«Wkln. ’ iifi‘l‘fi‘"? , . ‘ v Q .‘4‘. -\ hm“;- ‘ ...“.W "q L‘m r L ".3: 1-. A . _. ‘43:; #.zm'.‘ ~ - H ., .4 DI? 1‘ ‘r.', - . ..rw'” rm. ,c'u ....a’ - ... v. w. .. u . . a 7' inu .m rv- w '4‘. ‘ u u . v ~n ‘42:". .7 a -...f 1 . n . .m'. v.” r “'9' J 3‘. 0-P-' . - f». 1:; .. r) 'l 1‘ , In . 7', -a . 'é,‘ ' Ir ("a ,E .. ... ,..:'.m A .. x &>' ’3 .4. if I, L > .‘v A nn-nuuw "an.” as .,,_ . . -s ‘ .u ' . ' 'r'.i ,‘J‘J , ‘r. c “I"? s" .r' If a '1' .1” , .n "(at - $1.7 v: ....-.” ...,..::_:u, , V ...F .-.. ' ‘3’": ' " A {a : ..1 1"”: fat: 3.2". ’ .6” ”2'7”" -” ‘...”:T.' ‘l - ,r u .. r, 1""... r «T'- -,--~.-‘ r . c "Sf—3?“. ’ .... . v x.) I 1-: . .. a :V . ....Itx . -¢ if}: ...q n - afi" v :2"; {y .,. .,’- .. ,.,, 4n - ~}.' . x ‘45uwtn‘.n (v J ‘ :‘c'bam Huh, . sewn lmlllilflllllglylllwill '7‘“ This is to certify that the dissertation entitled Neuroendocrine Function and Regulation of the Tuberohypophysial Dopaminergic Neurons in the Intermediate Lobe of the Pituitary presented by Steven E. Lindley has been accepted towards fulfillment of the requirements for Ph.D. degeeh, Pharmacology WW Major professk Date August 10, 1989 "(Ilia/... All" 0' A ' 1-1 :m -, , . r 0‘1 1 M... , LIBRARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or More data due. DATE DUE DATE DUE DATE DUE ;__L__J —_|F——l MSU I: An Affirmative ActiorVEqual Opportunity lnditution l NEUROENDOCRINE FUNCTION AND REGULATION OF TUBEROHYPOPHYSIAL DOPAMINERGIC NEURONS TERMINATING IN THE INTERMEDIATE LOBE OF THE PITUITARY BY Steven E. Lindley 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 1989 ABSTRACT NEUROENDOCRINE FUNCTION AND REGULATION OF TUBEROHYPOPHYSIAL DOPAMINERGIC NEURONS TERMINATING IN THE INTERMEDIATE LOBE OF THE PITUITARY BY Steven E. Lindley The purpose of the present study was to characterize tuberohypophysial dopaminergic (THDA) neurons terminating in the intermediate lobe of the pituitary, and to evaluate their role in controlling both basal and stress-induced secretion of a pro-opiomelanocortin-derived peptide, a-melanocyte- stimulating hormone (a-MSH). Dopamine (DA) metabolite concentrations in the intermediate lobe were evaluated as an index of THDA neuronal activity. The major DA metabolite in the intermediate and neural lobes was 3,4-dihydroxyphenylacetic acid (DOPAC) . DOPAC was rapidly removed from the posterior pituitary after blocking its formation by pargyline. Blockade of DA reuptake by nomifensine resulted in only a slight decline in DOPAC concentrations indicating DOPAC is derived mainly from intraneuronal metabolism of unreleased DA. Changes in THDA neuronal activity induced by electrical stimulation or dopaminergic agonist or antagonist administration, were reflected in corresponding changes in the concentration of intermediate lobe DOPAC indicating that the concentration of ii DOPAC in this region is an index of THDA neuronal activity. Increases and decreases in THDA neuronal activity produced reciprocal changes in pdasma a-MSH concentrations which were blocked by pretreatment with the dopaminergic antagonist haloperidol . Although a-MSH administration produced a rapid increase in tuberoinfundibular dopaminergic neuronal activity, a-MSH failed to alter THDA neuronal activity either acutely or after a 12 hr delay indicating THDA neurons are not activated by a-MSH. Physical restraint increased plasma a-MSH concentrations and decreased THDA neuronal activity. Administration of the B-adrenergic antagonist propranolol blocked the stress-induced increase in a-MSH but had no effect on THDA neuronal activity indicating B-adrenergic activation is necessary for the stress-induced increase in a-MSH secretion. On the other hand, pretreatment with the dopaminergic agonist apomorphine prevented stress-induced a-MSH secretion and administration of the dopaminergic antagonist haloperidol potentiated fi- adrenergic agonist stimulation of a-MSH secretion. Furthermore, retrochiasmatic deafferentation blocked both the stress-induced changes in THDA neuronal activity and a-MSH secretion. Taken together, these results indicate that a decrease in THDA neuronal inhibitory tone in combination to fi—adrenergic stimulation mediates stress-induced increases in a-MSH secretion. iii to my wife, Bea, for all her support and patience, and in memory of my parents iv ACKNOWLEDGEMENTS I wish to extend my sincerest gratitude to Dr. Kenneth Moore and Dr. Keith Lookingland for lending their time, support and.guidance inedirecting'my scientific studies. Their constructive criticism, encouragement and direction have been essential in my scientific development. I would like to thank Drs. Glenn Hatton, Cheryl Sisk and Gregory Fink, who formed a helpful and constructive guidance committee. I wish to extend my thanks to Dr. Joseph Gunnet for his collaborative efforts and advice. I gratefully acknowledge all the individuals who provided technical support, especially John Moss, Chris Bommarito and Jill Stegman. Their high-standards and friendship were greatly appreciated. TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS INTRODUCTION I. Anatomical distribution of dopaminergic neurons A. Mesotelencephalic dopaminergic neurons B. Hypothalamic dopaminergic neurons C. THDA neurons D. Anatomy of the pituitary gland II. Neurochemistry of dopaminergic neurons A. Nigrostriatal dopaminergic neurons B. THDA neurons III. Neuroendocrine functions of DA in the neural and intermediate lobes of the pituitary A. DA in the neural lobe of the pituitary B. DA in the intermediate lobe of the pituitary C. Other regulators of intermediate lobe secretion IV. Differential regulation of THDA neuronal activity STATEMENT OF PURPOSE MATERIALS AND METHODS A. Animals B. Drugs vi GUI-FM 11 14 15 16 20 22 24 25 25 TABLE OF CONTENTS (continued) MATERIALS AND METHODS (continued) C. Surgical Manipulations 1. Intravenous Catheter 2. Intraventricular Cannula 3. Retrochiasmatic Deafferentation D. Electrical Stimulation E. Restraint Stress F. Tissue Preparation G. Neurochemical Analysis H. Radioimmunoassay of Prolactin I. Radioimmunoassay of a-MSH 1. Assay Material 2. Precipitation Method 3. Assay Sensitivity 4. Assay Characteristics J. Statistical Analysis EXPERIMENTS Section 1: Neurochemical Indices of THDA Neuronal Activity Introduction Results Discussion Section II: THDA Neuronal Regulation of o-MSH Sec- retion from the Intermediate Lobe of the Pituitary Introduction Results Discussion Section III: a-MSH Regulation of THDA Neuronal Activity Introduction Results Discussion Section IV: Role of THDA Neurons in Controlling o-MSH Secretion During Stress Introduction Results Discussion vii 26 26 26 27 28 29 29 30 32 36 37 37 44 44 46 48 51 55 65 65 71 78 79 88 92 92 102 TABLE OF CONTENTS (continued) SUMMARY CONCLUDING DISCUSION BIBLIOGRAPHY viii 106 108 110 10 11 LIST OF TABLES Serum a-MSH concentrations measured with differ- ent RIA methods. Effect of various assay alterations on the amount of tracer bound (% binding) and the assay sensit- ivity. Effect of NSD 1015 administration on serum a-MSH concentrations (pg/ml serum). Concentrations of DA, DOPAC and DOPA in the intermediate and neural lobes of the pituitary. Effect of GBL on the concentrations of DOPAC and DA in the intermediate lobe of the pituitary. Effect of arcuate nucleus stimulation on the con- centrations of DOPAC in the intermediate lobe and a-MSH in the serum. Intermediate lobe DOPAC and plasma a-MSH concen- trations at 03.00 and 11.00 hours. Time course of the effects of a-MSH on DOPAC and DA concentrations in selected regions. Effect of metaproterenol on the concentrations of a-MSH and prolactin in the serum and DOPAC in the median eminence and intermediate and neural lobes of the pituitary. Effect of propranolol and/or stress on DOPAC and DA concentrations in the intermediate lobe of the pituitary gland. Effect of stress on DOPAC and DA concentrations in the intermediate lobe of the pituitary in sham and retrochiasmatic deafferentated (RC) animals. ix PAGE 42 45 50 52 67 69 74 81 87 96 100 FIGURE 1 BA BB 8C LIST OF FIGURES Schematic of a sagittal section of the rat brain depicting the location of some of the NE (A1, A2, A5-A7) and DA (A8-A14, A16) containing perikarya. Schematic of a midsagittal view of the rat hyp- othalamus indicating the location of the tubero- hypophysial and tuberoinfundibular dopaminergic neurons. Schematic of a nigrostriatal DA neurons. The post-translational processing of POMC in the intermediate lobe of the rat pituitary. Diagrammatic representation of the dual regul- ation of adenylate cyclase activity and a-MSH release by fiz-adrenergic and D-2 dopaminergic receptors in the melanotrophs of the intermed- iate lobe. Effects of alterations in 2nd analytical elect- rode potential on the peak height generated by 500 pg of DA, DOPA or DOPAC. Sample HPLC chromatogram obtained following the injection of a mixture of 500 pg standards or intermediate lobe from control or NSD 1015- treated animals. Standard curve and serum dilutions generated in o-MSH radioimmunoassay using charcoal extrac- tion. Standard curve and serum dilutions generated in a-MSH radioimmunoassay using IgGsorb. Standard curve and serum dilutions generated in a-MSH radioimmunoassay using goat anti-rabbit gamma globulin. 12 17 21 33 34 39 41 43 LIST OF FIGURES (Continued) FIGURE 8D 10 11 12 13 14 15 16 17 18 Standard curve and plasma dilutions generated in a-MSH radioimmunoassay. Time course of the effect of pargyline on con- centrations of DOPAC in the intermediate lobe (IL) and neural lobe (NL) of the pituitary gland. The effect of nomifensine on DOPAC concentra- tions in the intermediate lobe (IL) and neural lobe (NL) of the pituitary. Comparison of the effects of electrical stimu- lation of the pituitary stalk on DOPA and DOPAC concentrations in the intermediate lobe (IL) and neural lobe (NL) of the pituitary. Comparison of the effects of haloperidol admin- istration on DOPA and DOPAC concentrations in the intermediate lobe (IL) and the neural lobe (NL) of the pituitary. Comparison of the effects of apomorphine admin- istration on DOPA and DOPAC concentrations in the intermediate lobe (IL) and neural lobe (NL) of the pituitary. The effects of administration of GBL on the con- centrations of DOPAC in the intermediate lobe of the pituitary and a-MSH in the serum. Time course of the effects of arcuate nucleus stimulation on the concentrations of DOPAC in the intermediate lobe of the pituitary and a-MSH in the serum. The effects of haloperidol on the stimulation- induced decrease in serum concentrations of a-MSH. Plasma o-MSH concentrations determined over a 24 hour period. Time course of the effect of a-MSH on DOPAC concentrations in the selected regions of the rat brain. xi PAGE 47 54 56 57 58 59 66 70 72 73 80 LIST OF FIGURES (Continued) FIGURE 19 20 21 22 23 24 25 26 27 Effect of a-MSH on DOPA concentrations in median eminence and the intermediate and neural lobes of the pituitary. Dose-response of a-MSH on DOPA concentrations in median eminence and the intermediate and neural lobes of the pituitary. Time course of the effects of a-MSH on plasma prolactin concentrations. Comparison of the effects of a-MSH and prolactin on the accumulation of DOPA in median eminence and the intermediate and neural lobes of the pituitary. Dose-response of the effects of propranolol on the stress-induced increase in a-MSH concentra- tions in plasma. Effects of restraint stress on intermediate and neural lobe DOPAC concentrations. Effect of apomorphine pretreatment on the stress- induced increase in plasma o-MSH concentrations. Effect of haloperidol on the metaproterenol- induced increase in plasma a-MSH concentrations. Effect of retrochiasmatic (RC) deafferentation on the stress-induced changes in plasma a-MSH concentrations. xii PAGE 82 83 84 86 93 95 97 98 101 ACTH CAMP CLIP COMT CRF DA DOPA DOPAC GBL HPLC HRP ICV IL fi-LPH a-MSH 3MT MAO MB NB NL NSD 1015 LIST OF ABBREVIATIONS adrenocorticotropic hormones adenosine cyclic 3',5'-monophosphate corticotropin-like intermediate lobe peptide catecholamine-O-methyltransferase corticotropin-releasing factor dopamine 3,4—dihydroxypheny1alanine 3,4-dihydroxyphenyacetic acid gamma-butyrolactone high performance liquid chromatography horseradish peroxidase homovanillic acid intracerebroventricular intermediate lobe of the pituitary gland B-lipotropin o-melanocyte stimulating hormone 3-methoxytyramine monoamine oxidase median eminence norepinephrine neural lobe of the pituitary 3-hydroxybenzy1hydrazine xiii LIST OF ABBREVIATIONS (Continued) POMC THDA TIDA pro-opiomelanocortin tuberohypophysial dopaminergic tuberoinfundibular dopaminergic xiv INTRODUCTION The dopamine (DA) -containing neuronal systems within the brain are more diverse and numerous than other catecholamine- containing systems (Moore and Bloom, 1978). Most of what is known about dopaminergic neurons has been learned from studies of the nigrostriatal system. Although the intermediate lobe of the pituitary has received a great deal of attention in recent years as an in vitro system for both the study of D-2 subtype DA receptors and pro-opiomelanocortin (POMC)-derived peptide secretion (for reviews Coté et al., 1982; Akil gt a;., 1984; Millington, 19B9), few studies have examined the in yigg characteristics and regulation of the tuberohypophysial dopaminergic neurons (THDA) that project to the intermediate lobe. This introduction summarizes the present knowledge about the anatomy and neurochemistry of dopaminergic neurons in general and THDA neurons in particular. It describes the structure, biochemistry and innervation of the intermediate lobe of the pituitary and what is presently known about the regulation of THDA neurons. 2 I. ANATOMICAL DISTRIBUTION OF DOPAMINERGIC NEURONS A. MESOTELENCEPHALIC DOPAMINERGIC NEURONS DA was first identified in the brain in 1957 by Montagu using the method of ethylenediamine condensation-induced fluorescence. In 1964, using the Falack-Hillarp technique of dry formaldehyde vapor-induced fluorescence of catecholamines in freeze-dried brain sections, Dahlstrom and Fuxe identified 12 discrete groups of catecholamine-containing perikarya within the brain and designated them as A1-A12. Further investigations have subsequently revealed four more DA cell groups, which have been designated as A13-A16 (for review; Bjorklund and Lindvall, 1984). The more caudal A1—A7 groups in the pons-medulla are norepinephrine (NE)-containing neurons, while the more rostral A8-A16 groups, extending from the mesencephalon to the olfactory bulb, are DA-containing neurons (figure 1). This anatomy has since been confirmed by the more sensitive glyoxylic acid-fluorescence technique and immunocytochemical identification of catecholamine- synthesizing enzymes, and has been reviewed (Moore and Bloom, 1978; Bjorklund and Lindvall, 1984). The perikarya of the major ascending dopaminergic neuronal systems, which are collectively known as the mesotelencephalic dopaminergic neurons, are located in the pars compacta of the substantia nigra (A8-A9) and in the more medial regions of the ventral tegmentum (A10) . These neurons can be divided into three groups; 1) nigrostriatal dopaminergic neurons which project Cor-bellow A8 ' Amway tllill'llllllflmm‘ll' 31‘1”" "’ A2 :21: 3:05“ allur‘ffuimn' POM WWII all”, [Isl/”I’ll”: \m)“‘ A A Dopamine f f Noropl. Figure 1. Schematic of a sagittal section of the rat brain depicting the location of some of the NE (A1, A2, A5-A7) and DA (As-A14, A16) containing perikarya. 4 primarily from A8 and A9 to the caudate-putamen, 2) mesolimbic dopaminergic neurons which project from A10 to various subcortical regions (such as the nucleus accumbens and olfactory bulb), and 3) mesocortical dopaminergic neurons which project from A8-A10 to various cortical regions (prefrontal, cingulate, entorhinal and pyriform). Because the nigrostriatal dopaminergic neurons comprise the largest group of dopaminergic neurons and, therefore, the easiest to study from a technical standpoint, much of what is known about dopaminergic neurons has been obtained through the study of this system. B. HYPOTHALAMIC DOPAMINERGIC NEURONS There are four groups of DA perikarya in the hypothalamus (All-A14) (figure 1). The largest percentage of dopaminergic neurons in the hypothalamus comprise the incertohypothalamic system, whose perikarya are located at the mesencephalic-hypothalamic junction (A11) , medial zona incerta (A13) and periventricular region of the anterior hypothalamus (A14) (Bjorklund e_t a1” 1975; Van den Pol gt _a_2_l_., 1984). Their axonal projections are not well defined, but are thought to consist mainly of short projections within the diencephalon. Some of the neurons originating in the A11 region project to the spinal cord (Bjorklund and Skagerberg, 1979). 5 The arcuate nucleus and adjacent periventricular area of the mediobasal hypothalamus (A12) contain the perikarya of tuberoinfundibular dopaminergic (TIDA) neurons (Bjorklund gt al., 1973). TIDA neurons, originating in both the rostral and caudal arcuate nucleus, have short axons that project ventrally to the external layer of the median eminence (figure 2). Most TIDA neurons do not form synapses but release DA from their terminals into the hypophysial portal blood systemmwhich transports it to the anterior lobe of the pituitary where it inhibits the secretion of prolactin (Ben-Jonathan, 1985; Gudelsky et al., 1981). There are several differences between TIDA and nigrostriatal dopaminergic neurons, including the absence of a high affinity DA uptake site (Annunziato and Weiner, 1980; Demarest and Moore, 1979b) and DA autoreceptor regulation (Demarest and Moore, 1979c) in TIDA neurons. C. THDA NEURONS The first report that described an innervation of the neural and intermediate lobes. of the pituitary ‘was the anatomical study of Ramon y Cajal (1894), who described nerve fibers that originated in the brain, passed through the pituitary stalk, and terminated in the neural and intermediate lobes of the pituitary. Dahlstrom and Fuxe (1966), using fluorescence histochemistry, identified the presence of monoamine-containing nerve fibers in the pituitary gland. Subsequent biochemical and spectofluorescence studies 6 demonstrated that this monoamine histofluorescence is due to the presence of both DA and NE fibers, but that DA is the predominant monoamine (Bjorklund e_t; 11., 1967). In 1973, Bjorklund and coworkers, using both electrolytic lesions and specific knife cuts in conjunction with fluorescence histochemistry, reported that the perikarya of THDA neurons are, like the perikarya of TIDA neurons, located in the arcuate nucleus, but are confined to the more rostral part of the arcuate nucleus and adjacent periventricular area (A12). The fibers of THDA neurons project ventrally through the median eminence and pass medially through the infundibular stalk to terminate in the neural and intermediate lobes of the pituitary (figure 2). Recent studies have suggested that the origin of THDA neurons may be different from that described by Bjorklund and coworkers (1973). Neonatal injections of monosodium glutamate which results in a substantial loss of arcuate nucleus DA neurons fails to decrease the DA content in the neural and intermediate lobes, suggesting that the arcuate nucleus may not be the origin of THDA neurons (Dawson gt a;., 1985). Luppi and coworkers (1986) retrogradely labeled THDA neurons in the cat by injection of horseradish peroxidase (HRP) into the combined neurointermediate lobe of the pituitary. When these neurons were examined in conjunction with tyrosine hydroxylase immunohistochemistry as a marker for DA neurons, they found that neurons containing both HRP and tyrosine hydroxylase were ZFUdJOmd mcououc owouocfieomoo Howmhneomanouonsu u dome uncououc UflmuoEHEomoc undonwocounwouonsu u «0H9 “mood amazon u 42 «onoa oumwooeuou:« u AH «huouwouqm uoHuouco n me .9555: UfimuUCHEmmoo Hod—unaccounwouonou can Hmwmacaom>nouonou ozu no coaumooH on» mowumowo:a masononuom>n you on» no sow> Hmuuwmomofie o no Ufiumsonom .w ouomwm 8 located in the rostral periventricular (A14) region and not in the arcuate nucleus. This finding was confirmed in the rat using wheat germ agglutinin as a retrograde label (Kawano and Daikoku, 1987). Taken together, these recent studies indicate that THDA neuron. perikarya are confined to the rostral periventricular region (A14). The reason for the discrepancy between these studies and those of Bjorklund and co-workers is not known. For the purposes of the present study, knowledge of the exact location of THDA perikarya was not necessary because the activity of these neurons was monitored by measuring DA synthesis and metabolism in their terminals in the intermediate and neural lobes. D. ANATOMY OF THE PITUITARY GLAND The pituitary gland is composed of three regions; the anterior, intermediate and neural lobes. Ontogenetically, both the anterior and intermediate lobes arise from the embryonic ectodermal Rathke's pouch whereas the neural lobe arises from neuronal growth of the floor of the diencephalon. The anterior lobe is a collection of different types of glandular cells which synthesize and secrete prolactin, follicle-stimulating hormone, luteinizing hormone, growth hormone, thyroid- stimulating hormone and adrenocorticotropic hormone (ACTH) (Genuth, 1983). The anterior lobe is not directly innervated by the brain, but is regulated by stimulatory and inhibitory 9 factors which are released from the median eminence into the hypophysial portal blood system. The neural lobe is composed primarily of dense bundles of the unmyelinated nerve terminals of vasopressin and oxytocin-containing axons whose cell bodies are located in the paraventricular and supraoptic nuclei of the hypothalamus. Individual axons are separated from one another by glial-like pituicytes. The neural lobe receives additional neuronal input centrally from the brain and peripherally from the autonomic nervous system. The intermediate lobe is composed of both glandular cells and nerve terminals. In the rat, it is a poorly vascularized compact structure consisting of 10-15 layers of closely packed polygonal endocrine cells, known as melanotrophs, which are separated into lobules by connective tissue (Eberle, 1988). Epithelial cells line the hypophysial cleft and contribute colloid material to the intermediate lobe. The melanotrophs of the intermediate lobe secrete a number of POMC-derived peptides, including a-melanocyte stimulating hormone (o-MSH) and fi-endorphin. The melanotrophs are heterogenous in color; darker cells have a higher secretory activity than lighter cells (Chronwall gt g;., 1988). Although the intermediate lobe is present in the human fetal pituitary, its size diminishes after birth, and it eventually disappears as the remaining melanotrophs intermingle with the cells of the anterior and neural lobes (Coates gt _t., 1986). 10 The intermediate and neural lobes differ in the type of dopaminergic neuronal innervation they receive (Baumgarten et g;., 1972: Luppi gt g;., 1986). In the neural lobe, dopaminergic varicose fibers are diffusely distributed and terminate in the proximity (within 8-12 nm) of neurosecretory axons and pituicytes, without the presence of true synaptic structures. It appears that all dopaminergic neurons that project to the intermediate lobe first.pass through the neural lobe. In the intermediate lobe, THDA neurons form numerous synaptic contacts with melanotrophs (Baumgarten gt gt., 1972; Luppi gt g;., 1986). A number of other neurotransmitters are located in neurons of the rat intermediate lobe, including NE, serotonin and gamma-aminobutyric acid (GABA). The origin of this innervation is not yet determined. The noradrenergic innervation has been reported to originate from either the central nervous system (Saavedra, 1985), peripheral sympathetic superior cervical ganglion (Bjorklund gt L1.” 1973) or both central and peripheral sites (Alper gt 11., 1980a) . The serotonergic innervation has been reported to originate from either cells of midbrain raphe and hypothalamic dorsomedial nucleus (Mezey gt g;., 1984) or from cells caudal to the dorsal and medial raphe nuclei but not the hypothalamic dorsomedial nucleus (Shannon and Moore, 1987) . GABA innervation originates in the central nervous system (Rabhi gt gt., 1987; Oertel gt g;., 1982) in descending projections 11 through the rostral hypothalamus (Tappaz gt gt., 1986). GABA has been co—localized with DA, suggesting it may be cc- released with DA from THDA neurons (Vuillez gt g;., 1987). II. NEUROCHEMISTRY OF DOPAMINERGIC NEURONS A. NIGROSTRIATAL DOPAMINERGIC NEURONS Concepts of DA synthesis, storage, release and metabolism in the central nervous system have been derived primarily from studies on nigrostriatal dopaminergic neurons. As depicted in figure 3, DA is synthesized from the amino acid precursor, L-tyrosine. L-tyrosine is present in the diet and, in addition, can be derived by conversion of dietary L- phenylalanine by phenylalanine hydroxylase found primarily in the liver (review, Cooper (gt .gt., 1982). L-Tyrosine is transported into the dopaminergic nerve terminal by an active uptake mechanism. Once inside the neuron, L-tyrosine is converted to erihydroxyphenylalanine (DOPA) by tyrosine hydroxylase, the rate limiting enzyme in DA synthesis. DOPA does not accumulate but is rapidly decarboxylated by aromatic L-amino acid decarboxylase to form DA. The newly synthesized DA can then be stored in synaptic vesicles or released in response to arrival of nerve action potentials. The concentrations of DA in the nerve terminal remain fairly constant despite alterations in the amount of transmitter released. This is because the activity of the 12 COMV WA \MAO 3MT DOPAC D POSTSYNAPTIC CELL BODY OR DENDRITE .1 TYROSINE -:;" DOPA —“'_g ' ' i C l TYROSINE Figure 3. Schematic of a nigrostriatal DA neurons. D -= dopamine; MAC 8 monoamine oxidase; COMT = catechol-o- methyltransferase: DOPAC = dihydroxyphenylacetic acid; EVA 8 homovanillic acid: 3MT = 3-methoxvtvramine. 13 rate-limiting enzyme tyrosine hydroxylase is regulated, in part, by end-product inhibition, such. that increases in cytoplasmic DA concentrations decrease tyrosine hydroxylase activity, and vice versa. When DA is released, it can activate DA receptors on dendrites or cell bodies of postsynaptic neurons. DA may also activate presynaptic autoreceptors, which inhibit both synthesis and release:of DA (Carlsson, 1975; Nowycky and Roth, 1978). The actions of DA at receptor sites are terminated by its removal from the synaptic cleft by a high-affinity transport system which transports DA back into the nerve terminal (Snyder and Coyle, 1969). Once back inside the neuron, DA can be repackaged in synaptic vesicles for re- release, or metabolized to inactive products. The main enzymes involved in the metabolic degradation of DA are monoamine oxidase (MAO) and catechol-O- methyltransferase (COMT) (Rosengren, 1960; Sharman, 1973; for review, Westerink, 1985). MAO, which is localized largely on the outer surface of mitochondrial membranes, converts DA to its corresponding aldehyde which is then rapidly oxidized to dihydroxyphenylacetic acid (DOPAC) . DOPAC can be further metabolized by COMT, which is located in glial cells, and catalyzes the methylation of DOPAC to form homovanillic acid (HVA). DA that is not recaptured by the presynaptic terminal may be converted first by COMT to 3-methoxytyramine (3MT) which may then be further metabolized by MAO to form HVA. 14 B. THDA NEURONS Most of the principles of DA synthesis, release, and metabolism derived from the nigrostriatal dopaminergic neurons also hold true for THDA neurons (for review, Holzbauer and Racke, 1985). Both tyrosine hydroxylase and aromatic:L-amino acid. decarboxylase activity' have Ibeen identified in 'the neurointermediate lobe (Saavedra gt 1., 1975; Demarest gt _t., 1979a; Johnston gt gt., 1984; Back gt gt., 1987). MAO, but not COMT, can be detected in homogenates of the neurointermediate lobe (Saavedra gt_gt., 1975), although both MAO and COMT activity can be detected in the isolated neurointermediate lobe it vitro (Racké and Muscholl, 1986). DA is released from the isolated neurointermediate lobe under basal conditions and.is increased.after electrical stimulation of the pituitary stalk (Holzbauer gt g;., 1982; Holzbauer and Racké, 1985). DA synthesis in THDA. neurons, as in :nigrostriatal dopaminergic neurons, is regulated by end-product inhibition. It has been shown that an increase in DA concentrations in the neurointermediate lobe after administration of a MAO inhibitor decreases the rate of DA synthesis, whereas a reserpine-induced decrease in DA concentrations increases DA synthesis (Demarest gt gt., 1979c: Demarest gt gt., 1981). One of the major differences between nigrostriatal and THDA.neurons is the apparent absence of aihigh affinity uptake site in the neurointermediate lobe. Studies which examined_the 15 uptake of [dHJ-DA in homogenates of neurointermediate lobes demonstrate a Km of approximately 1.6 uM in comparison to 0.58 uM for the striatum (Demarest and Moore, 1979b). The presence of only a low affinity uptake site has also been observed by others (Annunziato and Weiner, 1980), and indicates that the amount of released DA that is converted to DOPAC may be lower in the neurointermediate lobe than in the striatum. Because DOPAC concentrations are thought.to reflect, at least in part, released DA that has been taken back up into the neuron (Roth gt gt. , 1976) , this raises the question of whether DOPAC concentrations in the neural and intermediate lobes accurately reflect the activity of THDA neurons, as it does in the nigrostriatal dopaminergic neurons (Roth gt g;., 1976; Westerink, 1979) III. NEUROENDOCRINE FUNCTIONS OF DA IN THE NEURAL AND INTERMEDIATE LOBES OF THE PITUITARY. A. DA IN THE NEURAL LOBE OF THE PITUITARY There is considerable confusion concerning the effect of DA on the regulation of vasopressin and oxytocin secretion from the neural lobe (for review Pittman g a_l_., 1983). Conflicting results from ig vivo studies, in which DA agonists or antagonist are administered.peripherally, may be due to the presence of more than one site of action of DA that alters vasopressin and oxytocin release. DA applied directly to the isolated neural lobe has been reported to increase basal 16 vasopressin and oxytocin release (Bridges gt 1., 1976) and to decrease electrically stimulated vasopressin release (Lightman gt g;., 1982). Racké and coworkers (1982), using specific DA agonists and antagonists on isolated neurointermediate lobes, demonstrated that activation of D-l subtype DA receptors facilitate whereas D-2 subtype receptors inhibit electrically stimulated, but not potassium stimulated, secretion of vasopressin from the isolated neurointermediate lobe, suggesting a complex modulation of vasopressin release by DA. The absence of true DA synapses in the neural lobe has led to the suggestion that the actions of DA released from THDA neurons in the neural lobe may be delayed (Holzbauer and Racké, 1985). B. DA IN THE INTERMEDIATE LOBE OF THE PITUITARY Both the corticotrophs of the anterior lobe and the melanotrophs of the intermediate lobe synthesize the peptide POMC. POMC is initially cleaved, possibly by POMC converting enzyme (Loh e_t gt” 1985), to produce adrenocorticotropin (ACTH), B-lipotropin (fi-LPH) and the N-terminal fragment (fig 4). In the intermediate lobe, ACTH is then further processed to corticotropin-like intermediate lobe peptide (CLIP) and a-MSH, the N-terminal fragment is metabolized into gamma-MSH, and fi-LPH is converted to gamma-LPH and fi-endorphinwm fi- endorphinhn is further metabolized to fi-endorphinpv.and B- endorphinrui(for reviews, Eipper and Mains, 1980; Akil gt 17 «Ni .xmmma .Hamzcounu new couocflaafiz Eouuc eflnmuoocmun u ozone «onwumon DDOH oumaooauoucw mxwalcwmouuoowuuoo u quo “camoupomflaun u media “ocofiuon Uflnmouuoofiuuouocmuom u 55¢ “ocwumom ocflchn u on .Uoo>moau Uwvhaoououmoccu mo Upwm 0:» Runs msouuo one .xumuwouwn you on» no UDOH upmaouauuucw on» Ca 020m no ocwmmmuoum HMEOflvoncmuuuumon was .e munowm $.30 tends 5.55.9.2 foamfiée D EAEE .. \r/ twat; «It 2 Aseeixny‘ \Cx . a E so...» > 78? fivumfld “'SWIA “Wrfllw. m ...-lag $40 I . % geyxxxxxxxxxxd 534! a up memesTween Rowena ”fix/xx wxxx/x.7/.//y/xx/¢% i i ( 2ezn/zzwzzzyw_ 18 _t., 1984). In addition, peptide acetyltransferase acetylates both fi-endorphin peptides and a-MSH; acetylation increases the behavioral effects (induced grooming behavior and improved visual discrimination) and melanocyte stimulating properties of a-MSH and eliminates the opiate analgesic actions of 6- endorphin peptides (Akil gt _t., 1981; O'Donohue e_t g” 1981). All of the end-products of POMijrocessing (figure 4) are secreted from, the intermediate lobe. They appear' to be contained within the same synaptic vesicles, with very small amounts of precursors such as ACTH also being secreted (review Eberle, 1988). The secretion appears to occur in.a continuous, not a pulsatile, manner (Wilson and Harry, 1980). In addition, diurnal variations in ‘the secretion of a-MSHZ have been observed, but different patterns and magnitudes of variation have been reported, with both a monophasic (Wilson gt gt., 1979; Millington gt g;., 1986a) and a biphasic (Usategui gt gt., 1976; Monnet gt gt., 1981) pattern of secretion observed. The processing of POMC in corticotrophs of the anterior lobe differs from that of the intermediate lobe. In the anterior lobe, ACTH is not converted to o-MSH and CLIP, and fi-LPH is only partially converted to fi-endorphinyyp In addition, acetylation does not occur in the anterior lobe (Eipper and Mains, 1980; Akil gt g;., 1984). It had been demonstrated in studies dating back to 1941 that the intermediate lobe is under inhibitory control from 19 the hypothalamus. Sectioning of the pdtuitary stalk, transplanting the pituitary or lesioning the hypothalamus all produce skin darkening in Xenopus larvis and increase plasma a-MSH levels in the rat (review Eberle, 1988). In 1974, Bower and coworkers demonstrated that DA applied directly to the neurointermediate lobe it vitro decreases the secretion of a- MSH. Subsequent studies have indicated that in ytttg administration of dopaminergic agonists decrease while dopaminergic antagonists increase plasma a-MSH concentrations (Usategui gt gt., 1976: Penny and Thody, 1978). The secretion of other POMC-derived peptides of the intermediate lobe (fi- endorphin, ACTH, CLIP, pro-gamma-MSH and gamma-LPH) which are co-secreted.with a-MSH, is also inhibited by DA (Farah.gt gt., 1982; Jackson and Lowry, 1983; Randle e_t _l_., 1983). DA inhibits intermediate lobe secretion in a variety of ways, including inhibition of; 1) intermediate lobe spontaneous electrical activity (Davis and Hadley, 1976; Douglas and Taraskevich, 1978), 2) the number of melanotrophs with high secretory activity (Chronwall gt gt., 1988), 3) POMC processing enzymes levels (Millington gt g;., 1986b; Mains gt g;., 1985), and 4) POMC mRNA concentrations (Beaulieu gt_gl., 1984). This indicates that the mechanisms involved in the synthesis, processing and release of intermediate lobe peptides are interrelated in that all are subject to dopaminergic control. 20 The dopaminergic receptors in the intermediate lobe have been classified as the D-2 subtype (Munemura gt g;., 1980; Stefanini gt _J_.., 1980), according to the classification system of Kebabian and Calne (1979). The receptor is negatively coupled to adenylate cyclase, whereby binding of DA decreases the production of adenosine cyclic 3',5'- monophosphate (cAMP), and thereby decreases the secretion of a-MSH (Coté gt g;., 1982) (fig 5). C. OTHER REGULATORS OF INTERMEDIATE LOBE SECRETION Other neurotransmitters can alter secretion from the intermediate lobe. Epinephrine, released from the adrenal medulla, activates BZ-adrenergic receptors on melanotrophs to stimulate a-MSH secretion (Bowers gt gt., 1974; Berkenbosch e_t g” 1981; Kvetnansky e_t_ gt, 1987). The fiz-adrenergic receptor is positively coupled to adenylate cyclase, so that activation increases cAMP production and, in turn, thereby increases the secretion of a-MSH (Coté gt _t., 1982) (fig 5). GABA has a biphasic effect on both melanotroph spontaneous electrical activity (Taraskevich and Douglas, 1982) and a-MSH secretion (Tomiko gt gt” 1983). Administration of GABA to melanotrophs ig vitro produces a brief stimulatory phase followed by inhibition. This dual action of GABA has been attributed to GABA receptor subtypes; GABAfizreceptors produce a rapid release of a-MSH and GABAB 21 DOPAMINE EPINEPHRINE ( APOMORP! llNE l i METAPROTERENOL ) PROPRANOLOL orMSl-l Figure 5. Diagrammatic representation of the dual regulation of adenylate cyclase activity and o-MSH release by 52- adrenergic and D-2 dopaminergic receptors in the melanotrophs of the intermediate lobe. N, - stimulatory guanyl nucleotide site: N, - inhibitory guanyl nucleotide site: cyclase - adenylate cyclase; cAMP - adenosine cyclic 3',5'- monophosphate; ATP - adenosine S'-triphosphate (adapted from Coté fit all, 1982) . 22 receptors produce a sustained suppression (Demeneix gt gt., 1986). Other compounds have a weak effect of a-MSH secretion. High doses of serotonin have been reported to have a small stimulatory effect, whereas acetylcholine has a small inhibitory effect (for reviews, Tilders gt gt., 1985; Eberle, 1988) . Administration of corticotropin releasing hormone (CRH) can stimulate the secretion of a-MSH from melanotrophs both vivo (Proulx-Ferland gt gt., 1982) and it vitro (Meunier IS I“ _t., 1982), but much higher concentrations are required than is necessary to stimulate anterior lobe corticotroph secretion (Vale gt g;., 1983). IV. DIFFERENTIAL REGULATION OF THDA NEURONAL ACTIVITY Due to technical difficulties in separating the neural and intermediate lobes, most. of the early neurochemical studies on THDA neurons were performed in the entire neurointermediate lobe; THDA neurons in the two different lobes were considered to function in unison. With an improvement in technical capabilities, Lookingland and coworkers (1985a) developed a microdissection method to analyze the neural and intermediate lobes.separately, and discovered that THDA neurons in the two lobes have different properties. Studies using the entire neurointermediate lobe had indicated that release of DA from. THDA neurons is 23 regulated by a dopaminergic-receptor mediated mechanism (Demarest.gt gt., 1979c), but.when the neural and intermediate lobes were analyzed separately, this dopaminergic-receptor regulation of DA turnover was discovered to be restricted to the intermediate lobe (Lookingland gt gl., 1985a). Likewise, morphine had no effect on DA turnover in the entire neurointermediate lobe (Alper gt gt., 1980b), but when the neural and intermediate lobe were analyzed separately, morphine was found to inhibit DA turnover in the neural but not. in. the intermediate lobe (Lookingland and coworkers (1985b). Together, these results indicate that THDA neurons terminating in the two different lobes of the pituitary are regulated differently, and that. a change in DA release confined to one lobe may be masked by the lack of change in the other lobe if the entire neurointermediate lobe is analyzed. Therefore, conclusions based on studies performed on the entire neurointermediate lobe may not be accurate. In the present study the neural and intermediate lobes were analyzed separately. STATEMENT OF PURPOSE THDA neurons project from the mediobasal hypothalamus through the pituitary stalk to terminate in the neural and intermediate lobes of the pituitary. Although the intermediate lobe has received a great deal of attention in recent years, little is known about the regulation of THDA neurons in the intermediate lobe. The purpose of the present study was to test the hypophysis that the activity of THDA neurons is correlated with reciprocal changes in a-MSH secretion and that o-MSH can feedback to activate THDA neurons. In addition, it was determined if a decrease in THDA neuronal activity during stress is necessary for the stress-induced increase in intermediate lobe peptide secretion. As an initial step, DA metabolite concentrations in the intermediate lobe were evaluated as an index of THDA neuronal activity. 24 MATERIALS AND METHODS I. Methods A. Animals All of the experiments were conducted using male Long- Evans rats (initial weight 200-225 g: obtained from either Charles River Laboratories (Wilmington, MA) or Blue Spruce Farms (Altamont, NY) maintained under conditions of controlled temperature (22° C) and lighting (illumination 0700 to 1900 hr) with food (Wayne Labox) and tap water supplied ad libitum. In experiments involving restraint as a manipulation, animals were housed two per cage, while in all other experiments, animals were housed four per cage. Experiments were conducted no earlier than 3 days after arrival of the animals to allow for accommodation. B. Drugs a-MSH (Peninsula Laboratories, Inc., Belmount CA), apomorphine hydrochloride (Eli Lilly Co, Indianapolis, IN), gamma-butyrolactone (GBL; Sigma Chemical Co, St Louis, MO), nomifensine maleate (kindly supplied by Dr. S. Fielding: Hoechst—Roussel Pharmaceuticals, Somerville, NJ), m-hydroxy benzylhydrazine hydrochloride (NSD 1015; Sigma), pargyline hydrochloride (Sigma), rat prolactin (NIDDK Rat PRL-B-6, 25 26 kindly supplied by Dr. S. Raiti and Dr. A. Parlow, National Hormone and Pituitary Program) and urpropranolol hydrochloride (Sigma) were dissolved in 0.9% saline. Haloperidol (Sigma) was dissolved in 0.3% tartaric acid and metaproterenol hemisulfate (Sigma) in 0.9% saline with 6 mg% ascorbic acid. The anesthetic Equithesin was prepared by stirring 42.51 g chloral hydrate, 9.72 g pentobarbital sodium (Sigma) and 21.26 9 magnesium sulphate in 443 ml of warm propylene glycol. After these compounds were completely dissolved, 120 ml of 95% ethanol was added and the total volume was brought to 1000 ml with water. All drugs were administered as indicated in the legends of the appropriate figures or tables, with doses calculated as the respective salts. C. Surgical Manipulations 1. Intravenous Catheter In experiments requiring intravenous drug injections, animals were implanted with a polyethylene catheter (PE-50; Becton Dickson, NJ) inserted into the right atrium via the right jugular vein under ether anesthesia 2 days prior to injections. 2. Lntraventricular Cannula Intracerebroventricular (icv) injections were administered to freely-moving rats via cannula guides which 27 were implanted 5 days prior to the experiment. Rats were anesthetized with Equithesin (3 mg/kg, i.p.) and positioned in a stereotaxic apparatus with the incisor bar set 2.4 mm below the horizontal plane. A 23-gauge stainless-steel guide cannula was implanted 1.4 mm lateral to bregma and 3.2 mm below dura (Paxinos and Watson, 1982) and anchored to the skull with stainless-steel screws and dental cement. A stainless-steel stylet was used to occlude the guide cannula prior to and following icv injections. At the time of the experiment, drug or'vehicle‘was injected.to freely-moving rats over a period of 1 min in a volume of 5 pl delivered from a lo-Ul Hamilton microsyringe connected to a 30-gauge stainless- steel injector which protruded 1 mm beyond the tip of the guide cannula and into the lateral ventricle. 3. Retrochiasmatic Deafferentation Retrochiasmatic deafferentation of the mediobasal hypothalamus was performed 7 days prior to experimentation using a modified Halasz knife (height, 2.0 mm; radius 1.5 mm) with the aid of a stereotaxic instrument (David Kopf Instruments, Tujunga, CA). The animals were anesthetized with Equithesin (3 ml/kg) and placed in a stereotaxic frame with the incisor bar 8 mm below the intraaural line. The knife was lowered at the midline, 1.5 mm posterior to bregma. With the vertical blade in the rostral position, it was lowered to the base of the brain at the caudal border of the optic chiasm. The knife was then rotated 90° to the left and right 2 times 28 to produce a semicircular cut. Sham operations consisted of lowering the knife 5 mm through the midline without rotation. The deafferentations were verified postmortem in frozen brain sections; animals in which bilateral knife cuts were not visible lateral to the arcuate nucleus were not used (10 % of the animals). D. Electrical Stimulation For stimulation of either the pituitary stalk or arcuate nucleus, rats were anesthetized with GBL (1000 mg/kg; i.p.) and placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA). A coaxial bipolar stainless-steel electrode (NE- 100 for pituitary stalk, SNE-loo for arcuate nucleus; Rhodes Medical Instruments, Inc., Woodland. Hill, CA), with. the cathodal center and anodal shaft contacts exposed 0.25 mm, were placed in the appropriate region, using the atlas of Paxinos and Watson (1982). The position of the stimulation site was confirmed at the time of sacrifice by examining frozen frontal sections of each brain for the location of the electrode track. The pituitary stalk was stimulated with cathodal monophasic pulses of 1 msec duration, 300 DA intensity, at a frequency of 10 Hz. The arcuate nucleus was stimulated with twin biphasic pulses of 1 msec duration, 200 DA peak-to-peak intensity, at a frequency of 10 Hz, unless otherwise noted. Current was generated by Grass stimulators (Model S9 for pituitary stalk, Model S9 and SD-9 for arcuate 29 nucleus, Grass Instruments, Quincy; MA) and. continuously monitored through an oscilloscope. In sham-stimulated animals, the electrode was positioned in either the arcuate nucleus or pituitary stalk for the appropriate time without application of the current. E. Restraint Stress Stressed animals were briefly anesthetized with diethyl ether (approximately 2 mins exposure) and restrained in the supine position with adhesive tape for 30 min. Non-stressed control animals were removed from their cages and killed immediately by decapitation. F. Tissue Preparation Following appropriate treatments, animals were decapitated and trunk blood was collected in tubes on ice containing 100 pl 15% EDTA and 240 pg bacitracin. Brains were removed from the skull and a frontal cut was made with a razor blade just posterior to the mammillary bodies and the portion of the brain anterior to the cut frozen on aluminum foil over dry ice. Pituitaries were placed on glass slides and frozen immediately over dry ice. The frozen brain and pituitaries were stored over dry ice for no more than 72 hours before dissection. The brains were sliced in a cryostat into 600 pm frontal sections.beginning’at.approximately'A.9.2 (Paxinos and Watson, 1982). The striatum, nucleus accumbens and median 30 eminence were dissected with stainless steel cannulae from frozen sections using a modification (Lookingland and Moore, 1984) of the micropunch technique of Palkovits (1973). With the aid of a dissecting microscope, the neural and intermediate lobes of the pituitary were dissected from the whole frozen pituitary gland using a stainless steel cannula according to the technique of Lookingland and coworkers (1985a). The dissected tissue was placed in 60 pl of 0.1 M phosphate-citrate buffer (pH 2.5) containing 15 % methanol and stored at -20° C until assay. Trunk blood samples were centrifuged (model DPR-6000, International Equipment Co, Needham, MA) on the day of the experiment at 2000 rpm for 20 mins. Plasma.was removed, placed in a glass vial containing 250 p1 saturated sodium citrate, and stored at - 20° C until assayed. G. Neurochemical Analyses On the day of the assay, tissue samples were thawed, sonicated for 3 sec (Sonicator Cell Disrupter, Heat Systems- Ultrasonics, Plainview, NY) and centrifuged for 1 min in a Beckman Microfuge. Tissue pellets were dissolved in 1 N NaOH and assayed for protein (Lowry gt gt., 1951). DOPAC, DOPA and DA concentrations in the tissue extract supernants were determined by high-performance liquid chromatography (HPLC) with electrochemical detection. Fifty pl of the tissue extract supernatant was injected onto a Cw 31 reverse phase analytical column (5 pm spheres; 250 x 4.6 mm: Biophase ODS, Bioanalytical Systems, Inc., West Lafayete, IN) which was protected by a precolumn cartridge filter (5 pm spheres; 30 x 4.6 mm). The HPLC mobile phase consisted of 0.05 M sodium phosphate, 0.03 M citric acid buffer adjusted to pH 2.7, with 0.1 mM disodium ethylenediamine-tetraacetic acid, 0.035% sodium octyl sulfate and 25% methanol. Depending on the condition of the column, the components of the mobile phase were slightly adjusted to maintain separations of the compounds of interest and still minimize the total retention time (Chapin gt gt., 1986). Striatum, nucleus accumbens and median eminence catecholamines were detected using an electrochemical detector (LC4A, Bioanalytical Systems, Inc.) equipped with a TL-5 glassy carbon electrode set.at.a potential of +0.75 V relative to a Ag/AgCl reference electrode. Because the neural and intermediate lobes of the pituitary contain lesser amounts of DOPA, DOPAC and DA, a more sensitive electrochemical detection system was used. This system consisted of a single coulometric electrode conditioning cell in series with dual electrode analytical cells (models #5021 and 5011, respectively, ESA, Bedford, MA). The current signal from the second electrode of the analytical cell was monitored by a Hewlett-Packard 3390A Integrator (Hewlett—Pachard, Avondale, PA). Use of coulometric electrodes increases the sensitivity of the measurement of the compounds of interest, and. with the 32 analytical electrodes set up in an oxidation-reduction mode, only compounds oxidized at the first coulometric electrode and then reduced at the second electrode are detected, resulting in increased selectivity and reduced signal noise. In order to maximize the analyses of the catecholamines using this system, a voltammogram (current-to-voltage curve) was generated. In figure 6, the potential of the second analytical electrode was varied while the potentials of the first analytical and conditioning electrodes were held constant (+0.15 and +0.4 V, relative to internal reference electrodes, respectively). From this voltammogram, the lowest potential that produced a maximum response (-0.31 V) was selected for use in subsequent studies. The amounts of DOPA, DOPAC and DA in each sample were determined by comparing peak heights measured by the integrator with those of the standards run the same day. The lower limit of sensitivity of this assay for DOPA, DOPAC and DA was approximately 8 pg per sample. Sample chromatograms of a mixture of 500 pg standards and intermediate lobe samples from both control and NSD 1015-treated animals are shown in figure 7. H. Radioimmunoassay of Prolactin Prolactin concentrations in the plasma were measured by a double antibody radioimmunoassay using the procedures and reagents supplied through the NIDDK Rat Pituitary Hormone 33 0-——¢>IM\ 30 ._. DOPA A—A DOPAC ta+:::::::::::::::::°\\\\\ “*~—e e\\\\xouchn um u <4Hmlm «ocflfimmoc u «o “pace Ufluoooaacmcm>xoupwcflo n ommoo Amcficoamahcmnmaxouoxcflonv . n u soon Amcflunmocflmwuo: n mz uHoo>oaxcm£9>xouo>£wpue.m u ommoo .mamfiflcm ACHE om x.d.fl “oxxma code emummuunmaoa amz no Houucoo scum whoa oumfloosuoucw no menace—mum mm com mo ousuxfie o no :ofluooncun on» ocwsoHHou oocflmuno mfimumoumsouco 04mm mamfimm .s Unseen m. 35 m. O. m . o more}. m. - w 33 35895.32. ..LHS O V 35925. ocean a o 3.2.2. m. o. m . u . m -| . . ... o S o H A a 1 w m m. . N o 3 o Q d . v v M O .o . .3 5 (5 one up 39.32. 35925 e rum 00b Owkdiwm... ..m_o_ DmZ is DQaEZOQ 36 Distribution program by Drs. S. Raiti and A.F. Parlow. These reagents included an antiserum to rat prolactin produced in rabbits, a rat prolactin reference preparation (rPRL-I-S) to serve as a standard and a rat antigen which was labeled with 125Iodine (Amersham, Arlington Heights, IL) using the lactoperoxidase technique (Chard, 1982). Rat 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 the addition of second antibody, the mixture was centrifuged and the precipitate counted in a Micromedic gamma counter (Model 4/600). The intra- and inter-assay coefficients of variation were approximately 10% and 17% respectively. I. Radioimmunoassay of a-MSH In order to investigate THDA neuronal regulation of intermediate lobe secretory activity, it was necessary to obtain an index of melanotroph POMC-peptide secretion. Various forms of fi-endorphin are secreted from both the intermediate and anterior lobes of the pituitary and most B-endorphin radioimmunoassays do not differentiate between anterior lobe and intermediate lobe derived B-endorphin. The plasma concentrations of a-MSH, on the other hand, is a specific marker of intermediate lobe secretory activity, since plasma a-MSH is derived exclusively from the intermediate lobe (Akil 37 gt g;., 1984; Eberle, 1988). Therefore, a radioimmunoassay for a-MSH was established to monitor intermediate lobe secretion. 1. Assay Materials Antiserum containing immunoglobulin G (IgG) produced in rabbits against a-MSH conjugated to bovine thyroglobulin was kindly supplied by Dr. Greg Mueller (Uniformed Services University for the Health Sciences, Bethesda, MD). The antiserum was tested for cross-reactivity in Dr. Mueller's lab and found to cross-react on a equal molar basis with desacetyl-a-MSH and diacetyl-a-MSH; it did not detect up to 30 ng/tube of any of the following: deaminated a-MSH, fi-MSH, ACTH 1-10, ACTH 1-13, or ACTH 1-24, fi-endorphin peptides, or E-lipotropin. Synthetic a-MSH, for use as both assay standard and tracer was purchased from Peninsula Laboratories (Belmount, CA). The a-MSH for use as tracer was iodinated (125 I) using a modification of the chloramine-T method of Greenwood and Hunter (1963). Iodinated.a-MSH (in.0.05% triflouroacetic.acid) was separated from free iodine using a C18 Sep-Pak cartridge (Waters Associates, Milford, MA) in the presence of increasing concentrations of acetonitrile. This same procedure was used to repurify the tracer after 1 week. Tracer was stored at -20° C and was used for no more than two weeks. 2. Precipitation Method The radioimmunoassay procedure was a modification of that used by Pettibone and.Mueller (1984). In their procedure, 38 tracer, antiserum and sample were all added on the first day and allowed to incubate at 4° C for 4 days. This procedure was initially adapted in our lab. In establishing the assay in our lab, the selection of a method to separate free a-MSH from IgG-bound a—MSH presented some difficulty. Three separation methods were compared; 1) charcoal extraction, the method used by Pettibone and Mueller (1984), absorbs free but not IgG-bound a-MSH, thereby precipitating the free a-MSH, 2) Staphylococcus aureus cells containing protein A (tradename IgGsorb, The Enzyme Center, Inc., MA), which binds to the constant region of all IgG molecules, precipitating the IgG- bound a-MSH and, 3) goat anti-rabbit IgG antisera (Arnel Products), which binds to rabbit IgG. When tested in the a-MSH assay, the three methods produced different results. As part of the tests of these procedures, the presence of non-specific interference with IgG binding to a-MSH was examined. This was done by determining if increasing amounts of serum produced a parallel displacement of antibody binding as compared with the standard curve; non-parallelism indicates that some factor is altering the IgG-a-MSH binding interaction, presumably by non-specific binding interference (Chard, 1982). When charcoal extraction was tested, it resulted in a high intra-assay coefficient of variation (28%) and, as shown in figure 8A, dilutions of serum were not parallel to the standard curve, indicating non- specific binding interference. IgGsorb (protein A), on the 39 100 T. 00 I” 200 [11 of Serum % Tracer Bound o l n a A a n n n l V A 1 V ' I I 10 20 00 40 00 00 70 00 00100 800 pg of cx—MSH Figure 8A. Standard curve and serum dilutions generated in a- MSH radioimmunoassay using charcoal extraction. Standards or serum, tracer, and.anti-aMSH.antiserumwwere incubated for five days at 4° C. Dextran-coated charcoal (200 pl of suspension) was added to each tube which were then centrifuge, decanted and the precipitated absorbed free hormone was counted in a gamma-counter. 40 other hand, resulted in a lower intra-assay coefficient of variation (12%) than charcoal extraction and serum dilutions were parallel to the standard curve (figure 8B), but serum a- MSH concentrations obtained with IgGsorb differed from literature values by a factor of 10 (table 1). Evidently, some factor in the serum was interfering with the ability of protein A to precipitate the IgG bound a-MSH thereby producing artificially high values. Goat anti-rabbit IgG antisera resulted in a small intra-assay coefficient of variation (9%) , no non-specific binding interference (figure 8C), and serum a-MSH concentrations obtained with this method agreed with literature 'values (table 1). 'Therefore, this ‘method. was selected for use in future assays. Use: of goat anti-rabbit IgG' antisera requires the addition of normal rabbit serum to the assay in order to increase the size of the precipitation complex. Dilution curves of both.goat anti-rabbit Ingantisera and normal rabbit serum were performed to determine the lowest dilutions that produced a complete precipitation of bound complex (determined in the presence of excess anti-aMSH antiserum). Twenty-four hours after the addition of goat anti-rabbit IgG, the mixture was centrifuged and the precipitate counted in a Micromedic gamma counter (Model 4/600). The lowest dilutions that produced complete precipitation were a 1:200 dilution of goat anti-rabbit IgG antiserum and a 1:400 dilution of normal 41 I 100 ‘1 80C % Tracer Bound o l l l 4 I A A n n v v v r I v v v I 50 00 70 00 00 100 000 800 400 000 pg of a—MSH Figure 8B. Standard curve and serum dilutions generated in a- MSH radioimmunoassay using IgGsorb. Standards or serum, tracer, and anti-aMSH antiserum were incubated for five days at 4° C. IgGsorb solution (200 p1 of 1:5 dilution) was added to each tube which were incubated at 4° C for 30 min. After addition of 2.5 ml phosphate buffered saline, the tubes were centrifuged, decanted, and the precipitated bound hormone was counted in a gamma-counter. 42 TABLE 1 SERUM a-MSH CONCENTRATIONS MEASURED WITH DIFFERENT RIA METHODS METHOD a-MSH (pg/ml) Literature Range 140-360 Charcoal Extraction 250 IgGsorb 1200 Goat anti-rabbit IgG 230 Values obtained from single determination of the same serum pool 43 50 100 200 pl of Serum % Tracer Bound o : : : : : : ' : ‘ ' I 10 80 80 40 so 00 10 so 00 100 pg of a—MSH Figure 8C. Standard curve and serum dilutions generated in a- MSH radioimmunoassay using goat anti-rabbit gamma globulin. Standards or serum, tracer, and anti-aMSH antiserum were incubated for four days prior to addition of the goat anti- rabbit IgG antiserum (1:100 dilution) at 4°C. The tubes were incubated for an additional 24 hrs. After addition of 2.5 ml phosphate buffered saline, the tubes were centrifuged, decanted, and the precipitated bound hormone was then counted in a gamma-counter. The serum dilutions represent 50, 100 and 200 pl. 44 rabbit serum, and.theseiconcentrations‘were‘used.in.subsequent studies. 3. Assay sensitivity After selection of the precipitation method, a number of manipulations were carried out in an attempt to increase the sensitivity of the assay. Initially, an antibody dilution curve (incubation of fixed amount of tracer with different concentrations of antiserum) was performed in a 4 day assay with no preincubation and 10,000 cpm of tracer. From this curve, dilutions were selected to be tested in a standard curve to determine the effect on assay sensitivity. As shown in table 2, the 1:24,000 dilution produced the best sensitivity but very low binding (11 %), whereas the 1:6000 dilution produced 30 % binding without a large loss of assay sensitivity. Further investigation using a 1:6000 dilution of antiserum demonstrated that greater sensitivity was achieved with 5000 cpm vs 10,000 cpm of tracer and a 5 day assay in which tracer was added on day 2 vs a 4 day assay (table 2). Therefore, a 5 day assay with 1:6000 dilution antiserum and 5000 cpm of tracer was used in subsequent studies. 4. W In initial investigations (sections I and II), a-MSH was measured in serum, but upon the suggestion of Dr» Mueller, plasma that was treated with 240 pg of bacitracin (a protease inhibitor) was used. Addition of bacitracin resulted in an decrease in plasma a-MSH concentrations (from 267 i 24 pg/ml 45 TABLE 2 EFFECT OF VARIOUS ASSAY ALTERATIONS ON THE AMOUNT OF TRACER BOUND (% BINDING) AND THE ASSAY SENSITIVITY Assay Sensitivity Alteration % Binding (pg of a-MSH) Dilution of anti-aMSH antiserum 1:6000 31 13 1:12,000 21 11 1:24,000 11 3 Dilution of tracer 5000 cpm 30 14 10,000 cpm 27 30 4 day assay no preincubation 24 11 5 day assay preincubation 19 6 Values are derived from standard curves produced with the different manipulations. Assay sensitivity is the amount of a-MSH that displaces.20 % of the total amount of bound tracer. 46 in non-treated plasma to 178 i 27 pg/ml in bacitracin-treated plasma), while addition of large quantities of bacitracin did not alter antiserum binding in control binding tubes. This lowering of a-MSH concentrations by bacitracin may be due to inhibition of metabolism of ACTH into a-MSH in the blood. Plasma samples from.animals in‘which.the pituitary gland was removed exhibited no detectable a-MSH concentrations when tested in the assay, indicating that plasma does not contain any non-specific binding characteristics. Figure 8D is a recent standard curve plotted against increasing concentrations of bacitracin-treated plasma, indicating the absence of non-specific binding interference. Using the assay procedure described, the intra-assay variability was 9% while the inter-assay variability was 13%. Using an aliquot of 250 pl of plasma and a sensitivity limit of 80% binding of total binding, the assay can detect a little as 30 pg/ml. Routinely, dilutions of 100 and 200 p1 of plasma were used. J. Statistical Analyses Statistical analyses of single comparisons were conducted using Student's t-test. Statistical analyses between 3 or more groups were conducted using analysis of variance followed by Student-Newman-Keuls Test (Steel and Torrie, 1979). Differences were considered significant if the probability of error was less than 5% 47 60 too 200 p1 of Plasma 100 1- l l j 75 Tracer Bound .0 5 = = = n n n n I l v v v v I v 10 80 30 40 00 00 70 00 00 100 800 pg of a—MSH Figure 8D. Standard curve and plasma dilutions generated in a-MSH radioimmunoassay. Standards or plasma and anti-aMSH antiserum (1:6000 dilution) were incubated for 24 hrs at 4°C before addition of tracer (5000 cpm). After three more days, goat anti-rabbit IgG antiserum (1:200 dilution) was added. The tubes were incubated for an additional 24 hrs. After addition of 2.5 ml phosphate buffered saline, the tubes were centrifuged, decanted, and the precipitated bound hormone was then counted in a gamma-counter. RESULTS Section I. NEUROCHEMICAL INDICES OF THDA NEURONAL ACTIVITY The i vivo activity of major ascending dopaminergic neurons, such as those that comprise the nigrostriatal dopaminergic system, have been extensively examined with electrophysiological techniques that record edectrical activity from. their ‘tightly' packed ;perikarya located in discrete nuclei of the ventral midbrain. Dopaminergic neuronal activity can also be monitored with neurochemical techniques. The term neuronal activity as used in this thesis refers the secretory activity of dopamine from the nerve terminal. This is normally coupled to the rate of action potentials arriving at the nerve terminals (impulse flow) but may become uncoupled under certain circumstances (i.e. presynaptic regulation). Three of the more commonly used neurochemical indices include measurements in the striatum of: 1) DA turnover, or the rate of decline of DA concentrations after blockade of DA synthesis by administration of a-methyl tyrosine, an inhibitor of tyrosine hydroxylase, 2) DA synthesis, or the rate of accumulation of the immediate precursor of DA, DOPA, after administration of the decarboxylase inhibitor, NSD 1015, and 3) the concentrations of DA metabolites. All three of these techniques have been shown to be valid estimates of 48 49 nigrostriatal dopaminergic neuronal activity (Roth gt ggp, 1973; Murrin and Roth, 1976; Walters gt gt., 1973; Roth gt g;., 1976; Westerink gt g;., 1979), as well as TIDA neuronal activity (Gunnet _t a_l_., 1987; Lookingland gt at” 1987a; Lookingland gt gl., 1987b) and have proven to be useful for monitoring the activity of these neurons. The perikarya of THDA neurons are too diffusely distributed among non-dopaminergic neurons and the anatomically related TIDA neurons to allow easy electrophysiological recordings of their impulse flow. Therefore, investigators have relied (n1 neurochemical techniques. THDA neuronal activity is reflected by changes in both turnover and synthesis of DA in the neural and intermediate lobes of the pituitary; activation of THDA neurons by electrical stimulation of the arcuate nucleus increased both DA turnover and the rate of DOPA accumulation in the neural and intermediate lobes (Gunnet gt gt., 1987). However, there are disadvantages in using these two techniques. First, both require a minimum amount of time (30- 60 min) and cannot be used to detect rapid changes in neuronal activity. A second problem, specific to THDA neurons, is that both a-methyltyrosine (Penny and Thody, 1978) and NSD 1015, increase the secretion of a-MSH (table 3). This is probably due to a decrease in the amount of DA released from the nerve terminal after blockade of DA synthesis. These results 50 TABLE 3 EFFECT OF NSD 1015 ADMINISTRATION ON SERUM a-MSH CONCENTRATIONS (pg/ml serum) EXPERIMENT CONTROL 8 0 5 1 220 550 2 160 450 3 220 420 Values were obtained from determinations of pooled serum (8-10 rats/pool) from 3 different experiments. 51 indicate that measurements of plasma a-MSH levels cannot be made in the same animals that have received a-methyltyrosine (to estimate DA turnover) or' NSD 1015 (to estimate DA synthesis). In addition, it was possible that the increase in intermediate lobe peptide secretion could alter THDA neuronal activity. Therefore, experiments were designed to determine if DA metabolite concentrations in the intermediate lobe reflected THDA neuronal activity. In these experiments, the i vivo characteristics of the metabolism of DA to DOPAC were investigated.in the intermediate and neural lobes. The effects of electrophysiological and pharmacological procedures that are known to alter THDA neuronal activity were examined on DOPAC concentrations in ‘these regions and compared. with changes in the rate of DOPA accumulation after administration of NSD 1015 (DA synthesis). RESULTS In agreement with previous reports (Holzbauer gt g;., 1984; Lookingland gt gt., 1985a), the concentration of DA in the intermediate lobe was almost twice that in the neural lobe (table 4). This is consistent with the higher density of tyrosine hydroxylase containing nerve fibers in the intermediate vs neural lobe (Luppi gt g;., 1986). When the rate of DA synthesis was estimated by determining the accumulation of DOPA after administration of the decarboxylase 52 TABLE 4 CONCENTRATIONS OF DA, DOPAC AND DOPA IN THE INTERMEDIATE AND NEURAL LOBES OF THE PITUITARY Intermediate Neural Lobe Lobe DOPA 1.32 i 0.09 0.78 i 0.07 DA 16.1 i 1.1 9.8 i 0.8 DOPA/DA 0.086 t 0.004 0.080 t 0.004 DOPAC 1.13 i 0.04 0.64 i 0.07 DA 18.9 i 0.8 9.2 i 1.1 DOPAC/DA 0.060 t 0.003 0.070 t 0.003 Animals in which DOPA accumulation was measured received NSD 1015 (100 mg/kg: i.p.) 30 min prior to decapitation. Values (pg/pg protein) represent mean i 1 S.E. of 7-8 animals. 53 inhibitor NSD 1015, DOPA.accumulation in the intermediate lobe was almost twice that in the neural lobe. When expressed as a ratio of DOPA/DA (or the rate of DA synthesized over the amount of DA stored), the ratios were approximately the same in the two regions. These results are consistent.with.previous reports of similar rates of DA turnover in the intermediate and neural lobes (Lookingland gt g;., 1985a; Lookingland and Moore, 1985b). DOPAC was present in both the intermediate and neural lobes, but the the dopamine metabolite HVA was not detected in either lobe (sensitivity limit about 0.6 pg HVA/pg protein). DOPAC concentrations in the intermediate lobe were almost twice those in the neural lobe, but when expressed as a DOPAC/DA ratio (or the amount of DA metabolized over the amount of DA stored), the values were approximately the same in the two regions. Following the intravenous administration of the MAO inhibitor pargyline, there was a rapid decline (within 5 min) in DOPAC concentrations in both the intermediate and neural lobes (figure 9). The elimination of DOPAC during the first 10 min had a half life of 4.8 min in the intermediate lobe and 8.6 min in the neural lobe (determined from plot of log DOPAC concentration vs. time). The contribution of reuptake and metabolism of released DA to DOPAC concentration was examined by administering the DA uptake blocker nomifensine. This drug produced a modest 54 E .2 2 ,Q U E ‘\ D C 2 O. O C) .L I l J. O O 5 IO l5 MINUTES AFTER PARGYLINE Figure 9. Time course of the effect of pargyline on concentrations of DOPAC in the intermediate lobe (IL) and neural lobe (N10 of the pituitary'gland. Vehicle (0.9% saline: 2 ml/kg) was administered i.v. 15 min prior to decapitation (0 time) or pargyline (50 mg/kg) was administered 5, 10 or 15 mins prior to decapitation. Each symbol represents the mean DOPAC concentration and the vertical line 1 SE of 4-10 animals. Solid symbols represent those values that are significantly different from those of vehicle-treated animals (p < 0.05). 55 decline in DOPAC concentrations in the intermediate lobe (33% decline after 30 min) but was without effect in the neural lobe (figure 10), indicating that the uptake and metabolism.of released DA into'THDA nerve terminals makes only a small contribution to DOPAC concentrations in the intermediate lobe and no contribution to neural lobe DOPAC concentrations. The effect of alterations in THDA neuronal activity on DOPAC concentrations in the posterior pituitary was compared with changes in the rate of DA synthesis. As depicted in figure 11, stimulation of the pituitary stalk increased DOPA concentrations by 208% in the intermediate lobe and 155% in the neural lobe. DOPACiconcentrationS‘were also increased” but to a lesser magnitude: 173% in the intermediate lobe and 142% in the neural lobe. Administration of dopaminergic agonists decrease and dopaminergic antagonists increase the rates of DA turnover within the intermediate lobe but not the neural lobe (Lookingland gt gt., 1985a). In the intermediate lobe, the dopaminergic antagonist haloperidol increased both DOPA (177%) and DOPAC (174%) concentrations (figure 12), while the dopaminergic agonist apomorphine decreased both DOPA (62%) and DOPAC (81%) concentrations (figure 13). These drugs failed to alter DOPA or DOPAC concentrations in the neural lobe. 56 ELCDF lL NL E .2 l5” TL- ' O . . § ’1‘ >i< -\ '0' .. E l"— C) 8‘ . __._ - Q0.5 , - C). . l ' i [_‘_] J 0 I5 30 0 IS 30 MINUTES AFTER NOMIFENSINE Figure 10: The effect of nomifensine on DOPAC concentrations in the intermediate lobe (IL) and neural lobe (NL) of the pituitary. Animals were injected.with.vehicle (acidified 0.9% saline; pH 0.5; 2 ml/kg, i.p.) or nomifensine (25 mg/kg i.p.) 30 min prior to decapitation. Each column represents the mean DOPAC concentration and the vertical line 1 SE of 8-10 animals. *, Values that are significantly different from those of vehicle-treated animals (p < 0.05). 57 25' . 25' * 2.0 - I/ 2-0’ / A E Z .5 o .. '6 4 ° 5 l I: _ ,/ 3. L5 " G- g ? Ci '5 7’ * E \ Z E, S' i. , l c r- ' ,/// . .. / v I .O p z I O r... ”27/ //// 2 a. Z / d. o x g Q 127! f ‘ O 5 . f 0'5" // /x » % // / , ///// 7/ xix/ 5% 2g 2 O ”742’; [/37 O h [L NL Figure 11: Comparison of the effects of electrical stimulation of the pituitary stalk.on DOPA and DOPAC concentrations in the intermediate lobe (IL) and neural lobe (NL) of the pituitary. Animals were anesthetized with GBL (1000 mg/kg; i.p.), placed in a stereotaxic frame 20 min later and the pituitary stalk was stimulated electrically for 30 min (1 msec square wave pulse; 300 pA intensity: 10 Hz). Animals in which DOPA accumulation was measured received NSD 1015 (100 mg/kg; i.p.) 30 min prior to decapitation. Control animals received sham stimulation for 30 min. Each column represents the mean DOPA or DOPAC concentration and the vertical line 1 SE of 8-10 animals. *, Values that.are significantly'different from those of sham-stimulated animals (p < 0.05). 58 1 25 : VEHICLE HALOPERIDOL 213 DOPA (ng/mg protein) DOPAC (no/mg protein) 3 Figure 12: Comparison of the effects of haloperidol administration on DOPA and DOPAC concentrations in the intermediate lobe (IL) and the neural lobe (NL) of the pituitary. Animals were injected with either vehicle (0.3% tartaric acid: 1 ml/kg; s.c.) or haloperidol (0.1.mg/kg; s.c.) 2 hrs prior to decapitation. Animals in which DOPA accumulation was measured received NSD 1015 (100 mg/kg; i.p.) 30 min prior to decapitation. Each column represents the mean DOPA or DOPAC concentration and the vertical line 1 SE of 7- 8 animals. *, Values that are significantly different from those of vehicle-treated animals (p < 0.05). 59 7;: g | VEHICLE ° 0 ‘- - ' ' " .5 - 7.2127 E "5 TL g ' ///§ APOMOR. E E 2' B. :5 LO" l 5 L0- 5 % ' 2 '< ' %Z/ n. a. f 8 05~ g / 8 0.5- % ._ 0. xx Figure 13: Comparison of the effects of apomorphine administration on DOPA and DOPAC concentrations in the intermediate lobe (IL) and neural lobe (NL) of the pituitary. Animals were injected with either vehicle (0.9% saline; 1 ml/kg: s.c.) or apomorphine (2 mg/kg: s.c.) 45 mins prior to decapitation. Animals in which DOPA accumulation was measured received NSD 1015 (100 mg/kg; i.p.) 30 min prior to decapitation. Each column represents the mean DOPA or DOPAC concentration and the vertical line 1 SE of 7-8 animals. * Values that are significantly different from those of I vehicle-treated animals (p < 0.05). 60 DISCUSSION The results of the experiments in this section indicate that although the density of THDA neurons is greater in the intermediate lobe than in the neural lobe, the rates of DA synthesis, when expressed as a ratio of the DA content, are similar in the two lobes. This is in agreement with previous reports demonstrating that the rates of DA turnover in the intermediate and neural lobes of the pituitary are comparable (Lookingland e_t a_1. , 1985a; Lookingland gt a_1. , 1985b) . These neurochemical results suggest that the basal rates of.activity of THDA neurons terminating in the two lobes are similar. The concentration. of HVA. was. below the limits of detection by our assay (0.6 pg/pg protein) in the intermediate lobe and neural lobe of the pituitary. Racké and Muscholl (1986) reported that the concentration of DOPAC in the combined neurointermediate lobe was twice that of HVA. On the other hand, these same investigators reported that almost twice as much HVA as DOPAC was released from isolated neurointermediate lobe it ytttg. This would suggest that HVA is eliminated more rapidly than DOPAC from these tissues. THDA neurons are similar in this respect to TIDA neurons terminating in the median eminence; HVA is also non-detectable in this region (Lookingland gt gt., 1987b). Thus, these two hypothalamic neuronal systems are different from that in terminals of nigrostriatal dopaminergic neurons in_ the striatum where HVA is a major metabolite (Westerink gt g;., 61 1979), suggesting HVA is either transported more slowly from nigrostriatal dopaminergic neurons or is formed at a more rapid rate. After blockade of its formation, DOPAC concentrations declined rapidly in both lobes. DOPAC may be removed by either active transport into the blood or by rapid conversion to HVA (Westerink, 1985), which can then be transported into the blood. The rapid elimination of DOPAC from these tissues indicates DOPAC concentrations reflect newly metabolized DA. Blockade of the uptake of DA produced only a 33% decline in DOPAC concentrations in the intermediate lobe and was without effect in the neural lobe. This suggests that DOPAC in THDA nerve terminals is derived mainly from the intraneuronal metabolism of unreleased DA rather than DA that has been released and recaptured by these neurons. Because MAO activity is not exclusively’ present in. dopaminergic neurons (Agid gt gt., 1973; Demarest gt gt., 1980), DOPAC may also be derived from DA that has diffused into non- dopaminergic neurons or glial cells, but this process would be expected.to be slow and therefore have a small contribution to total DOPAC concentrations. The minor contribution of released DA to the total pool of DA metabolized to DOPAC in the neural and intermediate lobes may be due to a lack of high affinity uptake sites for DA (Demarest and Moore, 1979b; Annunziato and Weiner, 1980). The decline in DOPAC in the intermediate but not the neural 62 lobe after blockade of DA uptake may be due to a difference in DA uptake sites between the two lobes; the intermediate but not the neural lobe may contain a high affinity uptake site for DA. Because previous studies only examined uptake sites in the whole neurointermediate lobe (Demarest and Moore, 1979b; Annunziato and Weiner, 1980), the presence of uptake sites confined to the intermediate lobe may have been masked. The present results are in agreement with the 19 yittg results of Racké and coworkers (1986, 1987) who suggest that DOPAC produced in the neurointermediate lobe is derived mainly from the intraneuronal metabolism of newly synthesized DA. Dopaminergic agonists decrease and antagonists increase the rate of turnover of DA in the intermediate lobe, but not in the neural lobe, suggesting that the activity of only those THDA.neurons projecting to the intermediate lobe are regulated by dopaminergic receptor-mediated mechanisms (Lookingland gt g;., 1985a). The results of the present study are consistent with these earlier reports in that the dopaminergic agonist apomorphine decreased and the dopaminergic antagonist haloperidol increased the DOPAC concentrations and rates of DOPA accumulation in the intermediate lobe, but had no effect on these measurements in the neural lobe. In contrast, it yitrt studies have indicated that dopaminergic neurons in both the intermediate and. neural lobes of the pituitary are inhibited by dopaminergic receptor regulation (Racké gt g;., 1988). The reason for this discrepancy is not known, although 63 the igfivitro studies were done on electrically stimulated THDA neurons in the presence of a MAO inhibitor, a DA uptake blocker, and the opioid antagonist naloxone. Therefore, interactions may be occurring in this in vitro system that do not normally occur i vivo. On the other hand, Racke and coworkers suggest that endogenous opioids may inhibit neural lobe THDA neurons tn 11.92, thereby masking DA receptor regulation in it _vi_trt studies. Further investigation is needed to clarify these results. Activation of THDA neurons by electrical stimulation of the pituitary stalk increased the rate of DA synthesis and the concentrations of DOPAC in the intermediate and neural lobes. Pharmacological changes in THDA neuronal activity also resulted in corresponding changes in both DOPA accumulation and DOPAC concentrations in the intermediate lobe. Taken together, these results indicate that DOPAC concentrations in the intermediate and.neural lobes reflect the activity of THDA neurons terminating in these two regions. Therefore, THDA neurons are similar to both nigrostriatal dopaminergic and TIDA. neurons in 'which DOPAC iconcentrations also reflect changes neuronal activity (Roth gt gt., 1976; Westerink gt _a_., 1979; Lookingland gt 1., 1987a; Lookingland g_t _a_t., 1987b) The overall magnitude of the changes in DOPA were larger than the corresponding changes in DOPAC indicating that DA synthesis is a more sensitive index of THDA neuronal activity 64 than DOPAC concentrations. This is probably because DOPAC concentrations reflect a fraction of newly synthesized DA that is not packaged in synaptic vesicles for release, while DOPA accumulation reflects all of the newly synthesized DA. Despite these limitations, estimations of THDA neuronal activity in the intermediate and neural lobe by measuring DOPAC concentrations is more advantageous for determining rapid changes and for concurrent measurement of plasma a-MSH concentrations. In conclusion, DOPAC concentrations in the intermediate and neural lobes of the pituitary reflect the activity of THDA neurons projecting to this region. Concentrations of this metabolite can be a useful index of THDA neuronal activity when it is desirable to measure rapid changes in neuronal activity and/or when concurrent measurements of blood concentrations of POMC-derived peptide hormones are to be made. On the other hand, manipulations may alter the rate of DOPAC formation (i.e. alter MAO activity, DOPAC removal, etc.) without altering dopaminergic neuronal activity (Westerink, 1985), so in subsequent key experiments more than one neurochemical technique was employed. Section II. THDA NEURONAL REGULATION OF A-MSH SECRETION FROM THE INTERMEDIATE LOBE OF THE PITUITARY The secretion of POMC-derived peptides, including a-MSH, is regulated, at least in part, by tonic dopaminergic inhibitory tone (Bower gt 1., 1974; Penny and Thody, 1978; Tilder gt g;., 1985; Newman gt 1., 1987). Much of what is known about the inhibitory effects of DA on intermediate lobe secretion is based on studies performed in vitro, but little information is available regarding the direct role of THDA neurons in regulating the secretion of a-MSH secretion i_n_ yiyg. In this section, studies were conducted.toldetermine‘the effects of altering THDA neuronal activity on a-MSH concentrations in serum. In addition, the presence of diurnal variations in a-MSH secretion and THDA neuronal activity was examined to determine if THDA neuronal activity is correlated with a-MSH secretion over time. RESULTS GBL inhibits the activity of nigrostriatal dopaminergic neurons (Walters gt _t., 1973; Roth gt gt., 1976) and TIDA neurons (Demarest t g;., 1979c; Lookingland gt gt., 1987a). As shown in figure 14 and table 5, 35 min following the administration of an anesthetic dose of GBL (1000 mg/kg) there was a decrease in both DOPAC concentrations and the DOPAC/DA ratio in the intermediate lobe and a concomitant increase in a-MSH concentrations in the serum, suggesting that GBL 65 66 2.02 400 2 I: VEHICLE ..5- GE'- .00 _L L0 200- U CYMSH (pg/ml) d? ”p ‘9 .0? DOPAC (no/mg protein) IOO 9 1&5 a '0 0 .0 0 %%2 09 o ‘% 0 .0 0 ' 0.0' ‘ 0 ”NAPTQ 0999 9099 9909 pay» .&3 .0 V 0 .0 A! t o‘- OJ. Figure 14: The effects of administration of GBL on the concentrations of DOPAC in the intermediate lobe of the pituitary and a-MSH in the serum. Animals were injected ip with either vehicle (0.9% saline; 1 ml/kg) or GBL (1000 mg/kg) 35 min prior to sacrifice. Each column represents the mean and the vertical line 1 SE of seven to eight animals. *, Values that are significantly different from those of vehicle injected animals (p < 0.05). 67 TABLE 5 EFFECT OF GBL ON THE CONCENTRATIONS OF DOPAC AND DA IN THE INTERMEDIATE LOBE OF THE PITUITARY DOPAC DA DOPAC/DA 0.075 i 0.007 H- O \) VEHICLE 1.03 i 0.11 13.9 0.046 i 0.006 * H- O 01 GBL 0.66 i 0.07 * 14.5 Animals were treated as described in figure 14. DOPAC and DA concentrations expressed as pg/pg protein and represents the mean and.1 SE of 7-8 animals. *, Values that are significantly different from those of vehicle—injected animals (p < 0.05). 68 decreases impulse flow in THDA neurons. GBL had no effect on the concentration of DA in the intermediate lobe (table 5), similar to it reported lack of effect on median eminence DA concentrations (Demarest gt gt., 1979c). Bilateral stimulation of the arcuate nucleus increases the synthesis and turnover of DA in the intermediate lobe in GBL-anesthetized rats (Gunnet gt_gt., 1987). To determine the appropriate stimulation intensity for maximal activation of THDA neurons, the arcuate nucleus was stimulated in GBL- treated rats at 100, 200 and 400 pA for 15 min, and intermediate lobe DOPAC concentrations and serum a-MSH concentrations wereI< 3 I2 4- . -J =I< g * O. 2_ . 01. g L J J _I O 30 60 90 MINUTES AFTER aMSH Figure 2 1 . Time course of the effects of a-MSH on plasma Animals were injected icv with prolactin concentrations. either vehicle (5 pl 0.9% saline, 0 time) 60 min or o-MSH (20 pg) 30, 60 or 90 min prior to decapitation. Trunk blood was collected in tubes on ice containing 0.1 ml 15% EDTA and 240 pg bacitracin. Values represent the mean i 1 SE of 6-8 animals. * Values that are significantly different from those of vehicle-treated animals (p < 0.05). 85 (Gudelsky gt al., 1976, Moore and Demarest, 1982). It was of interest, therefore, to determine if THDA neurons, which tonically inhibit the secretion of a-MSH from the intermediate lobe of the pituitary, are also activated in a delayed manner by this hormone. Accordingly, the acute (30 min) and delayed (12 hr) actions of prolactin and a-MSH were compared on the synthesis of DA in the median eminence and intermediate and neural lobes of the pituitary. The results of this experiment are summarized in figure 22. Consistent with the data presented in figures 19 and 20, 30 mins after a-MSH administration DOPA accumulation was increased in the median eminence but not the intermediate or neural lobes of the pituitary. Prolactin did not alter DA synthesis in any region at this time. Twelve hours after administration, prolactin increased DOPA accumulation in the median eminence but not in the intermediate or neural lobes of the pituitary. In contrast, a-MSH failed to alter DA synthesis in any region at this time. Activation of BZ-adrenergic receptors on melanotrophs increases secretion of POMC-derived peptides (Bowers gt al., 1974; Berkenbosch e; a_1., 1981, Cote’ e_t g” 1982). To investigate if peripheral increases in a-MSH activate TIDA neurons, intermediate lobe secretion was increased by administration of the fiZ-adrenergic agonist metaproterenol. As shown in table 9, metaproterenol increased plasma a-MSH levels, but did not alter the concentration of either median 86 10F " 30 min. [ * l2 hour D VEHICLE I D VEHICLE 8 . * GMSH 0 OMS" - mount-nu I momma """/"V ,/,,..,, ,1 ,v, . , , (I . / I / - . , -’ :4 ’ 7/}; //’;I ?; "/" ' ‘IHI/I- / /’/l I I r I . %I 1”, 12,6 1' I 4 (5 // . \‘ \\‘-;-'~L -.'Z-l ‘:>\.‘<::.:.:.:~:‘._‘.:.j.j DOPA (09 I ma mold») c. I /,,- :s ‘ 51:12:; \\ NL ME Figure 22. Comparison of the effects of a-MSH and prolactin on the accumulation of DOPA in median eminence and the intermediate and neural lobes of the pituitary. Animals were injected iCV'With either vehicle (5 pl 0.9% saline), a-MSH (20 pg) or prolactin (10 pg) 30 min or 12 hrs prior to decapitation. All animals received NSD 1015 (100 mg/kg; i.p.) 30 min prior to decapitation. Each column represents the mean DOPA concentration (pg/pg protein) and the vertical line 1 SE of 5-9 animals. * Values that are significantly different from those of vehicle-treated animals (p < 0.05). 87 TABLE 9 EFFECT OF METAPROTERENOL ON THE CONCENTRATIONS OF a-MSH AND PROLACTIN IN THE SERUM AND DOPAC IN THE MEDIAN EMINENCE AND INTERMEDIATE AND NEURAL LOBES OF THE PITUITARY Dose of Metaproterenol (mg/kg) 0 30 100 a-MSH 91.9 i 7.5 160.7 i 23.2 * 141.7 1 9.4 * Prolactin 5.4 i 1.5 8.0 i 1.6 7.2 i 2.1 DOPAC MB 7.93 i 0.53 7.91 i 0.60 6.50 i 0.36 IL 1.29 i 0.06 1.36 i 0.07 1.47 i 0.11 NL 0.60 i 0.04 0.54 i 0.04 0.72 i 0.04 Animals were injected s.c. with either vehicle (0.9% saline w/ 6 mg% ascorbic acid; 1 ml/kg) or metaproterenol (30 or 100 mg/kg) 30 min prior to decapitation. Values expressed as pg/ml plasma for a-MSH, ng/ml plasma for prolactin and pg/pg protein for DOPAC and represent the mean i 1 SE of 6-8 animals. *. Values that are significantly different from those of vehicle-treated animals (p < 0.05). 88 eminence DOPAC or plasma prolactin, indicating peripheral a- MSH does not alter TIDA neuronal activity. In addition, the metaproterenol-induced increase in melanotroph secretion did not alter intermediate lobe DOPAC concentrations. DISCUSSION The studies in this section reveal that central administration of a-MSH failed to alter intermediate lobe DOPAC concentrations or DA synthesis either acutely or after a 12 hr delay, indicating that, unlike feedback activation of TIDA neurons by prolactin, a—MSH does not alter THDA neuronal activity. In addition, increases in the secretion of other melanotroph POMC-derived peptides by' metaproterenol also failed to alter intermediate lobe DOPAC concentrations, indicating THDA neurons are not responsive to acute feedback activation from any of the secretory products it regulates. In contrast to its lack of effect on THDA neuronal activity, central administration of a-MSH produced a prompt increase in both DOPAC concentrations and DA synthesis in the median eminence. These changes reflect an increase in TIDA neuronal activity (Gunnet gt g_., 1987; Lookingland gt g;., 1987a; Lookingland. gt ,gl., 1987b). Concurrent with the increase in TIDA neuronal activity, a-MSH administration caused a sustained decrease in plasma prolactin concentrations. This is in agreement with previous reports in both female (Khorram gt g_., 1982; Khorram gt g;., 1985) and 89 male rats (Newman gt g;., 1985). The ability of a-MSH to decrease plasma prolactin concentrations by increasing TIDA neuronal activity is consistent with the observation that a- MSH fails to decrease prolactin secretion in animals pretreated with the dopaminergic antagonist spiroperidol (Khorram gt g;., 1982). Lichtensteiger and coworkers (Lichtensteiger and Lienhart, 1977; Idchtensteiger and Monnet, 1979; Lichtensteiger'and.Schlumpf, 1986) suggested that the increase in intensity of catecholamine fluorescence in the arcuate nucleus after a-MSH administration was due to activation of THDA neurons. The present study, using techniques that differentiate between TIDA and THDA neurons, extends these results by demonstrating that a-MSH increases TIDA but not THDA neuronal activity. a-MSH appears to selectively activate TIDA neurons since it failed to alter DOPAC concentrations in the striatum or nucleus accumbens, an indicator that a-MSH does not influence the activity of nigrostriatal and mesolimbic dopaminergic neurons. a-MSH increased DA synthesis in the median eminence within 30 mins, whereas activation by prolactin takes several hours and is thought to involve synthesis of a protein (Moore and Demarest, 1982) . This protein may be tyrosine hydroxylase, although hyperprolactinemia does not increase the mass of tyrosine hydroxylase in the median eminence (Gonzalez and Porter, 1988). a-MSH must increase TIDA neuronal activity via 90 a different (more rapid) mechanism than prolactin that does not involve protein synthesis, possibly by directly altering neuronal membrane permeability. a-MSH is located within neurons in the central nervous system (Dubé gt g;., 1978; O'Donohue gt_g;., 1979; Jacobowitz and O'Donohue, 1978). fi-Endorphin, which is synthesized together with a-MSH in the same neurons (Watson and Akil, 1980), inhibits the activity of TIDA neurons (Gudelsky and Porter, 1979) and increases the concentration of prolactin in plasma (Rivier gt g;., 1977; Van Vugt and Meites, 1980). a- MSH antagonizes the fi-endorphin-induced increased prolactin secretion (Khorram g a_l., 1986; Wardlaw e_t a_1., 1986). Together with the present results, this suggests that a-MSH and B-endorphin have antagonistic actions on TIDA neuronal activity. It has been suggested that a-MSH’ may act to counteract the actions of B-endorphin on prolactin secretion during stress (Khorram gt g;., 1986). Previous studies have demonstrated that administration of antisera to a-MSH increases plasma prolactin concentrations (Khorram gt g;., 1984), implicating a role for endogenous a- MSH in the tonic inhibition of prolactin secretion. In the present study, elevation of peripheral a-MSH levels by metaproterenol did not alter either TIDA neuronal activity or plasma prolactin concentrations. This is in agreement with previous results which demonstrated that intravenously administered a-MSH does not decrease prolactin concentrations 91 (Khorram g gt. , 1982) . These results indicate that endogenous a-MSH from central neurons and not intermediate lobe-derived a-MSH inhibits prolactin secretion, as suggested by Khorram and coworkers (1984). THDA neurons terminating in the neural lobe are similar to TIDA neurons in that both lack dopaminergic receptor- mediated regulation mechanisms and are inhibited by opiates (Lookingland e_t a_1. , 1985a; Lookingland e_t gt. , 1985b) . In the present study, prolactin produced a delayed activation of DA synthesis in TIDA neurons, but failed to alter DA synthesis in the neural lobe. Thus THDA neurons terminating in the neural lobe are dissimilar to TIDA neurons in that their activity is not regulated by prolactin. In conclusion, central administration of a-MSH does not alter THDA neuronal activity, suggesting these neurons are unresponsive to either an acute or delayed feedback regulation by a-MSH. In contrast, a-MSH activates TIDA neurons, thereby decreasing the secretion of prolactin from the anterior lobe of the pituitary. Section IV. ROLE OF THDA NEURONS IN CONTROLLING a-MSH SECRETION DURING STRESS Various stressful manipulations, such as physical restraint and mild electric footshock, increase secretory activity of melanotrophs in the intermediate lobe (Usategui _e_t a_l. , 1976; de Rotte gt gt., 1982: Berkenbosch gt gt. , 1984; Lookingland and Moore, 1988). Epinephrine released from the adrenal medulla has been reported to be responsible for the stress-induced increase in a-MSH secretion (Berkenbosch gt gt., 1984; Kvetnansky'gt gt., 1987). It has also been observed that restraint stress decreases THDA neuronal activity in the intermediate lobe (Lookingland and Moore, 1988) so that a decrease in the inhibitory tone of DA on the melanotrophs may also play a role in the stress-induced release of a-MSH. The purpose of the studies in this section was to determine the role of changes.in THDA neuronal activity in the stress-induced increase in a-MSH secretion. RESULTS In agreement with earlier reports (Berkenbosch gt gt., 1984; Lookingland and Moore, 1988) physical restraint of rats increased the concentration of a-MSH in plasma (figure 23). Pretreatment with the fl-adrenergic receptor antagonist propranolol failed to alter basal levels of a-MSH, but at a dose of 10 mg/kg, blocked the stress—induced increase in circulating levels of a-MSH (figure 23). Concurrent with the 92 93 [:1 —Control - -Stress CD (D O (I! O 0‘ O O O O I g I g F a-MSH (pg/ml plasma) to m ' H ...I. 0| 0 0' O O O O I I I I o 5 10 Propranolol (mg/kg;i.p.) ' Figure 23: Dose-response of the effects of propranolol on the stress-induced increase in a-MSH concentrations in plasma. Animals were treated with either propranolol (5 or 10 mg/kg; i.p.) or 0.9 % saline vehicle (1 ml/kg;i.p.) 50 min prior to decapitation. Stressed animals were initially anesthetized with diethyl ether and then restrained in a supine position for’ 30 mins prior* to Idecapitation. Non-stressed. control animals were removed from their cages and decapitated immediately. Each column represents the mean plasma a-MSH concentration and the vertical line 1 SE of 7-8 animals. *, Values that are significantly different from those of non- stressed animals (P < 0.05). 94 stress-induced increase in secretion of a-MSH there was a decrease in DOPAC concentrations in the intermediate but not the neural lobe of the pituitary (figure 24), reflecting a decrease of THDA neuronal activity in the intermediate lobe, as has been reported previously (Lookingland and Moore, 1988) . This latter effect was not altered in animals pretreated with a dose of propranolol (10 mg/kg) that prevented the increased secretion of a-MSH in restrained animals (table 10). These results suggest that activation of B-adrenergic receptors is necessary for stress-induced secretion of a-MSE from the melanotrophs. Considering these results, it was questioned whether a decrease in THDA neuronal activity is important for the stress-induced increase:in.a-MSHIsecretionJ As shown in figure 25, the stress-induced increase in plasma levels of a-MSH was prevented if dopaminergic receptors were activated by the administration of apomorphine. The injection of this dopaminergic agonist reduced basal concentrations of a-MSH in plasma, and blocked the stress-induced secretion of this hormone. Taken together, the results in figures 23-25 suggest that increased activation of fi-adrenergic receptors and decreased activation of dopaminergic receptors on melanotrophs both contribute to the increased circulating levels of a-MSH in stressed animals. Results of a pharmacological study in non-stressed rats are consistent with this suggestion (figure 26). Subcutaneous h. 95 [:3 -Control - —Stress F DOPAC (pg/pg protein) S Intermediate Neural Lobe Lobe Figure 24: Effects of restraint stress on intermediate and neural lobe DOPAC concentrations. Stressed-animals were initially anesthetized with diethyl ether and then restrained in a supine position for 30 mins prior to decapitation. Non- stressed control animals were removed from their cages and decapitated immediately. Each column represents the mean DOPAC concentration and the vertical line 1 SE of 8 animals. *, Values that are significantly different from those of non- stressed animals (P < 0.05). TABLE 10 EFFECT OF PROPRANOLOL AND/OR STRESS ON DOPAC AND DA CONCENTRATIONS IN THE INTERMEDIATE LOBE OF THE PITUITARY GLAND Treatment DOPAC DA DOPAC/DA Saline Control 1.56 i 0.10 23.2 i 1.0 0.067 t 0.004 Stress 1.22 i 0.06 * 23.9 i 1.9 0.052 t 0.003 * Propranolol Control 1.34 i 0.05 20.5 i 0.7 0.066 t 0.002 Stress 1.11 i 0.04 * 21.6 i 1.3 0.052 t 0.002 * Animals were treated with either i.p.) or 0.9% saline vehicle (1 mg/kg; i.p.) 50 min prior to decapitation. propranolol (10 mg/kg; Stressed animals were initially anesthetized with diethyl ether and then restrained in a supine position for 30 mins prior to decapitation. Non-stressed animals were removed from their cages and decapitated immediately. DOPAC and DA are expressed as pg/pg protein and represent values obtained from 7-8 animals. *, Values that are significantly different from those of non-stressed animals (P < 0.05). 97 [:1 —Control " - —Strels g l I * * 'P F“:— I I a—IISH (pg/ml plasma) § Saline Apomorphine Figure 25: Effect of apomorphine pretreatment on the stress- induced increase in plasma a-MSH concentrations. Animals were treated with either apomorphine (2 mg/kg; s.c.) or 0.9 % saline vehicle (1 ml/kg: i.p.) 45 min prior to decapitation. Stress animals were initially anesthetized with diethyl ether and then restrained in a supine position for 30 mins prior to decapitation. Non-stressed animals were removed from their cages and decapitated immediately. Each column represents the mean plasma a-MSH concentration and the vertical line 1 SE of 4-8 animals. *, Values that are significantly different from those of non-stressed saline- treated animals (P < 0.05). 98 EJ-Saflne Vehicle - -l[etaproterenol 350-, ., 1? 300.. ** .. 5.... .. n. a zoo-- .. 3...... .. a 100" I . .. l a 50" .. 0 . Tartarlc Acid Haloperldol Vehicle . Figure 26: Effect of haloperidol on the metaproterenol-induced increase in plasma a-MSH concentrations. Animals were treated with either haloperidol (0.1 mg/kg: s.c) or its vehicle (0.3 % tartaric acid) and either metaproterenol (30 mg/kg; s.c.) or its vehicle (0.9 % saline containing 6 mg % ascorbic acid) 30 min prior to decapitation. Each column represents the mean plasma a-MSH concentration and the vertical line 1 SE of 8 animals. *,‘Values that are significantly different from those of saline vehicle/tartaric acid vehicle treated animals, **, Value significantly different from all other groups (P < 0.05) . 99 injection of a maximally effective dose of metaproterenol (30 mg/kg) (table 9) increased circulating levels of a-MSH by 175%. Haloperidol (0.1 mg/kg, s.c.), a potent dopaminergic antagonist, also increased the plasma concentration of a-MSH (199% of control). When administered concurrently these drugs produced a much larger increase in the plasma concentrations of a—MSH (304% of control). The results of this study suggest that stimulation of the fi-adrenergic receptors and blockade of the dopaminergic receptors act synergistically to enhance the secretion of a-MSH from the melanotrophs. In order to eliminate the effects of stress on THDA neurons, an attempt was made to severe the afferent neurons responsible for the stress-induced decrease in THDA neuronal activity. Severing of retrochiasmatic (RC) afferent neuronal inputs to the mediobasal hypothalamus blocks the stress- induced decrease in TIDA neuronal activity in female rats (Barton, 1988). Therefore, the effect of RC deafferentation on the stress-induced changes in the concentrations of intermediate lobe DOPAC and plasma a-MSH were examined. RC deafferentation decreased basal intermediate lobe DOPAC concentrations (p < 0.05), but did not affect the DOPAC/DA ratio, suggesting RC deafferentation may be eliminating some of the THDA neurons projecting to the intermediate lobe. As shown in table 11, stress decreased both DOPAC concentrations and the DOPAC/DA ratio in the intermediate lobe and this effect was eliminated by RC deafferentation. Concurrent with 100 TABLE 11 EFFECT OF STRESS ON DOPAC AND DA CONCENTRATIONS IN THE INTERMEDIATE LOBE OF THE PITUITARY IN SHAM AND RETROCHIASMATIC DEAFFERENTATED (RC) ANIMALS. Treatment DOPAC DA DOPAC/DA SHAM Control 2.00 i 0.1 20.4 i 0.7 0.098 i 0.004 Stress 1.47 i 0.12 * 21.4 i 1.6 0.070 1 0.005 * RC Control 1.61 i 0.14 17.2 i 2.0 0.091 t 0.008 Stress 1.40 i 0.10 17.1 i 1.2 0.083 1 0.005 Animals received either retrochiasmatic or sham surgery 7 days prior to experimentation. Stressed animals were initially anesthetized with diethyl ether and then restrained in a supine position for 30 mins prior to decapitation. Non- stressed animals were removed from their cages and decapitated immediately. DOPAC and DA are expressed as pg/pg protein and represent 7-10 animals. *, Values that are significantly different from those of non-stressed animals (P < 0.05). 101 l:|—Control - -—Stress 3001 A C! E 250" " 03 C5 '23. zoo-- ‘- "é \ 150" .- g v 100“ ,_.J__‘ " m 00 2 50-- '- I 5 O I Sham RC Cut Figure 27: Effect of retrochiasmatic (RC) deafferentation on the stress-induced changes in plasma a-MSH concentrations. Animals received either RC or sham surgery 7 days prior to experimentation. Stressed animals were initially anesthetized with diethyl ether and then restrained in a supine position for 30 mins prior to decapitation. Non-stressed animals were removed from their cages and decapitated immediately. Each column represents the mean plasma a-MSH concentration or and the vertical line 1 SE of 7-10 animals.*, Values that are significantly different from those of non-stressed animals (P < 0.05). 102 this blockade, RC deafferentation attenuated the stress- induced increase in plasma a-MSH concentrations (figure 27). DISCUSSION The objective of the studies in this section was to investigate the role of THDA neuronal inhibition in the stress-induced increase in intermediate lobe secretory activity. In agreement with previous results indicating that there is a rapid increase in both B-endorphin and a-MSH secretion with various forms of stress (Usategui gt gt., 1976; de Rotte gt _t., 1982; Tilders gt gt., 1985; Lookingland and Moore, 1988), restraint stress (immobilization in the supine position) increased plasma a-MSH concentrations. Both in the present and in previous studies (Berkenbosch gt gt., 1984), fi-adrenergic antagonism did not affect basal a-MSH secretion, but blocked the restraint stress-induced increase in plasma a-MSH concentrations. This indicates that fi-adrenergic receptor activation does not play an important role in the control of basal a-MSH secretion, but is necessary for the stress-induced activation of a-MSH secretion. Epinephrine secreted from the adrenal medulla is, at least in part, responsible for this stimulation, because removal of the adrenal medulla attenuates the stress-induced increase in plasma a-MSH concentrations (Kvetnansky gt g_., 1987). Restraint stress also decreases DOPAC concentrations and the DOPAC/DA ratio in the intermediate lobe, indicating a 103 stress-induced decrease in THDA neuronal inhibitory tone in the intermediate lobe, as has been reported previously (Lookingland and. Moore, 1988). The inhibitory' effect of restraint on THDA neuronal activity is not mediated via 8- adrenergic receptor activation since it was not influenced by propranolol. The importance of the inhibitory tone of DA on stress- induced secretion of a-MSH was examined by determining the effects of dopaminergic agonists and antagonists. Pretreatment with the dopaminergic agonist apomorphine prevented the stress-induced increase in plasma a-MSH concentrations, indicating that maintenance of dopaminergic inhibition overrides the release of a-MSH induced by activation of 3- adrenergic receptors. In non-stressed animals, administration of haloperidol increased a-MSH secretion and enhanced the £2- adrenergic agonist-mediated increase in a-MSH secretion beyond that attained with the fiZ-adrenergic agonist alone. The results of these studies indicate that a decrease in THDA neuronal inhibitory’ tone is necessary’ for' a ‘maximal 3- adrenergic-mediated stimulation of plasma a-MSH secretion. Under basal conditions, the retrochiasmatic cut decreased intermediate lobe DOPAC concentrations but not the DOPAC/DA ratio. The absence of a change in the DOPAC/DA ratio indicates that THDA neuronal activity is not altered by the cut, but that the decline in DOPAC concentrations may be due to the elimination of a small number of THDA neurons 104 originating' anterior' to ‘the, cut (rostral periventricular region) (Luppi gt gt., 1986: Kawano and Daikoku, 1987). The basal activity of TIDA neurons of male rats is also not altered by retrochiasmatic deafferentation (Barton gt gt., 1989). Severing afferent neuronal inputs passing through the retrochiasmatic region of the mediobasal hypothalamus attenuated the stress-induced increase in plasma a-MSH concentrations. Previous studies .have indicated. that retrochiasmatic deafferentation does not alter either the stress-induced increase in plasma concentrations of epinephrine (Kvetnansky t al., 1988) or the ability of the fi-adrenergic agonist isoproterenol to increase a-MSH secretion (Vermes Q g_l_. , 1981) . Therefore, attenuation of stress- induced increases in a-MSH secretion by retrochiasmatic deafferentation indicates that epinephrine is not the only mediator involved in stress-induced increases in a-MSH. Because retrochiasmatic deafferentation also blocked the stress-induced decrease in intermediate lobe DOPAC concentrations suggests that blockade of the stress-induced decrease in THDA neuronal activity was responsible for the attenuation of the stress-induced increase in a-MSH secretion. Taken together, the results of the studies in this section indicate that a decrease in THDA neuronal activity is necessary for the maximal stress-induced increase in intermediate lobe secretion. 105 The effects of both D-2 dopaminergic and B-2 adrenergic receptors on the melanotroph are mediated by adenosine cyclic 3',5',-monophosphate (cAMP); dopaminergic receptor activation diminishes whereas fi-adrenergic receptor activation stimulates adenylate cyclase activity (review Coté gt g” 1982). In dispersed neurointermediate lobe cells DA can completely abolish the effects of the fi-adrenergic agonist isoproterenol on adenylate cyclase activity and cAMP formation (Munemura gt gt., 1980: Cote gt _t., 1982). The ability of DA to block the tg vitro actions of fi—adrenergic stimulation is consistent with the i vivo results reported in the present study. Higher doses of CRH can directly stimulate the secretion of a-MSH from melanotrophs tg vitro (Vale gt gt., 1983). This CRH-induced increase in a-MSH release can be prevented by DA (Meunier g g” 1982; Saland _e_t g” 1988), indicating dopaminergic inhibition can block secretion of a-MSH induced by other stimulants in addition to fi-adrenergic agonists. In summary, both a decrease in THDA neuronal inhibitory tone and an increase in fi-adrenergic stimulation by epinephrine secreted from the adrenal medulla are involved in the control of a-MSH secretion during stress. SUMMARY The characteristics of THDA neurons terminating in the intermediate lobe of the pituitary gland were examined and their role in controlling both basal and stress-induced secretion of a-MSH was evaluated. The significant findings and conclusions of these studies are summarized below. a) The DA metabolite DOPAC, but not HVA, was found to be a major DA metabolite in the intermediate and neural lobes. DOPAC is rapidly removed from the posterior pituitary and appears to predominantly reflect the intraneuronal metabolism of unreleased DA. Alterations in THDA neuronal activity produced corresponding changes in DA synthesis and DOPAC concentrations in the intermediate and neural lobes. It was concluded that DOPAC concentrations in these terminal regions may be used as an index of THDA neuronal activity. b) Manipulations which decrease and increase intermediate lobe DOPAC concentrations were correlated with reciprocal changes in plasma a-MSH concentrations, indicating that THDA neurons play a major role in regulating the basal release of POMC-derived peptide secretion from intermediate lobe melanotrophs. c) Although THDA neurons regulate basal a-MSH secretion, THDA neurons are unresponsive to either an acute or delayed 106 107 feedback activation by a-MSH. In contrast, a-MSH administration increased TIDA neuronal activity thereby decreasing the secretion of prolactin from the anterior lobe. d) A decrease in THDA neuronal inhibitory tone as well as B-adrenergic activation by epinephrine is necessary for the full expression of the stress-induced increase in secretion of POMC-derived peptides from melanotrophs in the intermediate lobe of the pituitary. CONCLUDING DISCUSSION Although the physiological role of the intermediate lobe of the pituitary has not yet been determined, it serves as a useful system for studying neuroendocrine regulatory mechanisms because of its relative simplicity and homogenous structure. The THDA neurons projecting to the intermediate lobe are the only known DA neurons which synapse on an endocrine cell, allowing for the easy monitoring of the postsynaptic responses (a-MSH secretion). The studies in this thesis have demonstrated how DA neurons with different functions have evolved different regulatory mechanisms. For example, although both THDA and nigrostriatal dopaminergic neurons have a DA-mediated regulation, nigrostriatal but not THDA neurons are regulated by DA autoreceptors. THDA neurons terminating in the intermediate lobe are most similar to TIDA neurons terminating in the median eminence in that both are neuroendocrine neurons which tonically inhibit hormone secretion and have similar basal neuronal activities and an absence of DA autoreceptors, yet they differ in their responsiveness to hormonal regulation. The THDA neurons in projecting to the neighboring neural and intermediate lobes also differ in their responsiveness to DA-mediated regulation and stress. The 108 109 reasons for the differences between DA neuronal systems most likely reflect their different functional roles, and further investigation may reveal how these different factors contribute to these functions. These studies have also demonstrated how DA can act as a neuroendocrine regulator of POMC peptide secretion. DA does not act in isolation, but as demonstrated in this study, acts in concert with other factors, such as epinephrine, to regulate peptide secretion. As mentioned in the introduction, other regulatory factors, such as GABA and CRH may also regulate intermediate lobe secretion. The intermediate lobe, because of its simplicity, is an ideal system for the study the interaction of these neurotransmitters at a postsynaptic site. In addition, these studies have demonstrated how stress can regulate pituitary secretion through both hormonal and neural mechanisms. Further investigation of THDA neurons during stress may reveal the mechanism by which stress alters hypothalamic neuronal activity. In conclusion, investigation of the THDA neurons projecting to the intermediate lobe of the pituitary has revealed important information about both DA neuronal regulation and DA control of POMC-peptide secretion.and should continue to provide useful information in the future. 1! BI BLIOGRAPHY BIBLIOGRAPHY Agid, Y., Javoy, F. and Youdim, M.B.H.: Monoamine oxidase and aldehyde dehydrogenase activity in the striatum of rats after 6-hydroxy-dopamine lesion of the nigrostriatal pathway. Br. J. Pharmac. g§:175-178, 1973. Akil, H., Young, E. and Watson, S.J.: Opiate binding proper- ties of naturally occurring N- and C-terminus modified fi-endorphins. Peptides g:289-292, 1981. Akil, E., Watson, S.J., Young, E., Lewis, M.E., Khachaturian, H. and Walker, J.Mx Endogenous opioids: Biology and func- tion. Ann. Rev. Neurosci. 1:223-255, 1984. Alper, R.H., Demarest, K.T. and Moore, K.E.: Effects of surgi- cal sympathectomy on catecholamine concentrations in the posterior pituitary of the rat. Experientia ;§:134-135, 19803. Alper, R.H, Demarest, K.T. and Moore, K.E.: Morphine differen- tially alters synthesis and turnover of dopamine in central neuronal systems. J. Neural Transm. g§:157-165, 1980b. Annunziato, L. and Weiner, R.I.: Characteristics of dopamine uptake and 3,4-dihydroxyphenylacetic acid (DOPAC) formation in the dopaminergic terminals of the neurointermediate lobe of the pituitary gland. Neuroen- docrinology tt:8-12, 1980. Back, N., Soinila, S., Joh, T.H. and Rechardt, L.: Catecholamine-synthesizing enzymes in the rat pituitary: An immunohistochemical study; Histochemistryggg:459-464, 1987. Barton, A.C.: Anterior hypothalamic afferent regulation of tuberoinfundibular' dopaminergic neuronal activity. Doctoral dissertation, Michigan State University, 1988. Barton, A.C., Demarest, K.T., Lookingland, K.J. and Moore, K.E.: A sex: difference in ‘the stimulatory afferent regulation of tuberoinfundibular dopaminergic neuronal activity. Neuroendocrinology tg:361-366, 1989. 110 111 Baumgarten, H.G., Bjorklund, A., Holstein, A.F. and Nobin, A.: Organization and ultrastructural identification of catecholamine nerve terminals in the neural lobe and pars intermedia of the rat pituitary. Z. Zellforsch. mikrosk. Anat. m:483-517, 1972. Beaulieu, M., Goldman, M.E., Miyazaki, K., Frey, E.A., Eskay, R.L., Kebabian, J.W. and. Cote, T.E.: Bromocriptine- induced changes in the biochemistry, physiology, and histology of the intermediate lobe of the rat pituitary gland. Endocrinology ttt:1871-1884, 1984. Ben-Jonathan, N.: Dopamine: a prolactin-inhibiting hormone. Endocrine. Rev. g:564-589, 1985. Berkenbosch, F., Vermes, I., Binnekade, R. and Tilders, F.J.H.: Beta-adrenergic stimulation induces an increase of the plasma levels of immunoreactive a-MSH, B-endor- phin, ACTH and of corticosterone. Life Sci. _2_9_:2249-2256, 1981. Berkenbosch, F., Vermes, I. and Tilders, F.J.H.: The B- adrenoceptor-blocking drug propranolol prevents secretion of immunoreactive fi-endorphin and a-melanocyte-stimulat- ing hormone in response to certain stress stimuli. Endocrinology tt§:1051-1059, 1984. Bjorklund, A., Falck, B. and Rosengren, E.: Monoamines in the pituitary gland of the pig. Life Sci. §:2103-2110, 1967. Bjorklund, A., Moore, R.Y., Nobin, A. and Stenevi, U.: The organization of tuberbo-hypophyseal and reticulo-infun- dibular catecholamine neuron systems in the rat brain. Brain Res. fit:l71-191, 1973. Bjorklund, A., Lindvall, o. and Nobin, A.: Evidence for an incerto-hypothalamic dopamine neurone system in the rat. Brain Res. g2:29—42, 1975. Bjorklund, A. and Lindvall, o.: Dopamine-containing systems in the CNS. In flandbook of Chemical Neuroanatomy, Vol. 2, Classical Transmitters in the QNS, 2t,1, (A. Bjorkl- und and T. Hokfelt, eds.), Elsevier, Amsterdam/New York/Oxford, 1984, pp. 55-122. Bjorklund, A. and Skagerberg, 6.: Evidence for a major spinal cord projection from the diencephalic A11 dopamine cell group in the rat using transmitter-specific fluorescent retrograde tracing. Brain Res. 111:170-175, 1979. 112 Bower, A., Hadley, M.E. and Hruby, V.J.: Biogenic amines and control of melanophore stimulating’ hormone release. Science t§1:70-72, 1974. Bridges, T.E., Hillhouse, E.W. and Jones, M.T.: The effect of dopamine on neurohypophysial hormone release tg vivo and from the rat neural lobe and hypothalamus tg vitro. J. Physiol. 2§Q:647-666, 1976. Cajal, R.S.: Algunas contribuciones a1 conocimiento de los ganglios del encefalo. Alnn. Soc. Esp. Hist. gg:195—237, 1894. Carlsson, A.: Receptor-mediated control of dopamine metabo- lism. In Pre- and Postsynaptic Receptors, (E. Usdin and W.E. Bunny, eds.), Dekker, New York, 1975, pp. 49-63. Chapin, D.S., Lookingland, K.J. and Moore, K.E.: Effects of LC :mobile phase composition on retention ‘times for biogenic amines, and their precursors and metabolites. Current Sep. 1:68-70, 1986. Chard, T.: An Introduction to Radioimmunoassay and Related Technigtes. Elsevier, Amsterdam, 1982. Chronwall, B.M., Hook, G.R. and Millington, W.R.: Dopaminergic regulation of the biosynthetic activity of individual melanotrophes in the rat pituitary intermediate lobe: a morphometric analysis by light and electron microscopy and tg situ hybridization. Endocrinology t;;:1992-2002, 1988. Coates, P.J., Doniach, I., Hale, A.C. and Rees, L.H.: The distribution of immunoreactive a-melanocyte-stimulating hormone cells in the adult human pituitary gland. J. Endocrin. ttt:335-342, 1986. Cooper, J.R., Bloom, F.E. and Roth, R.H.: The Biochemical Basis of Neuropharmacology, Oxford University, New York/Oxford, 1982. Cote, T.E., Eskay, R.L., Frey, E.A., Grewe, C.W., Munemura, M., Stoof, J.C., Tsuruta, K. and Kebabian, J.W.: Bioche- mical and physiological studies of the beta-adrenorecep- tor and the D-2 dopamine receptor in the intermediate lobe of the rat pituitary gland: A review. Neuroen- docrinology ;§:217-224, 1982. 113 Dahlstrom, A. and Fuxe, K.: Evidence for the existence of monoamine-containing neurons in the central nervous system: I. Demonstration of monamines in the cell bodies of brain stem neurons. Acta Physiol. Scand. _6_2:1-55, 1964. Dahlstrom, A. and Fuxe, K.: Monoamines and the pituitary gland. Acta Endocrinologica §t=301~314, 1966. Davis, M.D. and Hadley, M.E.: Spontaneous electrical poten- tials. and. pituitary' hormone (MSH) secretion. Nature 261:422-423, 1976. Davis, M.D.: The hypothalmo-hypophyseal rat explant in vitto: endocrinological studies of the pars intermedia dopamin- ergic neural input. J Physiol. (Lond) 370:381-393, 1986. Dawson, R., Valdes, J.J. and Annau, Z.: Tuberohypophysial and tuberoinfundibular dopamine systems exhibit.differential sensitivity to neonatal monosodium glutamate treatment. Pharmacology tt:17-23, 1985. Demarest, K.T., Alper, R.H. and Moore, K.E.: DOPA accumulation is a measure of dopamine synthesis in the median eminence and posterior pituitary. J Neural Transm. 3g:183-193, 1979a. Demarest, K.T. and Moore, K.E.: Lack of a high affinity transport system for dopamine in the median eminence and posterior pituitary. Brain Res. 171:545-551, 1979b. Demarest, K.T. and Moore, K.E.: Comparison of dopamine synthesis regulation in the terminals of nigrostriatal, mesolimbic, tuberoinfundibular and tuberohypophyseal neurons. J. Neural Transm. g§:263-277, 1979c. Demarest, K.T., Smith, D.J. and Azzaro, A.J.: The presence of the type A form of monamine oxidase within nigrostriatal dopamine-containing neurons. J. Pharmacol. Exp. Ther. Zt§:461-468, 1980. Demarest, K.T. and Moore, K.E.: Type A monoamine oxidase catalyzes the intraneuronal deamination of dopamine within nigrostriatal, mesolimbic, tuberoinfundibular and tuberohypophyseal neurons in the rat. J. Neural Transm. 5;:175-187, 1981. Demeneix, B.A., Taleb, O., Loeffler, J. Ph. and Feltz, P.: GABA, and GABAa receptors on porcine pars intermedia cells in primary culture: functional role in modulating peptide release. Neurosci. t1:1275-1285, 1986. 114 De Rotte, A.A. and van Wimersma Greidanus, Tj. B.: Differen- tial secretion of a-melanocyte stimulating hormone into cerebrospinal fluid and blood in the rat. Front. Horm. Res. 2:131-141, 1982. Douglas, W.W. and Taraskevich, P.S.: Action potentials in gland cells of rat pituitary pars intermedia: Inhibition by dopamine, an inhibitor of MSH secretion. J. Physiol. (Lond.) 2§§;l71-184, 1978. Dubé, D., Lissitzky, J.C., Leclerc, R. and Pelletier, G.P.: Localization of a-melanocyte-stimulating hormone in rat brain and pituitary. Endocrinology t02:1283-1291, 1978. Eberle, A.N.: The Melanotropins: Chemistry. thsiotogy ggd Mechanisms of Action, S. Karger, Basel, 1988. Eipper, B.A. and Mains, R.E.: Structure and biosynthesis of pro-adrenocorticotropin/endorphin and related peptides. Endocrine Rev. t:1-27, 1980. Farah, J.M. , Malcolm, D.S. and Mueller, G.P.: Dopaminergic inhibition of pituitary fi-endorphin-like immunoreactivity secretion in the rat. Endocrinology t10:657-659, 1982. Genuth, S.M.: The hypothalamus and the pituitary gland. In Physiology, (R.M. Berne and M.N. Levy, eds), C.V. Mosby, St.Louis/Toronto, 1983, pp. 971-1012. Gonzalez, H.A. and Porter, J.C.: Mass and tg situ activity of tyrosine hydroxylase in the median eminence: Effect of hyperprolactinemia. Endocrinology t22:2272-2277, 1988. Greenwood, F.C.3 Hunter, W. M. and Glover, J. S.: The prepara- tion of I- labelled human growth hormone of high specific radioactivity. Biochem J. §_,:114-123, 1963. Gudelsky, G.A., Simpkins, J., Mueller, G.P., Meites, J. and Moore, R.E.: Selective actions of prolactin on catecho- lamine turnover in the hypothalamus and on serum LH and FSH. Neuroendocrinology 1;:206-215, 1976. Gudelsky, G.A. and Porter, J.C.: Morphine- and opioid peptide induced inhibition of the release of dopamine from tuberoinfundibular neurons. Life Sci. 2_5:1697-1702, 1979. Gudelsky, G.A.: Tuberoinfundibular dopamine neurons and the regulation of prolactin secretion. Psychoneuroendocrinol- ogy §:3-l6, 1981. 115 Gunnet, J.W., Lookingland, K.J., Lindley, S.E. and Moore, R.E.: Effect of electrical stimulation of the arcuate nucleus on neurochemical estimates of tuberoinfundibular and tuberohypophysial dopaminergic neuronal activities. Brain Res. g;5:371-378, 1987. Halasz, B. and Pupp, L.: Hormone secretion of the anterior pituitary gland after physical interruption of all nervous pathways to the hypophysiotrophic area. En- docrinology 11:553-562, 1965. Holzbauer, M., Muscholl, E., Racké, K. and Sharman, D.F.: Release of endogenous dopamine from single neuro-inter- mediate lobes of the rat hypophysis it vito following electrical stimulation of the stalk. J.Physiol. (Lond.) ggg:88-89p, 1982. Holzbauer, M., Racké, K., Mann, S.P., Cooper, T., Cohen, G., Krause, U. and Sharman, D.F.: Regional differences in the effect of pargyline on dopamine concentrations in the rat hypophysis. J. Neural Transm. §2:91-104, 1984. Holzbauer, M. and Racké, K.: The dopaminergic innervation of the intermediate lobe and of the neural lobe of the pituitary gland. Medical Biology 9;:97-116, 1985. Jackson, S . and Lowry, P.J. : Secretion of pro-opiocortin peptides from isolated. perfused, rat. pars intermedia cells. Neuroendocrinology t1:248-257, 1983. Jacobowitz, D.M. and O'Donohue, T.L.: a-Melanocyte stimulating hormone: Immunohistochemical identification and mapping in neurons of rat brain. Proc. Natl. Acad. Sci. USA 15:6300-6304, 1978. Johnston, C.A., Spinediq E., Negro-Vilary A.: Aromatic L-amino acid decarboxylase activity in the rat median eminence, neurointermediate lobe and anterior lobe of the pituitar- y: physiological and pharmacological implications for pituitary regulation, Neuroendocrinology t2:54-59, 1984. Kawano, H. and Daikoku, 8.: Functional topography of the rat hypothalamic dopamine neuron systems: retrograde tracing and immunohistochemical study. J. Comp. Neurol. z§§:242- 253, 1987. Kebabian, J.W. and Calne, D.B.: Multiple receptors for dopamine. Nature 277:93- 96, 1979. 116 Khorram, O., Mizunuma, H. and McCann, S.M.: Effect of a- melanocyte-stimulating hormone on basal and stimulated release of prolactin: evidence for dopaminergic media- tion. Neuroendocrinology 15:433-437, 1982. Khorram, O., Bedran de Castro, J.C. and McCann, S.M.: Physio- logical role of a-melanocyte-stimulating hormone in modulating the secretion of prolactin and luteinizing hormone in the female rat. Proc. Natl. Acad. Sci. USA fit:8004-8008, 1984. Khorram, O., Bedran de Castro, J.C. and McCann, S.M.: Stress- induced secretion of a-melanocyte-stimulating hormone and its physiological roLe in modulating the secretion of prolactin and luteinizing hormone in the female rat. En- docrinology tt1:2483-2489, 1985. Khorram, O. and McCann, S.M.: Interaction of a-melanocyte- stimulating hormone with fi-endorphin to influence anterior pituitary hormone secretion in the female rat. Endocrinology ttg:1071-1075, 1986. Kvetnanskyy R., Tilders, F.J.H., van.Zoest, I.D., Dobrakovova, M., Berkenbosch, F., Culman, J., Zeman, P. and Smelik, P.G.: Sympathoadrenal activity facilitates fl-endorphin and a-MSH secretion but does not potentiate ACTH secre- tion during immobilization stress.Neuroendocrinology 15:318-324, 1987. Kvetnansky, R., Makara, G.B., Oprsalova, Z., Dobrakovova, M. and Jezova, D.: Increased basal and stress-induced sympathetic activity in rats with lesion or deafferenta- tion of the medial basal hypothalamus. Biogenic Amines §:275-290, 1988. Lichtensteiger, W. and Lienhart, R.: Response of mesencephalic and hypothalamic dopamine neurones to a-MSH: mediated by area postrema? Nature 266:635-637, 1977. Lichtensteiger, W. and Monnet, F.: Differential response of dopamine neurons to a-melanotropin and analogues in relation to their endocrine and behavioral potency. Life Sci. 2§:2079-2087, 1979. Lichtensteiger, W. and Schlumpf, M.: Permanent alteration of peptide feedback on dopamine neurons after injection of a-melanotropin antiserum at a critical period of postna- tal development. Brain Res. t§§:205-210, 1986. 117 Lightman, Sz'L” Iyersen, L.L. and Forsling, M.L.: Dopamine and [o—ALA ,o-LEU ]Enkephalin inhibit the electrically stimu- lated neurohypophyseal release of vasopressin tg vitro: evidence for calcium-dependent opiate action. J. Neuros- ci. ;:78-81, 1982. Loh, Y.P., Parish, D.C., Tuteja, R.: Purification and charac- terization of a paired basic residue-specific pro- opiomelanocortin converting enzyme from bovine pituitary intermediate lobe secretory vesicles. J. Biol. Chem. ggg:7194-7205, 1985. Lookingland, K.J. and Moore, R.E.: Dopamine receptor-mediated regulation of incertohypothalamic dopaminergic neurons in the male rat. Brain Res. 304:329-338, 1984. Lookingland, K.J., Farah, J.M., Lovell K.L. and Moore, R.E.: Differential regulation of tuberohypophysial dopaminergic neurons terminating in the intermediate lobe and in the neural lobe of the rat pituitary gland. Neuroendocrinol- ogy 39:145-151, 1985a. Lookingland, K.J. and Moore, K.E.: Differential effects of morphine on the rates of dopamine turnover in the neural and intermediate lobes of the rat pituitary gland. Life Sci. t1: 1225-1229, 1985b. Lookingland, K.J., Gunnet, J.W. and Moore, R.E.: Electrical stimulation of the arcuate nucleus increases the metabo- lism of dopamine in terminals of tuberoinfundibular neurons in the median eminence. Brain Res. g;§:161-164, 1987a. Lookingland, K.J., Jarry, H.D. and Moore, R.E.: The metabolism of dopamine in the median eminence reflects the act1V1ty of tuberoinfundibular neurons. Brain Res. 419:303-310, 1987b. Lookingland, K.J. and Moore, R.E.: Comparison of the effects of restraint stress on the activities of tuberoinfun- dibular and tuberohypophysial dopaminergic neurons. In Pharmacology and Functional Regulation of Dopaminergic Neutogs, (P.M. Bert, G.N. Woodruff and D.M. Jackson, eds.), MacMillan, London, 1988, pp. 289-295. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem. t9§:265-275, 1951. 118 Luppi, P.H., Sakai, R., Salvert, D., Berod, A. and Jouvet, M.: Periventricular dopaminergic neurons terminating in the neuro-intermediate lobe of the cat hypophysis. J. Comp. Neurol. 215:204-212, 1986. Mains, R.E., Myers, A.C. and Eipper, B.A.: Hormonal, drug and dietary factors affecting peptidyl glycine a-amidating monooxygenase activity in various tissues of the adult male rat. Endocrinology tt§:2505-2515, 1985. Meunier, H., Lefevre, G., Dumont, D. and Labrie, F.: CRF stimulates a-MSH secretion and cyclic AMP accumulation in rat pars intermedia cells. Life Sci tt:2129-2135, 1982. Mezy, E., Léranth, C., Brownstein, M.J., Friedman, E., Krieger, D.T. and Palkovits, M.: On the origin of the serotonergic input to the intermediate lobe of the rat pituitary. Brain Res. ggg:231-237, 1984. Millington, W.R., Blum, M., Knight, R., Mueller, G.P., Roberts, J.L. and O'Donohue, T.L.: A diurnal rhythm in proopiomelanocortin messenger ribonucleic acid that varies concomitantly with the content and secretion of fi-endorphin in the intermediate lobe of the rat pituitary. Endocrinology tt§:829-834, 1986a. Millington, W.R., O'Donohue, T.L., Chappell, M.C., Roberts, J.L. and Mueller, G.P.: Coordinate regulation of peptide acetyltransferase activity and proopiomelanocortin gene expression in the intermediate lobe of the rat pituitary. Endocrinology tt§:2024-2033, 1986b. Millington, W.R. and Chronwall, B.M.: Dopaminergic regulation of the intermediate pituitary. In Neuroendocrine Perspec- tivesl Vot 7, (E.E. Muller and R.M. Machead, eds.) , 1989. Monnet, F., Reubi, J.C., Eberle, A., Lichtensteiger, W.: Diurnal variation in the release of a-MSH from rat hypothalamus and pituitary. Neuroendocrinology §;:284- 287, 1981. Montagu, K.A.: Catechol compounds in rat.tissues and in brains of different animals. Nature t§0:244-245, 1957. Moore, K.E. and Demarest, K.T.: Tuberoinfundibular and tuberohypophysial dopaminergic neurons. In Frontiers it Neutoeggocrinology, Vol 7, (W.F. Ganong and L. Martini, eds.), Raven, New York, 1982, pp 161-189. 119 Moore, R.E.: Hypothalamic dopaminergic neuronal systems. In Psychopharmacology: The Tgird generation of Progress, (R.Y. Meltzer, ed.), Raven, New York, 1987, pp. 127-139. Moore, R.Y. and Bloom, F.E.: Central catecholamine neuron systems: anatomy and physiology of the dopamine systems. Ann. Rev. Neurosci. t:129-169, 1978. Munemura, M., Coté, T.E., Tsuruta, R., Eskay, R.L. and Kebabian, J .W.: The dopamine receptor in the intermediate lobe of the rat pituitary gland: pharmacological charac- terization. Endocrinology t91:1676-1683, 1980. Murrin, L.C. and Roth, R.H.: Dopaminergic neurons: effects of electrical stimulation on dopamine biosynthesis. Mol. Pharmacol. t;:463-475, 1976. Newman, C.B., Wardlaw, S.L. and Frantz, A.G.: Suppression of basal and stress-induced prolactin release and stimula- tion of luteinizing hormone secretion by a-melanocyte stimulating hormone. Life Sci. ;§:1661-1668, 1985. Newman, C.B., Wardlaw, S.L., Stark, R.I., Daniel, 8.8. and Frantz, A.G.: Dopaminergic regulation of a-melanocyte- stimulating hormone and N-acetyl-fi-endorphin secretion in the fetal lamb. Endocrinology t;Q:962-966, 1987. Nowycky, M.C. and Roth, R.H.: Dopaminergic neurons: Role of presynaptic receptors in the regulation of transmitter biosynthesis. Proga Neuropsychopharmac. 2:139-158, 1978. O'Donohue, T.L., Miller, R.L. and Jacobowitz,D.M.: Identifica- tion, characterization and stereotaxic mapping of in- traneuronal a-melanocyte stimulating hormone—like immunoreactive peptides in discrete regions of the rat brain. Brain Res t1§:101-123, 1979. O'Donohue, T.L., Handelmann, S.E., Chaconas, T., Miller, R.L. and Jacobowitz, D.M.: Evidence that N-acetylation regu- lates the behavioral activity of a-MSH in the rat and human central nervous system. Peptides g:333-344, 1981. Oertel, W.H., Mugnaini, E., Tappaz, M.L., Weise, V.K., Dahl, A.L., Schmechel, D.E. and Kopin, I.J.: Central GABAergic innervation of neurointermediate pituitary lobe: Bioche- mical and immunocytochemical study in the rat. Proc. Natl. Acad. Sci. USA 12;675-679, 1982. Palkovits, M.: Isolated removal of hypothalamic or other brain nuclei of the rat. Brain Res gg:449-450, 1973. 120 Paxinos, G. and Watson, C.: the Rat Btgtn in Steteotaxic Coorginates, Academic, New York, 1982. Penny, R.J. and Thody, A.J.: An improved radioimmunoassay for a-melanocyte-stimulating hormone (a-MSH) in the rat: Serum and pituitary a-MSH levels after drugs which modify catecholamine neurotransmission. Neuroendocrinology z§:l93-203, 1978. Pettibone, D.J. and Mueller, G.P.: Differential effects of adrenergic agents on plasma levels of immunoreactive beta-endorphin and a-melanotrophin in rats. Proc. Soc. Exp. Biol. Med. t1§:168-l74, 1984. Pittman, Q.J . , Lawrence, D. and Lederis, K. : Presynaptic interactions in the neurohypophysis: endogenous modulato- rs of release. In The Neurohypophysi : Structurgt Function and Conttot. Progress in Brain Research, Vol 60, (B.A. Cross and G. Leng, eds.), 1983, pp. 319-332. Proulx-Ferland, L., Labrie, F., Dumont, D., Cote, J., Coy, D.H. and Sveiraf, J.: Corticotropin-releasing factor stimulates secretion of melanocyte-stimulating hormone from the rat pituitary. Science gt1:62-63, 1982. Rabhi, M., Onteniente, B., Kah, O., Geffard, M. and Calas, A.: Immunocytochemical study of the GABAergic innervation of the mouse pituitary by use of antibodies against gamma- aminobutyric acid (GABA). Cell Tissue Res. 251:33-40, 1987. Racké, K. Ritzel, H., Trapp, B. and Muscholl, E.: Dopaminergic modulation of evoked vasopressin release from the isolated neurohypophysis of the rat: possible involvement of endogenous opioids. Naunyn-Schmiedeberg's Arch. Pharmacol. ltg:56-65, 1982. Racké, K. and Muscholl, E.: Release of endogenous 3,4- dihydroxyphenylethylamine and its metabolites from the isolated neurointermediate lobe of the rat pituitary gland. Effects of electrical stimulation and of inhibi- tion of monoamine oxidase and reuptake. J .Neurochem. g§:745-752, 1986. Racké, K., Bdhm, E. and Muscholl, E.: The role of cytoplasmic (newly synthesized) dopamine for the spontaneous and electrically evoked release of dopamine and its metaboli- tes from the isolated neurointermediate lobe ofthe rat pituitary gland it Vitto. Naunyn-Schmiedeberg's Arch. Pharmacol. ttfi: 21-27, 1987. 121 Racké, K., Grosshans, A., Sirrenberg, S. and Ziegler, K.: Presynaptic regulation of the electrically evoked release of endogenous dopamine from the isolated neurointer- mediate lobe or isolated neural lobe of the rat pituitary tn Vitto. Naunyn-Schmiedeberg's Arch. Pharmacol. _331 :504- 511, 1988. Randle, J.C.R., Moor, B.C. and Kraicer, J.: Dopaminergic mediation of the effect of elevated potassium on the release of pro-opiomelanocortin-derived peptides from the pars intermedia of the rat pituitary. Neuroendocrinology g1:141-149, 1983. Rivier, C., Vale, W., Ling, N., Brown, M. and Guillemin, R.: Stimulation 1g vivo of the secretion of prolactin and growth hormone by fi-endorphin. Endocrinology 100:238-241, 1977. Rosengren, E.: On the role of monoamine oxidase for the inactivation of dopamine in brain. Acta Physiol. Scand. tg:370-375, 1960. Roth, R.H., Walters, J.R. and Aghajanian, G.K.: Effect of impulse flow on the release and synthesis of dopamine in the rat striatum, In Frontiers in Catecholamine Research, (E. Usdin.and S.H. Snyder, eds.), Pergamon, Oxford, 1973, pp. 567-574. Roth, R.H., Murrin, L.C. and Waltersw J.R.: Central dopaminer- gic neurons: effects of alterations in impulse flow on the accumulation of dihydroxyphenylacetic acid. Eur. J. Pharmacol. ;§:163-171, 1976. Saavedra, J.M., Palkovits, M., Kizery J.S., Brownstein, M; and Zivin, J.A.: Distribution of biogenic amines and related enzymes in the rat pituitary gland. J. Neurochem. 2_5_:257- 260, 1975. Saavedra, J.M.: Central and peripheral catecholamine innerva- tion of the rat intermediate and posterior lobes. Neuroendocrinology gQ:281-284, 1985. Saland, L.C., Gutierrez, L., Kraner, J. and Samora, A.: Corticotropin-releasing factor (CRF) and neurotransmit- ters modulate melanotropic peptide release from rat neurointermediate pituitary it ytttg. Neuropeptides t;:59-66, 1988. 122 Shannon, N.J. and Moore, K.E.: Determination of the source of 5-hydroxytryptaminergic neuronal projections to the neural and intermediate lobes of the rat pituitary gland through the use of electrical stimulation and lesioning experiments. Brain Res. gtg:322-330, 1987. Sharman, D.F.: The catabolism of catecholamines. Br. Med. Bull. ;Q:110-115, 1973. Steel, R.G.D. and Torrie, J.H.: Pringipals and Procedures of Statistics, McGraw-Hill, New York, 1979. Stefanini, E., Devoto, P., Marchisio, A.M., vernaleone, F. and Collu, Rt: [3H]-Spiroperidol binding to a putative dopaminergic receptor in rat pituitary gland. Life Sci. ;§:583-587, 1980. Snyder, S.H. and Coyle, J.T.: Regional differences in 3H- norepinephrine and H-dopamine uptake into rat brain homogenates. J. Pharmacol. Exp. Ther. t65:78-86, 1969. Tappaz, M.L., Kakucska, I., Paut, L. and. Makara, G.B.: Decreased GABAergic innervation of the pituitary inter- mediate lobe after rostral hypothalamic cuts. Brain Res. Bull. t1:711-716, 1986. Taraskevich, P.S. and Douglas, W.W.: GABA directly affects electrophysiological properties of pituitary pars inter- media cells. Nature 299:733-734, 1982. Tilders, F.J.H., Berkenbosch, F. and Smelik, P.G.: Control of secretion of peptides related to adrenocorticotropin, melanocyte-stimulating hormone and endorphin. In Fron- tiers of Hormone Research. Vol 14, (Tj.B. van Wimersma Greidanus, ed.), S. Karger, Basel, 1985, pp. 161-196. Tomiko, S.A., Taraskevich, P.S. and Douglas, W.W.: GABA acts directly on cells of pituitary pars intermedia to alter hormonal output. Nature 30;:706-707, 1983. Usategui, R., Oliver, C.,‘Vaudry, H., Lombardi, G., Rozenberg, I. and Mourre, A.: Immunoreactive a-MSH and ACTH levels in rat plasma and pituitary. Endocrinology 2§:189-196, 1976. Vale, W., Vaughan, J., Smith, M., Yamamoto, G., Rivier, J. and Rivier, C.: Effects of synthetic ovine corticotropin- releasingfactor,glucocorticoids,catecholamines,neuroh- ypophysial peptides, and other substances on cultured corticotropic cells. Endocrinology tt;:1121-1131, 1983. 123 Van den Pol, A.N., Herbst, R.S. and Powell, J.F.: Tyrosine hydroxylase-immunoreactive neurons of the hyothalamus: A light and electron microscopic study. Neurosci. t;:1117-1156, 1984. Van'Vugt, D.A. antheites, J.: Influence of endogenous opiates on anterior pituitary function. Fed Proc tg:2533-2538, 1980. Vermes, I., Berkenbosch, F., Tilders, F.J.H. and.Smelik, P.G.: Hypothalamic deafferentation in the rat appears to discriminate between the anterior lobe and intermediate lobe response to stress. Neurosci. Lett. 31:89-93, 1981. Vuillez, P., Carbajo Perez, S. and Stoeckel, M.E.: Colocaliza- tion of GABA and tyrosine hydroxylase immunoreactivities in the axons innervating the neurointermediate lobe of the rat pituitary: an ultrastructural immunogold study. Neurosci. Letters 1g:53-58, 1987. Wardlaw, S.L., Smeal, M.M. and.Markowitz, C.E.: Antagonism of fi-endorphin-induced prolactin release by a-melanocyte- stimulating hormone and corticotropin-like intermediate lobe peptide. Endocrinology tt2:112-118, 1986. Walters, J.R., Roth, R.H. and Aghajanian, G.K.: Dopaminergic neurons: similar biochemical and histochemical effects of gamma-hydroxybutyrate and acute lesions of the nigro- neostriatal pathway. J. Pharmacol. Exp. Ther. t§§:630- 639, 1973. Watson, S.J. and Akil, H.: a-MSH in rat brain: occurrence within and outside of fi-endorphin neurons. Brain Res. 182:217-223, 1980. Westerink, B.H.C.: The effects of drugs on dopamine biosyn- thesis and metabolism in the brain. In The Neurobiology of Dopamine, (A.S. Horn, J. Korf and B.H.c. Westerink, eds.) , Academic, London/New York/San Francisco, 1979, pp. 255-291. Westerink, B.H.C.: Sequence and significance of dopamine metabolism in the rat brain. Neurochem. Int. 1:221-227, 1985. Wilson, J.F. and Morgan, M.A.: Cyclic changes in concentra- tions of a-melanotropin in the plasma of male and female rats. J. Endocrinology fiz:361-366, 1979. Wilson, J.F. and Harry, F.M.: Release, distribution and half- life of a-melanotropin in the rat. J. Endocrinology §§:61-67, 1980.