THESHS Date 4!: _ ' 1 ‘ Emmi“ gm“ 3 1293 10178 9448 1 University *fi This is to certify that the thesis entitled REGULATION OF TUBEROHYPOPHYSEAL DOPAMINERGIC NEURONS presented by Richard H. Alper has been accepted towards fulfillment of the requirements for Pharmacology & Toxicology Major prof ssor August 12, 1981 0-7639 OVERDUE FINES: 25¢ per day per item RETURNING LIBRARY MATERIALS: Place in book return to remove charge from circulatton records REGULATION OF TUBEROHYPOPHYSEAL DOPAMINERGIC NEURONS By Richard H. Alper 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 1981 flsmqmv ABSTRACT REGULATION OF TUBEROHYPOPHYSEAL DOPAMINERGIC NEURONS BY Richard H. Alper Radioenzymatic assays for dopamine (DA) and dihydroxyphenylalanine (DOPA) were used to compare the regulation of DA synthesis in the posterior pituitary, median eminence and striatum, brain regions which contain terminals of the tuberohypophyseal, tuberoinfundibular and nigrostriatal DA systems, respectively. DOPA accumulation after decarboxylase inhibition was used as an in 3132 estimate of DA synthesis and neuronal activity. The DA concentrations in the posterior pituitary, median eminence and striatum of rats were unaltered following five days of water deprivation or 2% NaCl drinking water. DOPA accumulation in the posterior pituitary, but not in the median eminence or striatum, was increased following three days of dehydration. The plasma sodium concentration and hematocrit were increased with 12-24 hours dehydra- tion. DOPA accumulation in the posterior pituitary and the plasma sodium concentration, but not the hematocrit, were increased following five days of saline drinking. The data suggest that DA synthesis in the posterior pituitary is selectively increased following extended periods of hypernatremia. Richard H. Alper DOPA accumulation in the posterior pituitary was increased 24 hours after the administration of hypertonic saline or mannitol when rats were not permitted access to water. DOPA accumulation had re— turned to control 36 and 48 hours after saline, but increased again at 72 hours. These data suggest that tuberohypophyseal DA neurons are latently activated by consequences of marked intracellular dehydration induced by hyperosmotic stimuli. Three days of dehydration consistently increased DA synthesis in the posterior pituitary and the plasma sodium concentration. Both returned to control within 1-3 hours of rehydration; the hematocrit returned much slower. This suggests that increased tuberohypophyseal dopaminergic activity following dehydration is rapidly reversible and parallels plasma or cellular osmolality, but not plasma volume. In summary, only those DA neurons terminating in the posterior pituitary are regulated by intracellular osmolality. The tuberohypo- physeal DA neurons are slow to be activated, but their increased activity can be rapidly restored to control values by rehydration. The significance of the dehydration—induced activation of tuberohypo— physeal DA neurons cannot be ascertained until the functional role of these neurons is understood. to the memory of Dorothy Eligator Alper August 8, l920—August 18, 1976 ii ACKNOWLEDGEMENTS I would like to express my most sincere appreciation to Dr. Kenneth E. Moore for the research opportunities he has been able to offer for the last four years. I would also like to thank the members of my thesis committee, Drs. T.M. Brody, G.D. Fink and G.I. Hatton for their time and con— structive criticism. I am especially grateful to Greg Fink for the hours of helpful advice and discussion throughout the course of my research. I would like to offer special thanks collectively to all of the members of Dr. Moore's lab with whom I have had the pleasure to work. In particular, I feel extremely pleased to have been able to colla— borate closely with Dr. Keith Demarest. I received invaluable train— ing under his guidance. Also, I am very glad to have made a long— lasting friendship with my fellow classmate Craig Johnston. Finally, I emphatically thank the superb technicians with whom I have worked; Mrs. Sue Stahl and Mrs. Mirdza Gramatins. Sue Stahl for her patience and tireless effort, Mirdza Gramatins for her sincere warmth, kindness and understanding. iii TABLE OF CONTENTS Page DEDICATION ii ACKNOWLEDGEMENTS iii LIST OF FIGURES vii LIST OF TABLES viii INTRODUCTION 1 I. Neuroanatomy 4 II. Functions of Dopaminergic Pathways 12 III. Regulation of Neuronal Activity 16 A. Catecholamine Biochemistry 16 B. Biochemical Techniques 22 C. Regulatory Mechanisms 24 1. Receptor mediated regulation 24 2. Endproduct feedback regulation 26 3. Regulation by morphine and other opiates ----- 27 4. Endocrinological and other related regulatory mechanisms 28 a. Prolactin 28 b. ’ aéMelanocyte stimulating hormone -------- 29 c. Dehydration 31 PURPOSE 33 MATERIALS AND METHODS 34 I. Animals 34 II. Tissue Dissections and Extraction 34 III. Radioenzymatic Assays 36 A. Dopamine and Norepinephrine Determinations -------- 36 B. DOPA Determinations 39 IV. Preparation of Catecholfgfmethyltransferase 41 V. Experimental Treatments 43 VI. Plasma Analysis 44 iv TABLE OF CONTENTS (continued) VII. VIII. RESULTS Drug Treatments —— A. WMUOUJ Statistical Analyses a-Methyltyrosine-induced Decline of Dopamine and Norepinephrine DOPA Accumulation After NSD 1015 6-Hydroxydopamine Treatment Mannitol Infusion Vasopressin Infusions Equithesin Anesthesia I. II. III. Concentration and Synthesis of Catecholamines-----—---— A. Effects of Superior Cervical Ganglionectomy on Catecholamine Concentrations B. In_Vivo Estimates of Catecholamine Synthesis ------ 1. a-Methyltyrosine-induced decline of dopamine and norepinephrine 2. DOPA accumulation after NSD 1015 a. Effects of 6-hydroxydopamine on cate- cholamine concentrations and DOPA accu- mulation ' b. Effects of intravenous administration of NSD 1015 on DOPA accumulation Dehydration A. Effect of Dehydration on Dopamine Concentrations-- B. Effect of Dehydration on DOPA Accumulation -------- 1. Water deprivation in male and female rats--—- 2 Food deprivation 3. Time course of water deprivation 4 Comparison of water deprivation and saline drinking 5. Hypertonic saline injections 6. Mannitol infusion a. 1.5 ml of 20% mannitol b. 5.0 ml of 20% mannitol C. Effect of High Sodium Diet on DOPA Accumulation--- D. Summary Rehydration A. Effect of Saline and Water Rehydration on DOPA Accumulation Page 45 45 46 47 47 49 50 50 52 52 52 54 55 59 59 61 61 61 64 64 67 7O 73 75 84 84 88 91 91 93 93 TABLE OF CONTENTS (continued) Page B. Time Course of Water Rehydration on DOPA Accumula- tion 94 IV. Vasopressin 100 A. Effect of Continuous Vasopressin Infusion on DOPA Accumulation 100 DISCUSSION 103 I. Concentration and Synthesis of Catecholamines--—------ 103 II. Dehydration and Rehydration 107 A. Delayed Activation of the Tuberohypophyseal Dop- amine Neurons 107 B. Hyperosmolality and Tuberohypophyseal Dopaminergic Activity 111 C. Hypovolemia and Tuberohypophyseal Dopaminergic Activity 114 III. Possible Functions for the Tuberohypophyseal Dopamine Neurons 115 A. Vasopressin Release 116 1. Acute regulation 116 2. Augmentation 116 3. Feedback inhibition 118 4 Differential regulation of storage and re- leasable pools 120 5. Indirect regulation 121 a. Pituicyte function 122 b. Blood flow 123 B. Oxytocin Release 125 C. Prolactin Release 127 SUMMARY AND CONCLUSIONS 130 BIBLIOGRAPHY 134 vi LIST OF FIGURES Figure Page 1 Schematic diagram of the distribution of the dopaminer— gic nerves represented in mid-sagital views of the rat brain 3 2 Schematic representation of the catecholaminergic in— nervation of the median eminence-pituitary region of the rat as demonstrated by histofluorescent microscopy- 8 3 Schematic representation of catecholamine synthesis---- 19 4 The aMT-induced decline of dopamine and norepinephrine in the striatum, median eminence and posterior pitui- tary 57 5 DOPA accumulation in the striatum and posterior pitui- tary after intravenous administration of NSD 1015 in unanesthetized, restrained rats 63 6 Effects of water or food deprivation on DOPA accumula- tion in selected brain regions 69 7 Effects of hypertonic saline injection on DOPA accumu- lation in the posterior pituitary, and on plasma sodium concentration, and hematocrit 80 vii Table 10 ll 12 LIST OF TABLES Effects of superior cervical ganglionectomy on cate- cholamine concentrations in selected brain regions ----- Synthesis rates of catecholamines in selected brain regions calculated from the aMT-induced decline of catecholamines Effects of 6-hydroxydopamine on catecholamine concen- trations and DOPA accumulation in selected brain regions Effect of dehydration on the dopamine concentration in selected brain regions Effect of water deprivation on DOPA accumulation in selected brain regions of male and ovariectomized rats- Effects of water deprivation on dopamine and norepine- phrine concentrations in selected brain regions -------- Effects of water deprivation on DOPA accumulation in selected brain regions, and on plasma sodium and hema— tocrit Effects of water deprivation or saline drinking on catecholamine concentrations and DOPA accumulation in the posterior pituitary, and on plasma sodium, hemato- crit and plasma protein Effects of water deprivation on plasma sodium and hematocrit Effects of hypertonic saline injection on plasma so- dium, plasma osmolality and hematocrit Effect of hypertonic saline injection on DOPA accumu- lation in the posterior pituitary Effects of hypertonic saline injection on DOPA accumu- lation in the posterior pituitary, and on plasma so- Page 53 58 60 65 66 71 72 74 76 77 78 dium and hematocrit viii 82 LIST OF TABLES (continued) Table l3 14 15 16 17 18 19 20 21 22 23 24 Page Effects of hypertonic saline injection on plasma sodium and hematocrit in rats allowed free access to water---- Effects of hypertonic saline injection on DOPA accumu- lation in the posterior pituitary, and on plasma sodium and hematocrit Acute effects of hypertonic mannitol infusion on plasma sodium, plasma osmolality and hematocrit Effects of mannitol infusion on DOPA accumulation in the posterior pituitary, and on plasma sodium and hema- tocrit Acute effects of hypertonic saline and mannitol on plasma sodium, plasma osmolality and hematocrit -------- Effects of hypertonic saline and mannitol on DOPA accu- mulation in the posterior pituitary, and on plasma sodium, plasma osmolality and hematocrit Effects of high sodium diet on DOPA accumulation in the posterior pituitary, and on plasma sodium and hemato- crit Effects of dehydration and rehydration on DOPA accumu— lation in the posterior pituitary, and on plasma so- dium, hematocrit and plasma protein Effects of water deprivation and subsequent rehydration on DOPA accumulation in the posterior pituitary, and on plasma sodium and hematocrit Effects of water deprivation and subsequent rehydration on DOPA accumulation in the posterior pituitary, and on plasma sodium and hematocrit Effects of water deprivation and subsequent rehydration on DOPA accumulation in the posterior pituitary, and on plasma sodium and hematocrit Effects of continuous vasopressin infusion on DOPA accumulation in selected brain regions, and on plasma sodium and hematocrit 83 85 86 87 89 90 92 95 97 98 99 101 INTRODUCTION Histofluorescent and biochemical studies have revealed several distinct dopaminergic neuronal systems in the rat brain (reviewed by Lindvall and ijrklund, 1978; Moore and Bloom, 1978). The nigro- striatal dopamine (DA) system was the first to be studied in great detail. It constitutes the major ascending dopaminergic fiber tract in the brain (see Figure 1). The tuberoinfundibular DA system has been studied extensively using histofluorescent techniques. Bio— chemical analysis has become possible only recently with the develop- ment of sensitive microanalytical techniques capable of measuring the nanogram quantities of DA and norepinephrine (NE) which are present in the median eminence, which contains terminals of the tuberoinfundibu- lar DA neurons. The tuberoinfundibular dopaminergic pathway is known to be involved in the regulation of hormone secretion from the anterior pituitary gland (Weiner and Ganong, 1978). Mechanisms by which the activity of both the nigrostriatal and tuberoinfundibular DA neurons are regulated have been reviewed recently (Moore and wuerthele, 1979). A third group of DA neurons depicted in Figure l comprise the tuberohypophyseal system. Very little is known about this pathway even though it was revealed by early histofluorescent (Bjorklund, 1968; ijrklu d g£_§1., 1970; Smith and Fink, 1972) and electron Figure 1. Schematic diagram of the distribution of the dopaminer- gic nerves represented in mid—sagital views of the rat brain. The major ascending DA systems are presented in the upper figure: cp, caudate-putamen (striatum); ML, mesolimbic DA system; na, nucleus accumbens; NS, nigrostriatal DA system; ot, olfactory tubercle; sn, substantia nigra. The hypothalamic DA systems are shown in greater detail in the lower figure: AP, anterior pituitary; ar, arcuate nucleus; HP, hypophyseal portal system; NIL, neuro-intermediate lobe of the pituitary (posterior pituitary); pv, periventricular hypothalamic nucleus; TI, tuberoinfundibular DA system; TH, tubero— hypophyseal DA system. Modified from ijrklu d 35 31. (1973), MOore and wuerthele (1979), Moore and Bloom (1978) and Ungerstedt (1971). o .u 9:0": -......... P ‘ 00030-0. ”50*‘0‘lfl. A . “new. .. 2.”... .. oo'.\ .0 0“ 0 qutv .a O... ..00. I I .93. .. K“. .31.... .0 NH.‘ 0 u a a ‘- 0, u. o a... .W. s. . «PM o...- . Us» 0 .0 'pO-A . “321 0 c . . . hoe... 0..- J t3 QM.- -q.“ u . ".o \:-’ .‘o‘ I ' ‘A‘ Tl [I Figure 1 4 microscopic (Baumgarten 25 31., 1972) analyses. One reason is that sensitive analytical procedures required to measure the catecholamines contained in the posterior pituitary (the combined neural and inter- mediate lobes) have only recently been developed. A second reason is that no function has been ascribed to DA in the posterior pituitary. The present studies represent the first experiments designed specifically to characterize factors regulating the activity of the tuberohypophyseal DA neurons. The regulation of DA synthesis in the terminals of the tuberohypophyseal neurons was compared and contrasted to the regulation of DA synthesis in the terminals of the tuberoinfun- dibular and nigrostriatal DA neurons. The nigrostriatal system was included in these studies primarily because most of the information on the relationship between neuronal activity and DA turnover has been ascertained from studies conducted on this system. The tuberoinfundi— bular DA system was included in these studies to determine if DA synthesis in the terminals of the anatomically related tuberoinfundi- bular and tuberohypophyseal DA systems is regulated by similar mecha- nisms. I. Neuroanatomy The initial description of the monoamine-containing neurons in the the posterior pituitary was by ijrklund (1968). Several other subsequent reports using histofluorescent techniques (Bj6rklund gt 31,, 1970, 1973; Smith and Fink, 1972; Tilders g£H§1., 1979) or electron microscopy (Baumgarten gt al., 1972) have appeared in the literature. Although these neuroanatomical techniques provide invaluable information 5 they have several serious deficiencies, particularly when used to study the catecholaminergic innervation of the posterior pituitary. First, great variability in the fluorescent patterns observed through- out the posterior pituitary was reported in the initial studies (Bjdrklund, 1968; ijrklund gt_§l,, 1970). ijrklund and coworkers (1973) partially overcame this obstacle by administering admethyl NE to their rats prior to sacrifice. This treatment enhanced the fluor- escent intensity of all catecholaminergic (both dopaminergic and noradrenergic) neurons. A second methodological problem, one that could not be overcome, was the inability of histofluorescent tech- niques to clearly distinguish between DA and NE; both amines are present in the posterior pituitary. The investigators (Baumgarten 35 a1,, 1972; ijrklu d gt al., 1970) felt that the histofluorescence remaining in the posterior pituitary following bilateral removal of the superior cervical ganglia represented only DA—containing neurons. The NE in the posterior pituitary was postulated to be contained in terminals of peripheral sympathetic nerves. Holzbauer g£_al, (1980b) have used biochemical assays to demonstrate that approximately 50% of the noradrenergic innervation of the posterior pituitary originates in the superior cervical ganglia. This has been subsequently confirmed (see Results I.A). The histofluorescent descriptions of the dopami- nergic innervation of the posterior pituitary may not be completely accurate due to the interference of NE. In addition, newer anatomical techniques (e.g., horseradish peroxidase and immunocytochemistry) have not been applied successfully to study the tuberohypophyseal DA system. 6 Therefore, the anatomical descriptions that follow are based solely on results of histofluorescent and electron microscopic studies. The tuberohypophyseal DA neurons may actually be two distinguish- able systems (ijrklund_ E al., 1973). One component originates from a small group of cells in the most rostral regions of the arcuate nucleus (area A12; Dahlstrbm and Fuxe, 1964). These neurons innervate the entire intermediate lobe of the pituitary after passing through the median eminence and infundibular stalk. A second group of DA perikarya lies immediately caudal to this first group. These neurons also pass through the median eminence but innervate the neural lobe. As pointed out by ijrklund 35 31. (1974), one cell in the rostral arcuate nucleus may have terminals in the median eminence as well as the neural and intermediate lobes of the pituitary. For this reason, the arbitrary assignation of the names tuberohypophyseal and tubero— infundibular DA systems projecting to localized sites in the posterior pituitary and median eminence, respectively, may be misleading. The catecholaminergic innervation throughout the median eminence- pituitary region of the rat appears to be a continuum of terminals (Figure 2). There is no clear anatomical demarcation between the median eminence, the infundibular stem and the posterior pituitary. In the present studies the tuberohypophyseal DA system will be consi- dered as one fiber tract (in deference to BjBrklund gt 31., 1973) terminating in the posterior pituitary. The tuberoinfundibular DA system, terminating in the median eminence, will be considered as another (Cuello 35 al., 1973; see Materials and Methods II for de- tailed description). Figure 2. Schematic representation of the catecholaminergic innerva- tion of the median eminence-pituitary region of the rat as demonstrated by histofluorescent microscopy. Abbreviations: VIII, third ventricle; NL, neural lobe of the pituitary; PI, pars intermedia (intermediate lobe of the pituitary); PD, pars distalis (anterior pituitary). Taken from Bjorklund gt a1. (1973). \\ I. k 'V ’ I i c ‘ 7 {2‘ 1" ’, -9. VIII .,/.£g .. 3% }‘-\l:\ ‘ g ' a " 0. ~ ’ '4 7 h.“ . - ‘ O ' . 4;, , ' 71 , ‘ ¢’“I::’1C:£;\:- con "‘4' :4 i .1. ,‘O _ on “H: \fih “#qu W ‘3 I‘ in" M t.“ i 1"“.‘1 H \IIJ‘TL " I “Hi M "51““ I I Figure 2 9 Histofluorescent analysis has demonstrated that the cell bodies of the tuberohypophyseal DA neurons are contained within the medial basal hypothalamus (Tilders gt_§1,, 1979). The axons are predomi- nantly of a fine varicose type which form a plexus between the endo— crine cells of the intermediate lobe (ijrklu d gt al., 1970; Tilders §£_§1,, 1979). In the intermediate lobe the DA neurons may make direct contact on hormone secreting cells. These hormone secreting cells probably contain a-melanocyte stimulating hormone (MSH). DA most likely acts to tonically inhibit MSH secretion from the pituitary (for review see Tilders and Smelik, 1977). In the neural lobe the dopaminergic neurons have been reported to approximate (80-120 A), but not to make true synaptic contacts with (no membrane thickenings) neurosecretory axons and pituicytes (Baumgarten gt_al,, 1972). Tilders gt_§1, (1979) observed a plexus of DA terminals in the neural lobe (particularly the rostral aspects) around the neurohypophyseal capillary beds. An unusual feature of the nerve fibers in the posterior pituitary is the occurrence of large axonal swellings (greater than 2 u in diameter) filled with typical DA fluorescence (Baumgarten gt al., 1972; ijrklund, 1968). It was suggested that this fluorescence re- presents spontaneously degenerating DA axons and that tuberohypo— physeal DA fibers are undergoing continuous reorganization through degeneration-regeneration cycles. The significance of this is unclear, however. 10 The DA neurons which terminate in the median eminence are part of a large, diverse tuberoinfundibular neuronal system. The cell bodies of these tuberoinfundibular tracts reside primarily in the medial basal hypothalamus. This has been demonstrated with a variety of techniques, including Golgi methods (Bodoky and Rethelyi, 1977), surgical isolation (Halasz gt_§l,, 1965), histofluorescent methods (ijrklund gt_§1,, 1973) and microiontophoresis of horseradish peroxi- dase into the median eminence (Lechan g£_§l,, 1980). The anatomical distribution of the tuberoinfundibular DA neurons in the rat has been the subject of several recent reviews (Lindvall and ijrklund, 1978; Moore and Wuerthele,l979; Moore and Bloom, 1978). Histofluorescent studies have used selective mechanical and electrolytic lesions (ijrklund gt_al,, 1973, 1974) to suggest that the cell bodies of the tuberoinfundibular DA system lie in the arcuate and anterior periventricular hypothalamic nuclei (A Dahlstrbm and 12‘ Fuxe, 1964). These dopaminergic perikarya are not densely packed and constitute only a small fraction of the cells found in the arcuate nucleus (Bodoky and Rethelyi, 1977; Fuxe and kufelt, 1967; Renaud 35 .31., 1978). One report suggests that some DA neurons terminating in the median eminence originate in the ventral tegmental area (Kizer 35 21,, 1976b). There is, however, much histochemical (Jonsson g£_al,, 1972; Lbfstrbm gt 31., 1976) and biochemical (Brownstein 35 31., 1976; Gallardo gt 31., 1978; Gudelsky gt 31., 1978) evidence demonstrating that the DA concentration of the median eminence is not reduced following complete hypothalamic deafferentation. 11 The majority of the tuberoinfundibular DA neurons have short axons projecting in a dorsoventral orientation from all parts of the arcuate nucleus. These neurons terminate in corresponding parts of the external layer of the median eminence (ijrklund g£_§1,, 1973; Smith and Fink, 1972). In the external layer, the terminals are packed very densely in close proximity to the capillaries of the hypothalamo-hypophyseal portal system (Fuxe and kufelt, 1967). DA is released from these nerve terminals in the median eminence and trans- ported by the hypophyseal portal circulation to tonically inhibit prolactin secretion from the anterior pituitary gland (Gibbs and Neill, 1978; Weiner and Ganong, 1978). The tuberoinfundibular DA nerves also terminate in the lateral palisade zone of the median eminence. Here the DA-containing neurons are in close proximity to neurons containing luteinizing hormone releasing hormone (gonadotropin releasing hormone; Hdkfelt gt_§l,, 1976; Sladek 35 31., 1978) and tanycytes (Sladek and Sladek, 1978). The precise topographical projections of the nigrostriatal DA system have been recently reviewed (Moore and Wherthele, 1979; Mbore and Bloom, 1978). A brief description of this major ascending dopami- nergic pathway is provided because the relationships between the electrical activity (i.e., firing rate) and biochemical activity (i.e., DA synthesis and turnover) in dopaminergic systems has been obtained from studies on the nigrostriatal neurons. The axons forming the nigrostriatal DA pathway arise from cells mainly in the pars compacta of the substantia nigra (areas A and A 8 9 of the rat brain; Dahlstrbm and Fuxe, 1964), although a few may 12 originate in the adjacent ventral tegmental area (A10; Dahlstrom and Fuxe, 1964). The DA cell bodies in the substantia nigra are very densely packed, comprising approximately 80% of the total cells in this region (Andén g£_§1,, 1966). The fine unmyelinated DA axons (Lindvall and ijrklund, 1974) ascend from the substantia nigra through the medial tegmentum to traverse the lateral hypothalamic areas and the internal capsule (Ungerstedt, 1971; Moore gt_§l,, 1971). Some fibers terminate in the globus pallidus but most distribute throughout the striatum (the caudate-putamen). The axons undergo massive collateralization in the striatum forming synaptic-like con- tacts with small striatal dendrites (Hattori 32 31., 1973; McGeer gt 31,, 1975). The nigrostriatal DA projection has a highly specific and well organized topography (Carpenter and Peter, 1972; Moore et 31., 1971). II. Functions of Dopaminergic Pathways The anatomical descriptions of the tuberohypophyseal, tuberoinfun- dibular and nigrostriatal DA fiber tracts provided above document the diverse regions of the brain receiving dopaminergic innervation. DA would be expected to perform a variety of functions with such a wide- spread distribution throughout the central nervous system. Some of the functions postulated for DA in these diverse neuronal networks will be discussed briefly. The presence of DA in the posterior pituitary gland of a variety of mammals has been known for more than a decade. Fuxe and Hdkfelt (1967) proposed that catecholamines probably influenced the release of l3 hormones from the posterior pituitary. Recent technicological ad— vances such as immunocytochemistry, radioimmunoassay and high pressure liquid chromatography provide sensitive analytical techniques for studying the various peptides and hormones of the posterior pitui- tary. However, an unqualified function for DA in the posterior pitui- tary has yet to be elucidated. There is now substantial evidence for the tonic dopaminergic inhibition of MSH secretion from the intermediate lobe of the pitui- tary (reviewed by Tilders and Smelik, 1977). DA antagonists (halo- peridol, pimozide and sulpiride) rapidly and markedly increase, whereas DA agonists (bromocriptine and apomorphine) decrease the serum MSH concentration in rats (Penny_gtual., 1979; Penny and Thody, 1978; Usategui, 1976). These dopaminergic agents appear to act on DA receptors in the intermediate lobe and not in higher brain centers. DA, in a concentration dependent manner, inhibits the in yi££g_re— lease of MSH from isolated posterior pituitaries (Bower 25 al., 1974; Tilders gt 31., 1979). The addition of potassium into the incubation medium also inhibits MSH secretion in 31539, this results from potas- siumrinduced DA release (Tilders 25 31., 1979). These data imply that the tuberohypophyseal DA neurons inhibit MSH secretion. Nevertheless, other biogenic amines and polypeptides may also be involved in the regulation of MSH release from the posterior pituitary (Tilders and Smelik, 1977). The evidence for dopaminergic mediation or modulation of other posterior pituitary hormones is not as convincing (Brown gt_§1,, 1979). (30mplicating factors are that DA can influence hormone release from 14 two sites. The first is at the magnocellular neurosecretory neurons located primarily in the supraoptic and paraventrciular hypothalamic nuclei. The second site where DA could alter the release of posterior pituitary hormones is at the nerve terminals in the neurohypophysis. Some examples of contradictory reports will be provided. The addition of DA to isolated neural lobes may stimulate (Bridges 35 31,, 1976; Negro-Vilar, 1979) or not alter (Hisada g£_§l,, 1977) arginine vasopressin (AVP; antidiuretic hormone) secretion. Alternatively, in giyg_studies have reported that DA inhibits (Givant and Sulman, 1976) or does not affect (Kendler gt_§1,, 1978) the release of AVP into the blood. To date, the role of tuberohypophyseal DA neurons on tonic AVP secretion is uncertain. It is possible that the tuberohypophyseal DA neurons do not tonically regulate AVP secretion but are involved as modulators during stimulation of the hypothalamo—neurohypophyseal system. For example, in a recent abstract Robinson gt a}, (1981) demonstrated that the DA agonist bromocriptine augmented AVP secretion in humans administered hypertonic saline intravenously. This study needs to be confirmed. The data relating DA to oxytocin release is also contradictory. DA inhibits oxytocin release from hypothalamo-neurohypophyseal system explants (Seybold gt 31., 1978), stimulates oxytocin release from isolated neural lobes (Bridges 25 al., 1976), but may not be involved in the release of oxytocin in_y}yg_(Fuchs §£_al,, 1981; Russell gt a1” 1981). 15 The effects of DA on other posterior pituitary functions have not been studied. However, some hypothetical alternatives are included in Section III of the Discussion. The tuberoinfundibular DA neurons terminate very near the capil- lary loops of the hypothalamo-hypophyseal portal system which invagi- nate the ventral surface of the median eminence. Thus, DA released into the portal blood may be transported directly to the anterior pituitary gland to regulate hormone secretion. Consequently, suffi- cient quantities of DA have been found in the hypophyseal portal blood to tonically inhibit the release of prolactin from the anterior pitui- tary (Gibbs and Neill, 1978). The DA concentration in the portal blood is significantly greater than that found in the systemic circu- lation (Ben—Jonathan gt a1., 1977). It is currently believed that the principle function of the tuberoinfundibular DA neurons is in the tonic regulation of prolactin (and possibly luteinizing hormone) secretion from the anterior pituitary (for review see Weiner and Ganong, 1978). The nigrostriatal DA system is primarily involved in the regula- tion and coordination of motor functions. Degeneration of the nigro- striatal pathway is believed to be responsible for the akinesia and rigidity associated with Parkinson's disease (Hornykiewicz, 1966, 1977). The most specific lesion found in Parkinson's disease is the loss of melanin-containing neurons in the substantia nigra. The neurochemical correlate associated with this lesion is a marked depletion of the striatal DA concentration. In animal studies the 16 nigrostriatal DA neurons have been postulated to mediate the stereo- typed behaviors induced by DA agonists (e.g., dfamphetamine or apo- morphine) but not the locomotor activation induced by these drugs (for review see Moore and Kelly, 1978). III. Regulation of Neuronal Activity A. Catecholamine Biochemistry Impulse traffic in aminergic systems in the central nervous system can be studied using electrophysiological techniques. The activity of central DA and NE neurons can be estimated by recording from the cell bodies in the substantia nigra and pons-medulla, respec- tively. This is not a difficult task in the nigrostriatal DA system since the cell bodies in the substantia nigra are densely packed. Electrophysiological techniques have not been used to study the tuberohypophyseal DA neurons. Antidromic stimulation from the poste- rior pituitary to the arcuate nucleus could be used to identify cells belonging to the tuberohypophyseal DA system. The likelihood of recording either intracellularly or extracellularly from an identi- fiable DA perikarya in the arcuate nucleus is very small. The DA cells (particularly those projecting to the posterior pituitary and not the median eminence) constitute a very minor proportion of the total cellular population of the arcuate nucleus. Therefore, bio— chemical indices of DA turnover must be used to estimate dopaminergic activity in the posterior pituitary. Most of the neurochemical events occurring in DA nerve terminals have been studied in the striatum (see review by Moore and l7 Wuerthele, 1979). The relationships existing between neuronal acti— vity and the synthesis and release of DA from the nerve terminals in the posterior pituitary and median eminence are assumed to be similar to the events that have been previously described in the striatum. A schematic representation of catecholamine synthesis is depicted in Figure 3. Tyrosine is transported into DA neurons and converted to L-dihydroxyphenylalanine (DOPA) by the rate limiting enzyme tyrosine hydroxylase. This enzyme is regulated, in part, by endproduct feedback inhibition. Therefore, decreases in the releasable pool of DA will result in increases in DA synthesis; the converse is also true. Endproduct inhibition generally maintains the concentration of DA in the nerve terminals constant despite changes in the amount of DA released. DOPA is rapidly decarboxylated to DA by aromatic L-amino acid decarboxylase (DOPA decarboxylase). In dopaminergic neurons the newly synthesized DA can be stored in synaptic vesicles or released into the synaptic cleft in response to neuronal depolarization. In noradrenergic neurons, DA is hydroxylated to form NE by the enzyme dopamine B-hydroxylase. This enzyme is contained in the synaptic vesicles of noradrenergic neurons and is absent from dopaminergic neurons. NE, like DA, is released from nerve terminals in response to action potentials. Catecholamines are released into the synaptic cleft where they can interact with putative pre- and postsynaptic receptors. The amount of amine in the synapse is dependent on its rate of release and metabolism. High affinity uptake systems for both DA and NE are the 18 Figure 3. Schematic representation of catecholamine synthesis. Repre— sented to the left of the arrows are the enzymes involved in each reac— tion, to the right are inhibitors of these enzymes. The dashed line connecting DA and NE indicates that dopamine B-hydroxylase is present only in noradrenergic nerve terminals. This enzyme is not found in dopaminergic neurons. Tyrosine Hydroxylase Aromatic L-Amino Acid Decarboxylase Dopamine B—Hydroxylase 19 Tyrosine DOPA Dopamine <.--_-_-_ Norepinephrine Figure 3 a-Methyltyrosine 3-Hydroxybenzylhydrazine (NSD 1015) 20 major routes of inactivation for these amines (exceptions do exist, see below). Following reuptake, DA and NE are exposed to mitochon- drial monoamine oxidase and are deaminated to form 3,4—dihydroxy- phenylacetic acid (DOPAC) and 3,4—dihydroxyphenylethylglycol (DOPEG), respectively, in the brain. These deaminated catechols are further metabolized by catecholfgfmethyltransferase (COMT) to form homovanillic acid (from DOPAC) and 3-methoxy-4-hydroxyphenylethylglyco1 (MHPG; from DOPEG). In the striatum the concentration of DOPAC provides a useful index of alterations in the functional activity of the nigrostriatal DA neurons (Roth 35 31., 1976). That is, an increase in the striatal DOPAC concentration generally reflects an increase in nigrostriatal dopaminergic activity while a decrease in DOPAC reflects a decrease in neuronal activity. The utility of measuring DOPAC concentrations for biochemical indices of neuronal activity in various brain regions requires the presence of a high affinity DA uptake system. This system is not present in the posterior pituitary (Annunziato and Weiner, 1980; Demarest and Moore, 1979b) or the median eminence (Annun- ziato_g§mal., 1980; Demarest and Moore, 1979b). An inadequate reuptake mechanism may be partially responsible for the low DOPAC concentration reported in the posterior pituitary (Annunziato and Weiner, 1980; Umezu, Alper and Moore, unpublished observations) and the median eminence (Fekete £5 31., 1979; Umezu and Moore, 1979). The lack of a high affinity DA uptake system and the resultant low concentration of DOPAC explain why this DA metabolite is not a good index of neuronal 21 activity for the tuberohypophyseal and tuberoinfundibular DA neurons (Moore gt £13, 1979; Umezu and Moore, 1979). The concentration of MHPG or its sulfate metabolite has been used to estimate noradrenergic neuronal activity in the brain (Adér £5.2l3: 1978; Kohno gt al,, 1981; Korf 32 EA" 1973). To date, this technique has not been employed to estimate the activity of the nor- adrenergic neurons terminating in the posterior pituitary or median eminence (NE is virtually undetectable in the striatum). The events described above characterize DA biochemistry in the striatum. Most, but not all, of the same events occur in the median eminence (reviewed by Moore and wuerthele, 1979). One of the major differences is that the dopaminergic terminals in the median eminence end in close approximation to the capillaries of hypothalamo— hypophyseal portal system. Thus, dopamine released from these neurons does not act on receptors located across a synaptic cleft. Instead, this amine is thought to be transported to the anterior pituitary to activate DA receptors located on prolactin-secreting cells. The events occurring after DA is released in the posterior pituitary are not known. Putative DA receptors have been described in the posterior pituitary (Sibley and Creese, 1980; Stefanini £5 31., 1980a; but the lack of a high affinity DA uptake mechanism leads one to speculate that DA may be released directly into the neurohypophyseal circulation. Thus, there would be no need for a high affinity reuptake mechanism to inactivate DA in the posterior pituitary, analogous to the median eminence. 22 B. Biochemical Techniques Biochemical estimates of dopaminergic nerve activity rely on measurements of DA turnover and synthesis. The concentration of DA (and NE) in nerve terminals does not change appreciably even when impulse flow is markedly altered. It is assumed that synthesis of the amine is increased to replenish the amount released. Since the rate limiting step in catecholamine synthesis is the hydroxylation of tyrosine by tyrosine hydroxylase, ingigg_and in gitrg_estimates of the activity of this enzyme will generally reflect catecholamine synthesis and neuronal activity. Despite the number of assumptions that must be made to measure catecholamine turnover and synthesis (Weiner, 1974), useful information about the regulation of dopaminergic neurons has been obtained employing biochemical techniques. Two nonsteady state pro- cedures to estimate catecholamine synthesis and turnover rates in 2329. are the decline of catecholamine concentrations after synthesis inhi- bition (Brodie et al,, 1966; Spector gt_al,, 1965) and the rate of DOPA accumulation following decarboxylase inhibition (Carlsson 25 al., 1972; Carlsson and Lindqvist, 1973; Demarest and Moore, 1980) (see Figure 3). The rate of decline of both DA and NE after inhibition of tyrosine hydroxylase by a-methyltyrosine (aMT) follows first-order kinetics and is proportional to neuronal activity (Brodie g£_§l., 1966). When the activity of catecholamine neurons is increased, the rate of decline following aMT is similarly increased. This method 23 allows for separate estimations of both DA and NE turnover in brain regions such as the posterior pituitary and median eminence where both amines are present. Tyrosine hydroxylase activity can be estimated in_yi!g by quantifying the rate of accumulation of DOPA following the administra- tion of the centrally acting DOPA decarboxylase inhibitors benserazide (Ro4-4602) or 3-hydroxybenzylhydrazine (NSD 1015; see Figure 3). Roth g£_al, (1975) have demonstrated that the rate of DOPA accumulation in the striatum after the administration of a decarboxylase inhibitor is directly related to the activity of the nigrostriatal DA neurons; an increase in neuronal activity is reflected in an increase in the rate of DOPA accumulation. Since tyrosine hydroxylase is contained in all catecholami- nergic neurons, changes in enzyme activity reflect changes in DA and/or NE synthesis. This is not a problem in the striatum where the NE concentration is virtually zero. However, in the posterior pitui- tary and median eminence the ratio of DA to NE is about 3:1 (see Results, Tables 1-3). Kizer_egnal. (1976a) observed that in_yi££g tyrosine hydroxylase activity estimates DA synthesis, but not NE synthesis, in the median eminence. It will be demonstrated in Results Section I.B that the rate of DOPA accumulation after decarboxylase inhibition (an in vizg_measurement of tyrosine hydroxylase activity) reflects primarily DA synthesis in the posterior pituitary and median eminence. 24 C. Regulatory Mechanisms 1. Receptor mediated regulation The activity of the nigrostriatal DA neurons is altered by dopaminergic drugs, both agonists and antagonists. These drugs are believed to activate either postsynaptic DA receptors which regulate nigrostriatal activity through a long neuronal striatonigral feedback loop and/or presynaptic DA receptors (autoreceptors) located on the dopaminergic neurons (see reviews by Moore and Wuerthele, 1979; Nowycky and Roth, 1978). The location of the receptors mediating the effects of the dopaminergic agents on neuronal activity and DA synthe- sis is irrelevant for the purpose of this discussion. Acute systemic injections of DA antagonists (e.g., haloperidol, chlorpromazine, spiroperidol, pimozide) increase: 1) the nigrostriatal firing rate (Bunney gt 31., 1973a), 2) the release of DA in the striatum (Chéramy gt_al,, 1970), 3) the concentration of DOPAC in the striatum (Andén gt_§l,, 1964), 4) the aMT-induced decline of DA in the striatum (Gudelsky and Moore, 1977), and 5) the rate of DOPA accumulation in the striatum (Demarest and Moore, 1979a, 1980). Conversely, drugs that stimulate DA receptors (e.g., apomorphine, bromocriptine, piribedil, L-DOPA) decrease: l) nigrostriatal firing rate (Bunney gt al., 1973b), 2) striatal DOPAC concentrations (Roos, 1969), 3) the aMT—induced decline of DA in the striatum (Andén g£_al., 1967), and 4) the rate of DOPA accumulation in the striatum (Demarest and Mbore, 1979a). That is, DA antagonists increase whereas DA agonists decrease both electrophysiological and biochemical measure- ments of nigrostriatal dopaminergic neuronal activity. 25 The tuberohypophyseal DA system appears to be regulated, in part, by receptor mediated mechanisms. Demarest and Moore (1979a) have reported that haloperidol caused a moderate increase in the rate of DOPA accumulation in the posterior pituitary (45% as compared to an increase of 400% in the striatum). Apomorphine, on the other hand, caused only a moderate decrease in the rate of DOPA accumulation in the posterior pituitary when compared to the decrease observed in the striatum. Although the tuberohypophyseal DA neurons are regulated by receptor mediated mechanisms, DA synthesis in the posterior pituitary is not quantitatively as sensitive to dopaminergic drugs as is DA synthesis in the striatum. The tuberoinfundibular DA system, on the other hand, is unresponsive to acute injections of DA agonists or antagonists (Demarest and Moore, 1979a, 1980; Fuxe and kufelt, 1974; Gudelsky and Moore, 1976, 1977). Autoreceptors have been postulated to play an important role in modulating the synthesis of DA in the nigrostriatal DA system (Nowycky and Roth, 1978). The administration of y-butyrolactone (GBL) or baclofen, both of which inhibit impulse flow in the nigrostriatal DA system, increase the concentration and rate of synthesis of DA in the striatum (Carlsson 25 31., 1977; Demarest and Mbore, 1979a; Moore and Demarest, 1980; Walters g£_§l,, 1973). It is believed that com- plete cessation of impulse flow leads to the inhibition of DA released into the synaptic cleft. The presynaptic DA receptors are no longer activated leading to a disinhibition of tyrosine hydroxylase. The combination of an increase in synthesis and a decrease in release of DA causes marked elevations in the striatal DA concentration. 26 Both baclofen and GBL increase the concentration and the rate of synthesis of DA in the posterior pituitary but not in the median eminence (Demarest and Moore, 1979a; Moore and Demarest, 1980). Apparently, the tuberohypophyseal DA system, like the nigrostriatal DA system, is regulated, in part, by presynaptic autoreceptors. In summary, DA synthesis in the posterior pituitary and the striatum, but not in the median eminence, is regulated by blockade and activation of DA receptors. 2. Endproduct feedback regulation As noted in section III.A. of the Introduction, tyro- sine hydroxylase is regulated by endproduct feedback inhibition. This is true in the tuberohypophyseal, tuberoinfundibular and nigrostriatal DA systems. Demarest and Moore (1979a) have reported that reserpine decreased the DA concentration and increased the rate of DOPA accumu- lation (i.e., tyrosine hydroxylase activity) in the posterior pituitary, median eminence and striatum. In these same regions the monoamine oxidase inhibitor nialamide increased the DA concentration and de- creased the rate of DOPA accumulation. More recently, the same authors (Demarest and Moore, in press) have demonstrated that the selective Type A.monoamine oxidase inhibitor clorgyline mimicked the actions of nialamide, whereas the Type B monoamine oxidase inhibitor deprenyl did not. Thus, it was suggested that the intraneuronal deamination of DA in central dopaminergic pathways is catalyzed by Type A monoamine oxidase and that tyrosine hydroxylase in all central dopaminergic systems is regulated by endproduct feedback. 27 3. Regulation by morphine and other opiates The interactions of narcotic analgesics and, more recently, the opioid peptides with central catecholaminergic neuronal systems have been the subject of intensive investigation (see review by Iwamoto and Way, 1979). Briefly, it has been demonstrated that acute administration of morphine increased single unity activity in the substantia nigra (Nowycky gt 31., 1978), the rate of DOPA accumu- lation in the striatum (Alper g£_§l,, 1980; Garcia-Sevilla 25 al., 1978; Persson, 1979), the concentration of DOPAC in the striatum (Alper gt 31., 1980; Nowycky g£_§l,, 1978), the release of DA in the striatum (Chesselet gt_§l,, 1981) and the aMT-induced decline of DA in the striatum (Alper £5 31., 1980). On the other hand, morphine decreases the aMT-induced decline of DA in the median eminence (Alper g£_§l,, 1980; Deyo gt_§l,, 1979), the rate of DOPA accumulation in the median eminence (Alper gt .al., 1980) and the release of DA into the hypophyseal portal blood (Gudelsky and Porter, 1979). In contrast, DA synthesis and turnover were not altered in the posterior pituitary following morphine admini- stration (Alper gt 31., 1980). The results of these neurochemical studies reveal that morphine stimulates the nigrostriatal DA system, inhibits the tubero- infundibular DA system and does not influence the tuberohypophyseal DA system. None of these dopaminergic pathways are tonically regulated by the endogenous opiates since naloxone administration pg£_§g_does not alter DOPA accumulation in the striatum, median eminence or posterior pituitary (Alper gt al., 1980). 28 4. Endocrinological and related regulatory mechanisms Hypothalamic monoaminergic mechanisms regulating the release of hormones from the anterior and posterior pituitary gland have been reviewed by many authors (see, for example, Knowles and Vollrath, 1974; Weiner and Ganong, 1978). It is now realized that hormones of the pituitary may, in turn, influence various central catecholaminergic neuronal pathways OMoore‘gt_al,, l980a,b; Moore and wuerthele, 1979). Three endocrinological regulatory mechanisms will be discussed below. a. Prolactin. The dopaminergic inhibition of pro- lactin secretion from the anterior pituitary has been discussed pre— viously (see Introduction, Section II). It has been demonstrated using both histochemical and biochemical techniques that injections of rat or ovine prolactin systemically or into the cerebrospinal fluid (CSF) increase tuberoinfundibular dopaminergic neuronal activity 12—16 hours after the prolactin injections (Annunziato and Moore, 1978; kufelt and Fuxe, 1972; Wiessel 33 31., 1978). Prolactin has been implicated in the regulation of tuberoinfundibular but not nigro- striatal DA nerves. High doses of haloperidol latently increase DA turn- over in the median eminence and not the striatum following hypophysec- tomy or hypothalamic deafferentation (Demarest and Moore, 1980; Gudel- sky gt al., 1978). Furthermore, recent data suggest that the tubero- infundibular, but not the tuberohypophyseal, DA neurons are activated following estradiol benzoate administration (Moore §£_§l3, 1980b), intraventricular prolactin infusions (Johnston gt_al., 1980) and pituitary transplantation under the kidney capsule (Morgan and Herbert, 29 1980). All treatments are known to significantly elevate serum or CSF prolactin concentrations. In summary, prolactin is involved in the selective regulation of tuberoinfundibular dopaminergic neuronal activity. The tuberohypophyseal and nigrostriatal DA systems are unresponsive to hyperprolactinemia. b. a-Melanocyte stimulating hormone. DA inhibits the secretion of MSH from the intermediate lobe of the pituitary. It has been proposed, though certainly not unequivocally proven, that MSH secretion might be regulated through an inhibitory feedback loop (Kastin and Schally, 1967). One could speculate that the tuberohypo- physeal DA neurons are involved in MSH autoregulation, similar to the role of the tuberoinfundibular DA system in prolactin secretion. Lichtensteiger and coworkers (Lichtensteiger and Lienhart, 1977; Lichtensteiger and Monnet, 1978, 1979; Lichtensteiger g£_§l,, 1977) have examined the fluorescent intensity in the arcuate nucleus and substantia nigra following systemic injections of MSH. The results are quite complex but the basic interpretation put forth by the authors is that MSH increases the activity of the DA neurons in the arcuate nucleus and, to a slightly lesser degree, the activity of the nigrostriatal DA neurons. There are two serious flaws to these experiments. The first is that changes in fluorescent intensity in dopaminergic cell bodies in the substantia nigra and arcuate nucleus do not necessarily correlate with the amount of DA released from the terminals of these neurons. Secondly, the DA cell bodies in the 30 arcuate nucleus do not constitute a homogeneous population. The neurons project to different areas throughout the median eminence- posterior pituitary region (ijrklund g£_al,, 1973) and respond differ- entially to pharmacological treatments and endocrinological manipula- tions (Alper gt al., 1980; Moore gt 31., l980b). Subcutaneous injections of MSH cause a dose related increase in the rate of DOPA accumulation in the posterior pituitary, but not in the median eminence or striatum (Alper and Moore, unpub- lished observations; Moore E£.§ln’ l980b). The effect is rapid (ob- served only 60-90 minutes following MSH administration) and small though statistically significant (DOPA accumulation is increased 20- 25% above control). The small effect could be related to the obser— vations of ijrklund_ E El. (1973) on the divisions of the tuberohypo— physeal DA system. MSH might regulate only the activity of the DA neurons terminating in the intermediate lobe, but not the neural lobe, of the pituitary. The neural lobe contains more DA (and probably DOPA) than the intermediate lobe (Saavedra gt 31., 1975). The studies by Moore gt_al, (l980b) analyzed the combined neural and intermediate lobes for DOPA. The data do lend some support to the hypothesis that the tuberohypophyseal DA nerves are part of an MSH feedback loop and suggest that the tuberohypophyseal DA nerves can be influenced by a factor (MSH) that has no effect on tuberoinfundibular and nigrostriatal DA neurons. 31 c. Dehydration. The effects of dehydration on central catecholamines have been reported in several papers (Holzbauer _gtual., 1978, l980a,b; Torda gt 31., 1979). Torda and coworkers (1979) reported that water deprivation of 72 hours increased the DA concentration in the posterior pituitary; no other brain region was examined. Holzbauer gt a1. (1978, l980b) generally, although not always, observed an increase in the DA content of the posterior pitui- tary following 72 hours of water deprivation plus an additional 18-96 hours access to a 2.5% NaCl drinking solution. There was no change in the DA content of the striatum or medial basal hypothalamus. In contrast to reports by Torda gt_al, (1979), Holzbauer gt 31. (1978) observed no change in the DA content of rats deprived of water for 72 hours. Furthermore, the posterior pituitary content of DA was not altered in homozygous Brattleboro rats or their heterozygous controls following 72 hours of water deprivation and then 24 hours access to 2.5% NaCl (Holzbauer gt_al,, l980a). The authors of the papers cited above suggest that prolonged stimulation of the hypothalamo-neurohypophyseal system alters the functional state of the tuberohypophyseal DA neurons. Although the data on posterior pituitary DA concentrations are inter- esting, they are difficult to interpret. An increase in the steady state DA concentration in terminals of the tuberohypophyseal neurons could result from: 1) a decrease in the rate of release, 2) an in- crease in the rate of synthesis which exceeds release, or 3) a de- crease in the rate of intraneuronal metabolism. Therefore, it is 32 impossible to precisely determine functional changes in tuberohypo— physeal dopaminergic neuronal activity by measuring the concentrations of DA in the posterior pituitary. Furthermore, it was not determined if dehydration was selectively altering the tuberohypophyseal dopami- nergic system or if dehydration was affecting the metabolic activity in all dopaminergic pathways in the rat brain. PURPOSE The synthesis of DA in the terminals of tuberohypophyseal neurons is regulated, in part, by mechanisms which are also intrinsic to the ascending nigrostriatal and mesolimbic dopaminergic neurons termina- ting in various forebrain regions. In particular, tyrosine hydroxylase in the posterior pituitary is regulated through endproduct feedback inhibition and receptor mediated mechanisms. Tuberohypophyseal DA neurons in contrast to the anatomically similar tuberoinfundibular DA neurons are not regulated by the anterior pituitary hormone prolactin. Reports have suggested that the functional activity of the tuberohypo- physeal DA neurons might be altered during prolonged dehydration. It is unknown whether dehydration increases or decreases tuberohypophyseal dopaminergic neuronal activity. Furthermore, the mechanisms under- lying the dehydration—induced effects on the dopaminergic neurons terminating in the posterior pituitary have not been characterized. The purposes of the present studies are to: 1) demonstrate that the tuberohypophyseal.DA neurons are tonically active. This will be accomplished using tW°.lE@X£!2 biochemical estimates of DA synthesis and turnover, and 2) use a biochemical index of DA synthesis to characterize the responses of the tuberohypophyseal DA neurons to dehydration. 33 MATERIALS AND METHODS I. Animals Male and female Sprague-Dawley rats (150-250 g) were obtained from Spartan Research Animals, Haslett, MI, and maintained under 12 hour periods of light and dark (lights on from 0700 to 1900 h). Rats were housed 4 per cage and allowed free access to food and tap water for at least 3 days prior to the start of any experiment. II. Tissue Dissections and Extraction Rats were sacrificed by decapitation. The brain was carefully removed from the skull leaving the entire pituitary gland in the sella turcica. The pituitary gland was gently freed of connective tissue, removed from the skull and placed on a cold plate. The neural and intermediate lobes of the pituitary (the posterior pituitary) were teased from the anterior pituitary with a pair of fine forceps under a dissecting microscope. The median eminence was removed from the base of the hypothalamus with the aid of a dissecting microscope as described previously (Cuello g£_§l,, 1973). While holding the pituitary stalk with fine forceps, cuts were made with a pair of iris scissors at the lateral and frontal borders (defined by the presence of capillary loops). The average protein content of the entire median eminence dissected as 34 35 described was approximately 30 pg. This corresponds to 0.3—0.4 mg wet weight, assuming 0.11 mg wet weight/10 pg protein, as generally ob- served in brain tissue. In one experiment (see Table 5 in Results) the median eminence was dissected into rostral and caudal portions. The caudal median eminence (11:2 pg protein; n=8) was that portion of the pituitary stalk remaining with the brain but not securely attached to the tuber cinereum. The rostral median eminence (20:3 pg protein; n=8) was the tissue forming the floor of the third ventricle. The striatum (containing caudate, putamen and globus pallidus) was dissected using a modification of the method described by Glowin— ski and Iversen (1966). A frontal slice of brain tissue was dissected using a razor blade. The first cut was approximately 1 mm anterior to the optic chiasm. The forebrain was then discarded. A second razor cut was made through the brain just anterior to the optic chiasm. The remaining slice of tissue was approximately 1 mm thick. A small piece of striatum (3-5 mg wet weight) was dissected from this slice within the region delineated by the lateral ventricles as the internal limit and the corpus callosum as the external limit. The dissection as described included tissue from the head of the striatum exclusively. Upon dissection, brain tissues were immediately homogenized in appropriate volumes of ice cold 0.2 N perchloric acid containing 10 mg % disodiumethylenediamine-tetraacetic acid (EDTA). The homogenates were frozen at -15°C for analysis within two weeks; the samples were stable over this period of time. On the day of assay the homogenates were thawed on ice, centrifuged for 15 seconds in a Beckman 152 Micro— fuge to obtain a clear supernatant and 10 pl aliquots of the super- natants were analyzed for DA and NE or DOPA as described in Materials 36 and Methods Section III. The protein content of the remaining pellet was measured by the method of Lowry_gtual, (1951). The tissue con— centrations of DA and NE were expressed as ng catecholamine/mg pro- tein; the rate of DOPA accumulation was expressed as ng DOPA/mg protein/10 or 30 minutes (see below). III. Radioenzymatic Assays A. Dopamine and Norepinephrine Determinations DA and NE were assayed simultaneously using a modification of radioenzymatic assays described previously (Moore and Phillipson, 1975; Umezu and Moore, 1979). For catecholamine determinations, the posterior pituitary, median eminence and pineal gland were homogenized in 30 pl of 0.2 N perchloric acid containing 10 mg % EDTA; the striatum was homogenized in 100 pl of this same acid solution. The assay involved incubation of the catecholamines in the presence of partially purified COMT and S—adenosyl—L-[methyl-3H]methionine (3H-SAM) resulting in formation of the tritiated gfmethylated products of DA and NE, 3- methoxytyramine and normetanephrine, respectively. The labeled meta- bolites were separated from interfering gfmethylated catechols and unreacted 3H-SAM by extraction into an organic solvent, back extraction into acid and final separation by thin-layer chromatography. The DA and NE contents of the samples were calculated directly from standards after subtracting blank values; blanks were obtained by carrying duplicate 10 pl aliquots of the perchloric acid solution through the entire assay as described in detail below. A 10 pl aliquot of the tissue supernatant or combined DA and NE standard (in duplicate; 0.125-2.0 ng of both amines in 0.2 N 37 perchloric acid containing 10 mg % EDTA) was added to a 5 ml conical centrifuge tube. The centrifuge tubes in this and all subsequent steps were kept on ice. Twenty-five microliters of freshly prepared incubation mix were then added to each tube containing standard or tissue extract. The incubation mix for a 50 tube assay consisted of 84 p1 of 20 mM ethyleneglycol tetraacetic acid (EGTA), pH 7.2, 416 pl of partially purified COMT in 1 mM sodium phosphate, pH 7.0 (see Materials and Methods Section IV for details of enzyme preparation), 250 p1 of 3H—SAM (9-10 Ci/mmole, 250 pCi/ml; New England Nuclear Corp., Boston, MA), 84 pl of 8 mg pargyline HCl/ml 10% B-mercapto- ethanol and 540 p1 of l M Tris base containing 3 mM MgCl The final 2. pH of the incubation mix was 9.0-9.4, the optimum pH for this reaction (Nikodijevik gt a1., 1969). The COMT enzyme, 3H-SAM and Tris base were stored in small volumes at -15°C; the pargyline solution was prepared fresh daily. After addition of the incubation mix the tubes were briefly vortexed and placed in a water bath at 37°C for 60 minutes allowing the gfmethylation reaction to go to completion. The tubes were re- moved from the bath, placed on ice and 30 p1 of a mixture containing 5 volumes of 0.45 M borate buffer, pH 10.0, and 1 volume of methoxyamine carrier (5 mg/ml of both 3-methoxytyramine and normetanephrine plus 0.5 mg/ml of sodium metabisulfate in distilled water) were added; the tubes were vortexed quickly. In rapid succession, 20 pl of 1.5% tetraphenyl boron and 550 pl of toluene-isopentyl alcohol (3:2) were added; the tubes were vortexed and centrifuged. Four-hundred seventy- five microliters of the organic phase were transferred to 5 ml conical 38 centrifuge tubes containing 250 p1 of 0.45 M borate buffer, pH 10.0. The tubes were vortexed, centrifuged and 400 pl of the organic phase were then transferred to 5 ml conical centrifuge tubes containing 40 pl of 0.1 N HCl. Again, the tubes were vortexed and centrifuged. The organic phase was aspirated and the acid phase was washed with 250 p1 of water-saturated ethylacetate. The tubes were vortexed and centri- fuged; the organic phase was then aspirated. Twenty-five microliters of the acid phase were spotted on thin-layer chromatography plates precoated with silica gel (LK6D, Linear K, Whatman Inc., Clifton, NJ). The spots were allowed to dry for 30 minutes at room temperature and the chromatography plates were then developed for approximately 1.5 hours until the solvent front was about three cm from the top of the plate. The developing solvent was a mixture of methylamine-ethanol- chloroform (5:18:40). The spots were visualized after spraying with phenol reagent (Folin and Ciocalteu, Harleco, Gibbstown, NJ) diluted 1:1 with distilled water. The 3-methoxytyramine spot (Rf 0.78-0.82) and the normetanephrine spot (Rf 0.50-0.55) were scraped into scin- tillation vials. The tritiated amines were extracted into 0.5 ml of acetic acid-ethylacetate-water (3:3:1) for 30 minutes. After addition of 10 ml of toluene-ethanol (7:3) with 0.5% 2,5-diphenyloxazole (PPO; Research Products International Corp., Elk Grove Village, IL) radio- activity was determined in a Beckman LS 100 liquid scintillation counter (20-24% efficiency). The sensitivity of the assay, determined by the amount of DA or NE with counts twice those observed in the blank, showed slight daily variation. The background and sensitivity for DA were 70-100 cpm and 50-75 pg, respectively. For NE the corresponding values were 39 20-40 cpm as background, 30-50 pg for the lower limit of sensitivity. The assay was linear to at least 4 ng for both amines. B. DOPA Determinations DOPA was analyzed in brain tissue homogenates using a modi- fication of radioenzymatic assays described previously (Hefti and Lichtensteiger, 1976; Demarest and Moore, 1980). For DOPA determina- tions, the posterior pituitary and median eminence were homogenized in 20 pl of 0.2 N perchloric acid containing 10 mg % EDTA, whereas the striatum was homogenized in 100 pl of this same solution. The homo- genates were centrifuged in a Beckman 152 Microfuge to obtain clear supernatants. For radioenzymatic analysis DOPA was incubated in the presence of partially purified COMT and 3H—SAM resulting in the forma- tion of the tritiated gfmethylated product of DOPA, 3-methoxytyrosine. The labeled metabolite was separated from interfering gfmethylated catecholamines and unreacted 3H-SAM by cation exchange chromatography, adsorption onto activated charcoal and then anion exchange chromato- graphy. The DOPA content of each sample was calculated directly from standards after subtracting blank values. Blanks were obtained by carrying duplicate 10 p1 aliquots of the perchloric acid solution through the entire assay as described in detail below. Ten microliters of perchloric acid extracts of brain tissue from animals administered NSD 1015 (3-hydroxybenzylhydrazine dihydro- chloride; Sigma Chemical Co., St. Louis, MO; see Materials and Methods Section VII.B. for details) or standard (0.125-2.0 ng DOPA) were added to 5 ml conical centrifuge tubes on ice. Twenty-five microliters of fresly prepared incubation mix were then added to each tube. The 40 incubation mix was identical to that for the DA/NE assay except that a DOPA decarboxylase inhibitor, gfbenzylhydroxylamine (9 mg/ml 10% B— mercaptoethanol, prepared fresh daily), was substituted for pargyline. After incubation (37°C for 60 minutes) the reaction was stopped by placing the tubes on ice and adding 1.0 ml of ice-cold 0.1 M citrate buffer, pH 2. The samples were passed over cation exchange columns (15x5 mm; AG 50W+X4, H+ form, 200-400 mesh) which had been previously prepared with 3.0 ml of 0.1 M phosphate buffer, pH 6.5, and 1.5 ml of 0.1 M citrate buffer, pH 2.0. The columns were then washed with 5.0 ml of the same citrate buffer and 0.5 m1 of 0.1 M citrate buffer, pH 4.5; the radioactive 3-methoxytyrosine was eluted with 2.5 ml of 0.1 M citrate buffer, pH 4.5, and collected into 12x75 mm disposable tubes. The remaining steps were performed at room temperature. The 3-methoxy- tyrosine was adsorbed onto paraffin—treated activated charcoal (Asatoor and Dalgliesh, 1956) by the addition of 50 pl of a slurry consisting of approximately 2.5 g treated charcoal in 5 ml distilled water to each tube. The tubes were then vortexed and centrifuged, the aqueous supernatant was aspirated. The charcoal was washed with 2.5 ml of 0.5% acetic acid, and the 3-methoxytyrosine was eluted from the char- coal by vortexing with 1.0 ml of 5% phenol followed by centrifugation. The supernatant was transferred to test tubes (13x100 mm) containing 0.2 ml of 2 N HCl plus 1.0 m1 of 0.5 M piperazine. The phenol was extracted with 2.0 ml of water-saturated ethylacetate, vortexed and centrifuged. The organic phase was aspirated and the pH of the samples was adjusted to 10.0-10.5 by the addition of 3.0 ml of 0.2 M piperazine, pH 10.5. The samples were then passed over an anion 41 exchange column (15x5 mm; AG l—X2, OH- form, 200-400 mesh) which had been prepared with 5.0 ml of l N NaOH, 5.0 m1 of distilled water and 5.0 m1 of 0.2 M piperazine, pH 10.5. After addition of the samples, the columns were washed with 5.0 m1 of 0.2 M piperazine. The tri- tiated 3-methoxytyrosine was eluted in 3.0 m1 of 0.2 M piperazine, pH 6.0, and collected directly in scintillation vials. Fifteen milli- liters of ACS scintillation cocktail (Amersham/Searle Inc., Chicago, IL) were added; the radioactivity in each sample was determined by liquid scintillation spectrometry. Similar to the radioenzymatic assay for catecholamines, the sensitivity as defined by the amount of DOPA yielding counts at least twice the blank (125-600 cpm) varied daily. The sensitivity was generally between 50-150 pg DOPA. At this sensitivity endogenous concentrations of DOPA (i.e., in animals without NSD 1015 pretreat- ment) were not detectable in any brain region. The assay was linear to at least 2 ng of DOPA. To avoid interassay variation, individual brain regions from each experiment were analyzed for DA and NE or DOPA in the same assay. Furthermore, DA and NE concentrations were not determined in animals that were administered NSD 1015 as this treatment altered brain cate- cholamine concentrations (Demarest, Alper and Moore, unpublished observations). IV. Preparation of Catecholfgfmethyltransferase Partially purified COMT was prepared using a modification of the procedure as described by Moore and Phillipson (1975). All procedures 42 were carried out at 0°—4°C. Livers (140 g) from rats fasted overnight were homogenized in a Waring Blender (5 seconds) in 500 m1 of ice cold 1.1% KCl. Aliquots of this liver suspension were further homogenized in a glass homogenizer with a motor driven TeflonR pestle. The homo- genate was centrifuged at low speed (14,000 x g) for 10 minutes. The supernatant was transferred to ultracentrifuge tubes and spun at 95,000 x g for 60 minutes. The resulting supernatant was filtered through glass wool to remove most of the fat. Generally the procedure was stopped after this step and the K01 supernatant was refrigerated overnight. On the following day the pH was adjusted to 5.3 by adding ice-cold l N acetic acid. The acidified mixture was stirred for 10 minutes and then centrifuged. This and all subsequent centrifugations were carried out at 4°C at a force of 14,000 x g for 10 minutes. The supernatant was decanted and the pH adjusted to 6.8 with 0.5 M sodium phosphate buffer, pH 7.0. For every 100 m1 of solution, 17.7 g of solid ultrapure ammonium sulfate (Schwarz/Mann, Orangeburg, NY) were added slowly. The suspension was stirred gently for 10 minutes and then centrifuged. A second ammonium sulfate precipitation was performed on the supernatant fraction (16.2 g ammonium sulfate/100 m1 of supernatant). Following centrifugation the supernatant was discarded and the pellet was resuspended in 40 ml of cold 45% ammonium sulfate solution (14 g ammonium sulfate/50 ml of 0.1 M sodium phosphate buffer, pH 7.0). This was stirred for 15 minutes, the precipitate was removed by centrifugation and the supernatant was again discarded. This proce- dure was repeated with 20 ml of 33% ammonium sulfate (5 g/25 ml of 0.1 M 43 sodium phosphate buffer, pH 7.0). This time, however, the supernatant fraction was saved; 4.85 g solid ammonium sulfate was added slowly while stirring. The suspension was then stirred for an additional 15 minutes. After centrifugation the resulting pellet was dissolved in 4.0 m1 of 1 mM sodium phosphate buffer, pH 7.0, with 0.1 mM dithio— threitol to form the final enzyme solution. This solution was care- fully placed in dialysis tubing that had been soaked for 24 hours in a 1% EDTA solution. Dialysis was overnight at 4°C against 4 liters of the 1 mM phosphate buffer, pH 7.0. The partially purified enzyme solution was divided into 250 p1 aliquots and stored at -15°C. Fresh enzyme was prepared every 5 or 6 weeks; no change in activity was observed over this period of time. V. Experimental Treatments In general, rats were allowed 3 days to acclimate to the animal facilities prior to the start of any experiment. Throughout all treatments (except when noted) standard commercial rat chow (Wayne Lab Blox, Allied Mills, Chicago, IL) containing 0.39% sodium was available .ad libitum. The initial studies involved the effects of water depri— vation or the replacement of normal drinking water (tap water) with a 2% NaCl solution. When rats deprived of water for 3 days were rehy- drated for short periods of time (1-6 hours) they were first placed in individual cages to insure free access to water bottles; rats rehy- drated for periods longer than 6 hours were maintained 4 per cage. Hypertonic saline (15% NaCl; 5 m1/kg, s.c.) was administered to rats in several experiments. This is a very potent dispogenic stimulus. 44 In several experiments the animals were allowed free access to water after the injection of 15% NaCl, but in most experiments water bottles were removed from the cages following the hypertonic saline injection. As noted above, the animals were provided a normal rat chow. In one experiment the rats were deprived of food for 3 days and in another experiment they were provided a high sodium diet (High sodium diet, 3.15%, Ralston Purina Co., St. Louis, MO). The high sodium diet had the following composition; sodium chloride, 7.25%, casein, 21.0%; sucrose, 15.0%; solka floc, 3.0%; RP vitamin mix, 2.0%; RP mineral mix #11, 5.0%; dl-methionine, 0.15%; choline chloride, 0.2%; corn oil, 5.0%; lard, 5.0%; dextran, 36.4%. The sodium content of this food was approximately 10 times greater than the normal chow (3.15% sodium as compared to 0.39%). VI. Plasma Analysis Trunk blood was collected at the time of sacrifice in beakers containing 100 U heparin in 0.1 ml of 0.9% NaCl. The blood was imme— diately transferred to 12x75 mm test tubes and samples were taken in heparinized microcapillary tubes for triplicate hematocrit determina- tions. The blood was centrifuged (1100 x g, 10 minutes) at room temperature. Plasma was transferred to 10x75 mm test tubes, sealed with ParafilmR and frozen at -20°C for later analysis. Plasma sodium concentrations were determined in all samples by flame photometry (Instrumentation Laboratory Inc, 343 Flame Photometer). Plasma osmo— lality was determined using a Wescor 5100B Vapor Pressure Osmometer. 45 The plasma protein concentration was estimated by the method of Lowry g£_§l, (1951). The plasma analyses were used as determinants of plasma volume (Kutscher, 1971). VII. Drug Treatments A. a—Methyltyrosine-induced Decline of Dopamine and Norepine- phrine dMT inhibits tyrosine hydroxylase, the rate limiting step in catecholamine synthesis (Spector gt al., 1965). DA and NE concentra- tions in central and peripheral neurons decline with time following the administration of aMT. This rate of decline is proportional to neuronal activity, follows first order kinetics, and can be used to calculate synthesis rates and turnover times of tissue catecholamines (Brodie gt al., 1966). Catecholamine synthesis was estimated by quantifying the decline of endogenous DA and NE concentrations after synthesis inhi- bition with aMT (a-methyltyrosine methylester HCl; Regis Chemical Co., Chicago, IL). Rats were injected intraperitoneally (i.p.) with 250 mg uMT free base/kg (dMT was dissolved in distilled water) 0, 30, 60 or 90 minutes prior to sacrifice (see Figure 4). DA and NE concentra— tions were measured using the radioenzymatic assay as described in Section III.A. above. Rate constants (and the 99% confidence inter- vals) for the decline of the amines were calculated by least squares regression analysis (Goldstein, 1964) as described in detail by Brodie gt 31. (1966). The synthesis rates were calculated as the zero time catecholamine concentration (i.e., the steady state concentration) 46 multiplied by the rate constant for the appropriate amine and were expressed as ng catecholamine/mg protein/hour. B. DOPA Accumulation after NSD 1015. The rate of DOPA accumulation in the striatum (Carlsson and Lindqvist, 1973; Demarest and Moore, 1980) and in the median eminence (Demarest and Moore, 1980) after decarboxylase inhibition is an in yiyg_estimate of tyrosine hydroxylase activity and, therefore, DA synthesis. Roth 35 31. (1976) have shown a direct correlation between changes in the firing rate of the nigrostriatal DA neurons and changes in the rate of DOPA accumulation (i.e., DA synthesis) in the striatum. These changes occur in the absence of changes in the striatal con- centration of DA. It is therefore assumed that an increase in the rate of DA synthesis in the terminals of any DA neuronal system without a concomitant change in the DA concentration reflects an increase in neuronal activity. A time course and dose-response relationship for the accu- mulation of DOPA following the administration of the decarboxylase inhibitor NSD 1015 have been described previously (Carlsson and Lindqvist, 1973; Demarest and'Moore, 1980). When administered i.p., 100 mg NSD 1015/kg (100 mg/ml of 0.9% NaCl) caused a maximal inhibi- tion of DOPA decarboxylase and a linear accumulation of DOPA in the striatum and median eminence for at least 30 minutes. Except when noted, NSD 1015 was administered to the rats in a dose of 100 mg/kg, i.p., 30 minutes prior to sacrifice. In 2 experiments NSD 1015 was administered intravenously (i.v.) in a dose of 25 mg/kg via the tail vein in unanesthetized, restrained rats. This dose of NSD 1015 was 47 found to cause a maximal rate of DOPA accumulation which increased linearly for 10-20 minutes (see Results Section I.B.2.b.). Therefore, in experiments requiring i.v. administration, 25 mg NSD 1015/kg was injected 10 minutes prior to sacrifice. C. 6—Hydroxydopamine Treatment The catecholaminergic neurotoxin 6-hydroxydopamine HBr (6— OHDA; Sigma Chemical Co., St. Louis, MO) was infused into the lateral cerebroventricles to cause a selective depletion of brain NE. Male rats were anesthetized with Equithesin, placed in a stereotaxic frame and implanted bilaterally with 23 gauge stainless steel cannula guides with the tips located at bregma, i1.5 mm lateral and 2.8 mm below dura (Pellegrino and Cushman, 1968). The cannula guides were anchored to the skull by stainless steel screws and dental cement. One week after the implantation of the cannula guides, 3 pl of 6-OHDA (12.5 pg of free base) or its vehicle (0.9% NaCl containing 0.1% ascorbic acid) were infused bilaterally (l pl/minute) three times at 48 hour inter- vals through 30 gauge stainless steel cannulae which extended 1 mm below the tip of the guide cannulae. The injection cannulae were connected to a motor driven Hamilton syringe with polyethylene tubing. The animals were sacrificed 48 hours after the last infusion. D. Mannitol Infusion Mannitol is an osmotic diuretic (Mudge, 1975). This agent can be administered in sufficient quantities to cause a large increase in plasma, glomerular filtrate and tubular fluid osmolality, resulting in a large increase in urine flow. Mannitol is used clinically 48 because: 1) it does not penetrate cellular membranes, 2) it is not metabolized, and 3) it is rapidly excreted by the kidney (Lazorthes and Campan, 1972). Mannitol was infused intravenously to cause a rapid cellular dehydration as a consequence of an osmotic load. Mannitol was infused into the jugular vein of unrestrained rats (average weight of 200 grams) surgically implanted with a chronic polyvinyl jugular catheter exiting subcutaneously on the back of the animal as modified from the method described by Weeks (1962). The cannula consisted of a 30 mm length of polyvinyl tubing (0.01" inner diameter x 0.03" outer diameter) glued (Krazy Glue, Krazy Glue Inc., Chicago, IL) to a second 100 mm length of polyvinyl tubing (0.02" inner diameter x 0.06" outer diameter). Under Equithesin anesthesia the shorter piece of tubing was inserted into and tied to the jugular vein. The larger tubing was then passed subcutaneously behind the forelimb and was exited through a small incision at the base of the rat's neck. The cannula was secured to the skin with a drop of glue and was occluded with a 23 gauge needle. The patency of the cannula was tested one day prior to the experiment by injecting 0.1 m1 of methohexital sodium (BrevitolR, 10 mg/ml; Eli Lilly and Co., Indiana- polis, IN), a short acting barbiturate. The cannula was then flushed with 0.2 ml of 0.9% NaCl containing heparin (10 U/ml) and the animal was returned to its individual cage. The delivery system for the mannitol infusion was a glass syringe (fitted with a 23 gauge needle) driven by a Harvard infusion pump. Attached to the needle was a length of polyethylene tubing 49 sufficient to permit the rats free, unrestrained movement during the infusion. A short piece of 23 gauge stainless steel tubing was used to connect the polyethylene tubing to the cannula. Infusions of hyperosmotic sorbitol or mannitol stimulate drinking (Holmes and Gregersen, 1950). If the animals are permitted access to water after the infusion of mannitol they drink sufficient volumes of water to maintain an approximately normal state of cellular hydration. Therefore, immediately following the mannitol infusion, rats were returned to cages and not provided drinking water. In all mannitol experiments the time of sacrifice was determined from the end of the infusion. E. Vasopressin Infusions Synthetic AVP (Sigma Chemical Co., St. Louis, MO) was con- tinuously infused into rats for three days. The doses of AVP infused were 240 and 720 mU/rat/day. The lower dose has been reported to be the replacement dose of AVP necessary to restore normal urine volumes to Brattleboro rats deficient in AVP (G. Fink, personal communcation). The higher dose was chosen because rats deprived of water for two to three days exhibit plasma AVP concentrations 3-4-fold higher than normal (Dunn 35 31., 1973; Rougon-Rapuzzi 35 al,, 1978). It was felt that the infusion of 3 times more AVP then the replacement dose might approximate circulating concentrations of AVP observed in three day dehydrated rats. The AVP was delivered continuously for three days by means of AlzetR osmotic minipumps (Alza Corp., Palo Alto, CA). Under anesthesia a small length of polyvinyl tubing (0.01" inner diameter x 50 0.03" outer diamater) was inserted into and tied to the jugular vein of rats weighing approximately 300 grams: sham surgery consisted of jugular ligation. The tubing was threaded subcutaneously to a mini- pump which was secured under the skin of the animal's back. The flow rate of the pump was 1 pl/hour. AVP was diluted in 0.9% NaCl to the appropriate concentrations to allow for delivery of either 240 or 720 mU/rat/ day. F. Equithesin Anesthesia All surgical procedures (ovariectomy, superior cervical ganglionectomy, lateral ventricular cannulation, jugular catheteriza— tion and minipump implantation) were performed under Equithesin anes- thesia. Equithesin contains: chloral hydrate, 21.25 g; pentobarbi- tal, 4.86 g; magnesium sulfate, 10.63 g; propylene glycol, 221.7 ml; 95% ethanol, 60.0 ml; distilled water to 500 m1. Ovariectomies were performed on female rats administered 2 m1 Equithesin/kg, i.p.; all other surgery was performed on male rats administered 3 ml Equithe- sin/kg. VIII. Statistical Analyses All values presented represent the mean i 1 standard error of the mean (S.E.). Data were initially analyzed by a one-way analysis of variance; differences between means were determined by Student-Newman— Keuls' test with the level of significance set at p<0.01 (Steele and Torrie, 1960). 51 The GMT-induced decline of DA and NE were analyzed by regression analysis (Goldstein, 1964). The lines plotted are the best fit according to this analysis, and the rate constants (k) and 99% con— fidence limits were computed. RESULTS 1. Concentration and Synthesis of Catecholamines A. Effects of Superior Cervical Ganglionectomy on Catecholamine Concentrations Catecholamine-containing nerve terminals have been demon- strated in the median eminence-pituitary region of the rat and other mammals (for review see ijrklund gt 31., 1974). All of the DA in the posterior pituitary appears to be contained in the terminals of tuberohypophyseal neurons originating in the arcuate nucleus. On the basis of histofluorescent analysis it was initially proposed that NE in the posterior pituitary was in neurons of central origin (Bj6rklund, 1968) but it was later suggested that NE neurons were "exclusively of peripheral sympathetic origin" (ijrklu d 33 a1., 1970). The first study was designed to quantify the concentrations of DA and NE in the median eminence-pituitary region of the rat and to determine the contribution of the peripheral sympathetic nervous system to the noradrenergic innervation of this region. DA and NE were measured in selected brain regions seven days following bilateral superior cervical ganglionectomy (SCGx) or sham surgery, consisting of exposure but not removal of the SCG. The data are presented in Table l. The concentrations of DA and NE in the posterior pituitary of sham-control animals are in good agreement with those reported by 52 53 .oamaw Hmosam mawcfim m we vocaaumumo on oaooo umnu mz mo cowumuuamocoo Hmawcwa as» .«e .Aao.vmv Houuaoolamzm aoum unapoMMHv wauamowmwcmwm .« .mumu mHIoH aopm omnfiauoumo mm .m.m H H some can ucmmouaou mosam> .oofimauomm ou uoaum whom n mum» so vmauomnmm mp3 Showusm Bonn no maouoocoaawcmw Hmow>uoo uowuwmam *«m.o q.oaw.m III III camao Hmmaam *m.ona.a m.ona.~ m.onw.o m.onm.o spmuasuam poapmumom H.qu.Hq q.qun.~m o.mfim.HHH q.mpm.maa mococwam cmaooz III III w.muw.qm m.maa.ooa Boumfiuum kaouomaowawamo Emcm maouuchHchmu swam Adfimuoua wa\msv Assauonm wE\wcv mszmmmszmmoz mszuou uoauomnm mo muoowmm H mqm¢9 54 Annunziato and Weiner (1980) and by Saavedra 31 31, (1975), but are less than those reported by several other investigators (Holzbauer 33 31,, 1978, 1980a,b; Morgan and Herbert, 1980; Torda 33_31,, 1979). The amount of DA per posterior pituitary (152:10 pg protein) was 0.96:0.06 ng. SCGx did not alter the DA concentration in any region examined. The DA concentration in the pineal gland and the NE concen- tration in the striatum were below the sensitivity of the radioenzy— matic assay and are therefore not reported. The NE content of the median eminence was slightly, but not significantly, decreased by SCGx. This may represent the residual NE found in the median eminence following hypothalamic deafferentation (Brownstein 33H31., 1976; Gallardo 3£_31,, 1978). However, surgical sympathectomy substantially reduced the NE content of the posterior pituitary while totally de- pleting the pineal gland of the amine. These results suggest that all of the NE in the pineal gland and approximately one-third of the NE in the posterior pituitary is contained in terminals of peripheral sympa- thetic neurons. Holzbauer 35 31. (l980b) have similarly reported a 50% reduction in the NE concentration and no change in the DA concen- tration of the posterior pituitary following SCGx. Apparently the majority of the catecholaminergic neurons terminating in the posterior pituitary is of central origin with the concentration of DA being 2- to 3-fold greater than that of NE. B. 13HV133_Estimates of Catecholamine Synthesis There are several methods which may be applied to estimate monoamine synthesis 13,1123, Each has its own advantages and dis— advantages, but non-steady state methods do not yield absolute synthesis 55 rates (for review see Weiner, 1974). Two procedures commonly used to estimate catecholamine synthesis rates are the uMT-induced decline of DA and NE (Brodie 35 31., 1966) and the rate of DOPA accumulation after decarboxylase inhibition (Carlsson 33 31., 1972). The aMT- induced decline method was used to determine if the rate of DOPA accumulation could be employed as an 13fly133_estimate of DA synthesis in the median eminence and posterior pituitary. The difficulty with the latter method is that both brain regions have substantial nor- adrenergic innervation, and that DOPA is the amino acid precursor for both DA and NE (see Figure 3). l. a-Methyltyrosine-induced decline of dopamine and norepinephrine The relative rates of DA and NE synthesis in the posterior pituitary, median eminence and striatum were calculated from the rates of decline after the administration of aMT (Figure 4 and Table 2). The catecholamine concentrations presented in Table 2 represent the mean value of the zero time control (non-injected ani- mals). The rate constant is defined as the fraction of total catechol- amine lost per hour (fractional turnover rate; Brodie 3£_31,, 1966) and is a function of the slope of the corresponding line in Figure 4. The tuberohypophyseal DA neurons have a high tonic rate of activity as evidenced by a DA turnover rate of 0.53:0.23 hr-1 in the posterior pituitary. Both the amine concentration and the rate of decline (i.e., the rate constant) were greater for DA than for NE in the posterior pituitary and median eminence. When a ratio of DA synthesis to total catecholamine synthesis was calculated for median eminence 56 oum3 sBmuo mocfia ocH ucomouaou maooazm .mwmmamcm aowmmouwou So omumaooamo .mumu Nate aoum omsflauouow mm .m.m H H unommummu mosHH Hmowuum> mam momma .A.Q.H .wx\wa ommv 925 «o coaumuumwawsvm ofiu “comm mouocwa 00 ram 00 .0m .0 omoflwfiuomm mum3 mHmEHG< .Sumufinufia powumumoo cam monocwam cowooa .BSumHuum emu aw Ammaouwo ooHHHmV ocfiusamawaouoa was Ammaoufio aoQOV mafiamnoo mo osHHomw moosvcHIsz any .q ouowflm 57 q muowfim 5.2.0 amt; mw52=2 Om 00 on fil . . VKSCDFE m0.mm...w0m mug Q. .- .9 Om q r 00 on O 1 n. J ow 00 L Q uozmzim 2532 S. om om EDHSFPm ("Mud bw/ 60) samwvwouoalvo 58 .cOHumHucmoaoo ocHamHonooumo ouMumlmomoum onu x unnumcou dump can mamsvo oumH mHmmnuazm oca .momxamsm mcHH conmonoH >9 woaHauoumv mm Hm>HoucH mocooncoo Nam H some mau ucomouamu mucmumaoo mums one .Aq muomHm 9H Houudoo oaHu oumn onuv mumu ASHMuHoan HOHHoumon mam mococHao QMHvoav NH Ho AssumHuumv o Eoum ooaHanuov mm .m.m H H some mzu uaomounmu chHumHusoocoo oaHamHonooumo ooa III m.mm III 0N.OHmm.o III m.MH¢.mm azumHHum cm w.q m.wn NN.0H~H.O 9N.0Hmn.o w.HHo.mN N.nHm.qoa cocoaHam GMHooz mm 5.0 m.m mm.ono~.o m~.onmm.o m.ona.~ m.HHm.oa spmuasuam poHpmumom OOH x.mmmmm mz <9 mz <9 92 <9 AH9\nHououa wa\wav Aalusv AaHououa wa\wav 98 Boum ucoHoMMHo maucmonHame .s .mumu mic Eoum ooaHsHouoo mm .9.m H H some can ucmmounou mosam> .ooHMHHomm ou HOHHQ mmuscHE om A.9.H .wx\w8 OOHV maoa 9mz monouchHaom mums aOHumananoom <9O9 ocHaHmuoo ou pom: mama .aonswcH umma can Houmm musos we wooHMHHomm mums mHmSHam one .mHm>HoucH H50: we on AoHom oHQHoomm Na.o wcHaHchoo Homz Nm.o mo a: My oHoHno> Ho Aa: m\w: m.~Hv <9mOIo mo mGOHmomoH HmasoHHuam>muunH HonoumHHn m vo>HoomH mumm N.OHm.o m.OH<.c m.mHm.mm m.NHm.om III III asumHuum w.OHo.w n.9Hq.m m.oHo.om N.¢H¢.wm «m.HHH.mH m.MHm.mq oucocHEm cmHooz H.0Hm.a H.0Hm.a «.0Hm.w c.0Hn.m «N.OH<.H «.0Ho.m mucquuHm HOHHmumom <9molc oHoH£o> <9mouo oHoH£o> <9mOIo oHoHnm> ASHE om\aHououm wa\wcv AcHououm wa\wcv AcHououn wa\wcv onH<992900< <9o9 msz<909 mszmmmszmmoz mGOHwom chum owuooaom :H GOHumasanoo< <9o9 was mCOHumHucoocoo oaHEmHogooumo co maHammowhxouohmlc mo muoomwm m mqmv .m.m H H AaHououn wa\w:v mGOHumHuaoocoo <9o9 some anemones» .< Hoamm CH measHoo mam .AmumaHoHo ustH so onom mkumuHSHHm HOHHoumoav moHoHHo ooHHHw was AoumcHoHo ummH so mHmom manumHHumv mmHoHHo ammo 9 Honda s9 .A.>.H .mx\wa mmv mHOH 9mz Houmm moEHu mDOHum> um :OHumHsasoom <9o9 A9 .mHOH 9mz mo mmmoo mDOHHm> mo aonnmaH moocm>mHuGH onu Hmumm mousaHS om :OHumHsenoom <9O9 A< .mumu omaHmuume .ooNHuosumocmaa :H mHOH 9mz mo aOHumuuch IHEom msooo>muucH Houmm xumuHsan HOHHoumoa was EDHMHHum one CH sOHumHnasoom <9O9 .m mustm 63 m mHDme .8555 2.. .3}... 0.0. 82 5.7.? M22. 90. 82 won on 8 o. c on on as. on 3 ad. fi-le . a n at J . .. o. . I n o _ . . _ a N i 8 N I O. fimfiEDHE ; SEN—hon. 23:1um (:40de 6W0“) V600 64 estimated (Holzbauer 3£_31,, 1978, 1980a,b). The initial study was designed to examine a variety of dehydrating stimuli on the DA content of the posterior pituitary and to extend the experiment to other dopaminergically innervated brain regions. The results are presented in Table 4. The DA concentration of the striatum and median eminence was not altered by any treatment examined. The DA concentration of the posterior pituitary was increased only by water deprivation followed by saline drinking, similar to data previously reported (Holzbauer 33 31., 1978, l980a,b). B. Effect of Dehydration on DOPA Accumulation 1. Water deprivation in male and female rats Pharmacological treatments and endocrinological mani— pulations rarely alter the catecholamine concentration of any brain region due to the coupling of neuronal activity, and thus release of amine, to the rate of catecholamine synthesis. To estimate neuronal activity the rate of DOPA accumulation after decarboxylase inhibition was measured as an index of DA synthesis in selected brain regions of male and ovariectomized rats following three days of water deprivation (Table 5). Since these experiments were not performed or assayed together a direct male:female comparison is not possible. The data reveal, however, that three days of water deprivation did not alter DOPA accumulation in the striatum of male or female rats. In males, the median eminence was divided into rostral and caudal (pituitary stalk) regions; there was no significant difference in the rates of DA synthesis in these two regions. The rate of DOPA accumulation in female median eminence appeared greater than that in the male, but in 65 .Houucoo Eoum unonMMHo SHuamonHanm .« .mumu o aoum oosHa luouoo mm .m.m H H some onu uaomounou moon> .mumo m How Hoods wcHstHo Homz NN ou mmooom moum wooH>oun scan was m%mo N How Houm3 mo vo>HH9oo Ho .mhmw m Ho N How Houm3 wcHstHw Hmauoa mo omoumaH Howz NN .AHouucoov nouns was room on mmouom ooum vooH>oun mums mama «H.9Ho.HH m.ono.m o.ona.o w.o Hq.o sumuasuam uoapmumom m.qu.mHH m.mHN.om o.qu.HHH n.NHHm.ooH moswcHam smHooz o.mHo.oHH m.mHn.oHH m.qu.m0H m.q HN.moH anomHHum Amsme no Homz NN mafia Amsme mo amass NV 0 . . H Huaoo Amhmv NV aOHum>HH9m9 Houmz Humz SN Homz mm Aaamuoua wa\wav mzH2 .ooHMHHomm ou HOHHQ mousaHa om A.9.H .wx\wa OOHV mHOH 9mz monoumHaHaom mums mumu HH< .muson mm 909 HoumB mo oo>Humoo mums AcOHumehsmo ou HoHH9 whom n vmauomuom mp3 hummusmv mums ooNHBouooHHm>o Ho MHNZ *m9.onoo.9 99.0Hmo.9 «99.09mo.9 oo.on~m.o spmu9=u9m HoHHmumom In- In- m.on m.w 9.09 N.o 9me=mo .I. In- 0.99 m.n n.99 0.9 9mpumom H.9H m.~9 m.~9 9.59 -u- nu- muema9am cmHHmz m.o9 0.99 9.09 o.m H.on 9.99 m.oa 9.09 asumHHBm oo>Huam9luoum3 Houucoo om>Huao9lumum3 Houusoo m9mam9 m9m: 33:58 992530.999 @99an 209.399.9283 <98 mumm omNHEouomHum>o can on2 mo mGOHmom aHmum wouooHom 9H cOHHMHnanoo< <9o9 so GOHum>Humo9 Houmz mo uoommm m 9992.9 67 neither male nor female did water deprivation alter the rate of DOPA accumulation in this brain region. On the other hand, water depri- vation for 3 days significantly increased DOPA accumulation in the posterior pituitary of both males and females. The results of this experiment reveal that water depri— vation, in both male and female rats, increases the activity of the tuberohypophyseal DA neurons, but does not effect the nigrostriatal or tuberoinfundibular DA systems. It appears that some of the DA nerves terminating in the pituitary stalk (caudal median eminence) might be functionally related to those in the posterior pituitary and are stimulated by dehydration. There was often a tendency for water deprivation to cause a small but insignificant increase in DOPA accu- mulation in the median eminence when it was not separated into rostral and caudal portions (e.g., see Figure 6). This is presumed to be due to a small contribution of the tuberohypophyseal DA nerves to a por- tion of what has been arbitrarily defined as the "median eminence", for there is no clear demarcation between the median eminence and the posterior pituitary. Although a statistical comparison is not possible due to the nature of the experiments, there appears to be a sexual dimorphism in the normal rate of DOPA accumulation in the median eminence, but not in the striatum or posterior pituitary. The higher rate of DA synthesis in the median eminence of females has been con- firmed in a detailed series of experiments (Demarest 3£_31,, 1981). 2. Food deprivation Rats which are deprived of water exhibit a reduction of food intake. The data depicted in Figure 6 reveal that three days of 68 .AHo.v9v Honusoo 8099 ocouomeo zHucmoHMHame .« .qo.0Hnm.o .%HMuH=uH9 HOHHoumom “H.0HH.¢ .oocdeam smHoma “H.HHm.HH .EdumHHum uoHoS AGHE om\sHmuoua wa\ HouuSOO .Houucoo mo unmouoa mm oommouaxo mum mam mum» w aoum mocHauouoo mm AmocHH HmoHuHo>V .m.m H H some ucommummu msadHoo .moHMHHomm ou HOHHQ mmusaHS om A.9.H .wx\we OOHV mHOH 9mz omumum IHaHaom oHoB mums HH< .munon me 909 A99 moo>HH9oolo009v @009 no: ado nouns 90 A93 moo>HHva luoumsv Momma uoc won @009 .AU "Houunoov HoumB was @009 ou mmooom ovum omoH>oum mpo3 mumm .chHmoH chun wouooHom GH GOHumHsadoom <9O9 so cOHum>Huaoowooow Ho nouns mo uoomwm .o ouome o mpam99 69 $5.5»... mozmziu «0.550.. 258.2 552.5...” 9.5 93 0 E a; 0 on 03 U A. . on W . r._l. _ H 1 oo. 1 H _ fl 1 on. _ * meow (IOJW03 :0 °/.) V30? 70 water deprivation, but not three days of food deprivation, selectively increased DOPA accumulation in the posterior pituitary. That is, water deprivation pggugg, and not just a reduction in body weight due to decreased food intake (control = 269:5 g; water-deprived = 193:4 g; food-deprived = 211:3 g) is involved in activating the tuberohypo- physeal DA neurons. 3. Time course of water deprivation To determine changes occurring throughout the first 72 hours of water deprivation, catecholamine concentrations and DOPA accumulation in selected brain regions, and the plasma sodium concen— tration and the hematocrit were measured in rats sacrificed at 24 hour intervals. DA and NE were measured in rats not receiving NSD 1015 (Table 6), whereas DOPA accumulation and the plasma parameters were determined in rats administered NSD 1015 (Table 7). Seventy-two hours of water deprivation did not alter the DA or NE concentration of any brain region examined. DOPA accumulation remained constant in the striatum and median eminence throughout the duration of the experi- ment. However, the rate of DOPA accumulation in the posterior pitui- tary progressively increased, attaining statistical difference from control following 72 hours of water deprivation. The plasma sodium concentration and the hematocrit also steadily increased, reaching statistical significance by 24 hours of dehydration. These data clearly demonstrate that water deprivation increases DA synthesis only in the posterior pituitary. The increase in synthesis occurs at times when the DA concentration is unaltered. 7l unomouaou mmon> .mumu w Eoum moaHauouow mm .m.m H H some cap .GOHum>HHQoo Hmumz mo moOHHmm mnoHHm> Houmm omoHMHHomm ouo3 Ho AQOHum>HHQmo Hmum3 munon 0v noun? mam ooow Ou mmooom vmvH>oum mums mumm +l +| H.N 5.09 CON N.o m N n m w.~< +l +l mumuHsuHm HOHHoumom monocHam amHooz AaHouonm wa\mcv mszmmmszmmoz ONO 0‘an Chm +I+IH ole-q' h-CDCD F4 +I+IH o~oxa> tnHH909 HmumB mason chHwom chH9 wouomHom 9H mCOHumHusoocoo o mqmH99o9 nouns mo muoommm 72 .AHo.v9v Houuaoo oEHu o 8099 use luommHo xHuamonstHm .* .mumu CHIN aoum vmaHaHouoo mm .m.m H H coma MSH usmmoummu mosHm> .ooHMHHomm ou HOHHQ mouacHa om A.9.H .wx\wa OOHV mHOH 9mz monouchHaom muoB mumu HH< .:0Hum>H99wu Honda 90 mooHHon mSOHHm> Houmm voonHHomm oHoB Ho AcOHum>Hummo Houmz musos ov Houma ram @009 on mmooom mmum vooH>oum mums mumm 909900 009000 90 sq OH m mm «m OH m me em 0H 0 we 0 OH m N: HHMUOH<299 90.99 0.009 90.09 0.009 90.99 9.009 0.09 0.909 99\0000 +02 020090 909.0900.0 00.0909.0 00.0990.9 00.0900.9 000090090 009900000 0.09 0.0 0.09 0.0 0.09 0.0 0.09 9.09 00000900 009002 9.09 0.09 0.09 0.09 0.09 0.0 0.09 0.0 00009900 AsHa omxaHououm wa\wcv 209009020000 0000 09 00 00 0 90H00>H99o9 Munoz muoom uHHoouwamm mam EDHoom mammHm so mam .mGOmem chum wouomHom aH :OHumHnasoo< <9O9 so 90Hum>Hu9m9 nouns mo muommmm n mHm . 990 . v3 .mumu w 8099 vodHahouoo mm .m.m H H some .ooHMHHUMm ou HoHum muuaaHB om A.9.H .wx\wa OOHV mHOH 9mz ooumuchHEom mumu 9H woaHauouoo 0903 muouoamuma moamHmn oHsHm mam GOHumHnasoom <9O9 .Houm3 wstnHuo Hmahoa How vouauHumLSm Humz NN no coHum>HHQoo 90903 nonuHm mo whom m .Hmum3 mam @009 on mmooom oouw wooH>099 @903 mumu Houuaoo 0903 mucoaumouu use . - . . I . . - . 990 009900 00 0+99 9 999 0+90 0 99 0+00 0 2999000 020090 . - . . l . . l . 909900 009000 90 0 0+ 0 90 90 0+ 0 00 0 0+ 0 00 9900090292 . - . . - . . - . 9990900 «m ~+ m 99H «m H+ H HoH 0 0+ H 00H +mz H9909 90003 Houuaou CHououm mammHm mam uHuooumaom .asHoom mammHm so mam .humanuHm HOHHmumom mnu 9H :oHumHsafioo< <9O9 cam maoHumHuaoocoo maHEmHosomumo so wachHH9 maHHmm Ho aOHum>H99m9 Houm3 mo muoommm w 9HmH00000099m90 .« .0009 min ao9m 0099890000 00 .m.m H 9 0008 0:0 090009009 00=H0> .00900>99000 90003 mo 0009909 000990> 90000 000H099o0m 0903 90 A:OH00>99Q00 90003 09ao£ ov 90003 000 0oom 00 000000 0090 000H>o9m 0903 000m . - . . - . . - . . - . . + . 909900 000000 00 «0 0+0 0m 0m 0+0 09 +9 0+0 mm 00 0+0 9m 0 0+0 00 9900090290 . - . . - . . - . . + . . - . 99\0900 «0 0+9 909 00 9+0 909 00 9+0 009 9 9+0 009 0 0+0 909 +02 020090 00 00 00 N9 0 co000>fi900o 90003 09som 0990000a0m 000 abw0om 0800Hm do cow00>H9m0o 90003 No 0000mmm m MHm .9009009000 00890 0:O990> 00 0009999000 000 90003 00 000000 0000093 00w0o 99000 o0 00090009 .A.o.0 .wx\H8 mv H002 NmH 0093 00000009 0903 000m -u- 099000 999090 999990 nu- 99009 900900000 0999090200 020090 . - . . l . . u . . - . . - . . - . 909900 000000 90 0 0+0 90 0 0+9 90 0 0+0 00 0 0+0 00 +0 0+0 00 0 0+0 00 9900090299 «9.090.009 90.090.009 «0.090.909 90.090.009 90.099.009 0.090.009 9990900 +02 020090 009 009 099 00 00 0 900000900 9002 909 90090 0099 099oo0080m 000 >09H0Ho8mo 0800Hm .8090om 0800Hm 0o 0O90o0m09 009H0m 090O090090 mo 000099m OH m9m .009999000 00 90990 0000098 om A.0.9 .w0\w8 009v m909 sz 009000 1909800 0903 0009 990 .9009009000 00890 000990> 00 0009999000 0903 000 90003 00 000000 0000093 00w00 99000 00 00090009 .A.0.0 .w0\98 mv 9002 Nm9 0093 00000n09 0903 0009 00008990000 00090000 00900 09 99.0909.9 99.0999.9 . 99.O909.9 09.O99o.9 -n- 99.0909.9 9 9003990090 -u- 99.O9mo.9 mo.O999.9 90.099o.9 -u- 90.0990.o 9 900a990009 0o.0900.9 In- 99.09om.9 -u- 00.099o.9 99.O999.o 9 9000990090 A098 om\0900090 w8\w0v ZOHH582. 2505 Hammad-dug .r} 1 -J . 1 J * _ * * * * f . . F . L m m m m m m . (was. 3.8 3.838 529.5 253.... .3223: 24 36 48 72 l2 0 HOURS AFTER HYPERTONIC SALINE Figure 7 81 was not elevated following 12 hours of water deprivation. What is not apparent in Figure 5 is that the plasma sodium concentration in the animals administered 15% NaCl was elevated approximately 10 mEq/l for 1-2 hours immediately following the injection (again see Table 10). The effect of rapidly induced cellular dehydration (i.e., hypertonic saline injection) on DA synthesis and neuronal activity in the pos- terior pituitary was significantly different from water deprivation; 24 hours following the saline injection an increase in DOPA accumula- tion was observed. This increase was not sustained, for at 36 and 48 hours after the hypertonic saline injection DOPA accumulation had returned to control, only to be increased again at 72 hours by the continuing water deprivation. An experiment was designed to compare DOPA accumula- tion, plasma sodium and hematocrit in rats following 24 hours of water deprivation or a hypertonic saline injection (Table 12). The plasma sodium concentration and the hematocrit in the two treated groups were elevated to a similar extent at the time of sacrifice. DOPA accumu- lation in the posterior pituitary was increased only in those rats which received a subcutaneous injection of 15% NaCl 24 hours prior to sacrifice. In rats which were injected with 15% NaCl and permitted free access to water, there was a rapid (within 30 minutes) increase in the plasma sodium concentration and decrease in the hematocrit (Table 13). In direct contrast to rats administered 15% NaCl and then water—deprived, the rate of DOPA accumulation in the posterior pitui- tary, the plasma sodium concentration and the hematocrit in those 82 TABLE 12 Effects of Hypertonic Saline Injection on DOPA Accumulation in the Posterior Pituitary, and on Plasma Sodium and Hematocrit Water—deprived 15% NaCl Control (24 h) (24 h) DOPA ACCUMULATION . 0.64i0.08 0.83:0.12 1.30:0.13** (ng/mg protein/30 min) 3323??)Na+ 137.9 10.5 143.9 :0.6* 144.1 :0.7* figMgzgfififiTCells) 44.6 10.5 49.6 :0.6* 50.7 :0.5* Rats were provided free access to food and water (control), were water-deprived for 24 hours or were injected with 15% NaCl (5 ml/kg, s.c.), returned to their cages without access to water and sacrificed 24 hours later. All rats were administered NSD 1015 (100 mg/kg, i.p.) 30 minutes prior to sacrifice. Values represent the mean i l S.E. as determined from 8 rats. *, significantly different from control (p<.01). **, significantly different from both control and water- deprived (p<.01). 83 .AHo.vav Houuaoo mafia o Eouw uaouowwwv hauamoflmwamfim .« .m.m H H some map uammmnaou mmzam> .mumu wouwu locummama: no vofinomnmm mmz ousuoaam macaw Hmuanuo .cowuomncfi Houmm mounawa ONH cam on ad mums m Eoum no coauomfiaw umumm mmuoaHE co mam Aoafiu 0V down looms“ ou Hoaua mumu q Boum woamfimm mmz wooan madam Hmuanuo .umumB ou mmooom moum voww>oum conu cam A.o.m .wx\aa mv Homz Nma nuw3 vmuoomdfi muoB mumm . - . . - . . - . . - . Amaamo woxumm Nu «m H+m mq sq o+w mq so H+o qq w 0+0 me HHMUOH .oofimwuomm ou Rowan mousafia om A.Q.H .mx\w8 ooHv mHOH nmz monoumficfiawm muoz mumu HH¢ .GOHuothH mcwamm caucuumakn mfiu mafiaoaaom mudos «N mnu mom wo>fiummvlumum3 mm3 asouw nonuoam mawna undo: «N wcfi3oaaow man you umum3 ou mmooom mmum voww>oum mm3 mum“ wouomnaw mcHHmm ofiaouumamn mo asouw moo .A.o.m .wx\Ha my Homz Nma nufiB wouommcw mum3 no AHouwcoov “mums mam boom Ou mmooom omum vowa>oun mama mama . - . . - . . - . AmHHmo amxumm NV 40 0+ N am a o+ N we 0 0+ 0 cs eHmooaazmm . I . 0 III . . l . AH\UmBV 40 0+ s 44H m o+ H can m 0+ m 04H +42 azm .uouwmmuonu moaHu monHm> um vmonHuomm can Hoods ou mmmoom unosuHs mowmo uHosu ou vmaHSu low .AmmuncHa m\umu\Homz No.0 GH Ha m.Hv HouHaama NON zuH3 vomomaH wuw3.mumm . - . . - . . - . . - . . - . AmHHmu amxumm Nu m 0+0 m4 0 o+m 04 o H+N as N o+w H4 0 o+m H4 HHmooaazmm NHoom Hflmam Nwmmm HHHom afiqmm wa\amoav wHHH IHcHme ouo3 mum» HH< .AHo.vmV Houucoo Eoum ucmuommww hHuamoHMHame .* .mumu w Boum umaHahmumv mm .m.m .moHMHuomm ou HOHHQ mouoaHa om A.Q.H .wx\wa OOHV mHOH nmz powwow .umuwmouosu mmEHu mooHum> um wooHMHuomm mam nouns ou mmooum uoonuHa momma uHmsu ou coauauou .AmouscHa N\umu\Homz Nm.o aH HE m.Hv HoquamB NON nuHa womamaH who? mumm AmHHmu umxumva *m.OH 0.0m *0.0H m.mq «w.OH w.mq «0 OH q we q OH m mq HHMUOH0.01). b. 5.0 ml of 20% mannitol. To compare similar osmotic stimuli, rats were infused with 5.0 ml of 20% mannitol (5.5 mOsm/rat/8 minutes) or were injected subcutaneously with 15% NaCl (5.2 mOsm/rat). Also included in this experiment were 8 rats administered 15% NaCl and permitted free access to water 4 hours after the hyper- tonic saline injection. This was to determine if the elevation in plasma osmolality observed within the first 4 hours following the saline injection (see Table 10) triggered a 24 hour delayed increase in DOPA accumulation in the posterior pituitary (see Tables 12 and 14, and Figure 7). Acutely, mannitol markedly increased plasma osmo- lality and moderately increased plasma sodium concentrations (Table 17). A slight increase in hematocrit was also observed. The hyper- tonic saline injection caused a large elevation in plasma osmolality; this was attributable to a marked hypernatremia. In this experiment 15% NaCl administration did not alter the hematocrit (compare to the slight hypervolemia in Table 10). DOPA accumulation in the posterior pituitary, the plasma sodium concentration and osmolality, and the hematocrit were increased 24 hours after mannitol and hypertonic saline (Table 18). Also, the effects of the hypertonic saline injection were prevented by providing the rats access to water 4 hours after saline administra- tion. 89 .AHO.vmv Houucoo Eoum unmumwva zHucmonchHm .« .mumu w 804m wmcHaumumv mm .m.m H H amma onu uaommumou moaHm> .umummouonu mmBHu msoHnm> um OmoHMHuomm mam Houma ou mmmoom uaoaufis momma uHmSu ou OmaHSuou .A.o.m .mx\Ha mv Homz NmH no AmmuscHa w\umu\Homz Nm.O aH Ha mv HouHacma NON HmnuHo woumumHGHawm mum3 mumm . I . . I . . I . . I . . I . AmHHoo vmxomm NV 4 0+4 44 0 0+4 00 40 4+0 04 44 0+0 m4 0 0+0 00 H44004 .mowm4uomm ou uoHum mmusafia om A.Q.H .wx\wa OOHV mHOH Omz moumuchHavm o4m3 mumu HH< .mofim IHuomm Hausa aoHuomma4 oaHHmm onu Houmm muson 4 noumz ou mmmoom cm>4m mms Auoumz msHa Homz NmHv mumu mo mzoum moo .Hoummouonu mmaau mDOHnm> um onHMHuomm mam uoum3 ou mmmoom uaosufia momma uHmnu ou Omaunuou .AmouocHa w\um4\HOmz Nm.O :4 Ha O.mv Houfiaama NON Ho A.o.m .mx\Ha mv Homz NmH HosuHm wmumuchHawm ouoB mumm . I . . I . . I . 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AGHB OM\GHmuoum ma\wcv 40 0+44 0 440 0+44 4 444 0+44 4 40 0+44 0 204444222004 4400 uoumz 4040 4042 444 40040042 404 4040000 4042 444 uHuooumamm mam muHHMHoamO mammHm .ESHwom mammHm co mam .%umuH:uHm MOHuoumom msu :4 a04umHaaooo< .moHMHuomm ca 40449 mouscHa Om A.Q.H .mx\wa OOHV mHOH Omz wououquHavm ouo3 mHmaHam HH< .0840 mo mnumcmH msoHum> pom AHouuaoo 430:0 umu Hmauoc 04 +42 NOm.O ou woumaaoo mm +mz NmH.mv ume adHOom ans m 0mm mums mama 4.04 4.44 4.04 4.44 4.04 4.44 4.04 0.44 4444004mmwmmm2mw 4.44 4.444 0.04 4.444 4.44 4.444 4.04 4.444 +424wnwmmw 40.0440.0 04.0444.0 44.0404.0 44.0404.0 4040 mmwmwmwmmm0wa«wmw 4 0040 00400M 0442 00 4440 4 4040000 uHuooumawm mam ausom mammHm so 004 .zumuHsuHm HOHumumom 000 04 004044000004 0000 00 0040 004404 2440 00 4000444 mH MHm .moamauomm ca 40440 mmuaaaa on 4.0.4 .wx\wa OOHV maoa amz commumficfiavm 0403 mumu 44¢ .mzmw N Mom Jawuv 00 4042 MN no Hmum3 cm>4w c050 van 0400 m pom wo>aummv 40043 0403 0044 nonuo .0000 w you mafiuw on 4002 MN cm>fiw no Ammmv n no mv aofium>fiumow HmumB kn vmumuvhsmv 0403 mumm 4N.O4mm.o 04.04mo.4 44.0444.o {o4.04©w.o «N4.044N.4 44.0Hoo.o 448 ooa\wv szHomm <2m4uamwlumumz 4042 MN wm>fiumovlumum3 cm>aumowluoums afiououm mammam cam uwuooumamm .Edfivom mammam do 000 .44404304@ 404uoumom mnu 04 aofiumasaaoo< «mom :0 doaumuvhsom mam cOHumu%:mn mo muommmm ON mqm .004044000 04 40440 0040048 on 4.0.4 .wx\wa 004v m404 nmz 004040404800 0403 0404 44< .004u0>44000 40403 40 0400 m m04304Hom 04:00 me 40 «N .4 40m 0040404004 0403 0404 40 000040 .0400 m 404 40403 40 00>44000 0403 40 A404uaoov 40403 000 0004 04 000000 0044 0004>040 000m .0404 0 304m 0004840400 00 .m.m 4 4 0008 0:4 .I . .I . .I. .I . .I . 444400 4004040 40 «m 0+ 0 N4 04 0+ 4 N0 «0 o+w m4 *4 o+ N 4m 0 0+ N 04 HHMQOH<2mm .I. .I. .I. .I. .I. 4440000 0 0+ m om4 m 0+ 4 om4 m o+o MM4 «m 4+ 0 m04 0 o+ m mm4 +02 44m00l40u03 4044000 0044040400m 04=0m 444oou0a0m 000 8040om 08004m 00 000 .44044044& 40440400m 0:4 04 00440408500< 44Q0Q 40403 mo 0400mmm 4N mgmda 98 .A4o.v0v 4044000 8044 400400440 4440004440w40 .« .0404 04Im 8044 0004840400 00 .m.m 4 4 0008 0:4 400004004 00040> .004444000 04 40440 0040048 04 4.3.4 .wx\w8 mmv m404 nmz 004040404800 0403 0404 440 .00440>44000 40403 mo 0400 m w043044om 0400: N4 40 m .0 .m 404 0040404004 0403 0404 40 000040 .0400 m 404 40403 40 00344000 0403 40 44044000v 40403 000 0004 04 000000 0044 00043040 0403 040m . I . . I . . I . . I . . I . . I . 404400 004000 40 «0 4+ 0 00 *4 0+ 0 40 . *0 0+ 4 00 +0 0+ 0 00 «0 0+ 0 40 4 0+ 4 40 4400040200 . I . . I . . I . . I . . I . . I . 44400a0 0 0+ 4 404 0 0+ 0 404 0 0+ 0 404 0 0+ 4 N04 «0 0+ 0 004 0 0+ 0 004 +02 44M0Q 4044000 00440404400 04000 I404 3 4440040800 000 80400m 080040 00 000 .440440440 404404000 044 04 00440408000< <0on 00 00440404500 400000040m 000 00440344000 40403 mo 0400mmm NN MAM¢H 99 .A4o.v0v 4044000 .0404 04I4 8044 0004840400 00 .m.m 4 4 0008 044 400004004 .004444000 04 40440 0040048 04 4.3.4 .wx\w8 mmv m404 nmz 004040404800 0403 0404 440 .00440344000 40403 40 0400 m w04304404 04004 0 40 m .4 404 0040404404 0403 0404 40 000040 .0400 m 404 40403 40 00344000 0403 40 440440000 40403 000 0004 04 000000 0044 00043040 0403 040m 8044 400404440 4440004440040 .0 000403 o .I. o 0 II o o II o 0 0 al 0 0 .l o AmHHmo fimxomm NV «m 0+ 0 40 0m 0+ m mm «4 0+0 4m #0 0+ 0 0m m 0+ m we 9440040204 0.04 4.004 00.04 0.004 04.440.004 00.44 4.004 0.04 0.404 ,4440000 +02 000040 0 08 w 00.0440.0 00.0440.4 040.04mm.4 004.0440.4 00.0000.0 4040 044040004 4 0V 204904024000 0000 0 m 4 00344000I40403 4044000 00440404404 04004 4440040804 000 80400m 080040 00 000 .440440440 404404000 044 04 00440408000< «004 00 00440404404 400000040m 000 00440344000 40403 40 0400444 MN mqm<8 100 Although both DOPA accumulation in the posterior pituitary and the plasma sodium concentration are restored to control values at about the same time, a direct cause-and-effect relationship can be hypothe- sized from, but is not proven by, the data presented. IV. Vasopressin A. Effect of Continuous Vasopressin Infusion on DOPA Accumu- lation Tanaka g£_§l, (1977) reported that the intraventricular infusion of AVP increased the aMT-induced decline of DA in the median eminence and striatum (DA turnover in the posterior pituitary was not reported). Dehydration, however, increased the plasma AVP concentra— tion but did not alter the concentrations of AVP found in the CSF (Mens 35 al., 1980). An experiment was designed to examine the effect of systemic AVP administration on the activity of central dopaminergic neuronal systems. Two doses of synthetic AVP were infused intravenously at a rate of 24 ul/day using subcutaneously implanted AlzetR osmotic mini- pumps. The doses infused were 240 mU/rat/day as a physiological replacement dose and 720 mU/rat/day to approximate plasma AVP concen- trations observed following 2—3 days of dehydration. The results are summarized in Table 24. Neither dose of AVP altered the rate of DOPA acoumulation in the posterior pituitary, median eminence or striatum. The plasma sodium concentration, as expected, was slightly decreased by the higher dose of AVP (Chan, 1971; Smith g£_al,, 1979) whereas the hema- tocrit was unaltered by either dose of AVP. .A4o.v0v 4044000 8044 400404440 4440004440040 .0 .0404 m 8044 0004840400 00 .0.m 4 4 0008 004 400004004 000403 .004444000 04 40440 0040048 on 4.0.4 .wx\w8 004v 4404 nmz 004040404800 0403 0404 440 .004444000 04 40440 0400 m 0040044 003 0403 4040w0n 004 000 000440040000 0403 0408400 4044000I8000 00H .00800I4048 0440800 004004084 44000000400000 00400 0400 m 404 4400000304404 0000404 003 030 04400404m 101 . - . . I . . I . 404400 004000 40 0 0+0 40 0 0+4 40 0 0+ 0 40 4400040240 . I . . - . . I . A4\0mav «4 0+0 004 0 0+0 404 4 0+ 0 004 +02 020040 4.04 0.0 4.04 4.04 4.04 0.0 00404440 4.04 0.0 0.04 0.0 0.04 4.0 00000400 004002 40.0400.0 00.0404.0 00.0400.0 440440440 404404000 4048 0m\0404040 08\w0v 204404020000 0000 400400 004 400400 000 0000 UGOBUNNHH. 4440040800 000 80400m 080040 00 000 .0004000 04040 00400400 04 004404080000 0000 00 00400404 04000400003 0000044000 40 0400440 «N 04008 102 Infusions of AVP (Tanaka gt al., 1977) or an antibody to AVP (Versteeg gt al., 1979) directly into the CSF may alter DA turnover in the tuberoinfundibular and nigrostriatal neurons. However, the systemic infusion of AVP is without effect on DOPA accumulation in any region examined. These data are of interest because: 1) the CSF AVP concentration does not appear to be elevated concomitantly with the plasma AVP concentration. This has been demonstrated directly by measurement of the peptide (Mens gt al., 1980) and indirectly by measurement of neurochemical events (i.e., DA synthesis and turnover) induced by AVP. 2) DA synthesis in the posterior pituitary is not regulated by exogenous AVP infusions. This does not prove, however, that the rate of DOPA accumulation in the posterior pituitary is not stimulated by endogenously released AVP. The plasma AVP concentra- tions following dehydration and minipump implantation may not be comparable. DISCUSSION 1. Concentration and Synthesis of Catecholamines DA in the posterior pituitary gland of the rat is contained in nerve terminals of central origin, the cell bodies are most likely located in the arcuate nucleus (Bjorklund g£_§l,, 1970; Tilders 35 _al., 1979). The turnover rate of DA in the tuberohypophyseal neurons is similar to that in the nigrostriatal neurons. Since DA in the posterior pituitary is being continually synthesized, the steady state concentration remains constant and the intraneuronal metabolism to DOPAC is low, it must be concluded that this amine is being continu- ally released from the terminals of the tuberohypophyseal neurons. Once released DA can activate putative DA receptors (Sibley and Creese, 1980; Stefanini 25 al., l980a) which may (Ahn 33 al., 1979) or may not (Stefanini gt al., 1980b) be linked to adenylate cyclase. The post— synaptic DA receptors may be located on neurosecretory axons, pitui- cytes or vascular smooth muscle. Alternatively, DA released from the terminals of the tuberohypophyseal neurons can act as a neurohormone following its release into the capillary blood flowing from the neurohypophysis. DA synthesis in the posterior pituitary has been estimated in vivo using two biochemical techniques. All non-steady state methods 103 104 employed to estimate catecholamine synthesis and turnover have in- herent deficiencies yet it is agreed that all methods yield relative synthetic rates (Weiner, 1974). With prOper experimental design, the effects of pharmacological and physiological treatments on DA synthe— sis provide good biochemical indices of neuronal activity in dopami- nergic systems. The major advantage of the aMT technique over most others pre- sently in use is that both DA and NE synthesis rates can be determined following tyrosine hydroxylase inhibition. The disadvantages, however, are numerous. To accurately estimate catecholamine synthesis rates, 4 time points after aMT (from 0-90 minutes) are preferable. Eight rats are required for each time and all treatments must be compared to their appropriate control. Therefore, 64 rats are required to deter- mine the effect of just one treatment on DA and NE synthesis. Time and economic constraints limit the number of experiments that can be done employing this technique. The high dose of dMT required to completely inhibit tyrosine hydroxylase and the length of time of synthesis inhibition used experimentally probably cause effects secon- dary to the treatment being studied. Also, statistical analysis is quite difficult, particularly in the posterior pituitary. Comparisons between treatments are performed on the rate of decline (k) of the monoamines. This rate constant is the slope of the line (determined by regression analysis) of the natural logarithm of the catecholamine concentration versus time. The variability about each time point contributes to the variability of the slope (i.e., k). Ninety minutes after the administration of aMT, the amount of DA in control posterior 105 pituitaries is near the lower limit of the radioenzymatic assay. Therefore, the concentration of DA in the posterior pituitary will be extremely variable 60 to 90 minutes after aMT; this will be reflected in the confidence limits about the rate constant. If a treatment is expected to increase DA synthesis (i.e., cause a faster rate of de- cline following oMT), there generally will be an ever larger variation in the DA content of the posterior pituitary. A statistical compari— son is unlikely to show significance. Holzbauer_g£ El. (1978) first published results of a study uti- lizing the aMT-induced decline of DA in the posterior pituitary. The same method was used in this thesis only to validate the rate of DOPA accumulation after decarboxylase inhibition as a second in gi!g_esti- mate of DA synthesis in the posterior pituitary and median eminence. It was reported previously that in yi££g_tyrosine hydroxylase activity in median eminence homogenates reflects primarily DA synthesis (Kizer g£_§l,, 1976a). The ratio of DA to NE is similar in the median emi- nence and posterior pituitary. Accordingly, it was felt that an ig_ vi!g_measure of tyrosine hydroxylase, the rate of DOPA accumulation after the inhibition of DOPA decarboxylase, would estimate DA synthe- sis in both regions. Data verifying this assumption were presented in the Results Section I.B. Data were also presented demonstrating that DOPA accumulation can be measured as soon as 10 minutes after the intravenous administration of NSD 1015. This minimizes secondary effects of synthesis inhibition on the catecholaminergic systems. Since endogenous concentrations of DOPA are not detectable in brain tissue (Demarest and Moore, 1979a; 1980) no zero-time control is 106 required. The number of animals per experiment are reduced, allowing several treatments to be studied in each experiment and permitting easy statistical analysis. Estimating DA synthesis in the posterior pituitary and median eminence by measuring the rate of DOPA accumulation after decarboxy- lase inhibition is not without its limitations. For instance, DOPA accumulation estimates total catecholamine synthesis. Under normal circumstances, however, 90% of the catecholamines being synthesized in the posterior pituitary are DA. Changes in NE synthesis following pharmacological or endocrinological manipulations may be obscured in measurements of DOPA accumulation. A second deficiency in this technique is that decreases in DOPA accumulation cannot be observed readily in the posterior pituitary. Care should be taken to maximize the sensitivity of the assay. For example, the SAM can be purified as suggested by Bauce gt El. (1980) using the procedure of Glazer and Peale (1978). When the advantages and disadvantages of the two techniques are compared, it is obvious that the rate of DOPA accumulation after decarboxylase inhibition is the more appropriate in_yiyg_estimate of DA synthesis to characterize the regulation of the tuberohypophyseal DA system. The utility of this technique to study dopaminergic acti- vity in the posterior pituitary has been recently demonstrated (Alper gt_§l,, 1980; Alper and Moore, 1981; Demarest and Moore, 1979a). 107 II. Dehydration and Rehydration A. Delayed Activation of the Tuberohypophyseal Dopamine Neurons The activity of the tuberohypophyseal DA neurons appears to be regulated, in part, by cellular dehydration. There is controversy about thirst, AVP release and the relative importance of sodium? versus osmoreceptors (see for example, Andersson, 1977). The data presented in Results Section 11.3. clearly demonstrate that the mecha— nism responsible for increasing DA synthesis in the posterior pitui— tary is osmosensitive. The apparent delayed activation of the tubero- hypophyseal DA neurons (three days of water deprivation or 24 hours following an osmotic stimulus) is difficult to explain. This "delayed activation" of the tuberohypophyseal DA neurons may actually be an artifact of the in 1132 estimate of DA synthesis employed. Although DOPA accumulation is clearly increased in the posterior pituitary many hours after the administration of mannitol or hypertonic saline, or following three days of water deprivation, no direct measure of neuronal activity or DA release was employed. A small increase in the firing rate and subsequent release of DA from the terminals of the tuberohypophyseal neurons could markedly alter physiological responses, yet not cause a measurable increase in the synthesis rate of DA in the posterior pituitary. Assuming that the latent response of the tuberohypophyseal DA neurons is real, a simplistic view of this is that DA synthesis in the posterior pituitary can be increased only in a sluggish manner. Data presented by Demarest and Moore (1979a) do not support this possibility. The authors have demonstrated that several pharmacological 108 treatments (e.g., haloperidol, apomorphine, GBL) will moderately, but rapidly, alter the rate of DOPA accumulation in the posterior pitui- tary. The molecular regulation of tyrosine hydroxylase may be different in response to physiological and pharmacological manipula- tions. Briefly, the activity of tyrosine hydroxylase may be increased through induction, as observed following reserpine treatment (Reis gt El): 1974, 1975; Renaud_g£”§l., 1979), by increasing the affinity of the enzyme for its pteridine cofactor, as seen in the striatum after neuroleptic treatment (Zivkovic §£_al,, 1974, 1975), or by increasing the specific activity of the enzyme without altering the amount of immunoprecipitable tyrosine hydroxylase or the affinity of the enzyme for substrate or cofactor, as seen in the retina following long-term exposure to light (Iuvone g£_§l,, 1979). The pharmacologically induced acute changes in DOPA accumu- lation observed in the posterior pituitary (Demarest and Moore, 1979a) are probably due to an increase in the affinity of tyrosine hydroxy- lase for the pteridine cofactor. 0n the other hand, the sluggish response of in giyg_tyrosine hydroxylase activity (i.e., DOPA accumu- lation) in the posterior pituitary to osmotic stimulation by dehydra- tion, hypertonic saline or mannitol may involve enzyme induction. It should be noted at the outset of this discussion that induction of tyrosine hydroxylase has not been documented in the terminals of any dopaminergic fiber tract, although it has been suggested to occur in the median eminence (Johnston gt al., 1980). 109 DA synthesis is increased selectively in the median eminence 8 to 12 hours after the elevation of the prolactin concentration in the serum or CSF (see review by Moore g£_§1,, 19803). The sluggish response of the tuberoinfundibular DA neurons is dependent on protein synthesis (Johnston 35 a1., 1980) and induction of tyrosine hydroxylase has been postulated to be the mechanism involved. DOPA accumulation in the median eminence is elevated for approximately thirty hours following the administration of a high dose of haloperidol (Demarest and Moore, unpublished observations) resulting from.hyperprolactinemia induced by blockade of DA receptors located in the anterior pituitary gland (Gudelsky gt al., 1978; Demarest and Moore, 1980). However, the serum prolactin concentration is elevated for only a few hours follow- ing haloperidol (Demarest and Moore, unpublished observations). Thus, the high serum concentration of prolactin triggers events dependent on protein synthesis (induction of tyrosine hydroxylase?) to latently increase DA turnover in the median eminence. The increased tuberoin- fundibular DA activity is maintained long after the initiating stimulus has subsided. The administration of a hyperosmotic stimulus (saline or mannitol) causes a delayed activation of the tuberohypophyseal DA system in a manner reminiscent of the prolactin—induced effect on the tuberoinfundibular DA system. Three experiments suggest that intra- cellular dehydration selectively regulates the tuberohypophyseal DA neurons by a mechanism other than induction of tyrosine hydroxylase. First, hypertonic saline injections induce only short-term increases 110 in DA synthesis in the posterior pituitary. Long—term elevation would be predicted if enzyme induction had indeed occurred. Secondly, rats permitted access to water 4 hours after the administration of hyper- tonic saline do not respond with the characteristic latent increase in the rate of DOPA accumulation in the posterior pituitary. The stimulus to increase tuberohypophyseal dopaminergic activity appears to occur within the first 4 hours of saline injection when a marked elevation of plasma osmolality is observed. Had tyrosine hydroxylase induction actually been initiated during the rapidly developing cellular dehydra— tion, the subsequent availability of water would not have been expected to shut off the protein synthetic machinery, thereby inhibiting the effect of the saline injection on DOPA accumulation in the posterior pituitary. Thirdly, it is assumed that water deprivation and the acute osmotic stimuli activate tyrosine hydroxylase by the same mecha- nism. Three days of dehydration are required to increase DOPA accumu— lation in the posterior pituitary, whereas only 1—3 hours of rehydra— tion can reverse this effect. If enzyme induction was the reason for the slow onset of increased tuberohypophyseal neuronal activity, the inactivation of tyrosine hydroxylase during rehydration would be gradual, related to the turnover time of the enzyme protein. The arguments presented above assume that the activation and inactivation processes are mechanistically opposites. That is, if activation of tyrosine hydroxylase due to cellular dehydration requires protein synthesis, the inactivation involves protein degradation. This may be a false assumption. The delayed increase in tyrosine hydroxylase activity observed in the tuberohypophyseal DA neurons 111 resulting from osmotic stimulation could be due to enzyme induction. The restoration of normal enzymatic activity during rehydration could be a change in the affinity of the enzyme for substrate or cofactor. There are techniques presently available which permit the testing of this hypothesis. Finally, the latent increase in DA synthesis in the termi- nals of the tuberohypophyseal neurons might result indirectly from marked cellular hyperosmolality (see below) induced rapidly by the injection of a hyperosmotic solution or slowly by water deprivation. The increase in cellular osmolality may initiate a series of events which culminate in the selective increase in DOPA accumulation in the posterior pituitary. These events could include, but are not limited to, synthesis of tyrosine hydroxylase as discussed previously, deple- tion of the AVP or oxytocin content of the neural lobe, or synthesis or depletion of some unknown factor which is the immediate regulator of the DA nerves terminating in the posterior pituitary. As noted previously, once the events are initiated they can be reversed by permitting the rats access to water. B. Hyperosmolality and Tuberohypophyseal Dopaminergic Activity Even more perplexing than the delayed activation of tyrosine hydroxylase in the posterior pituitary is the time course of the effects of the hypertonic stimuli on DOPA accumulation. There is a delayed increase (24 hours) in the activity of the tuberohypophyseal DA neurons in rats administered hyperosmotic solutions. The increase is not maintained (control values observed 36 and 48 hours after the 112 hypertonic saline injection), but later reappears following 72 hours of water deprivation. There obviously is not a 1:1 temporal corre— lation between plasma osmolality and DOPA accumulation in the pos- terior pituitary. No effect is observed on DOPA accumulation from 1 to 4 hours after hypertonic saline, the period of peak plasma osmo— lality. The marked hyperosmolality lasts only a few hours until there is a redistribution of body water (a shift from intracellular to extracellular spaces) and until the renal excretory mechanisms can compensate for the osmotic load. The homeostatic mechanisms act very rapidly; the fluid balance parameters are stabilized about 4 hours after mannitol or hypertonic saline injection. A theoretical explanation for the effects of the hypertonic stimuli on DOPA accumulation in the posterior pituitary is outlined below. As the cellular osmolality surpasses a threshold value a series of events are triggered to increase DOPA accumulation in the posterior pituitary some 24 hours later. As the osmolality returns back through the threshold value towards normal (in the presence of continuing dehydration), the tuberohypophyseal DA system is inacti- vated, again with a 24 hour lag time. If this scheme is correct the osmo-receptive site must be exquisitely sensitive over a narrow range. When cellular dehydration is at least partially inhibited by providing water to rats administered 15% NaCl, the plasma sodium concentration rises rapidly and to a very similar degree as in those rats admini- stered saline but not allowed to drink. However, DOPA accumulation in the posterior pituitary is not increased when rats are given drinking water simultaneous to, or 4 hours following, the injection of hypertonic 113 saline. This argues against a receptor sensitive to changes in plasma, CSF or extracellular fluid osmolality in the regulation of tuberohypophyseal DA neuronal activity. Instead it supports regula- tion of DA synthesis in the posterior pituitary directly or indirectly by an intracellular osmoreceptor. The location of the osmoreceptive cells is unknown. Hypertonic stimuli may activate the tuberohypophyseal DA neurons by increasing intracellular osmolality and not by decreasing cellular volume. There is good evidence that the cerebral osmolality parallels the plasma osmolality with only a slight time delay (see Arieff g£_ 21,, 1977). Changes in the plasma osmolality, therefore, closely mirror changes in the osmolality of the cells regulating DA synthesis in the posterior pituitary if it is assumed that these cells are located centrally, possibly in the tuberohypophyseal perikarya in the arcuate nucleus. The central nervous system is partially protected from large decreases in volume during sustained hyperosmolality (Arieff and Guisado, 1976; Arieff 35 31., 1977) and extended dehydration (Jelsma and McQueen, 1967). Both CSF volume and total brain water decrease acutely following rapid hypernatremia, but return to normal after a period of hours to days (depending on stimulus and species). The osmole content of the brain and CSF is elevated even when the water content has been restored. All of the major osmotically active con- stituents of the brain are in their normal concentration. The brain cells need to maintain normal volume during sustained hypernatremia and do so by generating "idiogenic osmoles" to retain water. These 114 idiogenic osmoles have not yet been identified. The measurement of brain water content during prolonged hyperosmolar states will not be an index of cellular osmolality in the brain. It may be suggested, therefore, that cellular osmolality and not cellular volume is the factor involved in the regulation of the tuberohypophyseal DA neurons. C. Hypovolemia and Tuberohypophyseal Dopaminergic Activity Water deprivation causes not only hypernatremia but it also reduces plasma volume. The results of the present study indicate that hypovolemia is not essential for the increased DA synthesis in the posterior pituitary. Saline drinking does not produce hypovolemia but does increase DOPA accumulation. Furthermore, reversal of the plasma volume deficit pg£_§g_by allowing dehydrated rats access to a 2% NaCl drinking solution does not reduce the elevated rate of DA synthesis in the posterior pituitary of these rats. Plasma volume deficits were not considered to be vital to the water deprivation-induced effects on DA synthesis and were therefore omitted from the discussion above. This is not to say, though, that a rapid reduction in plasma volume (hemorrhage, for example) might not alter DA synthesis in the posterior pituitary. Essentially, the data suggest that DA synthesis and release in the terminals of only the tuberohypophyseal neurons may parallel, and be partially regulated by, cellular osmolality. This correlation occurs with a delay of 24 to 72 hours, depending on the intensity and the nature of the stimulus. 115 III. Possible Functions for Tuberohypophyseal Dopamine Neurons Synthetic, metabolic and receptive machinery for DA have been identified in the posterior pituitary. DA synthesis has been verified by the presence of tyrosine hydroxylase activity both in yigg_(see Results Section I.B.)vand in_yi£rg_($aavedra 35 al,, 1975). DA is utilized at a rapid rate (see Results Section I.B. and Holzbauer gt 31,, 1978) and may be enzymatically inactivated by monoamine oxidase (Saavedra 35 al., 1975; Demarest and Moore, in press). Although monoamine oxidase is present in both the neural and intermediate lobes, the concentration of DOPAC, the major deaminated metabolite of DA, is not a good index of DA release in the posterior pituitary (Annunziato and Weiner, 1980; Umezu and Moore, unpublished observa- tions). DOPAC formation is largely dependent on DA reuptake into the presynaptic terminal. Nigrostriatal DA neurons have a high affinity DA uptake mechanism which is not found in tuberoinfundibular or tuberohypophyseal neurons (Annunziato gt_§l,, 1980; Annunziato and Weiner, 1980; Demarest and Moore, 1979b). The lack of a high affinity DA transport system may explain why the DOPAC concentration in the posterior pituitary (Annunziato and Weiner, 1980; Umezu, Alper and Moore, unpublished observations) and the median eminence (Fekete 35 31,, 1979; Umezu and Moore, 1979) is very low. The data presented in the body of the thesis demonstrate that the tuberohypophyseal DA system can be selectively activated. DA synthe— sis in the posterior pituitary is increased when there are many other dynamic events occurring in the neural and intermediate lobes of the pituitary gland. Thus, DA release may be increased in the posterior 116 pituitary of dehydrated rats to mediate or modulate some pituitary function. A few possibilities will be discussed below. A. Vasopressin Release 1. Acute regulation One of the major hormones of the posterior pituitary is AVP. Water deprivation, saline drinking, volume depletion, hyper- osmotic states and a variety of other stimuli increase the secretion of AVP from the neurohypophysis (see Verney, 1947). water deprivation and saline drinking have similar effects on the pituitary and plasma concentrations of AVP (Jones and Pickering, 1969; Mens g£_al,, 1980). More detailed experiments have shown that small increases in plasma osmolality (1-2%) cause prompt increases in AVP secretion (Dunn gt 31., 1973). This is in contrast to the delayed effect of increases in plasma osmolality on DOPA accumulation in the posterior pituitary. Dunn et_al, (1973) have also demonstrated that 12 hours of water deprivation significantly increase the plasma AVP concentration in rats. Again, this can be contrasted to the 72 hours of water depri- vation required to consistently increase DA synthesis in the tubero- hypophyseal neurons. The tuberohypophyseal DA system is obviously not involved in the acute release of AVP during osmotic stimulation or dehydration. 2. Augmentation One possible function for the tuberohypophyseal DA neurons is to augment AVP release from the posterior pituitary. Following-long—term dehydration (four days) the storage pools of AVP in the neural lobe of the rat are virtually depleted (Rougon-Rapuzzi 117 E£;§l3: 1978). As dehydration continues, the release of AVP from the neurohypophysis is dependent on its rate of synthesis and transport from the hypothalamus. Gainer ‘gtual. (1977) have shown that neuro— physin precursors (MW 20,000) associated with AVP are transported from cells in the supraoptic nucleus into axons and nerve terminals in the neural lobe where they are processed into smaller releasable neuro- physins (MW 10,000). The rate of this conversion is markedly enhanced in animals provided 2% NaCl to drink for seven days (Russell gt_a1,, 1981) allowing for the immediate release of AVP as it enters the neurohypophyseal nerve terminals. The enzymes involved in neurophysin conversion and their regulatory mechanisms have not been identified. Also, the duration of dehydration required to increase the rate of pro-hormone conversion to releasable neurophysin has not been deter- mined; a temporal correlation with changes observed in the rate of DA synthesis in the posterior pituitary is not possible. However, the tuberohypophyseal DA system is activated when the posterior pituitary appears to be depleted of AVP. This increase in dopaminergic activity may stimulate either the axonal transport of the neurosecretory granules or the enzymatic conversion of the pro-hormone into its releasable form. Further evidence linking DA to the stimulation of AVP secretion has appeared in two abstracts. Negro-Vilar (1979) demon- strated that AVP secretion from isolated neural lobes was increased in the presence of DA [Bridges and coworkers (1976) earlier reported that DA slightly increased the in vi££g_secretion of AVP from the neural lobe]. Bromocriptine (a direct acting DA agonist), administered 118 orally, augmented AVP release in response to osmotic stimulation in man (Robinson gt_al,, 1981). Bromocriptine crosses the blood-brain barrier, therefore these data do not prove a pituitary site of action. Although these results have not been confirmed, a stimulatory role for the tuberohypophyseal DA neurons on AVP release is implied. The tuberohypophyseal dopaminergic pathway may not be involved in tonic regulation of hormone secretion (Kendler g£_al,, 1978), but it may be activated to augment AVP release following prolonged stimulation of the hypothalamo-neurohypophyseal system. 3. Feedback inhibition 0n the other hand, DA may function in the posterior pituitary in an AVP negative feedback loop. Bakker gt 21. (1975) reported that exogenously administered AVP increased the neural lobe content of the neurophysins without effecting the concentrations of AVP or oxytocin. The authors suggested that hormone release was decreased, intracellular degradation of AVP and oxytocin was increased, and the metabolism of the neurophysins was unaltered. The tuberohypophyseal DA system has been directly implicated in the autoregulation of AVP secretion (see abstract by Ferris_g£ual., 1979). The authors reported that low frequency stimu- lation of the arcuate nucleus in anesthetized rats decreased the spontaneous electrical activity in the neural lobe. This was blocked by the superfusion of pimozide (a DA antagonist) onto the neural lobe. Application of AVP on cells in the arcuate nucleus also reduced the electrical activity in the neural lobe. This was likewise blocked by superfusion of a neuroleptic onto the neurohypophysis. 119 Forsling and Lightman (1979), in another abstract, also proposed an inhibitory role for DA on AVP release. L-DOPA inhibited AVP secretion in man. This apparently occurred at the level of the neural lobe as pretreatment with carbidopa, a peripheral decarboxylase inhibitor, blocked the effect. It was not discussed, but L—DOPA is also converted to NE which, rather than DA, may inhibit AVP secretion. It has been reported that the neuroleptic chlorproma- zine stimulates AVP release by blocking DA receptors (Givant and Sulman, 1976). However, chlorpromazine has fairly potent anti-adrener- gic and anti-cholinergic properties. Using a more specific anti- dopaminergic drug, haloperidol, Kendler 23 31. (1978) demonstrated that DA does not tonically regulate AVP secretion in humans. The results presented in this thesis support a possible role for the tuberohypophyseal DA pathway in a proposed, though cer- tainly not proven, AVP autoregulatory feedback loop. Dehydration causes progressive increases in plasma AVP concentrations (Dunn gt ‘g1., 1973; Mens-gtnalf, 1980) which appear to be paralleled by similar progressive increases in the rate of DOPA accumulation in the posterior pituitary. Unfortunately, no data on plasma AVP concentrations have been reported following the hyperosmotic stimuli used experimentally in this thesis. According to the theory of Dunn 35 a1, (1973), hyper— tonic saline and mannitol injections would be expected to cause rapid but short bursts of AVP secretion. The plasma AVP concentration would be directly related to the plasma osmolality. The initial rapid increase in the plasma AVP concentration could feedback through the tuberohypophyseal DA system to latently inhibit its own release. This 120 feedback mechanism would be inactivated as the plasma AVP concentra- tion (reflected by the plasma osmolality) subsided. There is one serious flaw in this scheme. Three days of exogenous AVP infusion did not alter DOPA accumulation in the posterior pituitary (Table 24). However, the plasma AVP concentration in those rats administered exogenous AVP was not determined. It is unknown if either dose of AVP infused approximated the concentration of AVP in the plasma induced by three days of water deprivation or either of the hypertonic stimuli. Therefore, although weakened by these data, the hypothesis that the tuberohypophyseal DA neurons act sluggishly as feedback inhibitors of AVP secretion cannot be totally dismissed. More substantial evidence of AVP autoregulation, and then a positive correlation with DA syn- thesis in the posterior pituitary, is required. 4. Differential regulation of storage and releasable pools DA may act in the posterior pituitary to differentially regulate the release of AVP from the "storage pool" (as contrasted to the "readily releasable pool"). Sachs 35 a1, (1967) demonstrated that the initial release of AVP due to hemorrhagic hypotension was very rapid. Over time the rate of release markedly decreased and was not correlated to the AVP content of the posterior pituitary. A second hemorrhage 60 minutes after restoration of normal mean arterial pressure by reinfusion of blood stimulated AVP secretion but at a much reduced rate. The data suggest that following depletion of the readily releasable pool of AVP from the dog neurohypophysis, the release of the remaining hormone by appropriate stimuli will be at a substantially reduced rate. This observation was not related to the 121 pituitary content of AVP but apparently to an inhibitory mechanism located within the neural lobe. The tuberohypophyseal DA system may be that inhibitory mechanism. Sachs gt_§1, (1967) postulated that a factor in the neurohypophysis of anesthetized dogs inhibited AVP release 60 minutes after severe hemorrhage. Experiments have been performed demonstra- ting that DA synthesis in the posterior pituitary of rats is increased following water deprivation (72 hours) or hyperosmotic injections (24 hours). Any links between the studies by Sachs 35 31. (1967) and those reported in the body of this thesis are purely speculative since the experimental designs differ so widely. However, Sachs g£_§1, (1967) suggest "the possibility that extensive stimulation [of AVP secretion] leads to the exhaustion of some essential transmitter substance(s) or coupling factor...". Alternatively, it can be pro- posed that extensive stimulation of AVP secretion leads to the acti- vation of an inhibitory factor (i.e., DA) Which will reduce the rate of AVP release in the presence of ongoing stimulation. To test this hypothesis dogs could be bled, reinfused with blood and then hemor- rhaged a second time. One group of dogs would be administered a dopaminergic antagonist prior to the second hemorrhage. If DA was the inhibitory substance proposed to exist in the neural lobe, plasma AVP in the treated dogs would be expected to be elevated to higher concen- trations than in non-treated dogs after the second hemorrhage. 5. Indirect regulation Vasopressin secretion from the neurosecretory neurons into the systemic circulation requires neurovascular contacts and 122 adequate perfusion of the neural lobe. Changes in either of these could result in changes in plasma AVP concentrations. DA may in- directly modulate AVP secretion by quantitatively altering the neural- vascular interaction or the blood flow through the posterior pituitary. a. Pituicyte function. Tweedle and Hatton (1980a,b) have reported that the neurosecretory nerve endings in the neurohypo- physis are normally surrounded and enclosed by glial cells (pitui- cytes). Within 24 hours of water deprivation the number of neuro- secretory axons enclosed by pituicytes is decreased. This is rever- sible with 24 hours of rehydration. Wittkowski and Brinkman (1974) demonstrated that three days of dehydration increased the number of neurovascular contacts and decreased the number of gliovascular con- tacts in the posterior pituitary. This was due to an increase in the size of the nerve terminals contacting the perivascular space, not an increase in the number of neurosecretory nerve endings. These data suggest a great plasticity in the neural lobe of the pituitary which is particularly evident when the hypothalamo-neurohypophyseal system is highly active. The net result of the events occurring between the glial processes and the neurosecretory axons is to increase the accessibility of the AVP—containing terminals to the neurohypophyseal circulation. This is due to a redistribution of glial and neuronal elements in the posterior pituitary. Perhaps the tuberohypophyseal DA neurons are regulators of the pituicyte motility; DA could be either stimulatory or inhibitory. kufelt (1973) has provided data to support an inhi- bitory role for DA. He found that an infusion of DA into the lateral 123 ventricle of rats markedly decreased the number of nerve endings reaching the capillary basement membrane in the median eminence. The neurovascular contacts were disrupted by glial endfeet. DA could act similarly in the posterior pituitary as part of a hypothetical nega- tive feedback loop. DA could reduce the number of nerve terminals freed from the pituicytes, thereby inhibiting the rate of AVP secre- tion. If, on the other hand, kufelt's observations in the median eminence are the opposite of occurrances in the posterior pituitary, DA could augment AVP secretion by stimulating the retraction of the pituicytes from the neurosecretory nerve endings and the capillaries of the neurohypophysis. b. Blood flow. The studies of Baumgarten g£_al, (1972) demonstrated that catecholaminergic neurons terminated near perivascular spaces in the neural lobe of the rat pituitary. MDre recent studies (Tilders gt a1., 1979) suggested that the tuberohypo- physeal DA nerves were in close proximity to the neurohypophyseal capillaries. The DA nerve terminals appear to be situated in a posi- tion to regulate blood flow through the posterior pituitary. Blood flow to the rat posterior pituitary, but not the anterior pituitary or hypothalamus, is increased following 24 hours of water deprivation (Lichardus £5 31., 1977). The blood volume of the rat posterior pituitary is increased by stimuli (e.g., hemor- rhage, vagal stimulation, saline drinking) associated with the release of neurohypophyseal hormones (Sooriyamoothy and Livingston, 1972). The neurohypophyseal vasodilation is mediated partially by cholinergic, but not by noradrenergic, mechanisms (Sooriyamoothy and Livingston, 124 1972). Increased blood flow through the posterior pituitary is not correlated to AVP secretion. Twenty-four hours of water deprivation increase neurohypophyseal blood flow in AVP deficient Brattleboro rats (Kapitola 35 a1., 1977), whereas hemorrhage (a potent stimulus for AVP secretion) does not alter posterior pituitary blood flow in anesthetized sheep (Page EE.§l:’ 1981). Nevertheless, the effect of water deprivation and saline drinking to increase blood flow through the posterior pituitary could be mediated by the tuberohypophyseal DA neurons. Administration of exogenous DA causes vasodilation in pial arteries (Edvinsson g£_al,, 1978), renal arteries (reviewed by Gold— berg, 1972) and in the superior mesenteric artery (Clark and Mennin- ger, 1980). In all cases, specific DA receptors have been implicated. Unfortunately, the effect of DA on the neurohypophyseal vasculature has not been studied. From the literature cited above and the data presented in the body of this thesis, an interesting scheme of events can be proposed. DA synthesis and release are increased in the pos- terior pituitary during dehydration, when AVP secretion would be expected to be stimulated (Dunn gt 31., 1973; Mens gt a1., 1980). Postsynaptic DA receptors in the posterior pituitary are activated and decrease the responsiveness of B-receptors (Cote gt_al,, 1981). B- adrenergic receptors in the posterior pituitary are reportedly linked to adenylate cyclase (Stefanini 35 31,, l980b) which is located on the neurohypophyseal vasculature (Santolaya and Lederis, 1980). Further- more, posterior pituitary DA receptor activation inhibits basal and 125 stimulated adenylate cyclase activity (Cote 33 31., 1981). This may cause vasodilation to sustain or increase blood flow to the posterior pituitary even when the plasma volume is severely compromised. Maxi— mal delivery of AVP to the systemic circulation would be insured as a homeostatic mechanism to promote water reabsorption in the collecting duct of the kidney during severe dehydration. This hypothesis is based on several inferences from various sources, but it does offer a testable function for the dopaminergic neurons terminating in the posterior pituitary. For example, if DA was acting as a vasodilatory agent, haloperidol would be expected to decrease, whereas apomorphine and piribedil would increase, blood flow to the posterior pituitary. The haloperidol- induced response may be observed only in dehydrated rats; the tubero- hypophyseal DA system may have no tonic regulatory function. B. Oxytocin Release The second major hormone of the posterior pituitary is oxytocin. This peptide is released concomitantly with AVP during dehydration (Jones and Pickering, 1969), but is released with relative selectivity during parturition and lactation (Haldar, 1970). Speci— fically, oxytocin is required for milk ejection during suckling. Dopaminergic regulation of oxytocin secretion is not very clear. DA inhibits oxytocin release from cultured hypothalamo-neuro- hypophyseal system explants (Seybold 3E 31., 1978). The authors cite unpublished data suggesting that DA may stimulate oxytocin release from isolated neural lobes as had been reported previously (Bridges 33 31,, 1976). The dopaminergic involvement in oxytocin release has also 126 been studied during suckling. Oxytocin release in the milk ejection reflex may be purely neurogenic without any dopaminergic regulation (Fuchs 33 31., 1981; Russell 33 31., 1981) or it may be stimulated through a dopaminergic mechanism (Clarke 33 31., 1979). The latter study did not localize the site of DA to the posterior pituitary. A more direct approach to characterize the dopaminergic regulation of oxytocin secretion is to study biochemical indices of DA synthesis and turnover in the posterior pituitary. Holzbauer 33 31. (1978) used the aMT-induced decline of DA to suggest that DA turnover is increased in the posterior pituitary during lactation. This experi- ment, however, should be viewed with caution for several reasons. First, neither the number of pups suckled nor the duration of lacta- tion was constant for all rats. Secondly, the DA concentration in the posterior pituitary was measured at only one time (30 minutes) after the administration of dMT. The most appropriate use of the aMT- induced decline technique requires several time points and comparisons of rate constants. More recently the effect of the suckling stimulus on DOPA accumulation in the posterior pituitary has been studied (Demarest and Moore, unpublished observations; McKay 33 31., 1980). DOPA accumula— tion in the posterior pituitary was not altered following continuous suckling, 4 to 8 hours of pup deprivation, or 4 hours of pup depriva- tion followed by 30 minutes of suckling. These studies were performed in lactating rats twelve days post-partum.and all comparisons were to diestrous females. It should also be noted that the rate of DOPA 127 accumulation in the posterior pituitary is identical in lactating, diestrous and male rats (Demarest 3£_31,, 1981). Critical evaluation of the data suggest that the tubero— hypophyseal DA neurons do not influence, and in turn are not influenced by, the release of oxytocin. This is in contrast to the interaction between tuberoinfundibular DA neuronal activity and the release of prolactin in lactating rats (McKay 3E 31., 1980; Selmanoff and Wise, 1981). C. Prolactin Release Prolactin secretion from the anterior pituitary is under the tonic inhibitory control of DA released from the median eminence (Gibbs and Neill, 1978; Weiner and Ganong, 1978). Recently, Ben- Jonathan (1980) suggested that the tuberohypophyseal DA neurons may also be involved in the regulation of prolactin release. Little supportive evidence was presented, however. The presence of a common capillary bed uniting the anterior and posterior pituitaries (Bergland and Page, 1979), plus the anatomical juxtaposition of the dopamine nerve terminals to perivascular spaces in the posterior pituitary (Baumgarten 33 31., 1972; Tilders 3£_31,, 1979), make Ben-Jonathan's hypothesis tenable. Water deprivation increases DA synthesis in the posterior pituitary, but not in the median eminence. Progressive increases in the anterior pituitary DA concentration and concomitant decreases in the plasma prolactin concentration have been observed in rats deprived of water for one to three days (Alper and Moore, in preparation). 128 Marshall 3£_31, (1975), however, observed an increase in serum prolac- tin following dehydration. Dehydration may increase the amount of DA released directly into the neurohypophyseal capillaries or, due to the inadequate mechanisms to inactivate DA in the posterior pituitary, the excess amine being synthesized and released may overflow into the intrapituitary circulation. In either case, the amount of DA trans— ported to the anterior pituitary will increase during dehydration. Greater quantities of DA can thus be incorporated into the prolactin- secretory granules to inhibit prolactin release from the anterior pituitary (Gudelsky 33 31,, 1980; Nansel 3£_31,, 1979). These data do not necessarily imply that the physiological role of the tuberohypophyseal DA system is to regulate the secretion of anterior pituitary hormones. First, the data do not prove that the increased rate of DA release in the posterior pituitary is actually causing the inhibition of prolactin release; the two events may be unrelated. Prolactin release is influenced by many biogenic amines and peptides other than DA (see review by Weiner and Ganong, 1978). Secondly, if the tuberohypophyseal DA system played an important role in regulating prolactin secretion, DA synthesis in the posterior pituitary would be expected to be regulated much like it is in the median eminence. In fact, however, DA turnover in the posterior pituitary is .not increased following the infusion of prolactin into the CSF (Johnston 3E 31., 1980) or following hyperprolactinemia in- duced by pituitary transplants under the kidney capsule (Morgan and Herbert, 1980), while both treatments markedly enhance DA synthesis and turnover in the median eminence. 129 Teleologically the dehydration-induced decrease in the plasma prolactin concentration is an interesting phenomenon. Prolac- tin is a well known natriuretic and anti-diuretic hormone in many species, but its role on fluid and electrolyte metabolism in mammals is not completely understood (see review by Horrobin, 1980). If pro— lactin is an anti-diuretic hormone in rats, plasma prolactin concen- trations would be expected to increase during dehydration. Further investigations into the relationship between dehydration, the tubero- hypophyseal DA system and prolactin secretion could prove extremely interesting. SUMMARY AND CONCLUSIONS Sensitive radioenzymatic assays for DA, NE and DOPA were employed in preliminary investigations on mechanisms regulating DA synthesis in the posterior pituitary, terminals of the tuberohypophyseal DA system. DA synthesis was also estimated in the median eminence and striatum, terminals of the tuberoinfundibular and nigrostriatal DA systems, respectively. The results of the studies will be reviewed briefly. 1) The catecholamines DA and NE appear to be synthesized, stored and released in neurons terminating in the posterior pituitary gland of the rat. Both the concentration and synthesis rate of DA are much greater than for NE. Approximately 30-35% of the NE-containing neurons of the posterior pituitary are of peripheral sympathetic origin. Even though there is a substantial concentration of NE in the posterior pituitary, the rate of DOPA accumulation following the inhibition of DOPA decarboxylase (an 13 3133_estimate of tyrosine hydroxylase activity) reflects primarily DA synthesis. Thus, DOPA accumulation can be measured in the posterior pituitary as an 13_y133' estimate of DA synthesis (and therefore neuronal activity) after the administration of the decarboxylase inhibitor NSD 1015. 2) DOPA accumulation progressively increases in the posterior pituitary of water-deprived male rats, attaining statistical signi- ficance in both male and female rats deprived of water for three days. 130 131 The increased rate of DA synthesis is observed when there is no change in the DA concentration of the posterior pituitary. Furthermore, water deprivation does not increase the DA concentration or the rate of DOPA accumulation in the terminals of the tuberoinfundibular or nigrostriatal DA systems. These results suggest that water depriva- tion selectively increases the synthesis and release of DA in the posterior pituitary, the terminals of the tuberohypophyseal dopaminer- gic neurons. 3) DA synthesis in the posterior pituitary is increased by other stimuli related to water deprivation. The activity of the tuberohypophyseal DA neurons and the plasma sodium concentration are both increased following saline drinking; indices of plasma volume are unaltered. Also, 24 hours after the administration of hypertonic saline or mannitol, DOPA accumulation in the posterior pituitary is elevated above control. This effect appears to be related (either directly or indirectly) to a rapid and marked intracellular dehydra- tion induced by the hypertonic solutions. The effect of hypertonic saline administration on DA synthesis is blocked by providing rats free access to water. The reason for the delayed effect of osmotic stimuli on the tuberohypophyseal DA neurons is unclear. 4) The dehydration-induced increase in DA synthesis in the tuberohypophyseal neurons occurs after a long latent period. In contrast, within 3 hours of presentation of water to dehydrated rats, the rate of DOPA accumulation in the posterior pituitary is returned to control. The rapid reduction of the elevated tuberohypophyseal DA neuronal activity follows a temporal pattern similar to the reduction 132 in the plasma sodium concentrations, while the hematocrit is returned at a much slower rate. When water-deprived rats are provided 2% NaCl to drink, measurements of plasma volume return to control whereas the plasma sodium concentration and the rate of DA synthesis in the pos- terior pituitary remain elevated. These data implicate cellular osmolality and not plasma volume as a major factor associated with regulation of tuberohypophyseal DA neuronal activity. In conclusion, DA synthesis in the posterior pituitary is moder- ately increased by DA antagonists and decreased by DA agonists. These effects are similar to, though quantitatively less than, the marked effects of dopaminergic drugs in the striatum. The tuberohypophyseal and nigrostriatal DA neurons have presynaptic DA receptors to regulate DA synthesis. These presynaptic autoreceptors are absent from the tuberoinfundibular DA neurons. Neither the tuberohypophyseal nor the tuberoinfundibular DA systems have a high affinity DA uptake mecha- nism. DA synthesis in the median eminence is latently activated by increased serum or CSF concentrations of prolactin; this is not ob- served in the posterior pituitary. Furthermore, the administration of morphine does not alter DA synthesis in tuberohypophyseal system, the only central dopaminergic system to behave in this manner. 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