminence, arcuate nucleus, paraventricular nucleus and choroid plexus of the third ventricle) were sampled and the binding quantified from a minimum of 5 brain sections representing total and nonspecific binding. Values for specific binding were generated from a total of 3 brains by subtracting nonspecific binding values from corresponding total binding values. Values for specific binding, in all brain regions sampled, were tested using a one-way analysis of variance followed by the Student-Newman-Keuls test (Steel and Torrie, 1960), and differences with a probability of error of less than 5% were considered significant. K. Statistical Analyses Statistical analyses were conducted using the Student's t-test for compari- sons between two groups. Comparisons among 3 or more groups in experiments with one variable (i.e. deafferentation) were analyzed using a one-way analysis of variance. Experiments utilizing 2 variables (i.e. drug treatment and deafferenta- tion) were analyzed using a two-way analysis of variance, random factorial design. Between-group comparisons were made by the Student-Newman-Keuls test (Steel and Torrie, 1960). Differences with a probability of error of less than 5% were considered significant. RESULTS A. Role of Afferent Neuronal Input in the Regulation of Basal Tuberoinfundibu- lar Dopaminergic Neuronal Activity in the Female Rat 1. Effect of deafferentation of the mediobasal hypothalamus on basal tuberoinfundibular dopaminergic neuronal activity Studies were undertaken to determine the effect of complete and retrochiasmatic deafferentation on the rate of DOPA accumulation in the median eminence and neurointermediate lobe of the pituitary gland in female rats (Figure 11). A complete deafferentation of the mediobasal hypothalamus reduced the basal rate of DOPA accumulation in the median eminence with no effect in the neurointermediate lobe, the terminal region of the tuberohypophysial DA neurons. Similarly, retrochiasmatic deafferentation of the mediobasal hypothalamus, which represents the anterior portion of the complete deafferentation, selectively reduced the basal rate of DOPA accumulation in the median eminence of female rats. Retrochiasmatic deafferentation also reduced the basal rate of DA turnover in the median eminence of female rats (Figure 12) but did not alter steady-state concentrations of DA and NE and the rate of NE turnover in the median eminence. In addition, retrochiasmatic deafferentation reduced the concentration of the deaminated metabolite, DOPAC, in the median eminence without altering DA concentrations in this region (Table 2). Serum prolactin concentrations, as well, were unaltered by retrochiasmatic deafferentation. Thus retrochiasmatic deafferentation of the mediobasal hypothalamus decreases TTDA neuronal activity in the female rat as evidenced by the decreased rates of synthesis and turnover, and the metabolism of DA in the terminals of these neurons in the A7 48 E, ME NIL o a I6 r 2 - - a E B 6. l2 - ,_.l._. 1,5 - - Z 9. I" * g 8 I— * I 1- FJ-— I , —.l_ a 2 I‘L— 8 g 4 — ,5 - .. ‘3‘ Q o .. ,__. _I ,_._. o .. . .__. ._. - SHAM MBH RC SHAM MBH RC Figure 11. The effect of complete (MBH) and retrochiasmatic (RC) deafferenta- tion of the mediobasal hypothalamus on DOPA accumulation in the median eminence (ME) and neurointermediate lobe of the pituitary (NIL) of female rats. Columns represent the means and the vertical lines 1 SE of 6-8 determinations. 1‘Values significantly different from respective sham control rats, p < 0.05. 49 CONTENT TURNOVER '20 " - LOO I C] SHAM _I_ no Th I g, on I \\\\\\\\- CATECHOLAMINE, nq/mq protein RATE CONSTANT, III: I (I o \\\\\\\ \\\\\\* // NE 0 O l D > NE Figure 12. The effect of retrochiasmatic (RC) deafferentation on DA and NE content and turnover in the median eminence of female rats. The columns illustrating DA and NE content represent the means and the vertical lines 1 SE of 8 determinations, while those illustrating DA and NE turnover represent the means and the vertical lines 1 SE of the rate constant calculated from the decline in DA and NE concentrations determined in 8 animals decapitated 0, 30 and 60 minutes after alpha-methyl tyrosine (a total of 24 determinations). *Value significantly different from respective sham control rats, p < 0.05. 50 TABLE 2 The Effect of Retrochiasmatic Deafferentation (RC) on DOPAC and DA Concentrations in the Median Eminence and Serum Prolactin Concentrations of Female Rats DOPAC DA Prolactin ng/mg protein ng/mg protein ng/ml Sham 20.7 i 1.6 97 i 7.6 6.8 i 2.8 RC 15.0 i 1.4* 102 3; 9.8 6.6 + 1.6 Values represent the means i 1 SE of 6-8 determinations. *Value signifi- cantly different from sham control rats, p < 0.0 5. 51 median eminence. These results suggest that regions rostral to the retrochiasma- tic deafferentation exert a stimulatory influence on TIDA neurons in the female rat. Studies were undertaken to more completely characterize the effects of retrochiasmatic deafferentation on TIDA neuronal activity and circulating prolactin by examining a time course for the effect of deafferentation. DOPAC and DA concentrations in the median eminence were measured 1, 3, 7, 14 and 21 days after retrochiasmatic deafferentation (Figure 13). DOPAC concentrations were significantly reduced by 3 days and remained decreased for 21 days following deafferentation. A 30% to 35% decrease in DOPAC was observed at all time periods. Consistent with previous results (Figure 12), DA concentrations in the median eminence were unaltered at 7 days and at all other times following retrochiasmatic deafferentation (Figure 13). Circulating prolactin concentrations also remained unchanged at 1 and up to 21 days following retrochiasmatic deafferentation (Figure 14). These results suggest that retrochiasmatic deaf- ferentation acutely interrupts stimulatory afferent input to the TTDA neurons which is maintained for at least 21 days. Since the deafferentation-induced decrease in TIDA neuronal activity does not result in an increase in circulating prolactin concentrations, this procedure may interfere with neurons which contain prolactin-releasing factors or a reserve of DA may be present at the anterior pituitary. To determine more precisely the region rostral to the mediobasal hypothalamus mediating the stimulatory influence on TIDA neurons in the female rat, anterior hypothalamic knife cuts were made at the retrochiasmatic region and at 0.5 mm increments rostral into the anterior hypothalamus (Figure 15). Retrochiasmatic deafferentation (-1.5 mm to bregma) and placement of this knife cut 0.5 mm rostral into the anterior hypothalamus (-1.0 mm to bregma) decreased 52 I25? 3 O IOO- " a: 1.. z o o 75- - .§ 0 50- - E o o 25- - o- . , . , . . . . . SHAM l 3 7 I4 2| I25r - A IOO' - .J . ° f-L- I E z 75- - o 0 s5 sob . < o 25" - o. , _ __..._ ,____I. ,__1 ._ 1 SHAM I 3 7 I4 2| TIME AFTER DEAFFERENTATION (DAYS) Figure 13. A time course for the effect of retrochiasmatic deafferentation (open columns) on DOPAC and DA concentrations in the median eminence of female rats. Columns represent the means as a percent of the control (DOPAC and DA = 12.73 1 0.66 and 97.38 _t 9.60 ng/mg protein, respectively) and the vertical lines 1 SE of 6-8 determinations. *Value significantly different from sham control rats, p < 0.05. 53 I0 I ‘ 8 . . E \ o i: _ 6" - z . I- ' ‘ - . _| E? _L F o 4 ' . ‘ a: . 0. 2 - .. 0 b [—1 .— —_I .h—I .— «_I J SHAM I 3 7 l4 2! TIME AFTER DEAF FERENTATION (DAYS) Figure 14. A time course for the effect of retrochiasmatic deafferentation on serum prolactin concentrations in female rats. Columns represent the means and the vertical lines 1 SE of 6-8 determinations. Values are from the same animals used for determinations shown in Figure 13. 54 I ,5 '6I' +1.0 0 -IO . £3 E 1 rl_ 2' I r1 2 9 FL FL- : 8- * a. - 5' —I— r1. 2 8 o 4- - < E O SHAM +I.O +0.5 0.0 -0.5 —I.O -I.5 SITE OF HYPOTHALAMIC DEAFFERENTATION (mm from bregma ,AP) Figure 15. The effect of knife cuts made at the retrochiasmatic region (-1.5 mm to bregma) and at 0.5 mm increments rostral to this region on DOPA accumula- tion in the median eminence of female rats. Columns represent the means and the vertical lines 1 SE of 6-8 determinations. *Values significantly different from sham control rats, p < 0.05. 55 the basal rate of DOPA accumulation in the median eminence. Deafferentations made in more rostral regions (i.e., -0.5, 0.0, +0.5 and +1.0 mm from bregma), however, did not reduce basal TIDA neuronal activity. These results suggest that afferent input originating in or coursing through the caudal portion of the anterior hypothalamus mediates a stimulatory influence on the basal activity of TIDA neurons in the female rat. A number of neurons from different anterior hypothalamic nuclei have axons which project laterally and caudally before converging at the lateral retrochiasmatic area and entering into the median eminence (Palkovits, 1982). Since the afferent input which stimulates TIDA neurons in the female rat could originate in these more rostral areas and project to the TIDA neurons via this lateral route, the effect of deafferentation of the lateral retrochiasmatic area on basal TIDA neuronal activity was examined in female rats (Figure 16). Retro- chiasmatic deafferentation decreased the basal rate of DOPA accumulation in the median eminence while deafferentation of the lateral retrochiasmatic area had no effect. In agreement with results from the anterior hypothalamic deafferentation studies, these data provide evidence that the stimulatory afferent input to the TIDA neurons originates in the caudal portion of the anterior hypothalamus. 2. Effect of electrolytic lesions of anterior hypothalamic nuclei on basal tuberoinfundibular dopaminergic neuronal activity To attempt to identify the origin of stimulatory afferent input to TIDA neurons in the female rat, the effect of electrolytic lesions of nuclei located in the caudal region of the anterior hypothalamus was examined. In these experiments, the effect of electrolytic lesions of the suprachiasmatic nucleus (SCN), anterior hypothalamic area (AHA) and paraventricular nucleus (PVN) on the rate of DOPA accumulation in the median eminence was examined (Figure 17). A bilateral electrolytic lesion of the SCN decreased the basal rate of DOPA 56 24F .. . . ._.__ _._.I . SHAM RC RCAL DOPA ACCUMULATION, ng/mg protein SITE OF DEAF F ERENTATION Figure 16. The effect of retrochiasmatic (RC) deafferentation and deafferenta- tion of the lateral retrochiasmatic area (RCAL) on DOPA accumulation in the median eminence of female rats. Columns represent the means and the vertical lines 1 SE of 7-9 determinations. *Value significantly different from sham control rats, p < 0.05. 20F - I C '8 E Q 0' :5 E 2' * it 2" F"— r_|_ 9 IO' _ 1.. < _I D 2 3 5- - O < 5 D 0.. __I .___. _..I (___I .1 SHAM SCN AHA PVN SITE OF LESION Figure 17. The effect of electrolytic lesions of the suprachiasmatic nucleus (SCN), anterior hypothalamic area (AHA) and paraventricular nucleus (PVN) on DOPA accumulation in the median eminence of female rats. Columns represent the means and the vertical lines + 1 SE of 6-9 determinations. I"Values significantly different from sham cont-1:01 rats, p < 0.05. 58 accumulation in the median eminence. Electrolytic ablation of the PVN produced a similar decrease in DOPA accumulation in the median eminence while lesioning the AHA had no effect . These results suggest that stimulatory afferent input to the TIDA neurons in the female rat originates in the SCN and/or PVN. B. Afferent Regulation of Tuberoinftmdibular Dopaminergic Neuronal Activity in the Female Rat: Role of Prolactin 1. Effect of retrochiasmatic deafferentation on the delayed activation of tuberoinfundibular dopaminergic neurons by prolactin TIDA neuronal activity is believed to be primarily regulated by the stimulatory action of prolactin (Moore and Demarest, 1982). This action of prolactin is divided into two components: a delayed "induction" component and a rapid "tonic" component (Demarest gt 11., 1984d, 1986). Since the deafferenta- tion-induced decrease in TTDA neuronal activity may be the consequence of interrupting afferent input mediating either the delayed or rapid activation of TIDA neurons by prolactin, the effect of retrochiasmatic deafferentation on the ability of prolactin to stimulate TTDA neuronal activity was examined. The effect of retrochiasmatic deafferentation on the delayed activa- tion of TIDA neurons by prolactin was examined in female rats receiving an icv injection of rat prolactin (Figure 18). Retrochiasmatic deafferentation produced a characteristic decrease in the rate of DOPA accumulation in the median eminence. The administration of exogenous prolactin directly into the lateral ventricle increased median eminence DOPA accumulation in both sham-operated and retrochiasmatic-deafferentated rats. Although the overall magnitude of responsiveness of the TTDA neurons in the retrochiasmatic-deafferentated rats was reduced, the percent increase in median eminence DOPA accumulation was approximately the same for both groups. Similar results were observed when endogenous prolactin concentrations were elevated by haloperidol administration 59 2° E VEHICLE I - rPRL : 3.3 o b :1. o \5 l5- ** - on c E I: IO" ' < _l D 2 D 8 5- - < E O O o- SHAM Figure 18. The effect of retrochiasmatic (RC) deafferentation on the prolactin- induced increase in DOPA accumulation in the median eminence of female rats. Rat prolactin (rPRL, 10 11g/3 111) or its 0. 9% saline vehicle was administered via an m- ru ular cannula 12 hours prior to sacrifice. Columns represent the means and the vertical lines 1 SE of 8- 10 determinations. *Values significant- ly different from sham vehicle-treated rats; **value significantly different from RC vehicle-treated rats, p < 0.05. 60 (Figure 19). Haloperidol is a DA antagonist which blocks the tonic inhibitory action of DA on lactotrophs in the anterior pituitary gland and thereby increases secretion of prolactin. The increase in serum prolactin levels then stimulates TIDA neuronal activity (Demarest and Moore, 1980; Demarest gt 11., 1984d). Haloperidol produced a significant increase in the rate of DOPA accumulation in the median eminence of both sham-operated and retrochiasmatic-deafferentated female rats. Thus, although the overall magnitude of responsiveness of the TIDA neurons to the stimulatory actions of prolactin was decreased in the retrochias- matic-deafferentated rats, the percent increase in TIDA neuronal activity to prolactin was not altered by the knife cut. Thus, retrochiasmatic deafferentation significantly decreases the basal rate of TIDA neuronal activity in female rats yet is ineffective in blocking the delayed prolactin-induced activation of these neurons. 2. Effect of retrochiasmatic deafferentation on the tonic activation of tuberoinfundibular dopaminergic neurons by prolactin The effect of retrochiasmatic deafferentation on the rapid or "tonic" activation of TIDA neurons by prolactin was also determined. Following the administration of bromocriptine, a DA agonist, the concentration of prolactin in the serum is reduced, thereby removing the stimulatory influence of endogenous prolactin on TIDA neurons (Demarest gt 11., 1984d). As a consequence of lowered prolactin levels, the activity of TTDA neurons is decreased (Demarest gt 11., 1984d; 1985c). Bromocriptine administration significantly decreased the rate of DOPA accumulation in the median eminence of sham-operated female rats (Figure 20). Retrochiasmatic deafferentation also decreased the rate of DOPA accumulation in the median eminence and this was further reduced following bromocriptine administration. Prolactin reversed the bromocriptine-induced decrease in DOPA accumulation in the median eminence of both sham-operated rats and retrochiasmatic-deafferentated rats. Although the overall magnitude of 61 24 . c I [:l VEHICLE {3’ - HALOPER. 3' * a l8 - . E \ E” o * * 5 I: '2 " - < _l D 5 :1: D 8 6 - . < < 0. ° I D SHAM RC Figure 19. The effect of retrochiasmatic (RC) deafferentation on the haloperidol- induced increase in DOPA accumulation in the median eminence of female rats. Haloperidol (2.5 mg/kg, s.c.) or its 0.3% tartaric acid vehicle was administered 16 hours prior to sacrifice. Columns represent the means and the vertical lines 1 SE of 8 determinations. *Values significantly different from sham vehicle-treated rats; **value significantly different from RC vehicle-treated rats, p < 0.05. 62 24 C] VEHICLE f, BROMO. O a I8 _ - BROMO.+ rPRL o E * O C S :2 - . 1. < _l g 1 ' _ D S 8 6 ' i}? < u < ><‘ 5 ix“ ‘9 E :2 1 g :2: J o I Y‘( Figure 20. The effect of retrochiasmatic (RC) deafferentation on the bromocrip- tine-induced decrease and the prolactin-induced increase in DOPA accumulation in the median eminence of female rats. Bromocriptine (3 mg/kg, s.c.) or its 0.1% ascorbic acid vehicle was administered 24 hours prior to sacrifice. Rat prolactin (rPRL, 10 ug/3 111) or its 0. 9% saline vehicle was administered via an m- . rieular cannula 4 hours prior to sacrifice. Columns represent the means and the vertical lines 1 SE of 7-10 determinations. *Values significantly different from sham vehicle-treated rats; **value significantly different from RC vehicle-treated rats, p < 0.05. 63 the response of TIDA neurons to prolactin was decreased in retrochiasmatic- deafferentated rats prolactin produced similar percentage increases in the rate of DOPA accumulation in the median eminence of both groups. These results suggest that the rapid or "tonic" activation of TTDA neurons by prolactin is not mediated by rostral afferent input to the mediobasal hypothalamus. 3. Identification of prolactin binding sites in rat brain The target site for the prolactin-induced activation of TIDA neurons appears to be contained in the mediobasal hypothalamus since both complete (Gudelsky gt 11., 1978) and retrochiasmatic (Barton gt 11., 1988) deafferentations of this region do not block the stimulatory action of prolactin on these neurons. Thus, prolactin may exert its effects on TIDA neurons via an action on Specific receptors localized within the mediobasal hypothalamus. The present study was designed to identify the presence or absence of specific binding sites for prolactin in a discrete hypothalamic region(s) of rat brain following the i_n gi_ti_'_q labeling of these sites with 125I-ovine prolactin. Autoradiographs of tissue sections incu- bated in 0.4 nM 125 I-ovine prolactin exhibited an intense reaction over the choroid plexus of the lateral ventricle (Figure 21-A). Incubation of an adjacent brain section with labeled prolactin in the presence of excess unlabeled hormone inhibited the binding reaction in the choroid plexus (Figure 21-B). More caudal tissue sections also showed a reaction for prolactin binding in the choroid plexus of the third ventricle and in the median eminence (Figure 22-A). Incubation of an adjacent brain section with excess unlabeled prolactin inhibited the binding reaction in the choroid plexus and significantly reduced the binding in the median eminence (Figure 22-B). A similar pattern of total and nonspecific binding was observed when tissue sections were incubated in 0.04 nM 125I-ovine prolactin (data not shown). 64 duh-'- swam—«nag % 9:2 945 _.J D-P P H S NMQMWNGN N 'U Figure 21. Autoradiographs of consecutive frontal sections of rat brain taken 0.3 mm posterior to bregma with ect to the atlas of Paxinos and Watson (1982). (A) representsltfital binding of I-ovine prolactin, while (B) represents nonspeci- fic binding of ' I-ovine prolactin. The binding scale refers to Figures 21 and 22. 65 Figure 22. Autoradiographs of consecutive frontal sections of rat brain taken 2.8 mm posterior to bregma with gect to the atlas of Paxinos and Watson (1982). (A) representsltzqtal binding of I-ovine prolactin, while (B) represents nonspeci- fic binding of I-ovine prolactin. 66 Specific binding of ovine prolactin was quantified in selected brain regions (Table 3) and significant binding was observed only in the choroid plexus of the lateral ventricle and in the median eminence. Although specific binding was present in the choroid plexus of the third ventricle it was not significant when compared to the other brain regions which demonstrated no Specific binding for prolactin. These results suggest that prolactin may influence the activity of the TTDA neurons directly via a receptor-mediated interaction at the terminal region of these neurons in the median eminence. C. Role of Afferent Neuronal Input in the Regulation of Tuberoinfundibular Dopaminergic Neuronal Activity in the Male Rat 1. Effect of complete and retrochiasmatic deafferentation of the medio- basal hypothalamus on basal tuberoinfundibular dopaminergic neuronal activity The basal rate of DA synthesis and turnover in the median eminence is several times greater in the female than in the male rat (Demarest gt 11., 1981a; Gunnet gt a_l., 1986). Since the higher level of basal activity in the female may be propagated by stimulatory afferent input from the rostral hypothalamus it was important to determine whether a similar input regulates TIDA neuronal activity in the male. As demonstrated previously (Figure 10) complete and retrochiasma- tic deafferentations of the mediobasal hypothalamus decreased the basal rate of DOPA accumulation in the median eminence of female rats (Figure 23). The basal rate of DOPA accumulation in the median eminence of sham-operated male rats was much less than that of sham-Operated female rats and neither the complete nor the retrochiasmatic deafferentation reduced the rate of DOPA accumulation in the median eminence of male rats. Similarly, retrochiasmatic deafferentation reduced the basal rate of DA turnover in the median eminence of female rats but had no effect in male rats (Figure 24). Steady state concentrations of DA in the median eminence were not altered by retrochiasmatic deafferentation in either 67 TABLE 3 Specific Binding of 12'SI Ovine Prolactin in Various Regions of the Female Rat Brain Average . . . . Region Sample Area n Spec1f1c Binding (mmz) (fmol/mg protein) Choroid plexus Lateral ventricle 0. 10 6 11.8 t 2.1 * Third ventricle 0. 04 8 nd Striat um 6 . 34 12 nd Thalamus 5 . 17 18 nd Hippocampus 3 . 05 10 nd Amygdala 4.43 10 nd Hypothalamus Median eminence 0.05 9 2.0 i 0.2 Arcuate nucleus 0. 28 8 nd Ventromedial nucleus 0 . 30 6 nd Dorsomedial nucleus 0 . 70 6 nd Anterior hypothalamic area 0. 94 7 nd Paraventricular nucleus 0. 70 5 nd Values for specific binding represent the means of (n) determinations. Specific binding in the choroid plexus (lateral ventricle) and median eminence represent 43,018 dpm/mg protein and 7,312 dpm/mg protein, respectively, and represent 66% and 69% of the total binding. *, depicts significant Specific binding as compared to binding in all brain regions sampled. nd, statistically significant specific binding was got getectable. 68 FEMALE MALE .E 3 I6 1 r - O 5 TL \ g I2~ «~ - 5 it rt 3- 1’ ‘4'— " .. _I D L 2 _.1_ 8 ‘ . o 4- -- ~ < <1: 0. SHAM MBH RC SHAM MBH RC Figure 23. The effect of complete (MBH) and retrochiasmatic (RC) deafferenta- tion of the mediobasal hypothalamus on DOPA accumulation in the median eminence of female and male rats. Columns represent the means and the vertical lines 1 SE of 6-8 determinations. *Values significantly different from respective sham control rats, p < 0.05. 69 I20 ,— DA CONTENT 4F. DA TURNDVER E] SHAM 90- -.75- _1_. I no .5 ‘3 a. 60 - f .550 - s :- * \ 3' so - -.25 - o - , . o I- . 1 FEMALE MALE FEMALE MALE Figure 24. The effect of retrochiasmatic (RC) deafferentation on DA content and turnover in the median eminence of female and male rats. Columns illustrating DA content represent the means and the vertical lines 1 SE of 8 determinations, while those illustrating DA turnover represent the means and the vertical lines 1 SE of the rate constant calculated from the decline in DA concentrations determined in 8 animals decapitated 0, 30 and 60 minutes after GMT (a total of 24 determinations). *Value significantly different from respective sham control rats, p < 0.0 5. 70 female or male rats. In addition, steady state concentrations and the turnover of NE in the median eminence was not altered by retrochiasmatic deafferentation in the male rat (Table 4). These results suggest that regions rostral to the retrochiasmatic deafferentation exert a stimulatory influence on TIDA neurons in the female but not the male rat. 2. Effect of retrochiasmatic deafferentation on the prolactin-induced activation of tuberoinfundibular dOpaminergic neurons. Consistent with previous experiments, retrochiasmatic deafferentation decreased the rate of DOPA accumulation in the median eminence of female but not male rats (Figure 25). An icv injection of rat prolactin increased the rate of DOPA accumulation in the median eminence of both sham-Operated and retro- chiasmatic-deafferentated female and male rats. These results indicate that the ability of prolactin to stimulate TIDA neuronal activity is not blocked by retrochiasmatic deafferentation in either male or female rats. Thus, the deafferentation-induced decrease in basal TTDA neuronal activity in female rats appears to be the consequence of removing afferent input to these neurons which is independent of the action of prolactin. 3. Effect of retrochiasmatic deafferentation on tuberoinfundibular dop- aminergic neuronal activity in castrated female and male rats Gender differences in TIDA neuronal activity result, in part, from differences in the hormonal environment of the adult rat. In particular, estrogen has a stimulatory influence (Fuxe e_t_ g, 1969a; Eikenburg e_t_ a_l., 1977; Demarest and Moore, 1980) while testosterone has an inhibitory influence (Brawer g1 11., 1986; Nakahara gt a_l., 1976) on TIDA neuronal activity. Since differences in the actions of the gonadal steroids on TIDA neurons may be responsible for the ability to observe a retrochiasmatic deafferentation-induced decrease in TIDA neuronal activity in female but not male rats, the effect of deafferentation on gonadally- intact and castrated female and male rats was examined. 71 TABLE 4 The Effect of Retrochiasmatic Deafferentation (RC) on NE Content and Turnover in the Median Eminence of Male Rats Steady State Concentration Rate Constant. k . h ng / mg protem Sham 15.6 i 1.0 0.11 i 0.01 RC 12.5 i 0.6 0.17 i 0.01 Steady state concentrations represent the means i 1 SE of 8 determinations. The rate constants, an index of NE turnover, represent the means _+_ 1 SE calculated from the decline in NE concentrations determined in 8 animals decapitated 0, 30 and 60 minutes after aMT (a total of 24 determinations). 72 20 FEMALE .. MALE . ( 1* :1 VEHICLE - r PRL DOPA ACCUMULATION, no/mo protein 5 SHAM Figure 25. The effect of retrochiasmatic (RC) deafferentation on the prolactin- induced increase in DOPA accumulation in the median eminence of female and male rats. Rat prolactin (rPRL, lO 113/3 111) or its 0.9% saline vehicle was administered via an intracerebroventricular cannula 12 hours prior to sacrifice. Columns represent the means and the vertical lines 1 SE of 7-10 determinations. *Values significantly different from respective sham vehicle-treated rats; **values significantly different from respective RC vehicle-treated rats, p < 0.05. 73 Both ovariectomy and retrochiasmatic deafferentation decreased the rate of DOPA accumulation in the median eminence of female rats (Figure 26). Retrochiasmatic deafferentation, however, did not produce a further decrease in the rate of DOPA accumulation in the median eminence of ovariectomized rats suggesting that retrochiasmatic deafferentation and ovariectomy may decrease TIDA neuronal activity via a common neuronal pathway. This common pathway may involve a direct stimulatory action of estrogen at the level of the anterior hypothalamus. The effect of retrochiasmatic deafferentation on the rate of DOPA accumulation in the median eminence of intact and orchidectomized male rats is shown in Figure 27. Orchidectomy increased the rate of DOPA accumulation in the median eminence of male rats; retrochiasmatic deafferentation, however, did not alter the rate of DOPA accumulation in either intact or orchidectomized male rats. Thus, the inhibitory influence of testosterone in the adult male does not appear to be responsible for the failure to observe a deafferentation-induced decrease in TIDA neuronal activity, suggesting that the afferent input which stimulates TTDA neurons in the female rat may not be present and/or operational in the male rat. D. Inhibitory Afferent Regulation of Tuberoinfundibular Dopaminergic Neuron- al Activity in the Female Rat 1. Effect of complete deafferentation of the mediobasal hypothalamus on the stress-induced changes in tuberoinfundibular depaminergic neuronal activity and pituitary prolactin secretion TTDA neuronal activity is regulated, in part, by inhibitory afferents which impinge on these neurons to decrease their activity during stress. This decrease may facilitate the stress-induced increase in prolactin secretion. The origin of inhibitory afferents to the TTDA neurons was investigated by examining the effect of complete deafferentation of the mediobasal hypothalamus on the 74 :3 2% [:1 SHAM ‘ C) a - RC C! \E 15- - CD C ‘5 IO * 2 a _l D 2 8 5. 2 < 8 o O- INTACT OVX Figure 26. The effect of retrochiasmatic (RC) deafferentation on DOPA accumulation in the median eminence of intact and ovariectomized (OVX) female Columns represent the means and the vertical lines 1 SE of 7-10 rats. *Values significantly different from intact sham control rats, determinations. p < 0.0 5. 75 “T D SHAM ' I RC 1* .E 2 O L O. 0 E12- . O C 2‘ 98 l; .- .J D 2 D 84- - < < 35 J o O- lNTACT ORCHDX Figure 27. The effect of retrochiasmatic deafferentation on DOPA accumulation in the median eminence of intact and orchidectomized (ORCHIDX) male rats. Columns represent the means and the vertical lines 1 SE of 6-9 determinations. *Values significantly different from intact sham control rats, p < 0.05. 76 stress-induced decrease in TIDA neuronal activity and increase in plasma prolac- tin. As demonstrated previously (Demarest gt 94., 1985a), acute restraint stress decreased DOPA accumulation in the median eminence of sham-operated rats but failed to alter DOPA accumulation in animals which had received complete deafferentation (Figure 28). Furthermore, restraint stress decreased DOPAC concentrations in the median eminence of sham-operated rats, but failed to produce a decrease in DOPAC concentrations in the median eminence of rats receiving complete deafferentation (Figure 2.9). These results suggest that complete deafferentation of the mediobasal hypothalamus blocks the stress- induced decrease in TTDA neuronal activity. In addition to the decrease in TTDA neuronal activity, restraint stress increased circulating prolactin concentrations (Table 5). Complete deafferenta- tion of the mediobasal hypothalamus had no effect on basal prolactin concentra- tions in sham-operated rats, but this procedure attenuated the rise in prolactin due to restraint stress. These results suggest that complete deafferentation of the mediobasal hypothalamus may blunt the stress-induced increase in prolactin by blocking the stress-induced decrease in TIDA neuronal activity. In addition, complete deafferentation may interrupt neurons which contain prolactin-releasing factors whose increased activity may stimulate prolactin release during stress. 2. Effect of retrochiasmatic deafferentation on the stress-induced changes in tuberoinfundibular dopaminergic neuronal activity and pituitary prolactin secretion The prolactin response to various stressful stimuli is attenuated by interruption of neural connections originating rostral to the mediobasal hypothala- mus (Kawakami and Higuchi, 1981; Mioduszewski and Critchlow, 1981; Kaler e_t_ _a__l., 1984). Thus, inhibitory afferent input originating rostral to the mediobasal hypothalamus may project to and decrease THDA neuronal activity and increase prolactin secretion in response to stress. As such, the ability of retrochiasmatic 77 so C :73 T [j CONTROL g .STRESS o 24- . é 5’ C 2- 18- - 9 p. 3 3 12- - 2 D 8 < 6 ' < a J 8 0- SHAM MBH Figure 28. The effect of complete (MBH) deafferentation of the mediobasal hypothalamus on the stress-induced decrease in DOPA accumulation in the median eminence of female rats. Columns represent the means and the vertical lines 1 SE of 5-8 determinations. *Value significantly different from sham control rats, p < 0.05. 78 15 - {‘ E] CONTROL I STRESS c 12- . E 9 O. a, 9- . \E O C . 6- . 0 < O. O o 3- - 0- J SHAM MBH Figure 29. The effect of complete (MBH) deafferentation of the mediobasal hypothalamus on the stress-induced decrease in DOPAC concentrations in the Columns represent the means and the vertical median eminence of female rats. significantly different from sham lines 1 SE of 4-8 determinations. *Value control rats, p < 0.05. 79 TABLE 5 The Effect of Complete Deafferentation of the Mediobasal Hypothalamus (MBH) on the Stress-Induced Increase in Plasma Prolactin Concentrations ng/ml Control Stress Sham 2.8 :0.5 107 i 10.5* MBH 5.9 1 1.2 28 _+_ 7.1 ** Values represent the means i 1 SE of 5-8 determinations. *Value significantly different from sham control rats, **value significantly different from RC control rats, p < 0.05. 80 deafferentation to block the stress-induced decrease in TIDA neuronal activity and increase in prolactin was examined. Restraint stress and retrochiasmatic deafferentation decreased the rate of DOPA accumulation in the median emi- nence of female rats (Figure 30). Restraint stress did not decrease DOPA accumulation in the median eminence of retrochiasmatic-deafferentated rats. As demonstrated previously (Figure 13), retrochiasmatic deafferentation did not alter basal prolactin concentrations, however, deafferentation attenuated the stress- induced increase in prolactin (Table 6). These results suggest that the neuronal mechanism(s) which decreases TIDA neuronal activity and increases prolactin secretion during stress originates rostral to the mediobasal hypothalamus. 81 2T E] CONTROL I STRESS i DOPA ACCUMULATION, ng/mg protein SHAM Figure 30. The effect of retrochiasmatic (RC) deafferentation on the stress- induced decrease in DOPA accumulation in the median eminence of female rats. Columns represent the means and the vertical lines 1 SE of 8-10 determinations. *Values significantly different from sham control rats, p < 0.05. 82 TABLE 6 The Effect of Retrochiasmatic Deafferentation (RC) on the Stress-Induced Increase in Plasma Prolactin Concentrations ng/ml Control Stress Sham 0.9 10.1 73.1 1123.1" RC 6.3 12.0 21.6 i 6.3 Values represent the means i 1 SE of 7-10 determinations. *Value significantly different from sham control rats, p < 0.05. DISCUSSION A. Afferent Neuronal Regulation of Basal Tuberoinfundibular Dopaminergic Neuronal Activity in the Female Rat: Anterior Hypothalamic Origin Results of the present studies demonstrate that afferent neurons originating rostral to the mediobasal hypothalamus provide stimulatory input to TEA neurons in the gonadally-intact female rat. Retrochiasmatic deafferentation, a procedure which interrupts afferent connections from the anterior hypothalamus to the mediobasal hypothalamus, reduced basal neuronal activity of the TEA neurons in the female rat. This was demonstrated by a decrease in DA synthesis, turnover and metabolism in the terminal region of the TEA neurons in the median eminence. The deafferentation-induced decrease is specific for the TEA neurons since the tuberohypophysial DA neurons, which are related anatomically to the TEA neurons (Moore and Demarest, 1982), are not influenced by the knife cuts. A state of constant estrus is observed in animals receiving retrochiasmatic deafferentation (Halasz and Gorski, 1967; Brownstein fl a_l., 1976a). A similar state is observed in rats chronically treated with estrogen (Demarest st 11., 1984b). Both retrochiasmatic deafferentation and. long-term (18 days) estrogen treatment decrease basal TEA neuronal activity and reduce the effectiveness of the prolactin feedback mechanism in the female rat (Demarest gt a_l., 1984b; Barton gt 31., 1988). Estrogen-induced changes in TEA neuronal activity are biphasic; short-term (6 days) treatment with estradiol increases TEA neuronal activity while long term (18 days) treatment decreases the activity of these neurons (Demarest gt a_l., 1984b). The deafferentation-induced decrease in TEA 83 84 neuronal activity is observed 3 days after deafferentation at a time when estrogen would be expected to have a stimulatory influence on TEA neurons. Thus, it is unlikely that the elevated estrogen levels during the state of continuous estrus induced by retrochiasmatic deafferentation is responsible for the deafferentation- induced decrease in TEA neuronal activity. The decrease in TEA neuronal activity and the interruption of cyclicity following retrochiasmatic deafferentation are probably not the result of direct insult to the TEA neurons or of perturbations in the hypophysial vascular supply. Neither the complete nor the retrochiasmatic deafferentation altered TEA neuronal activity in male rats. Furthermore, the decrease in TEA neuronal activity following complete (Weiner e_t_ 11,, 1972; Gudelsky gt g, 1978) and retrochiasmatic (Barton _et 11., 1988) deafferentation in the female rat is not associated with an alteration in DA content in the median eminence suggesting that there is no loss of TEA neurons. In addition, the deafferentation-induced decrease in TEA neuronal activity is observed at 3 and up to 21 days after deafferentation, at a time when blood flow to the hypothalamus/anterior pituitary is maintained at normal levels (Nikitovitch-Winer and Goldman, 1986). The DA which is released from TEA neurons inhibits the secretion of prolactin from the anterior pituitary (MacLeod, 1976; Ben-Jonathan _e_t 31., 1977). Despite the decrease in TEA neuronal activity at 3 and up to 21 days after retrochiasmatic deafferentation, serum prolactin concentrations remained un- altered. Although Caligaris and Taleisnik (1976) reported a significant elevation in circulating prolactin concentrations, several investigators observed no change in circulating levels of this hormone following retrochiasmatic deafferentation (Siegel gt a_l., 1979; Siegel gt 31., 1980; Mioduszewski and Critchlow, 1981). Not only does the deafferentation-induced decrease in TEA neuronal activity fail to elevate circulating prolactin concentrations, but destruction of half of these 85 neurons also produces no change in serum prolactin concentrations (Demarest gt gl_., 1980; Fenske and Wuttke, 1976). Pretreatment with 6-hydroxydopamine produces a 50% decline in DA content in the median eminence yet no alteration in circulating prolactin. Thus, a reserve of DA at the anterior pituitary may compensate for the decrease in TEA neuronal activity after retrochiasmatic deafferentation as well as for the partial destruction of TEA neurons following 6- hydroxydopamine treatment. On the other hand, retrochiasmatic deafferentation may not only interfere with the regulation of basal TEA neuronal function, but may also interrupt neurons containing prolactin-releasing factors. A number of such neurons have cell bodies in the anterior hypothalamus with neuronal projections to the median eminence (for review, see Shin gt 11,, 1987). Thus, the deafferentation-induced decrease in TEA neuronal activity may be counter- balanced by a decrease in the activity of neurons which contain prolactin- releasing factors to result in no net change in circulating prolactin concentra- tions. In addition, DA released from the posterior pituitary has been reported to inhibit prolactin release (Peters gt a_l., 1981; Ben-Jonathan and Peters, 1982). An enhanced activity of the tuberohypophysial DA neurons, however, does not appear to compensate for the decreased TEA neuronal activity since neither complete nor retrochiasmatic deafferentation altered DA synthesis in the terminal region of the tuberohypophysial DA neurons. Several studies were done to determine the origin of stimulatory afferent input to the TEA neurons in the female rat. DA synthesis in the median eminence was decreased following retrochiasmatic deafferentation and in animals receiving deafferentations 0.5 mm rostral to the retrochiasmatic region. Place- ment of the deafferentation more rostral into the anterior hypothalamus and bilateral deafferentation of the lateral retrochiasmatic area did not alter the basal activity of TEA neurons. Thus, afferent input originating in or coursing 86 through the caudal region of the anterior hypothalamus mediates a stimulatory influence on TEA neurons in the intact female rat. The caudal portion of the anterior hypothalamus contains 3 distinct areas, the SCN, AHA and PVN. Bilateral electrolytic lesions of the SCN and PVN decreased the basal rate of DA synthesis in the median eminence, while bilateral lesions of the AHA had no effect. The afferent neuronal input which stimulates TEA neurons in the female rat may, therefore, originate in the SCN and PVN or in either the SCN or PVN and course through the other on route to the arcuate nucleus/median eminence. This latter route is most likely the one taken by stimulatory afferent neurons which project to the TEA neurons. Electrolytic lesions of the SCN and PVN each produced a 30% to 40% decrease in TEA neuronal activity. If unique afferent projections to the TEA neurons originated in both the SCN and PVN, deafferenta- tion of this input would be expected to produce a 60% decrease in TEA neuronal activity. Retrochiasmatic deafferentation generally produced a 30 to 40% decrease in TEA neuronal activity suggesting that stimulatory afferent input to the TEA neurons in the female rat originates in either the SCN or PVN. Anatomical evidence supports reciprocal connections between the SCN and PVN (Berk and Finkelstein, 1981; Silverman gt 9, 1981; Watts 3 a_l., 1987; Watts and Swanson, 1987), as well as from the SCN and PVN to the arcuate nucleus/median eminence complex (Swanson and Cowman, 1975; Swanson and Sawchenko 1980; Weigand and Price, 1980; Stephan _e_t a_l., 1981). Several neuropeptides are possible candidates for mediating the stimulatory afferent neuronal regulation of TEA neurons in the female rat. Atrial natriuretic factor in the median eminence is contained in neurons which originate in the PVN (Palkovits gt 11,, 1987). It has been suggested that the prolactin-inhibitory effect of atrial natriuretic factor is mediated by a dopaminergic mechanism (Samson gt a_l., 1988). Similarly, vasopressin, which is of known PVN origin, and neurotensin, 87 which is widely distributed throughout the CNS (Lazarus gt a_l., 1977) presumably stimulate DA release from TEA neurons and thereby inhibit prolactin secretion (Tojo gt a_l., 1986; DePaolo gt gl_., 1986). The role of these neuropeptides and the more classical neurotransmitters in mediating the stimulatory afferent neuronal regulation of TEA neurons remains to be elucidated. B. Stimulatory Afferent Input Does Not Mediate the Action of Prolactin on Tuberoinfundibular DOpaminergic Neurons 1. Delayed and tonic activation of tuberoinfundibular dopaminergic neurons in the absence of afferent input from the anterior hypothala- mus . TEA neuronal activity is believed to be primarily regulated by the positive feedback action of prolactin (Moore and Demarest, 1982). Increases in circulating concentrations of prolactin stimulate the synthesis and turnover of DA in the median eminence (Hb'kfelt and Fuxe, 1972; Gudelsky. gt a_l., 1976; Selma- noff, 1981) while decreases in prolactin are associated with a decline in these indices of TEA neuronal activity (Demarest and Moore, 1981a; Demarest g a_l., 1985c). The stimulatory action of prolactin on TEA neurons is divided into two interrelated components: a rapid "tonic" component and a delayed "induction" component (Demarest g g” 1984d; 1986). The rapid component mediates relatively short-term changes in TEA neuronal activity in response to acute increases or decreases in circulating prolactin concentrations. This rapid compo— nent can be demonstrated as early as 4 hours when one examines the effect of prolactin on TEA neuronal activity in a bromocriptine-treated rat. The delayed component is activated by long-term changes in prolactin and has been demon- strated by a variety of jg gtvg techniques where prolactin increased DA synthesis and turnover in the terminal region of the TEA neurons after a delay of 12 to 16 hours (Ho'kfelt and Fuxe, 1972; Gudelsky gt gt, 1976; Johnston gt gl_., 1980). 88 Results of the present studies indicate that the retrochiasmatic deafferentation-induced decrease in TEA neuronal activity is not the conse- quence of interrupting afferent input mediating either the rapid or delayed components of prolactin's actions. Despite the decrease in basal TEA neuronal activity following retrochiasmatic deafferentation, manipulations which increase prolactin levels increase activity of the TEA neurons, while those which decrease prolactin levels are associated with a further decrease in TEA neuronal activity in the deafferentated female rat. For example, 12 to 16 hours after a single injection of either prolactin or haloperidol respectively, DA synthesis in the median eminence was increased by approximately the same relative amount in both sham-operated and retrochiasmatic-deafferentated rats. In addition, remov- ing the tonic stimulatory influence of endogenous prolactin on TEA neurons by pretreating the animals with bromocriptine, decreased median eminence DA synthesis in sham-operated and retrochiasmatic-deafferentated rats. This de- crease was reversed by prolactin administration, which produced similar percent- age increases in the rate of DA synthesis in the median eminence of both groups. These results demonstrate that both the delayed and rapid activation of TEA neurons by prolactin is still operating when afferent input from areas rostral to the mediobasal hypothalamus is removed. Although prolactin increases TEA neuronal activity proportionally in retrochiasmatic-deafferentated and sham-operated rats, TEA neurons in animals receiving retrochiasmatic deafferentation cannot be stimulated to the same absolute magnitude. This reduced magnitude of response in deafferentated rats resembles that observed in an aged rat (Demarest gt gl_., 1987). Aging produces a similar decrease in basal TEA neuronal function as does retrochiasmatic deaffer- entation and, although these neurons in the aged rat are still responsive to changes in prolactin, they cannot be stimulated to the same absolute magnitude. 89 The aging-induced decrease in basal TIDA neuronal function is associated with a decrease in DA content (Demarest gt g}, 1980; 1982; Estes and Simpkins, 1980) presumably the result of a decrease in the number of viable terminals of TEA neurons (Hoffman and Sladek, 1980; Selemon and Sladek, 1981). Retrochiasmatic deafferentation is not associated with a decrease in DA content suggesting that the TEA terminals are intact. Rather, the deafferentation-induced decrease in TEA neuronal function is apparently the result of a decrease in stimulatory input to the TEA neurons. 2. Site of action of prolactin Deafferentation studies (Gudelsky gt _a_.I_., 1978; Barton gt gl__., 1988) provide evidence to suggest that the target site for the prolactin-induced activation of TEA neurons is contained in the mediobasal hypothalamus. This is consistent with the identification of specific binding sites for prolactin in the median eminence of the female rat brain. Autoradiographic reactions were 1251_ observed in both the median eminence and choroid plexus after labeling with ovine prolactin and the intensity of the reactions was significantly reduced after coincubation with excess unlabeled ovine prolactin. The demonstration of prolactin binding sites in the choroid plexus is in agreement with previous reports (Walsh gt a_l., 1978; 1984; 1987; Dube gt a_l., 1980). Prolactin receptors on choroid plexus appear to be present on epithelial cells (Walsh gt glfl 1978; 1984) where they function to transport prolactin from the circulation to the cerebrospinal fluid (CSF; Walsh gt a_l., 1984; 1987). It has been suggested that a prerequisite step for all prolactin-brain interactions lies in the receptor-mediated uptake and transport of prolactin from the vasculature to CSF via the choroid plexus. For example, prolactin in the CSF can increase the activity of the TEA neurons in the mediobasal hypothalamus (Annunziato and Moore, 1978). In turn, the dopaminergic neurons of the TEA system inhibit 90 prolactin secretion from the anterior pituitary (MacLeod, 1976; Gudelsky and Porter, 1980; 1981). The autoregulation of prolactin secretion via TEA neuronal activation may be mediated by prolactin in the CSF (Nicholson g gt, 1980). Increases in prolactin in the plasma produce a delayed elevation of CSF prolactin (Belchetz gt gt” 1982; Kalin gt _a_1., 1982). Thus, the increase in CSF prolactin may be responsible, in part, for the delayed activation of TEA neurons by prolactin (HSkfelt and Fuxe 1972; Gudelsky gt g” 1976). On the other hand, the median eminence is outside the blood-brain barrier providing free access of this region to prolactin in the circulation. In fact, prolactin is in higher concentra- tions in the hypophysial portal vasculature than in the systemic circulation and this is due, in part, to retrograde flow of prolactin from the pituitary to the hypothalamus (Oliver e_t _a_l., 1977; Mezey and Palkovits, 1982). Nevertheless, the exact mechanism by which prolactin activates the TEA neurons has yet to be elucidated. The present results suggest that prolactin influences the activity of the TEA neurons directly via a receptor-mediated interaction at the terminal region of the TEA neurons in the median eminence. VanHouten and coworkers (1980) identified receptors in the rat median eminence which they termed "lactogen receptors" since they were identified using 125I—human growth hormone as the ligand, and antagonized by unlabeled human growth hormone and ovine prolactin. Binding of human growth hormone was found in both the external and internal layers of the median eminence and unlabeled hormone inhibited binding in the external layer but not in the internal layer. The findings in the present studies are consistent with this observation in that incubation with unlabeled prolactin did not prevent but only reduced 125 I~prolactin binding in the median eminence. The binding signal for prolactin which persists in the presence of excess unlabeled hormone may represent nonspecific binding of prolactin to the 91 internal zone of the median eminence at the arcuate nucleus border. The specific binding for prolactin may, therefore, represent binding to receptor sites in the external zone of the median eminence, a region known to contain projections of the TEA system (Bjiirklund gt gl_., 1973b). Together with the observation that prolactin can augment dopamine release from median eminence synaptosomes (Gregerson and Selmanoff, 1988), these results suggest the possibility of a direct interaction of prolactin on the terminals of the TEA neurons. Previous attempts to identify prolactin binding sites in rat hypotha- lami using 125 I-prolactin as the radiolabeled ligand have been unsuccessful. Traditional i3 v_i_t_t-g competitive binding assays (DiCarlo gt a_l., 1983), '2 3119 autoradiographic analysis of whole hypothalami (Dube gt__a_t., 1980) and '2 trilq autoradiographic analysis of the median eminence (Walsh gt gt” 1978; 1987) failed to demonstrate significant specific binding for prolactin. The difficulty in demonstrating specific prolactin binding sites in earlier '2 gittg procedures is most likely due to a low concentration of receptors in the rat hypothalamus, as well as to their discrete localization in the median eminence. Autoradiographic reactions were observed in rat median eminence both 5 minutes (Walsh g gl_., 1978) and 1 hour (Walsh gt 31., 1987) after the g m administration of lzsl-ovine prolactin. Unfortunately, the intensity of nonspecific binding was so high that it may have obscured identification of specific prolactin binding sites in this region. In addition, the total binding of prolactin in the median eminence was low suggesting that prolactin was not reaching its site of action. The rapid metabolism of prolactin (Koch gt gl_., 1971) may have contributed to this finding or perhaps a longer time after the .12 gtvg administration of prolactin is required for the transport of prolactin into the CSF for subsequent interaction of this hormone at binding sites in the median eminence. The potential requirement for prolactin transport into CSF is not a problem in the present :12. vitro analysis and 92 the concentration of labeled prolactin in the incubation solution, as well as the incubation parameters, were optimized in the present study to reduce the amount of nonspecific binding. C. Sexual Difference in the Afferent Neuronal Regulation of Tuberoinfundibu- lar Dopaminergic Neuronal Activity: Influence of the Gonadal Steroids Afferent neuronal input originating rostral to the mediobasal hypothalamus stimulates TEA neurons in gonadally-intact female but not male rats. That is, retrochiasmatic deafferentation, a procedure which interrupts neuronal connec- tions from regions rostral to the mediobasal hypothalamus, reduced DA synthesis and turnover in the terminal region of the TEA neurons in the median eminence of intact female but not male rats. TEA neurons appear to be primarily regulated by prolactin and, in the female rat, these neurons are more sensitive to the stimulatory actions of this hormone (Demarest and Moore, 1981a). Thus, an increased sensitivity to and a continuous activation by prolactin has been suggested to be the basis for the higher level of basal activity of TEA neurons in the female rat. Results of the present studies demonstrate that a prolactin-independent afferent neuronal input may also play a role in propagating the sexual difference in TEA neuronal activity since retrochiasmatic deafferentation decreases the basal activity of TEA neurons only in the intact female rat. Results of the present studies suggest that the hormonal environment may play a role in the retrochiasmatic deafferentation-induced decrease in TEA neuronal activity in intact female rats. For example, ovariectomy decreased the rate of DA synthesis in the median eminence of female rats and retrochiasmatic deafferentation did not produce a further decrease. The decrease in TEA neuronal activity following ovariectomy may be due to a removal of the 93 stimulatory action of prolactin on the TIDA system or to removal of the stimulatory action of estrogen at some region(s) in the hypothalamus. The DA agonist, bromocriptine, decreases TEA neuronal activity by reducing the stimula~ tory action of prolactin on TEA neurons (Demarest and Moore, 1981a), and this decrease is further reduced following retrochiasmatic deafferentation. Thus, bromocriptine and retrochiasmatic deafferentation together exert an additive effect in decreasing TEA neuronal activity. Bromocriptine reduces circulating levels of prolactin thereby reducing the stimulatory action of this hormone on TEA neurons while retrochiasmatic deafferentation removes stimulatory afferent input to the TEA neurons which is not mediated by prolactin. Since ovariectomy and retrochiasmatic deafferentation do not exert additive effects in decreasing TEA neuronal activity they may decrease the activity of these neurons via a similar mechanism. This mechanism may involve a direct stimulatory action of estrogen at the level of the anterior hypothalamus. In fact, a moderate to high density of estrogen receptors have been identified in the PVN and SCN/preoptic nucleus of the female rat (Rainbow fl gt, 1982). The inability to observe a retrochiasmatic deafferentation-induced decrease in TEA neuronal activity does not appear to be influenced by the hormonal environment of the adult male rat. The TEA neurons in the male rat have a lower basal rate of activity. Following castration, DA synthesis in the median eminence of male rats was increased to a level similar to that observed in an intact female. In the castrated male, however, retrochiasmatic deafferentation did not decrease DA synthesis in the median eminence. Thus, the inhibitory influence of testosterone in the adult male rat is not responsible for the failure to observe a deafferentation-induced decrease in TEA neuronal activity. Retro- chiasmatic deafferentation removes stimulatory input to the TEA neurons in the intact female rat which is either not present or not functional in the male rat. 94 The absence of stimulatory afferent input to TEA neurons in the male rat may be due to sexual differentiation resulting from neonatal androgen exposure. For example, male rats castrated 12 hours after birth display a level of TEA neuronal activity similar to that of an adult intact female (Demarest gt gt, 1981). Conversely, females receiving testosterone on day 5 after birth display a male profile of TEA neuronal activity in adulthood. Thus, neonatal androgen exposure may result in the sexual differentiation of stimulatory afferent neuronal input to the TEA neurons which may then result in a higher (female) or a lower (male) rate of neuronal activity. D. Inhibitory Afferent Neuronal Regulation of Tuberoinfundibular Dopaminer- gic Neuronal Activity: Anterior Hypothalamic Origin Inhibitory afferent neuronal input originating rostral to the mediobasal hypothalamus appears to mediate the stress-induced decrease in TEA neuronal activity and increase in plasma prolactin concentrations in the female rat. Restraint stress decreased DA synthesis and metabolism in the median eminence of sham-operated female rats yet failed to decrease DA synthesis and/or metabolism in the median eminence of rats which had received either complete or retrochiasmatic deafferentation of the mediobasal hypothalamus. In addition, both deafferentation procedures markedly attenuated the stress-induced elevation in plasma prolactin. Although the origin of inhibitory afferent input to the TEA neurons has yet to be identified, several studies have been done to examine the role of neural connections to the mediobasal hypothalamus in mediating the prolactin response to stress. The increase in circulating prolactin in response to ether stress (Krulich gt gt, 1975), heat stress (Siegel gt Q” 1979) and 10 minutes of leg restraint (Mioduszewski and Critchlow, 1981) was blocked by complete deafferentation and 95 attenuated by retrochiasmatic deafferentation. In the present study, the prolac- tin response to stress was attenuated by both complete and retrochiasmatic deafferentation. The discrepancy in the findings of these early studies and those of the present study may result from the different stressors used to elicit the prolactin response. Demarest gt gt (1985a) have observed a graded increase in the stress-induced rise in prolactin with increasing intensities of stress. It is possible, therefore, that the graded prolactin response to stress is the conse- quence of a gradual recruitment of different hypothalamic and/or pituitary mechanisms which stimulate prolactin-releasing factors and/or inhibit prolactin- inhibiting factors. The recruitment of additional mechanisms may explain why the increase in prolactin in response to a 30 minute period of intense restraint is only attenuated by complete deafferentation of the mediobasal hypothalamus. The hypothalamic PVN plays a pivotal role in the regulation of prolactin secretion in response to a variety of stimuli. PVN lesions attenuate the stress- induced increase in prolactin secretion (Meyerhoff gt gt, 1987; Minamitani g gt, 1987). In addition, PVN lesions attenuate prolactin release in lactating rats (Kiss gt a_l., 1986) and in rats which have received an injection of the serotonin precursor, S-hydroxytryptophan (Minamitani gt a_l., 1987). Serotonin stimulates prolactin release (Caligaris and Taleisnik, 1974; Chen and Meites, 1975) presumab- ly by stimulating the release of a prolactin-releasing factor (Clemens gt a_l., 1978). Although cell bodies for many prolactin-releasing factors are located in the PVN (Mezey and Kiss, 1985; Brownstein gt a_l., 1982; Shin fl gt, 1987), only vasoactive intestinal polypeptide (VIP) is reported to mediate prolactin secretion induced by serotonin (Shimatsu _e_t gt, 1984). Anti-serum generated against VIP attenuates both the serotonin-induced (Shimatsu gt gt, 1984) and the stress- induced (Kaji gt a_l., 1985) increases in prolactin secretion. These findings are in agreement with those of Demarest gt gt (1985a) who found that the action of 96 restraint stress to increase prolactin secretion as well as to decrease TEA neuronal activity is mediated by a serotonergic mechanism. It is interesting to speculate that stress stimulates a serotonergic projection to the PVN to either stimulate an inhibitory afferent or inhibit a stimulatory afferent (ie. the one which maintains basal TEA neuronal activity in the female rat) to then produce a decline in TEA neuronal activity. Simultaneously, serotonin may stimulate VTP- containing neurons to enhance the release of VIP from identified terminals in the median eminence (Ceccatelli gt a_l., 1988) which then potentiates the elevation in circulating prolactin levels associated with stress. VIP may, in fact, influence TEA neuronal activity directly via a presynaptic mechanism similar to that which has been reported for galanin (NordstrOm gt gt, 1987). The stress-induced increase in plasma prolactin was not entirely blocked by the loss of afferent input to the mediobasal hypothalamus. These results suggest that stressful stimuli can act within the mediobasal hypothalamus or at the level of the pituitary to mediate, in part, the stress-induced rise in prolactin. The posterior pituitary contains both DA and a prolactin-releasing factor (Murai and Ben-Jonathan, 1987; Hyde _et gt, 1987) which may play a pivotal role in the regulation of prolactin secretion during certain physiologic conditions. Extracts of posterior pituitary stimulate prolactin secretion tit _v_itr_o (Hyde fl a_l., 1987) and removal of the posterior pituitary interferes with the suckling-induced increase in prolactin secretion (Murai and Ben-Jonathan, 1987). Taken together, these results suggest that a decrease in the DA tone of the posterior pituitary or stimulation of a prolactin-releasing factor from this region may mediate, in part, the increase in prolactin secretion in response to stress. In addition, recent reports have demonstrated that VIP is synthesized by the pituitary gland (Aranout gt gt, 1986) and it appears to act there to stimulate prolactin secretion (Hagen gt gt, 1986; Nagy gt _a_l_., 1988). Thus, restraint stress may increase the activity of a prolactin- 97 releasing factor (VIP) at the level of the anterior pituitary to elevate circulating prolactin concentrations. SUMMARY AND CONCLUSIONS Interruption of afferent input originating rostral to the mediobasal hypotha- lamus decreases the basal rate of activity on TEA neurons in gonadally-intact female rats. This is evidenced by a decrease in the synthesis, turnover and metabolism of DA in the median eminence. Deafferentations made 1 mm rostral to and bilateral cuts of the retrochiasmatic area failed to alter basal TEA neuronal activity suggesting that afferent neuronal input to these neurons originates in the caudal region of the anterior hypothalamus. Electrolytic lesions of the SCN and PVN produced a similar percentage decrease in TEA neuronal activity as did retrochiasmatic deafferentation. Thus, afferent neuronal input originating in the SCN or PVN mediates a stimulatory influence on TEA neurons in the intact female rat. Although prolactin appears to be the primary physiological factor which regulates TEA neuronal activity, the stimulatory action of this hormone on TEA neurons is still functional when afferent input from areas rostral to the medio- basal hypothalamus is removed. These findings together with the identification of specific binding sites for prolactin in the median eminence provides evidence for a direct action of this hormone on the terminals of the TEA neurons. The deafferentation-induced decrease in TEA neuronal activity in female rats may operate via the same neuronal mechanism that functions to decrease TEA neuronal activity following ovariectomy. Retrochiasmatic deafferentation and ovariectomy decreased TEA neuronal activity yet both procedures together produce no further decrease suggesting that these manipulations act via a similar 98 99 neuronal pathway. This common pathway may involve a direct action of estrogen at the level of the anterior hypothalamus. The neuronal mechanism which stimulates TEA neuronal activity in intact female rats is either not present or not Operational in intact and orchidectomized male rats. Even after removal of the inhibitory influence of testosterone (when TEA neuronal activity in the male approximates that observed in an intact female) retrochiasmatic deafferentation had no effect. The absence or quies- cence of stimulatory afferent input to the TEA neurons in the male could be the consequence of neonatal androgen exposure. Inhibitory afferent neurons which decrease TEA neuronal activity during stress, as well as additional afferent inputs to the median eminence which may regulate prolactin secretion during stress, originate rostral to the mediobasal hypothalamus. Complete and retrochiasmatic deafferentation of the mediobasal hypothalamus blocked the stress-induced decrease in TEA neuronal activity and attenuated the rise in prolactin. The stress-induced increase in prolactin which persists after complete and retrochiasmatic deafferentation may result from alterations in prolactin-releasing and/or inhibiting factors at the level of the mediobasal hypothalamus and/or anterior pituitary. In summary, the TEA neurons are not simply part of a hormonal/neuronal feedback system controlling prolactin release independent of afferent neuronal influences. 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