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Pharmacology & [ox1coIogy flaw degree in Major professor Date véfl ‘27 z /952 MSU is an Affirmative Action/Equal Opportunity Institution 0-12771 MSU LIBRARIES “ RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. Mum... ‘5. 5, THE SYNTHESIS AND METABOLISM OF 5—HYDROXYTRYPTAMINE IN DISCRETE REGIONS OF THE RAT BRAIN DURING VARIOUS PHARMACOLOGICAL, ENVIRONMENTAL AND ENDOCRINOLOGICAL MANIPULATIONS By Craig Alan Johnston 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 1982 ‘v‘ v“ o - ABSTRACT The Synthesis and Metabolism of S-Hydroxytryptamine in Discrete Regions of the Rat Brain During Various Pharmacological, Environmental and Endocrinological Manipulations by Craig Alan Johnston A simple, sensitive and rapid method using high performance liquid chromatography (HPLC) coupled with electrochemical detection was de— veloped for the concurrent measurement of picogram quantities of 5- hydroxytryptamine (S-HT), S—hydroxyindole-B-acetic acid (S-HIAA) and 5— hydroxytryptophan (S-HTP) in the median eminence (ME), medial preoptic nucleus (MPO), striatum (ST), suprachiasmatic nucleus (SCN) and arcuate nucleus (AN) of a single rat brain. S-HTP in these brain areas prior to decarboxylase inhibition is essentially zero. NSD 1015, an inhibitor of decarboxylase, caused a linear accumulation of 5-HTP in these brain areas for at least 30 minutes. In the present study the concentration of S-HIAA was taken as an index of 5-HT metabolism, and the rate of 5- HTP accumulation as an index of 5—HT synthesis. Pargyline increased 5—HT and decreased S—HIAA concentrations and probenecid increased 5-HIAA in all brain regions. Reserpine increased 5-HT synthesis and metabolism at 2 and 24 hr and chlorimipramine or fluoxetine decreased S-HT synthesis and metabolism in the selected brain regions. L—Tryptophan increased S—HT synthesis and metabolism in the Craig Alan Johnston SCN, MPO, AN and ST. Mbrphine increased S—HT synthesis and metabolism in the MPO, AN and SCN through an opiate-receptor mediated mechanism. Restraint stress increased serum prolactin and 5-HT synthesis in the MPO and SCN. S-HT synthesis and metabolism was increased in the ME of lactating rats. Suckling increased serum prolactin and 5—HT synthesis and metabo- lism in the MPO. Early in pregnancy S—HT synthesis was increased in the AN and SCN at a time coincident with the nocturnal surge of prolactin. The diur- nal surge of prolactin was associated with an increase and decrease in S-HT synthesis in the AN and SCN, respectively. Later in pregnancy, when the nocturnal surge does not occur, S-HT synthesis was no longer stimulated in the AN but was in the SCN. Serum prolactin and 5—HT synthesis and metabolism in the SCN increased and 5—HT synthesis in the ME decreased on the afternoon of proestrus. to the greatest gift of my life, my lovely wife, Sharon; and to our Lord, Jesus Christ ii ACKNOWLEDGEMENTS The author takes this opportunity to acknowledge and give glory to the guidance, comfort and love constantly supplied by Jesus Christ, the Holy Spirit and God, the Father that has allowed me to accomplish the degree of Ph.D. despite my many shortcomings. The author also wishes to acknowledge the love, guidance and support of his wife, Sharon Rae; and his mother, E. Jean, father, Clayton A., and sister, Debra J. Johnston. He expresses his deepest gratitude and sincere appreciation for the guidance, patience, concern, development, constant support, love and warm friendship always afforded he and his wife by Dr. Kenneth E. Moore and his family. He acknowledges the constructive criticism, advice and time offered him by the other members of his graduate committee, Dr. Theodore M. Brody, Dr. Gerard L. Gebber, Dr. Glenn 1. Hatton and Dr. Joseph Meites. He acknowledges the guidance, support, consideration and friendship of Dr. John D. Fernstrom in allowing him his first exposure to scienti— fic research and helping him begin to attain his professional goals. He expresses his deepest appreciation to Dr. Keith T. Demarest for his advice, friendship and collaboration on several experiments through— out the author's graduate training. He wishes to thank Dr. Gail D. Riegle for his advice, facilities and his willing collaboration on several experiments. He recognizes and treasures the love, friendship, concern and con- stant support (financial and otherwise) of Mr. and Mrs. Alfreds and Mirdza Gramatins. He deeply appreciates the excellent technical assistance and friendship of Mrs. Susan Stahl and Miss Susan Redding. He thanks Mrs. Muriel Shinaver, Miss Diane Hummel, Mrs. Marty Burns and Miss Debbie Fish for their excellent secretarial and organizational skills as well as their consistently enjoyable sense of humor. He values greatly and wishes to acknowledge the advice, special friendship and unique closeness shared with Dr. Richard H. Alper, Dr. David Doolittle, Karen Lawson-Wendling, Dr. Jann A. Nielsen and Dr. Suzanne M. Wuerthele during his graduate‘training. He appreciates the professional counsel, advice and concern offered him by Dr. Gregory D. Fink, Dr. William H. Lyness and Dr. Janice L. Stickney. He values the friendships of Nancy Duda, Dr. Douglas C. Eikenburg, David T. Mokler and Dr. Katsushi Yamada which were formed during the years of his graduate training. iv TABLE OF CONTENTS Page DEDICATION ii ACKNOWLEDGEMENTS iii LIST OF TABLES viii LIST OF FIGURES x INTRODUCTION--—- 1 I. Justification for Selection of Regions 5 II. Neuroanatomy of 5-HT Neurons 7 III. Neurochemistry of 5—HT Neurons 12 IV. Methods for Estimating 5-HT Neuronal Activity —————————— l7 STATEMENT OF PURPOSE 24 MATERIALS AND METHODS 26 I. Animals and Blood Collection 26 II. Biochemical Procedures 26 A. Dissections 26 B. High Performance Liquid Chromatography Technique—— 31 C. Preparation of Mobile Phase 39 D. Radioimmunoassay 39 E. Estimation of 5-HT Neuronal Activity —- 40 F. Drugs 41 1. Miscellaneous Drugs 41 2. Solutions and Drugs Used in the Preparation of the Mobile Phase for the HPLC Analysis——-- 42 3. Compounds Used as Standards for HPLC Analysis 42 G. Statistics 42 H. Specialized Methods Used in Experiments Involving Environmental and Endocrinological Manipulations—— 43 l. Restraint Stress 43 2. Proestrous Surge 43 3. S—HTP Accumulation Time Course in Female Rats 44 4. Pregnancy 44 5. Lactation/Suckling 44 TABLE OF CONTENTS (continued) RESULTS AND DISCUSSION I. Measurement of 5—HT, S—HIAA and S—HTP in Selected Regions of the Rat Brain Using HPLC with Electrochemi- cal Detection II. Pharmacological Verification that HPLC with Electro— chemical Detection can Detect Altered Metabolic Acti— vity in 5—HT and DA Neurons in Selected Regions of the Rat Brain A. B. G. Effect of NSD 1015 on S—HTP Accumulation in Selected Brain Regions of Male and Female Rats-—-— Effect of Pargyline or Probenecid on DA, DOPAC, 5-HT and S-HIAA Concentrations in Selected Regions of the Rat Brain Effect of Reserpine on S—HT Metabolism and Synthe— sis in Selected Regions of the Rat Brain —————————— Effect of Chlorimipramine or Fluoxetine on S-HT Metabolism and Synthesis in Selected Regions of the Rat Brain Effect of Tryptophan on S-HT Metabolism and Syn- thesis in Selected Regions of the Rat Brain ——————— Effect of Morphine on S-HT Metabolism and Synthe— sis in Selected Regions of the Rat Brain —————————— 1. Effect of Various Doses of Morphine on S-HT Synthesis in Selected Regions of the Rat Brain 2. Effect of Morphine on 5-HT Metabolism and Synthesis in Selected Regions of the Rat Brain 3. Effect of Morphine and Naloxone on S-HTP Accumulation in Selected Regions of the Rat Brain Discussion of Pharmacological Manipulations ——————— III. Effect of Restraint Stress on S—Hydroxytryptaminergic Neuronal Activity and Serum Prolactin A. B. Effect of Restraint Stress on 5-HT Metabolism in Selected Regions of the Rat Brain — Effect of Restraint Stress on 5—HT Synthesis in Selected Regions of the Rat Brain and Serum Pro- lactin IV. Effects of Suckling and Pregnancy on S—Hydroxytryptami— nergic Synthesis and Metabolism in Selected Brain Regions and Serum Prolactin in the Female Rat —————————— vi Page 46 46 51 51 52 61 64 66 69 72 74 77 81 82 83 84 90 .......-.--I|‘i TABLE OF CONTENTS (continued) Page A. Evidence for Involvement of 5—HT Neurons in the Suckling-induced Release of Prolactin 90 1. Effect of Suckling on 5—HT Metabolism in Selected Regions of the Rat Brain 92 2. Effect of Suckling on S-HT Synthesis in Selected Regions of the Rat Brain and on Serum Prolactin 93 B. Evidence for Involvement of 5-HT Neurons in Pro— lactin Secretion During Pregnancy 97 1. Serum Prolactin and 5-HT Metabolism in Selected Regions of the Rat Brain During Day 6 of Pregnancy 99 2. S-HT Synthesis in Selected Regions of the Rat Brain During Day 6 of Pregnancy 103 3. S—HT Synthesis in Selected Regions of the Rat Brain at 4:30 and 12:00 hr on Day 8 and Day 13 of Pregnancy 103 V. 5-Hydroxytryptamine Metabolism and Synthesis in Selected Brain Regions and Serum Prolactin Throughout Proestrus in the Female Rat 107 A. Serum Prolactin and 5-HT Metabolism in Selected Brain Regions During Proestrus in Female Rats ————— 109 B. 5—HT Synthesis in Selected Brain Regions During Proestrus in Female Rats 113 SUMMARY AND CONCLUSION 116 BIBLIOGRAPHY— 124 vii Table 5a 5b 10 ll 12 LIST OF TABLES Literature values for 5—HT, 5—HIAA and 5-HTP accumula— tion in selected regions of the rat brain Quantification of 5-HT, 5-HIAA and 5-HTP Concentrations in Selected Regions of Rat Brain Using High Performance Liquid Chromatography with Electrochemical Detection——— Time Course of S-HTP Accumulation Following NSD 1015 (100 mg/kg, ip) in Male Sprague—Dawley Rats Time Course of 5—HTP Accumulation Following NSD 1015 (100 mg/kg, ip) in Female Long—Evans Rats Effect of Probenecid and Pargyline on DA, DOPAC, 5-HT and 5—HIAA in Selected Regions of the Rat Brain ———————— Effect of Probenecid and Pargyline on DA, DOPAC, 5-HT and 5—HIAA in Selected Regions of the Rat Brain ———————— Effect of Reserpine on 5—HT Metabolism and Synthesis in Selected Regions of the Rat Brain Effect of Chlorimipramine or Fluoxetine on 5-HT Metabo— lism and Synthesis in Selected Regions of the Rat Brain Effect of Tryptophan on 5—HT Metabolism and Synthesis in Selected Regions of the Rat Brain Effect of Various Doses of Morphine on 5—HTP Accumula- tion in Selected Regions of the Rat Brain- Time Course of Effect of Morphine on 5-HTP Accumulation in Selected Regions of the Rat Brain Effect of Morphine on 5-HT Metabolism and Synthesis in Selected Regions of the Rat Brain Effects of Morphine and Naloxone on the Rate of 5—HT Synthesis in Selected Regions of the Rat Brain ————————— viii Page 47 49 53 54 58 59 63 65 67 73 75 76 78 LIST OF TABLES (continued) Table 13 14 15 16 17 18 19 20 21 Page Effect of Acute Restraint Stress on 5—HT Metabolism in Selected Regions of the Rat Brain 85 Effect of Acute Restraint Stress on 5—HTP Accumulation in Selected Regions of the Rat Brain 86 Effect of Acute Suckling on 5—HT Metabolism in Selected Regions of the Rat Brain 94 Effect of Acute Suckling on 5—HTP Accumulation in Selected Regions of the Rat Brain— 95 5-HT Metabolism in Selected Regions of the Rat Brain at Various Times of Day 6 of Pregnancy 102 5—HTP Accumulation in Selected Regions of the Rat Brain at Various Times of Day 6 of Pregnancy 104 5—HTP Accumulation in Selected Regions of the Rat Brain at 4:30 and 12:00 hr on Day 8 and Day 13 of Pregnancy-— 106 5-HT Metabolism in Selected Regions of the Rat Brain at Various Times on Proestrus 112 5—HTP Accumulation in Selected Regions of the Rat Brain at Various Times on Proestrus 114 Figure LIST OF FIGURES Page Schematic horizontal section (a) of rat brain at the level of the medial forebrain bundle and frontal sec- tions at (b) the level of the anterior commissure and (c) 2500 u caudal to the anterior commissure 9 Schematic representation of a 5—hydroxytryptaminergic nerve terminal 13 Photographs of three frontal sections with examples illustrating location of punches 28 Principles of electrochemical detection— 35 HPLC chromatograph resulting from an injection of a mixture of authentic standards (light peaks) and a single ME sample (dark peaks) 37 Accumulation of 5—HTP in the suprachiasmatic nucleus (SCN, (D) and striatum (ST, (D) from male Sprague- Dawley rats at various times after NSD 1015 (100 mg/kg, ip) 55 Serum prolactin during day 6 of pregnancy 100 Serum prolactin on proestrus 110 INTRODUCTION S—Hydroxytryptamine (5—HT, serotonin) neuronal systems are believed to play a role in a wide variety of functions in the brain. A partial list of these functions includes regulation of sleep-wake cycles, sexual and feeding behaviors, locomotion, aggression, body temperature, noci- ception, memory and cardiovascular systems. 5-HT neurons projecting to the hypothalamus appear to be involved in regulating the release of several pituitary hormones (see reviews: Wilson, 1974; Knowles and Vollrath, 1974; McCann and Ojeda, 1976, 1979; Muller gt_al,, 1977; Weiner and Ganong, 1978; Sawyer, 1979). It has been suggested that 5—HT is a neurotransmitter involved in the stimulation of prolactin release from the anterior pituitary. This conclusion has been based primarily on resultsobtained from experiments using 5—HT precursors and putative 5—HT receptor antagonists. When added in vit£9_to pituitary glands of normal rats 5—HT is without an effect on prolactin secretion (Birge et al,, 1970; Lamberts and MacLeod, 1978) suggesting that the site of interaction of 5—HT with the secretion of prolactin occurs at the level of neural circuits located upstream to the pituitary lactotrophs. However, exactly how, when and where activation of 5-HT neurons controls physiological fluctuations in prolactin secretion is still a very con— troversial subject. Much of the literature on this subject is difficult 2 to interpret because it is based largely on lesion studies, gross neuro- chemical analyses and pharmacological studies using drugs that are systemically administered. Furthermore, different investigators often test endocrine effects of 5—HT under different, not necessarily com— parable experimental conditions. Pharmacological studies are limited in their usefulness because of two additional reasons. First, drugs affecting S—HT transmission in one area of the brain often do so else— where in the brain and can thus cause simultaneous changes in many systems that may affect pituitary secretion either directly or indirect— ly. Secondly, nearly all drugs which are currently thought to interact with 5-HT neuronal systems lack specificity. For example, high doses (3100 mg/kg) of the 5-HT precursor, 5-hydroxytryptophan (S-HTP), can enter catecholamine terminals, be decarboxylated to form 5—HT and dis- place endogenous catecholamines (Ng 25 al., 1972). p-Chlorophenylala— nine (PCPA) inhibits S—HT biosynthesis but also affects the metabolism of catecholamines by competing with the uptake of tyrosine (the pre— cursor for dopamine (DA) and norepinephrine) into catecholaminergic neurons (wurtman, 1974). Furthermore, many of the putative 5—HT recep— tor antagonists may not be active in the central nervous system, and many exert agonistic and antagonistic effects at other aminergic recep— tors (Lamberts and MacLeod, 1978; Besser_e£flal., 1980). Even some of the seemingly more specific 5-HT receptor antagonists, such as meter- goline or methiotepin, are specific for 5-HT receptors only in a very narrow concentration range. An important first piece of evidence for the involvement of 5—HT neurons in a particular behavioral, pharmacological or endocrinological 3 event can be obtained by measuring 5—HT neuronal activity during the event. 5-HT neuronal activity in the whole brain and large brain regions has been examined throughout several experimental paradigms (for examples see reviews: Wilson, 1974; Knowles and Vollrath, 1974; McCann and Ojeda, 1976, 1979; Mfiller_g£flal., 1977; Weiner and Ganong, 1978; Sawyer, 1979). However, whether results from experiments examining this activity in large areas of the brain will identify functionally impor- tant changes occurring in discrete brain regions is unknown. Relatively few reports of studies designed specifically to charac— terize 5-HT neuronal activity in discrete hypothalamic nuclei exist. This is primarily due to technical problems associated with the rela— tively poor histochemical procedures for this amine (Parent, 1981), and the lack of sensitivity of analytical techniques for analyzing 5—HT, 5— HTP, and its major metabolite, 5—hydroxyindole-3-acetic acid (5—HIAA). The recent development of sensitive radioenzymatic (Tappaz and Pujol, 1980) and high performance liquid chromatographic (HPLC) (Krstulovic and Matzura, 1979; Meek and Lofstrandh, 1976) assays for 5~HTP and assays using HPLC in combination with electrochemical detection (Loullis_e£ al,, 1979) or radioimmunoassays (Delaage and Puizillout, 1981) for measuring 5—HT and 5-HIAA has made it possible to employ biochemical techniques to estimate 5-HT neuronal activity in areas such as the striatum, hypothalamus and mediobasal hypothalamus. No technique per— mitted the concurrent measurement of 5-HT, S-HIAA and 5-HTP in a sample of discrete hypothalamic nuclei such as the suprachiasmatic or medial preoptic nucleus from a single rat brain. Therefore, one of the primary objectives of the present study was to develop an assay capable of 4 detecting 5-HT, 5—HIAA and 5—HTP in discrete hypothalamic nuclei of the rat brain that had been implicated in the regulation of prolactin secretion. The capability to measure S—HT neuronal activity in dis- crete hypothalamic nuclei generates more questions than it answers initially. Much evidence already exists to suggest that 5-HT neurons not only may be involved in the regulatory control of the secretion of several hormones but also that 5—HT neuronal systems may functionally interact with several other neurotransmitter/neuropeptide/neurohormonal systems (Meites et_al,, 1963; Weight and Salmoiraghi, 1968; Nicoll, 1971; Meites, 1973; Ford_e£nal., 1974; Knowles and Vollrath, 1974; Wilson, 1974; MacLeod, 1976; McCann and Ojeda, 1976, 1979; Bruni et_al,, 1977; Mfiller_etflal., 1977; Baumgarten 35 al., 1978; Weiner and Ganong, 1978; Holaday and Loh, 1979; Iwamoto and Way, 1979; Meites ggwal., 1979; Sawyer, 1979; Koenig et_al,, 1980; Moore and Johnston, 1982). Thus, the measurement of neuronal activity in these 5—HT systems only supplies the means to begin to examine the functional roles and inter- actions of 5—HT neurons in the brain. In.addition it would be advan— tageous if the method could also measure DA and its major metabolite, 3,4—dihydroxypheny1acetic acid (DOPAC). Because steady—state conditions as well as drug effects on the dopaminergic neuronal systems in discrete brain areas have been more fully characterized than on 5-HT neuronal systems, the measurement of DA and DOPAC concentrations served as a check on pharmacological effects as well as providing further verifi— cation that the regions examined were indeed the areas that had been selected to be investigated. Such a verification proved very important 5 because no information on the concentration of 5—HIAA or S—HTP accumu— lation in some of the hypothalamic nuclei was available. I. Justification for Selection of Regions The discrete hypothalamic regions chosen for the present analysis included the suprachiasmatic nucleus, the medial preoptic nucleus, the arcuate nucleus and the median eminence. In addition, a sample of the striatum was also taken as an example of an extrahypothalamic region. All of these regions contain substantial quantities of 5—HT as well as the rate—limiting enzyme in 5—HT synthesis, tryptophan hydroxylase (Saavedra et_al,, 1974; Brownstein et_al., 1976a). The suprachiasmatic nucleus (SCN) has been shown (mostly via lesion studies) to be essential for entrainment of many types of circadian rhythms including the prolactin surges associated with the estrous cycle at the time of ovulation, and the diurnal and nocturnal prolactin surges observed in pregnancy and pseudopregnancy (Brown—Grant and Raisman, 1977; Bethea and Neill, 1979, 1980; Dunn et_al., 1980; Yogev and Terkel, 1980). This nucleus also appears to be an important modulator or relay station of preoptic efferents projecting to the mediobasal hypothalamus, and is believed to exert inhibitory control on prolactin secretion (Kimura and Kawakami, 1978). The SCN receives some of its innervation from the tuberal hypothalamus and sends efferents through the ventral periventricular and arcuate nuclei into the ventral tuberal area to terminate throughout the periventricular and arcuate nuclei as well as the internal and subependymal median eminence (Moore, 1979). Further— more, S-HT concentrations in the SCN decrease following acute immobili— zation stress for up to 3 hours (Palkovits e£_al., 1976). 6 The medial preoptic nucleus (MPO) is both anatomically and func— tionally connected with the arcuate nucleus (Whitehead and Ruf, 1974; Renaud and Martin, 1975). Axons project from cell bodies in the MPO to innervate the external layer of the median eminence (Terasawa and Sawyer, 1969; Dyer and Saphier, 1981). Stimulation of the MPO causes ovulation, increases plasma concentrations of prolactin and electrical activity in the arcuate nucleus, and inhibits neuronal activity in the median eminence. Lesioning the MPO induces pseudopregnancy which is characterized by diurnal and nocturnal prolactin surges (Freeman and Banks, 1980). The MPO not only appears to contain neurons which are inhibitory to the nocturnal surge of prolactin but that are also stimu— latory to the diurnal surge (Freeman and Banks, 1980). A bilateral lesion of the MPO blocks ovulation and the associated surges of lutein— izing hormone and prolactin without affecting the follicle stimulating hormonal surge (Terasawa gt_al,, 1980; Kimura and Kawakami, 1978). The arcuate nucleus (AN) contains the cell bodies of several differ— ent neurons which project to the tuber cinareum, including the tubero— infundibular dopamine (TIDA) neurons (Bjorklund and Nobin, 1973) which functionally act to tonically inhibit the secretion of prolactin from the pituitary (MacLeod, 1976). It has been suggested that if 5—HT neurons inhibit TIDA neurons; one possible site for this interaction is in the AN. In addition, recent studies have revealed the existence of 5—HT containing cell bodies in the arcuate nucleus (Smith and Kapers, 1975; Kent and Sladek, 1978). The median eminence (ME) represents the final common neural pathway to the anterior pituitary. It contains nerve terminals of several 7 tuberoinfundibular neuronal systems including those of the TIDA neurons and possibly those thought to contain a presently unidentified prolactin releasing factor. The ME not only receives projections from the AN, but also from the SCN and MPO (Terasawa and Sawyer, 1969; Moore, 1979; Dyer and Saphier, 1981). Finally, the ME is a storehouse of putative neuro— transmitter substances and hormones which regulate anterior pituitary hormonal release (Palkovits, 1980). The striatum (ST) was selected as the extrahypothalamic control region for several reasons. The ST contains well defined innervations of both DA— and 5-HT—containing neurons (Dahlstrfim and Fuxe, 1964; Azmitia and Segal, 1978; Ungerstedt, 1971). The ST is primarily inner- vated by S—HT neurons originating in the dorsal raphé nucleus (Dray, 1981; Parent, 1981), with a minimal projection from the median raphé nucleus (Conrad 55 al., 1974; Miller e£_al., 1975; Azmitia and Segal, 1978; Jacobs g£_al,, 1978; Parent, 1981). Lastly, aminergic neuronal responses in the ST to several pharmacological, environmental and endocrine paradigms have been frequently studied and provide a large amount of background information for comparison (Yarbrough et_al., 1971, 1973; Carlsson £5.3lra 1972a; Gudelsky and Moore, 1976; Kueng et a1,, 1976; Mena £5 31., 1976; Palkovits 33 al., 1976; Karoum at 31., 1977; Demarest and Moore, l979a,b; Marco and Meek, 1979; Umezu and Moore, 1979; Alper et_al,, 1980; Moore gt 31., 1980a,b; Demarest §£.al., 1981a,b). II. Neuroanatomy of 5—HT Neurons Current knowledge of the anatomy of 5—HT neurons is derived pri— marily from the results of innumerable investigations in the rat (see review by Steinbusch et_al,, 1981). This knowledge stems primarily from results of studies using the Falck and Hillarp fluorescence histochemi- cal method (Falck at al., 1962) which permits visualization of 5-HT at the cellular level. Other techniques that have added valuable knowledge to our present understanding of 5—HT neuroanatomy include the use of tritiated 5-HT uptake as a marker for 5—HT neurons (Descarries §t_al., 1975) and the use of immunohistochemical techniques to visualize trypto- phan hydroxylase (Pickel e£_al., 1976, 1977) and 5—HT (Steinbusch_et 31,, 1978; H5kfe1t_eg.al., 1979). The distribution of 5-HT neurons in the brain has been extensively reviewed by Azmitia (1978). The anatomy of this distribution to areas of interest in the present study is shown schematically in Figure 1. Initially, Dahlstrom and Fuxe (1964) demonstrated that, in the rat, most S—HT nerve cell bodies are located in the brain stem raphé. Originally, 9 major groups of 5—HT—containing cells were identified and designated B1_B9' Perikarya in at least four and probably five of these nuclear groups project to the forebrain (McGeer e£_a13, 1978; Parent g£_al,, 1981). The most medial ascending 5—HT pathway arises primarily from cell groups B7 and B8 although some fibers come from cell groups B5 and B6 and project ventrally through the decussation of the superior cere— bellar peduncles before passing rostrally within the medial forebrain bundle (MFB) to terminate in several hypothalamic, preoptic and septal areas. A second, slightly more laterally located pathway also arises from cell bodies located mainly in the midbrain raphé of groups B7 and B8 with small contributions possibly being contributed from cell groups B5 and B6' Fibers of this 5—HT ascending system also follow the MFB and Figure 1. Schematic horizontal section (a) of rat brain at the level of the Medial Forebrain Bundle and frontal sections at (B) the level of the anterior commissure and (c) 2500p caudal to the anterior com- missure (modified from Saavedra 35 al., 1974 and Palkovits §t_al,, 1980). Abbreviations: A, amygdala; AC, anterior commissure; AN, arcuate nucleus; CI, internal capsule; F, fornix; MB, mammillary body; ME, median eminence; MFB, medial forebrain bundle; NDM, dorso— medial nucleus; NHA, anterior hypothalamic nucleus; NIST, interstitial nucleus of the striae terminalis; NPO, preoptic nucleus (1 = lateral, m = medial, and p = periventricular); NVM, ventromedial nucleus; oc, optic chiasm; OT, optic nerve; PM, premammillary nuclei; RN, raphé nuclei (individual cell groups B5-9); S, preoptic suprachiasmatic nucleus; SCN, suprachiasmatic nucleus; st, striae terminalis; ST, striatum. 10 01' "\ ANTER‘OR -'—-- ,1 p-lI- I'll I ’I"--""l ‘.l 4 04 \. “\ €39.20 '0- 25004 O n x”) a“ V POSTERIOR DORSAL. \ 53.630 ~)C Figure l In 11 then sweep dorsally along the cingulate gyrus and laterally to terminate in the hippocampus branching along the way to innervate all cortical areas. A third system originates from cell group B9 with some contri— bution from B7 and B8 also possible and ascends slightly lateral to the MFB to terminate primarily in the corpus striatum. S-HT neurons inner— vating the SCN originate in the rostral part of the medial (B8) and dorsal (B7 and B6) raphé (Héry 35 al., 1978). 5-HT innervation to the MPO primarily originates in the median raphé nucleus (Azmitia and Segal, 1978). S—HT fibers destined to terminate in the tuberoinfundibular area form a compact bundle in the mesencephalon, just above the interpeduncu- 1ar nucleus, prior to entering the hypothalamus (Palkovits_gt_al,, 1977). Taking advantage of the specific 5—HT uptake system (Shaskan and Snyder, 1970), Calas 3.3 21- (1974) identified 3H—labelled 5-HT-contain— ing neurons distributed throughout the ME, although they were more abundant in the external layer. Although 5—HT neurons terminating in the ME and other intrahypo— thalamic areas are generally considered to originate in the dorsal midbrain raphé, the results of some experiments indicate that there may also be another source. The relatively high concentrations of 5-HT and tryptophan hydroxylase found in the ME following lesions to the raphé or surgical isolation of the medial basal hypothalamus (Brownstein 35 .al., 1976b; Palkovits §E_al,, 1977) suggest the presence of 5-HT neurons in the latter brain region. Neuronal perikarya in the arcuate nucleus of the rat hypothalamus which contain yellow histofluorescence possess— ing spectral characteristics consistent with that of a 5—HT fluorophor have been identified using microspectrofluorometric and histopharma— cological techniques (Kent and Sladek, 1978). Beaudet and Descarries 12 (1979) have demonstrated possible S-HT perikarya in the pars ventralis of the dorsomedial hypothalamic nucleus utilizing radioautographic techniques following intraventricular perfusion with tritiated 5—HT. 5—HT autofluorescent granules have been demonstrated by Smith and Kappers (1975) in neuronal perikarya of the arcuate and ventromedial hypothalamic nuclei of rat brains. ‘Microspectrofluorimetric techniques have also revealed the presence of 5—HT in the area of the tanycytes in the ME (Sladek and Sladek, 1978). III. Neurochemistry of 5—HT Neurons A schematic representation of the events thought to be involved in the synthesis, release and inactivation of 5—HT at neuronal terminals within the brain is depicted in Figure 2. Tryptophan, the essential amino acid precursor of 5—HT, is actively transported into 5-HT neurons where it is hydroxylated to form S-hydroxytryptophan (5—HTP). This reaction is catalyzed by the rate-limiting enzyme in the synthesis of 5—HT, tryptophan hydroxylase (tryptophan-S—monooxygenase). Although the precise manner by which tryptophan hydroxylase is regulated is not completely understood, the rate of this hydroxylation in viva appears to depend not only upon the kinetic characteristics and concentration of the enzyme itself, but also upon the local concentrations of its three substrates: L—tryptophan, reduced pteridine cofactor (perhaps tetra- hydrobiopterin, see Hamon_g£nal., 1979) and oxygen. Under normal physiological circumstances these substances are not present in satur- ating concentrations (Tappaz and Pujol, 1980; Lovenberg gt 31., 1968). Some changes in the rate of hydroxylation of tryptophan that cannot be l3 .amfiaou Imhuu%xouw%;im .mHmIm mmawamumkhuhx0HU%£lm .Hmlm mufiom oHuoomIMIoHowaflmxouwhnlm .<munn< .Hmafiaumu m>uma UHwHoGHSMummuuhxouwkalm m mo doaumuammonmmu oflumemnom .N mudwwm N wunwflm WIPEJII Finn “253 9r 1% angdeavfi... K g < ffi 14 15 attributed to changes in the concentration of tryptophan hydroxylase have been attributed to result from alterations in the concentration of available pteridine cofactor (Gal, 1974). Kuhn et_al, (1980) suggest that the catalytic activity of tryptophan hydroxylase is dependent upon the oxidation—reduction status of —SH groups and iron binding sites, which are probably located at the catalytic (tryptophan substrate binding) site of the enzyme. The fact that the enzymatic activity of tryptophan hydroxylase (Km value for tryptophan of about 34 uM; Hamon et 31,, 1981) is not saturated by the concentration of tryptophan under normal physiological conditions (approximately 20 uM; Hamon e£_al., 1981) implies that raising or lowering brain tryptophan concentrations within its physiological range alters the saturation of the enzyme, and thus the rate of 5—HT synthesis (Fernstrom and Wurtman, 1971). There— fore, alterations in the availability of 'free' tryptophan in the brain induced by dietary, hormonal, environmental and/or pharmacological manipulations can influence brain 5—HT synthesis (Curzon gt_al,, 1972; Curzon and Knott, 1977; Pardridge, 1977; Fernstrom and Faller, 1978). Tryptophan is the only essential amino acid found in the plasma that is largely (80—90%) bound to albumin rather than existing in the free form (Knott and Curzon, 1972). Thus, any factor(s) influencing the binding of tryptophan to plasma albumin can alter the 'free' fraction of plasma tryptophan and, in turn, its availability for transport into the brain (Curzon and Knott, 1977). L-Tryptophan is taken up into the brain by the same stereospecific system that transports several other large, neutral amino acids (tyro— sine, phenylalanine, leucine, isoleucine and valine) into the brain 16 (Pardridge, 1977; Fernstrom and Faller, 1978). Therefore, tryptophan availability, and hence, the concentration in the brain can be in— fluenced by altering the serum concentrations of any one of these competing amino acids (see discussion in Curzon and Knott, 1977). Extracerebral metabolism of tryptophan may also regulate (at least in part) the availability of tryptophan to the brain. Liver tryptophan pyrrolase, the initial enzyme for tryptophan degradation via the kynure— nine pathway, can be induced indirectly by contraceptive treatment (NisticB and Preziosi, 1970) or directly by corticosteroids (Knox and Auerback, 1955; Nisticd and Preziosi, 1969; Scapagnini ggnal., 1969; Curzon and Green, 1969), tryptophan or structurally-related compounds (Sourkes and Townsend, 1955). Such an induction of tryptophan pyrrolase can effectively shunt plasma tryptophan toward the liver kynurenine pathway and away from the brain. Finally, tryptophan availability in the brain for 5—HT formation might be altered by changes in the rate of cerebral protein synthesis as tryptophan is required as a precursor for both processes (Curzon and Knott, 1977). There is some evidence that the rate of 5—HT synthesis may not only depend upon tryptophan availability to the brain but also upon the efficiency of the neuronal tryptophan carrier system (Hamon and Glow— inski, 1974; Hamon_g£ El}: 1977) which exhibits a diurnal variation similar to the circadian rhythm in the concentration and metabolism of 5-HT that has been observed in the rat brain (Héry_gg.§l, 1974; Quay, 1968). l7 L—5—hydroxytryptophan synthesized in S-HT neurons is rapidly de- carboxylated to form 5-HT by a pyridoxal phosphate-dependent enzyme, L— aromatic amino acid decarboxylase (L-AAAD; possibly the same enzyme that converts L-dihydroxyphenylalanine to DA). The specific activity of L-AAAD is 70-100 times greater than tryptophan hydroxylase, indicating that the rate-limiting step for 5—HT synthesis is tryptophan hydroxyla— tion (Peters gt_al,, 1968). Newly synthesized 5-HT may be stored in synaptic vesicles or re- leased from the nerve terminals in response to nerve impulses, electri— cal stimulation or drugs (Mfiller_g£_al., 1977). Following its release from the nerve terminal into the synaptic cleft S-HT is free to interact with postsynaptic 5—HT receptors. Activation of these receptors is terminated when S-HT is either metabolized by extraneuronal monoamine oxidase (MAO) (Sjoerdsma ggflal., 1955) or is transported back into the nerve terminal by a stereospecific active uptake mechanism (Shaskan and Snyder, 1970). Within the neuron 5-HT is oxidatively deaminated to form the intermediate metabolite, 5-hydroxyindole acetaldehyde, which under- goes immediate oxidation to form the end-product of 5-HT degradation, 5- hydroxyindole—3—acetic acid (S-HIAA). This acid metabolite is then removed from the brain by a probenecid—sensitive acid transport mecha- nism. IV. Methods for Estimating 5—HT Neuronal Activity It is difficult to obtain information on the basic processes in- volved in the regulation of 5—HT release in the central nervous system of mammals because simple peripheral S-HT neuronal systems do not exist 18 as they do for the catecholaminergic systems. Furthermore, functional similarities between the peripheral and central 5-HT receptors do not appear to exist as they do for the catecholamines (i.e., they do not show the same sensitivity to the same drugs; Jalfre, 1974). Several types of 5-HT receptors may exist in the brain (Weight and Salmoiraghi, 1968; Tebecis and DiMaria, 1972; Bourgoin g; 31., 1978). Lastly, 5—HT systems in the brain appear to be organized in a more diffuse manner, in general, than central DA systems. There appears to be a relationship between nerve impulse flow and 5—HT synthesis, release and turnover. Stimulation of 5-HT neuronal cell bodies in the raphé nuclei causes an increase in S-HT synthesis (Eccles— ton.g£_al,, 1970; Shields and Eccleston, 1972; Herr gt_al,, 1975), turnover (Andén.g£_al,, 1964; Aghajanian 3E al., 1967; Sheard and Aghajanian, 1968; Kostowski gt al,, 1969; Eccleston 25 al., 1969, 1970; Carlsson 2; al,, 1972b; Shields and Eccleston, 1972), and release (Aghajanian et al., 1972; Ashkenazi_g£_al,, 1972; Bramwell and Conye, 1976; Héry_gtfl§l., 1979) in distant S-HT nerve terminal areas. Further- more, evidence obtained from studies in which the anterior raphé nuclei have been acutely lesioned or axons descending from the raphé through the spinal cord have been transected also supports the contention that nerve impulses play an important role in the control of 5-HT synthesis as both procedures produce a rapid decrease in the rate of tryptophan hydroxylation in distal areas (Carlsson ggflal., 1973; Herr and Roth, 1976). Correlations between synthesis and release of 5—HT have also been made (Héry.gg.al., 1970, 1972, 1977; Hamon_g£_al., 1974a,b). Finally, it appears that 5—HT may be functionally contained in two l9 compartments and that the newly synthesized S—HT "pool" or compartment may be preferentially released in response to nerve stimulation (Héry §E_al,, 1970; Sheard and Aghajanian, 1968; Shields and Eccleston, 1972). Even though much evidence supports a correlation between nerve impulse flow and 5—HT synthesis, metabolism and release; synthesis and degradation of 5-HT in the CNS may not always be exclusively linked to 5-HT neuronal activity (Ternaux g£_§l,, 1976; Aghajanian, 1972; Héry gghgl,, 1972). Nevertheless, changes in the rates of synthesis and metabolism of 5—HT have been used to estimate the activity of 5—HT neurons (e.g., Héry_§£“al., 1972; Neckers and Meek, 1976). Some of the neurochemical methods that have been employed to estimate S-HT neuronal activity include measurements of: i) the relative concentrations of 5—HT and 5-HIAA. 5—HIAA con- centrations alone, or S-HIAA/S-HT ratios, have been related to turnover and used as an estimate of 5—HT neuronal activity in a number of physiological or pharmacological paradigms (Tag- liamonte EEHE£°9 1971; Héry g; 31., 1972). An increase in 5—HIAA concentrations or the 5—HIAA/5—HT denotes an increase in 5-HT neuronal activity. When using this method, caution must be taken to note whether the steady—state 5-HT concen- tration is changing. ii) rates of accumulation of labelled 5-HT and 5-HIAA following systemic or intracerebroventricular (icv) administration of radioactive tryptophan (Neff_eg.al., 1971). This method depends upon the measurement of the intraneuronal precursor pool (assumed to equal the plasma precursor pool) and often iii) 20 includes the assumption that there exists a single neuro— transmitter pool. Another assumption that is often not evaluated carefully is that the unmeasured precursor pool does not change throughout the experiment. This assumption is critical to interpretation of data obtained with non-steady state conditions used in isotopic experiments. Finally, attempting to examine S-HT activity in discrete areas of the rat brain such as the ME by isotopic methods is very difficult due to the size of the region, sparse 5—HT innervation, accessibility of the isotope and length of time necessary for labelled precursor to accumulate in quantities substantial enough to measure without having to pool tissues from several animals. rates of accumulation of 5—HT (Lin e£_§13, 1969; Neff 35 31., 1967) or disappearance of S-HIAA (Tozer 3E.al,, 1966) after the administration of an inhibitor of MAO such as pargyline. Assumptions included in these steady—state systems are that the synthesis of 5-HT is equal to its metabolism to S-HIAA and that the S-HIAA produced reflects the amount of 5—HT used functionally in the brain. Furthermore, it is assumed that the drug employed to inhibit MAO does not affect the synthesis and disposition of 5-HT itself, or the release of 5—HIAA from the neuron. Evidence has been presented that pargyline may cause an increased conversion of 5-HTP to 5—HT, and that intraneuronal 5—HT may inhibit the rate of hydroxylation of tryptophan once the amine reaches some critical concentration 21 (weber, 1966; Carlsson and Lindqvist, 1973). These methods also assume that MAO is inhibited completely and that 5—HT is converted entirely to 5-HIAA. iv) rates of accumulation of 5-HIAA after administration of the acid transport inhibitor, probenecid (Neff 25 al., 1967; Meek and Werdinius, 1970). This method also assumes that the drug administered does not affect the synthesis or disposition of 5+HIAA itself. v) the activity of tryptophan hydroxylase. This has been measured ipyzitrg_following treatment of animals ip;yiyg_(Kizer gt_al,, 1976c; Palkovits gt §L°, 1976) or in vivg_by measuring the rate of accumulation of 5-HTP after the administration of an L—AAAD inhibitor (NSD 1015; Ro4-4602). In the absence of an L—AAAD inhibitor the concentration of 5—HTP in brain is essen— tially zero, but it increases linearly with time once the decarboxylating enzyme is inhibited (Carlsson §£_al,, l972a,b). This method measures the direct product of trypto- phan hydroxylation (the rate—limiting step in 5—HT synthesis) and thus the synthetic rate of tryptophan hydroxylase, without having to assume anything about the number of metabolically active 'pools' of 5—HT. Only one measurement is necessary (5-HTP concentration) so that 5-HT synthesis can be measured even if steady—state concentrations of 5-HT are changing. Limitations of this method include the fact that NSD 1015 also inhibits L-AAAD in catecholaminergic neurons which could secondarily affect 5-HT synthesis. It is also possible that 22 the accumulation of an intermediate substrate in an abnormal way might disturb the normal metabolic activity in the neuron. In some cases 5—HT continues to be synthesized and metabolized to 5—HIAA even after impulse traffic in S-HT neurons ceases and may be metabolized by MAO without first being released (Carlsson and Lindqvist, 1973). Thus, it is difficult to know what proportion of synthesized 5—HT is actually released and what proportion is merely metabolized within the neuron. Nevertheless, as already discussed, the evidence does support a relationship between 5-HT neuronal activity, 5-HT syn- thesis and metabolism in the brain. In the studies to be reported two methods for examining 5—HT neuronal activity have been utilized. First, the concentrations of 5-HT and 5—HIAA and the ratio ([5—HIAA]/[5-HT]) have been studied as an estimate of metabolism. Secondly, the accumulation of 5-HTP following inhibition of L—AAAD by NSD 1015 was measured as an index of 5—HT syn- thesis. Following development of a method sensitive enough to measure 5-HT, 5-HIAA and 5—HTP in the SCN, MPO, AN, ME and ST the validity of the method was tested using pharmacological manipulations. The activity of 5—HT neurons was then evaluated during several endocrinological, en— vironmental and pharmacological paradigms in which the secretion of prolactin from the anterior pituitary is undergoing dynamic change. These included determining the effects of morphine, restraint and suckling, all of which increase the secretion of prolactin, as well as the physiological surges of prolactin that occur in the afternoon of 23 proestrus during the normal ovarian estrous cycle and twice daily during early pregnancy. Each of these paradigms will be introduced in their appropriate section under Results and Discussion. STATEMENT OF PURPOSE 5-HT neuronal systems are believed to exert a modulatory role in the regulation of prolactin release from the anterior pituitary. Al— though S—HT has no effect on the pituitary pg; se, indirect pharmaco— logical evidence (mostly obtained using S-HT agonists and antagonists) suggests that 5—HT neurons play a stimulatory role in situations where dynamic changes in prolactin secretion are occurring. An important first piece of evidence for the involvement of 5—HT neurons in the secretory control of prolactin from the anterior pituitary could be obtained by examining 5-HT neuronal activity throughout experimental paradigms where dynamic changes in prolactin secretion are occurring. Although such an examination would not provide information concerning a cause and effect relationship between S-HT neuronal activity in parti— cular areas of the brain and prolactin secretion, it could provide information as to what areas should be examined in the future. At the beginning of this project no single technique which would permit the quantitation of 5—HT, 5—HIAA and 5—HTP, in the ME, SCN, MPO, AN and ST of a single rat brain existed. The objective of the studies was to: i) Develop a method utilizing high performance liquid chromato- graphy coupled with electrochemical detection sensitive enough 24 ii) iii) 25 to measure 5-HT, 5—HTP and 5-HIAA in discrete regions and nuclei (ME, MPO, SCN, AN, ST) of a single rat brain. Evaluate the validity, sensitivity and capability of the method by examining the 5—HT neuronal responses occurring in these regions following the administration of various drugs with known pharmacological actions that might be expected to alter the dynamics of 5-HT neurons. Examine S—HT synthesis and metabolism in these discrete brain regions after various pharmacological, environmental and endocrinological manipulations which alter the secretion of prolactin from the anterior pituitary. MATERIALS AND METHODS 1. Animals and Blood Collection Male Sprague-Dawley rats (Spartan Research Animals, Inc., Haslett, MI) weighing 200-275 g were utilized in all experiments where pharma- cological manipulations alone were investigated or where the effects of restraint stress on 5-hydroxytryptaminergic neuronal activity were examined. Female Long—Evans rats (Charles River Breeding Laboratories, Wilmington, MA) weighing 225—300 g were used in all experiments where 5- hydroxtryptaminergic neuronal activity was examined during pregnancy, lactation/suckling, and throughout the day of proestrus in the ovarian cycle. All animals were maintained in air-conditioned (22i1°C) and light controlled (12 h light cycle, 07.00—19.00 h) rooms and were allowed access to food (wayne Lab-Blox, Allied Mills, Chicago, IL) and water'§d_libitum. Blood samples for the determination of serum concentrations of prolactin were collected from the trunk following decapitation. The samples were stored at 4°C for 24 h, centrifuged at 1,000 g for 30 min, and the serum was frozen at -20°C until assayed. All animals were sacrificed between 10.00 and 12.00 h unless otherwise designated. II. Biochemical Procedures A. Dissections Following decapitation rat brains were quickly removed from the skull and placed dorsal side down on a cold plate (Thermoelectrics 26 27 Unlimited). The median eminence was removed from the hypothalamus with the aid of a dissecting microscope and fine scissors (Cuello gt EL" 1973). While grasping the infundibular stalk with fine forceps cuts were made along the tuberoinfundibular sulcus on both sides to the rostral border of the median eminence. When dissected in this manner the average protein content of the whole median eminence was approxi— 'mately 24 pg. The remaining brain was cut just caudal to the hypothala— mus and the anterior portion of the brain was frozen on dry ice until frontal sections could be prepared using a sliding microtome fitted with a freezing stage. A modification of the procedure described by Palko- vitz (1973) was used to dissect the other discrete brain areas using the atlas of Konig and Klippel (1967) as a guide. Sections of the appro— priate thickness and location were mounted on conventional glass slides and frozen on dry ice. Examples of the 3 frontal sections and location of punches can be seen in Figure 3. The most rostral section was taken at the level of the crossing of the commissura anterior and included structures between A7020—6720 (300 pm thick). From this section a bilateral punch was taken of both the striatum (nucleus caudatus puta— men) and the medial preoptic nucleus (nucleus preopticus medialis). The next section taken included structures between A6220-5720 (500 pm thick). From this section a single punch extending across the midline of the section just above the optic chiasma was taken to include the suprachiasmatic nucleus (nucleus suprachiasmaticus) bilaterally. The final section (A4920—3920; 1000 pm thick) was used to obtain a single punch containing the arcuate nucleus (nucleus arcuatus). Needles for the punches were made from stainless steel hypodermic tubing (Small adv 28 Figure 3. Photographs of three frontal sections with examples illu- strating location of punches. Slices were removed according to KBnig and Klippel (1967); rostral to caudal: A) represents a 300 pm slice (A7020—A6720) from which punches of the ST (arrow with asterisk) and MPO (arrow) were removed bilaterally. B) Represents a 500 um slice (A6220—A5720) from which a single punch of SCN was removed (arrow). C) Represents a 1000 um slice (A4920-A3920) from which AN was removed (arrow). 29 Figure 3 30 Parts, Inc., Miami, FL). Two punches were used. A 14 gauge tubing (0.063", i.d.) was used to construct a crescent-shaped punch to dissect the arcuate nucleus. An oblong punch was constructed from a 17 gauge (0.042", i.d.) tubing for use in dissecting the striatum, medial pre— optic nucleus and the suprachiasmatic nucleus. The punch containing the arcuate nucleus may have also contained small amounts of the pars ven— tralis of the ventromedial nucleus. The punch for the suprachiasmatic nucleus undoubtedly contained small amounts of the tractus infundibu— laris and possibly a small contribution from the periventricular nucleus of the hypothalamus. Similarly, the punch containing the medial pre— optic nucleus probably also contained small contributions from the periventricular preoptic and median preoptic nuclei as well as a small portion of lateral preoptic nucleus. The approximate protein contents for the bilateral striatal and medial preoptic nucleus punches as well as the single suprachiasmatic and arcuate nuclei punches are 100, 80, 40 and 110 pg, respectively. All tissue samples were homogenized directly into 30 pl of the mobile phase used for the high performance liquid chromatographic analysis described below (B). The homogenates were diluted to a total volume of 100 pl with additional mobile phase and then centrifuged for 30 sec in a Beckman microfuge. Ninety of the resulting 100 pl of supernatant was then filtered through glass wool (previously washed with 600 p1 of mobile phase) in order to remove particulate matter and diluted to a final volume of 600 pl with addi- tional mobile phase. Five hundred microliters of this final volume was then injected directly onto the chromatography column. The protein content of the homogenate pellet of each sample was analyzed as 31 described by Lowry gt a1. (1951) and all data is expressed as ng of the compound of interest per mg protein. B. High Performance Liquid Chromatography Technique Several neurochemical methods have been employed to measure the activity of dopaminergic and 5—hydroxytryptaminergic neurons in the rat brain. For the purposes of the present studies 5—hydroxytryptami— nergic neuronal activity was estimated by measuring the ratio of metabo— lite to neurotransmitter (S-HIAA/S-HT), and the accumulation of the amino acid precursor of 5-HT, 5-hydroxytryptophan, following inhibition of L—aromatic amino acid decarboxylase. Until recently it had not been possible to measure S—HT, 5—HIAA or 5—HTP in discrete brain regions primarily because of a lack of sensitivity in available analytical techniques. Development of sensitive radioenzymatic (Tappaz and Pujol, 1980) and high performance liquid chromatographic (HPLC) (Krstulovic and Matzura, 1979; Meek and Lofstrandh, 1976) assays for 5—HTP and assays using HPLC in combination with electrochemical detection for measuring 5—HT, S—HIAA and 5+HTP (Loullis eg_al,, 1979) have recently made it possible to employ biochemical techniques to estimate S-HT neuronal activity in areas such as the striatum, hypothalamus and mediobasal hypothalamus. No technique allowed for the measurement of 5-HT, 5-HIAA, S—HTP, DA and DOPAC in a sample of ME, SCN, MPO, AN or ST from a single rat brain. Thus, the development of an assay using HPLC coupled with electrochemical detection that would possess the selectivity to allow the concurrent analysis of all of these compounds without sacrificing the sensitivity required to measure them in such discrete nuclei was attempted. 32 Several modifications of a mobile phase used originally by Felice SE a1. (1978) were examined for their effects on retention times for each of the compounds of interest (Nielsen and Johnston, submitted). A mobile phase that allowed separation and concurrent measurement of 5-HT, 5-HIAA, S-HTP, DA, DOPAC and HVA in a single run of approximately 20-24 min was selected. A retention time of 20—24 minutes was required in order to resolve all of the compounds of interest in a single sample of a discrete hypothalamic nuclei. The final mobile phase consisted of a 0.1 M citrate—phosphate buffer (pH 3.6) containing 8% (v/v) methanol, 0.032% (W/V) sodium octyl sulphate (SOS) and 0.1 mM disodium ethylene- diaminetetraacetate (EDTA). A.model LC—40 (Bioanalytical Systems, West Lafayette, IN) liquid chromatography system with either a TL—3 carbon paste electrode or a TL—S glassy carbon electrode thin—layer transducer connected to a LC-2A amperometric controller was equipped with a 30 cm x 3.9 mm, i.d. C18—pBondapak reverse phase column (Waters Associates, Milford, MA). Samples and standards were injected into a 6—port valve (Rheodyne, Berkeley, CA) equipped with a 500 p1 loop. Flow rate of the mobile phase was 2.0 ml/min maintaining column pressure at approximately 2000 psi. All chromatographic experiments were performed at ambient temper- ature in an electrically shielded room. The speed of the Omniscribe RYT B-SOOO-D strip chart recorder (Houston Instruments, Austin, TX) was 5 cm/lO min. The working electrode was set at +0.75 mV relative to a Ag+/AgCl reference electrode. The electronic controller was set at 5 nA/volt and the recorder at 1.0 and 0.1 volts full scale providing 5 nA and 0.5 nA full scale chromatographs on the RYT dual pen chart recorder. P deuce ‘9/ 30229.5: ‘ViZ/fli/x a! 4‘s \\\\ 1 “MN \ Ne 33 The stationary phase of the Cls-pBondapak column is composed chains of totally porous 10 pm silica particles to which N—alkyl Cl8 have been bonded. This type of column generally separates compounds through hydrophobic interactions and since the catecholamines are very polar molecules, they are not retained significantly by this type of The result is a poor resolution of the charged com— Stationary phase. Thus, by pounds which are often inseparable from the void volume. itself, normal reverse phase liquid chromatography is often unusable for nunnoamine analysis (especially the catecholamines). The alternative to normal reverse phase liquid chromatography :iss the ion—pair or "soap" chromatography modification which utilizes the sszajzze stationary phase (column) but adds, in dilute concentrations, a c:c>111pound capable of forming an ion pair with the analyte(s) of interest. I3<>jth‘:3. dry has been advanced that the octyl sulphate partitions onto the mod ified silica surface to give it an anionic character and create a (1371:)‘isiitnic ion—exchange column in physical (not chemical) appearance. IIC) £3; :iLUtZively charged analyte ions could then partition onto the stationary I>11 Esg‘Ei by a conventional ion exchange process resulting in their 34 resolution. In reality, probably both theoretical mechanisms contribute to the resulting separation. The use of microparticulate reverse phase "soap" chromato— graphy for analysis of monoamines is ideal in many respects. The reten— tion of the various cationic monoamines and their amino acid precursors Can be varied to suitable values simply by modifying the concentration of the ionic detergent in the mobile phase. The reverse phase column is nnare versatile in that under the proper conditions it can be used to seeparate both polar and neutral compounds. Furthermore, the reverse— 13116156 procedure is faster, more efficient and slightly more sensitive tzllain the earlier pellicular cation exchange catecholamine method and aa;1_].ows concurrent analysis of the catecholamine, DA; the indoleamine, 55-—£EIT; the amino acid precursor of 5—HT, 5—HTP; and the acid metabolites (3.1? .DA.(DOPAC and 3~methoxy-4-hydroxyphenylacetic acid (HVA)) and 5—HT C's—HIAA). Some of the principles of electrochemical detection can be bet ter understood by examining Figure 4. At the top of Figure 4 a ES‘CI 1_1‘€=—~Inatic of the TL—3 thin—layer transducer containing the carbon paste rxrc>”1:‘ls;:ing electrode is illustrated. The eluent from the C18 pBondapak C:<):Ir‘1;t1nn flows in this diagram from left to right across the face of the VJC):I:-15:L:ing electrode (w) and then up towards the reference electrode. On tZIIIE: :lower part of this figure an example of the oxidation process using DA . €5l—=s the orthodihydroxy constituent and a schematic enlargement of the t2]:l:iL:1:1_ 1 . . . ayer cell are shown. The detector IS operated in an amperometric nuc>aoo muoB :m: %n woumoHodH moSHm> moose nHmuoua wa\wd udmmoummu mHmIm you monHm> .uoanHnaH mmMmeopuoumw m mo GOHuoonaH HmoaouHumaosuaH men wcHBOHHom CHE om .aHmuoum ma\wa uaomoumou < m.mNIH.wH ¢.w N.HH N.Hm1n.mH N.o¢ «.mN m.HH H.m m.NH m.HNIo.NH ¢.OH 0.5 m.OH w.wHIn.NH m.mH o.HH m.oHIq.m ome .HOmnm mam NmmmmH ul.ll. wan ..Hm um mHHH>mmlmHoumo mumH Hum: mam ovum: mman ..HM uM oommHumu wan .GomHan mam mHNaoMomz wan usmm maul HmHmathB ammH ..Hm uM muoxowz noan ..HM uM Ionumosoum nmmH ..HM uM ImuH>omem ome ..HM HM whmsaHmm owaH ..HMi uM dmaHdw man ..HM uM mmH3opo onaH ..HMi HM mquomem mmmH ..HM HM Mom or am> «maH ..HM uM muvm>omm mHmIm ¢ musumumuHH H mHm on moumoHoaH .III .H mHan mom “undue IHouHH map :H omuuomou AdHououm wa\wnv mGOHumuuamuaoo mo owcmn msu pammoummu mmSHm> omzmHHnsm .AQH .mx\wa OOHV mHOH sz mo GOHuowmaH may umumm SHE om monommoa mos mHmIm .mmlom n z aHououa Hoe end .om u z maxim Hoe .HmumN u z < o.NIq.o III III III III mmaHm> omanHnsm m.o H m.m N.e H H.e e.o H m.a H.o H e.m m.o H H.H deHde wa\we Ho.o me.o Ho.o Hem.o no.0 HmH.o oo.o HHe.o No.0 HHH.o dHeadm\me eem-m o.oI¢.m III III III H.m mode> omanHndm N.o H m.e m.o H m.mH ~.H H e.o~ m.o H e.NN m.o H e.NH eHdHOHe Ha\wd eo.e Ham.o HH.o HeH.H wo.o Hem.o N.o H H.m mo.o Hmm.o dHeaHm\me Haemum m.HHuH.m N.NHIe.H N.oeue.w e.em-~.e w.HNIe.H edeHee edemHHeee N.o H H.“ e.o H e.m o.H H H.eN m.o H e.mH e.o H H.5H eHmHOHe we\wd no.0 Hmo.o Hm.o Hee.o No.0 Hmo.H N.o H o.m Ho.o Hmm.o dHeadm\me Harm e.H H H.me o.H H H.Hm o.H H e.ee m.e H e.m0H N.H H H.wN Awac dHeedm\eHdHoee Hm om: zom za m: mGOwam aHmHm GOHuoouon HmUHEmnoouuomHm nuHB hemmuwoumaounu stwHH oudmfiuomumm :me wchD anum umm mo maOHmom wmuUMHmm dH maOHumHuauoaoo mHmIm one ¢ Houudoo Scum AHo.owmv unmummmHv hHuamonHame wH umnu dem> .< .w u z ..m. m H some udmmmummu mode> .monHHomm \na ouommn GHE oo umuoonaH we? AmH .wx\wa oomv uHumcmnoum no AmH .wx\we mwv maHH%wumm 58 Hm.HH e.em He.HHw.NH mH.OHNm.H N m Ho.om eHdHHHBOHe He.HH w.NH HH.HHH.mm HH.OHmm.H o e Hm.me ddHHHdee e.HH e.NN H.HHH.mH HN.OHem.H m H He.om HOHHeoo za Hm.OH H.eH H.HHH.HH Hm.HH e.aH e a HH.mm eHddednoee He.OH H.e HH.HHN.HN «H.0H m.H m mHHe.HHH ddHHHdee N.HH H.NH o.HHe.wH e.OH e.m H e He.OOH HOHHeoo a: «as. OHme. m H.0Hm.m H.HH N.HH N H HH.em eHededeOHm Hmm. OHHm. m HH.OHo.HH «H.0H m.H H e Hm.omH deHHHdee He. OHmm. e e.OHo.w e.eH m.OH e H He.es HOHHdou Hm HHHmum Helm uaeoe an unmaummHH GOmem AeHdHopm we\wdv mHHHOHHHmz Ho msz< HHOdem edHedem eH «HHmum use Helm .oHeoe .Hm do deHHHmHee dHeHm Hem one H0 mm mHm Houuaoo aoum AHo.mev uanoHHHw kHucmoHHHame mH udfiu osHm> .x .w u z ”.m.m H some unmmoummn mmDHm> .oUHHHuomm ouommn oHa.oo umuommoH was AmH .wx\wa,oomv wHomdonoua Ho AQH .wx\wa mnv maHthHmm Hm.NHm.HN H.0Hw.w HOH.OH¢N.N mm.OHem.m eHddedeoee Hm.HHm.w HH.OHH.OH HH.OHmo.H Hm.OHNm.m ddHHHdee o.HH~.mH e.OHm.H HH.OHmm.H NH.OHmH.e HOHHdoo om: HH.HHm.mN H.NHH.mN we.0Hmm.m e.HH e.mH eHddddeoee HH.HHm.HH He.HHw.me mm.OHeH.m H.HH H.HH ddHHHdee H.0HN.HH m.NHH.mN me.OHmH.H H.HH H.mH HOHHeoo 20m HaHmum Helm oamoe He uaoaummuH GOHwom AdeHoee me\mev mHHHomeamz Ho mzH2< dHeHm Hem and Ho HHOdem ededem eH HHHmum Hem Helm .uHmom .He no ddHHHmHee Hem eHdddHBOHe «0 HddHHm nm mHmHH 60 ST, ME and AN but not in the SCN or MPO. In general, these results fit very well with what might be predicted from the known actions of these drugs and previously reported results (Tozer ggngl., 1966; Weber, 1966; Macon.§£;§l,, 1971; Maickel gt al., 1974; Wilk SE 21', 1975a,b; Roth 22 al,, 1976; Karoum g; 1977; Umezu and Moore, 1979) with a couple of exceptions. First, the DA neuronal system in the SCN appeared to be very resistant to the effects of pargyline and probenecid. Secondly, the concentration of DOPAC in the AN is near to the limit of sensitivity of the assay and did not show a significant change to any treatment in this experiment. Trends towards change in AN DOPAC were consistent with effects of these drugs observed in other areas. The primary objective of this experiment was to determine whether HPLC with electrochemical detection could detect changes in steady-state concentrations of 5-HT and 5-HIAA induced by pargyline and probenecid in the selected brain regions. 5—HTP accumulation was not measured in these experiments because NSD 1015 pg£_§g alters steady—state concentrations of 5-HT and 5-HIAA. In addition, DA and DOPAC were measured in this experiment because investigations on dopaminergic neuronal systems in the selected brain regions are much more numerous and would help confirm the location of the dissections. 5-HIAA appears to require a probenecid-sensitive acid-trans- port system for its removal in all five brain areas examined whereas DOPAC required such a transport system only in the ME, MPO and possibly AN (included because of the previously mentioned problem of sensitivity for detecting changes in DOPAC concentrations in the AN). The lack of probenecid on DOPAC concentrations in the ST agree with observations 61 by other investigators (Wilk.EE“§i°I 1975a,b; Karoum egnal., 1977; Umezu and Moore, 1979) but the lack of effect in the SCN has not pre- viously been reported. The effect of pargyline on DA, 5—HT, DOPAC and 5—HIAA concentrations in the ST and ME agree with those of other in— vestigators (Tozer e£_el,, 1966; Weber, 1966; Macon EE”§1°’ 1971; Maickel eguel., 1974; Wilk.et_al,, 1975a,b; Roth.et_el,, 1976; Karoum 55,313, 1977; Umezu and Moore, 1979). The results concerning the effects of pargyline in the MPO, AN and SCN agree with the known actions of this drug. The resistance of the DA system in the SCN to change following pargyline or probenecid administration was unexpected and the reason for such a resistance remains unknown. These results demonstrate the capability of HPLC with electro- chemical detection to detect pharmacologically-induced changes in the concentration of 5-HT and 5-HIAA in discrete brain regions. Further- more, the concentrations of DA and DOPAC and the effects of pargyline or probenecid on those concentrations agree with previous investigations and help confirm the correct location of the dissections. C. Effect of Reserpine on 5-HT Metabolism and Synthesis in Selected Regions of the Rat Brain Reserpine has been shown to decrease S—HT concentrations and storage, and to increase 5—HT synthesis, S—HIAA concentrations and tryptophan hydroxylase activity in rat brain (Tozer_etflel., 1966; Zivkovic SE El}: 1973; Sze e3 El': 1976; Sanders-Bush and Massari, 1977; Saner and Pletscher, 1978). The effects of reserpine (2 mg/kg, i.p.) on the concentrations of 5—HT and S—HIAA as well as 5-HTP accumulation were examined at 2 and 62 24 hours following its administration (Table 6). Reserpine disrupts the ability of neuronal storage vesicles to bind 5-HT and other putative amine neurotransmitters. Thus, over time, 5—HT concentrations decrease, 5-HIAA concentrations increase as more 5-HT is exposed to intraneuronal MAO, and 5—HTP accumulation might increase if synthesis in the neuron attempts to maintain 5—HT concentrations. As shown in Table 6, follow— ing reserpine 5-HT concentrations decreased and S—HIAA concentrations either increased or remained at normal values at both 2 and 24 hours in all five brain regions. Therefore, the ratio of [5-HIAA]/[5-HT] was dramatically increased at both 2 and 24 hrs post-reserpine. These results suggest that 5-HT synthesis is maintained despite the loss of binding capacity of the storage vesicles. This is consistent with the 5-HTP accumulation data which show 5-HT synthesis in all five regions was increased at both 2 and 24 hrs following the administration of reserpine. Thus, both indices of 5-HT neuronal activity (5-HTP accumu- lation and [5-HIAA]/[5-HT]) increase in the ME, ST, SCN, MPO and AN following reserpine treatment. These results extend those obtained by other investigators who have found that reserpine decreases 5-HT concentrations in the rat brain while the formation of 5-HIAA, 5-HT synthesis and 5—HT turnover are all increased (Tozer eg_el,, 1966; Zivkovic_eg.el., 1973; Sze e£_el,, 1976; Sanders—Bush and Massari, 1977; Saner and Pletscher, 1978). Further- more, the results using the present technique show that reserpine in- fluences the concentration of 5—HT, 5-HIAA and the rate of accumulation of S-HTP in discrete hypothalamic regions in a manner similar to that in larger brain areas. 63 .AHo.OVeV mHouudoo .daHH o. scum HddeHHHe HHdedeHmHamHm .H .A.e.H .mx\me OOHV mHOH emz Hddmd eHa om AHuz “.m.m H some maHe om\cHououm wa\wav QOHumHaasoom memlm mo moon onu muaomoummu ode> Somme .Aeuz ”.m.m H some deouowm we\wov SOHuouuaooooo < sommm Hm.OHe.e Hee.0HNm.N He.0Hm.N Hm.OHN.H Hm HH.OH¢.H HHN.OHHN.H «H.0He.m Hm.OHm.N N H.0Hm.H OH.OHee.o H.0Hm.m m.OHe.m o oez HH.HHH.eH HHH.OHHH.H HH.NHe.me He.OHm.m em Hw.OHH.eH Hem.HHom.e Hm.mHH.om He.NHm.NH N H.0He.OH mH.OHmN.H o.MHo.mN e.HHH.NN o Zom HH.OHw.e HHe.OHHH.m m.HHm.HN «H.0Hm.e em He.OHN.H Hme.OHHm.m He.~Hm.om He.OHN.m N H.0Ho.m mN.OHmm.H m.OHm.m~ e.OHm.eH o z< HN.OH~.N «Hm.0Hoo.m m.HHm.NH HH.oHH.e Hm Hm.OHm.m HHo.HHem.e HH.HHN.ON Hm.HHm.e N H.0He.H oH.OHeH.o m.OHH.OH e.HHH.mH o a: o.HHH.e HHm.OHmo.m Hm.0Hm.H HH.OHH.N em m.OHo.e HmN.OHeH.m HH.OHm.H HN.OHm.N N H.0HN.m no.0HmH.o N.OHH.H m.OHe.e o Hm A.e.H .mx\ma NV eeemum Hm|m\< :omm .Amuz m.m.m H some moHouonm ma\wav aOHumuuaooaoo <4Hmlm Ho Hmlm mucomoumou msHm> Somme .oonHHomm muommn GHE mm wouomnaH mos A.Q.H .wx\we OHV ocHuoxonHm was moHHHHUMm muommn aHa om wmuoode mmB A.Q.H .wx\wa my moHamHmHaHHOHSU HH.OHH.N mo.OHme.H e.oHe.eH N.on.m ddHaeHeHeHHOHeo HH.OHO.N Hmo.OHHH.H e.OHo.mH e.OHo.NH deHdeosHm N.OHN.H Ho.OHmm.H m.OHo.mH H.0Hm.m HOHHdoo ems Hm.OHe.e HHO.OHHN.H H.NHm.mN m.HHH.HH deHadHeHaHHOHeu «H.0HH.m Heo.OHmN.H «m.HHe.NN H.0He.mH ddHHexoeHe m.OHm.o 0H.0Hmm.H H.NHm.Hm m.OHo.ON HOHHdoo zom HH.OHH.N Heo.OHom.o Hm.HHm.mH N.HHm.OH ddHEdHeHaHHOHeo HH.OHm.N so.OHmH.H H.NHm.HN H.0Hm.mH ddHdeodHe H.0Hm.e HH.OHHm.H m.NHH.m~ H.HHH.mH Hopudoo z< «n.0Hm.m HHO.OHem.o N.HHo.e HH.NHm.mN deHadHeHaHHoHeo H.0Ho.e «No.0Hmm.o m.OHH.m Hm.HHo.HN deHdeosHe m.OHm.H no.0Hme.o m.OHH.m o.NHm.mH Hopudoo me HH.OHN.N «Ho.OHmm.o H.0Ho.m m.OHH.m ddHaeHeHeHHOHao H.0Hm.N «Ho.0Hem.o m.OHm.m m.OHm.m ddHHdedHa H.0He.m mo.OHNm.o H.0Hm.m H.0Ho.m HOHHdoo Hm amemnm Hmum\< sommn .Am I z ”.m.m H coma mcHououa wa\wcv aoHumHuaooaoo ¢ .ooHHHHomm ou HOHHQ SHE om %Hmsooamusoeam wouoomaH mp3 oHoH£o> muH Ho ostmHoz «.0Hm.q «N.OHH.© m.OHq.q H.0Hm.m N.DHN.H omz 0.0Ho.m «m.0Hq.qH w.oHo.w 0.0Hm.n w.on.OH zom H.0Hq.m «c.0HN.o «H.0Hm.m 0.0Hq.m m.on.q z< H.0Hw.¢ m.OHo.m n.0HN.m H.0H¢.m m.OHm.q m: m.OH¢.H m.OHm.q m.OHm.m n.0Hm.m q.OH©.m Hm 0.0N o.OH o.m m.N 0.0 SOmem Amx\wav odHnmuoz mo omon aHmHm umm men we mGOkum wouoonm zH GOHuMHseboo< mHmIm do ocHamHoz mo momom mDOHHm> Ho noowmm m MHm Loam .HHo.oveV Houueod scum HcdeHHHe HHHemoHHHeme .H .A.a.H .me\ma OOHO mHOH emz added eHa on HA I z m.a.m H dame n ko "z momom H came maHououm wa\wdv GOHumHudoodoo < sommm .oonHuomm ou HOHHQ GHE om hHmdooumunonom wouooncH mp3 oHoH£o> muH Ho wa\wfi OHV oaHanoz HN.OHm.H mm.OHae.N HHe.mH H.0HH.H ddHaeHoz H.0Hm.m mH.OHeH.H OHN.mH H.0Hm.H HOHHaoo om: Hm.OHm.N NH.OHoH.H mHm.m~ N.HHm.m~ daHeeHez H.0Hm.m mo.OHHo.H HHm.mN H.HHo.eN HoHHaoo zom HN.OHN.e «mo.OHmH.H OHo.eN H.OHN.eH deHeeHoz n.0HH.m mo.0HeH.H OHm.om H.0Hm.HH Hoaaeoo za H.0HH.m «mo.OHmH.H OHm.mH e.OHm.mH adHeeHoz H.0He.m No.0HHm.o OHo.NH m.OHa.eH HeHHdoo m2 m.OHo.m Hmo.OHee.H on.m H.0He.e aeHeeHoz H.0Hm.m Ho.OHHa.o OHN.H m.on.H Hoaddoo Hm eaemum Hm-m\< aonm ucwummHHw %HuomonHame .« .Awnz “.m.m H amoa mcHa om\oHououm wa\w:v GOHumHseboom mHmIm Ho mums ofiu udomoummn mmsHm> .oonHHomm ou HOHHQ an: em AA: Aime 85 33 amz fie. e38 EH 3d; mHmaHae HHH .8323... 8 HOHHQ aHa,mq oHoH£o> mcHHmm Ho A.m.H .wx\wa q no H.0v odoNOHmc auHB Ho\wcm oonHHomm ouonn H: H oHoH£o> ooHHMm nuHB Ho A.o.m .wx\wa OHV mcHsmHoa £uH3 mouooncH mum3 mumm N.DHH.m m.OHN.m N.OHh.m «d.OHm.m N.DH©.m omz w.OHw.m m.OHO.OH m.OHm.m HH.OH®.MH m.OHm.OH 20m m.OHw.m q.OH©.m m.OHm.m «H.0Hm.q m.OHm.m Z< H.HH0.0H m.OHm.m w.HHm.m w.HHm.w m.HHO.n m2 N.DHH.M m.OHN.m m.OHN.m ©.OH@.¢ ©.OHN.M Hm Awhowcmmmwm%mz ANVQMMMMMMMM Aqv odoHOHmz oaHamHoz Houudoo SOHwom GHMHm umm wan Ho maOHwom wouomHom SH mHmonuehm HmIm mo oumm man do epoxOHmz was ooHamHoz Ho muoommm NH mHm6) after 30 Values represent 5-HT or 5-HIAA concentration (ng/mg protein; mean treatment. 10118 f restraint stress, ether exposure alone or no prev min 0 *, significantly different from control value (p<0.01). 86 .AHo.ovmV mHouudoo woumoHquHoH£o> doum udmuomHHu mHudondemHm .« .udodumodu m50H>mud od do oddmOQNo nosed .mmouum udeHumoH mo moudde on sound Aw I z ”.m.m H dome "mouddHa om\dHououd we \wdv doHuoHdddoom mamlm me down mnu udmmoddmd mode> .ooHHHHOdm ou HoHHd mouddHE om A.d.H .wx\wd OOHV mHOH mmz Ho dOHuoondH do mo>Hmowu mHmaHdm HH< ~.0Hw.m HH.OHH.H HdeHumom + “deem H.0Hm.m m.OHo.m Honum N.OHH.m H.0HN.m Houudou om: «N.0Hm.mH «m.OHw.HN udedumom + Honum 0.0Hm.NH o.HH©.HH Honum «.0HN.NH n.0Hm.NH Houudoo zom H.0Hq.m m.OHm.q udedumom + Honum N.0Hm.m m.OHo.m Hosum H.0Hm.m n.0Hm.m Houudoo z< m.OHq.H m.0Ho.q udeHumom + Hosum «N.on.H H.0Hm.¢ Honum m.OHo.m m.OHm.m Houudoo m2 H.0Ho.N H.0Hm.m udeHumom + Hodum N.DHm.N N.0Hm.m Hmzum N.OHm.m N.DHH.m HOHudoo Hm NH udmeHHmde HH udmaHHodxm udwaummHH dOHwom deHm udm ozu Ho mdonom umuoonm dH dOHudeddoo< mHmIm do mmmuum udeuummm oudo< Ho uomwmm «H MHQHH 87 stress. Actual concentrations for control, ether and ether plus re- straint serum prolactin (ng/ml serum, mean i SE) were 15.6i2.9, 10.4: 1.1 and 54.7i17.7, respectively. These results support the hypothesis that 5—HT neuronal acti- vity in certain discrete nuclei of the rat brain may be stimulated by restraint stress. In particular, restraint stress appears to increase 5-HT synthesis and metabolism in the SCN and possibly MPO. Chlman.eg 31, (1980) reported an increase in the concentration of 5—HT in the ME at 5 and 15 min following the introduction of restraint stress. The failure to observe such changes in the present study may be attributable to the fact that the present experiments were performed using a longer (30 min) duration of immobilization. Chlman.eg 31. (1980) could no longer detect a significant change in 5-HT concentrations in the ME following 30 min of restraint. The same investigators also reported increases in 5-HT in the SCN following 5—150 min restraint without affecting 5-HT concentrations in the AN. Their findings in the SCN could have resulted from an increased synthesis of 5-HT, a decreased release of 5—HT or several other possible explanations. The present results suggest that 5—HT synthesis and metabolism increased in the SCN and possibly the MPO. What proportion of this increased synthesis and metabolism produces a functionally important increase in the release of 5-HT into the synapse and what proportion simply represents a conversion of excess 5-HT to 5-HIAA intraneuronally by MAO without ever being released is not known. However, a relation between nerve impulse flow and 5—HT synthesis and release has been demonstrated (Aghajanian 33 31., 88 1972; Ashkenazi e£_31,, 1972; Shields and Eccleston, 1972; Herr 33 31,, 1975; Bramwell and Gonye, 1976; Héry 33 31., 1979). Mueller e£_313 (1976) observed an increased 5-HT turnover in the whole hypothalamus following 15 min of immobilization but did not examine hypothalamic 5—HT turnover at 30 min. In contrast, Morgan 35 31. (1975) could not find changes in 5-HIAA concentrations or 5-HT turnover in the diencephalon (hypothalamus plus thalamus) following 30, 60 or 90 min immobilization. The discrepancy between these findings and the present data can probably be attributed to the much larger brain area being examined by Morgan and coworkers. Certainly, the lack of effect on diencephalic 5-HT does not discount the possibility that alterations in S-HT neuronal activity in discrete regions like the SCN and MPO are occurring. The lack of effect of 5,7-DHT pretreatment on the increase in prolactin secretion resulting from ether vapour stress (wuttke 33 31., 1977; Baumgarten e£_31,, 1978) does not necessarily preclude the possi- bility that 5-HT neuronal systems in discrete areas may be playing a role in the prolactin response to restraint stress, let alone any of the other endocrine events occurring during stress. First, recovery of function does not necessarily have to be identical with the extent of 5— HT neurons present in the area if supersensitivity of the 5-HT post— synaptic receptors involved in the response has occurred. In that case, the normal hormonal secretory response could be elicited despite drama— tic decreases in 5-HT innervation to the area. Furthermore, the minimum amount of 5—HT innervation required to maintain a functional hormonal response (allowing for a sufficient reserve capacity) is not known. 89 WUttke.3£H31. (1977) evaluated 5-HT integrity by measuring hypothalamic 5—HT uptake and synthesis of 5-HT in the.ME following 5,7—DHT treatment and found both parameters were reduced by about 70% which possibly might not have been severe enough. Furthermore, the present results suggest 5-HT in the ME may not be involved in the endocrine responses occurring during stress. Thus, the indices of 5-HT integrity examined by Nuttke and coworkers may not have been appropriate. Baumgarten e£_31, (1978) did not examine the effect of 5,7—DHT pretreatment on 5—HT concentra- tions in the mediobasal hypothalamus or ME. Neither study examined possible non—specific effects of 5,7-DHT treatment on other neurotrans— mitter systems that may be involved in the regulation of endocrine responses to stress. Finally, it is not presently well understood to what extent endocrine responses to different types of stress (such as ether vapour and restraint) are mediated by the same neuronal pathways. Therefore, the lack of effect of a 5,7—DHT pretreatment on an ether vapour—induced prolactin surge does not necessarily prove that a re— straint-induced prolactin surge would also be unaffected. Although both serum.prolactin and 5—HT neuronal activity in the SCN and MPO are increased following 30 min of immobilization stress, these results should not be interpreted as illustrating a cause-and- effect relationship between increases in 5-HT neuronal activity and secretion of prolactin in response to restraint stress. The data only indicate that restraint stress increases 5-HT neuronal activity in the SCN and possibly MPO. Immobilization is known to produce an activation of several components of the pituitary—adrenocortical system. In rats, increases in plasma ACTH, corticosterone and prolactin (Morgan 33.31,, 90 1975; Seggie and Brown, 1975; Kvet‘r'lansky 33 31., 1976) as well as a decrease in growth hormone (Seggie and Brown, 1975) have been reported following stress. Inhibition of 5-HT reuptake as well as direct sti— mulation of 5—HT receptors in the brain have been reported to stimulate the pituitary—adrenocortical system (Fuller 33 31., 1976; Meyer 33 31., 1978). Furthermore, 5-HT has been shown to stimulate release of corti- cotrophin releasing factor from isolated hypothalami (Jones eE_31,, 1976; Buckingham and Hodges, 1977). Therefore, the increases in 5-HT neuronal activity observed in the SCN and MPO may be related to another of these endocrine responses or perhaps some event that has not yet been identified. Investigations concerning questions like these should be the subject of future studies. Interestingly, Moore and Eichler (1972) reported the SCN participates in the regulation of the circadian adrenal corticosterone rhythm. Thus, the SCN might represent a good location for the beginning of future investigations into the possible role of 5—HT in the control of plasma corticosterone responses to immobilization stress. IV. Effects of Suckling and Pregnaney on 5—Hydroxytryptaminergic Synthesis and Metabolism in Selected Brain Regions and Serum Prolactin in the Female Rat A. Evidence for Involvement of 5-HT Neurons in the Suckling- induced Release of Prolactin Results of studies in the lactating rat suggest that 5-HT neuronal systems are involved in the suckling-induced release of pro— lactin. In fact, serotonin facilitation of prolactin responses to the 91 suckling reflex in lactating rats represents the most documented illu— stration of 5-HT involvement in a neuroendocrine reflex. The dramatic increase in serum prolactin concentrations observed 5-10 min after the onset of the suckling stimulus (Nicoll, 1971) is maintained until the suckling stimulus is removed (Kordon 33 31., 1973). This response can be abolished by prior blockade of 5-HT biosynthesis by p-chlorophenyl- alanine (PCPA) (Kordon 33 31., 1973) or of its receptors by methysergide (Gallo 3£_31,, 1975) and can be re-established following PCPA treatment by administering the immediate precursor for S-HT synthesis, 5-HTP (Kordon 33 31., 1973; Héry EE”§£°, 1976). Transient secretory episodes of oxytocin, which are also induced by suckling, are equally inhibited following 5-HT depletion (Moss and Richard, 1979). 5-HT neuronal pathways involved in this effect probably project from the raphé nuclei to the mediobasal hypothalamus across the medial forebrain bundle because transaction of this tract impairs the suckling- induced activation of neurosecretory neurons (Averill and Purves, 1966). The bundle of Schultz has also been reported to play an important role in the reflex (Beyer and Mena, 1965). Results from experiments examining the neurotransmitter content of various brain regions during suckling also support an important role of 5-HT innervation to the hypothalamus in the regulation of prolactin secretion during lactation. 'Mena 35_31, (1976) and Coppings and McCann (1981) observed a decrease in hypothalamic S-HT concentrations in paral— lel with an increase in 5-HIAA concentrations within 5 min of the suckling stimulus that was maintained as long as suckling was continued. The activity of tryptophan hydroxylase is also increased in the basal 92 hypothalamus by suckling (Carr and Jimenez, personal communication). In contrast, the 5-HT concentration in other brain areas remains unaffected, as does hypothalamic noradrenaline CMena 33 31., 1976). The physiolo- gical significance of these neurochemical results are supported by the fact that the 5-HT response does not occur under weaning or pre—weaning conditions where the pups have been separated from their mother for a period of 24 hr rather than 8 hr or less (Mena 33 31., 1976). Grosvenor and Mena (1971) have reported that prolactin is no longer released by suckling under similar conditions. Furthermore, Rowland 33_31, (1978a) reported that administration of p-chloroamphetamine (PCA), a serotonin neurotoxin, into the third ventricle depleted hypothalamic 5-HT by 43% and resulted in a 98% incidence of pup mortality. In contrast, PCA infusion into the lateral ventricle depleted hypothalamic 5—HT by 23% and resulted in a 48% incidence of pup mortality. Coincident with the hypothalamic 5-HT depletion was a reduction in serum prolactin, and subcutaneous injections of prolactin into the treated females sharply reduced the number of pup deaths. From these results the authors con— cluded that 5-HT neurons in the hypothalamus mediate the suckling— induced release of prolactin necessary for the maintenance of lactation. These results suggest that hypothalamic 5—HT neurons are activated during suckling. Therefore, the effects of suckling on 5—HT synthesis and metabolism in the ME, AN, SCN, MPO and ST were examined in Long- Evans lactating female rats. 1. Effect of Suckling on 5-HT Metabolism in Selected Regions of the Rat Brain 5-HT metabolism in the ST, ME, SCN, MPO and AN was measured in lactating Long—Evans rats who had been 1) deprived of their 93 pups for 4 hr (non—suckled), 2) deprived of their pups for 4 hr and then allowed to be suckled for 30 min (Suckled) and in 3) non—lactating diestrous controls. The results of this experiment are shown in Table 15. Suckling did not effect 5—HT or 5-HIAA concentrations in the ST, SCN, ME or AN. Both lactating experimental groups demonstrated tenden— cies toward increased 5-HT and 5-HIAA concentrations in the ME. Lacta— tion tended to decrease 5-HT concentrations and increase S-HIAA con- centrations in the AN reaching statistical significance in the suckled group. Lactation by itself did not alter 5—HT or 5-HIAA concentrations in the MPO but the addition of a suckling stimulus caused an increase in both 5-HT and S-HIAA. These results suggest that the state of lactation may alter 5—HT metabolism to some extent in the ME and AN. Furthermore, suckling increases 5-HT metabolism in the MPO. 2. Effect of Suckling on 5—HT Synthesis in Selected Regions of the Rat Brain and on Serum Prolactin The effects of lactation alone (non-suckled, 4 hr pup- deprived) or suckling stimulus (4 hr pup—deprived, 30' suckled) on 5—HTP accumulation in the ST, ME, SCN, MPO and AN were compared to a non— lactating diestrous control. The results from this experiment are shown in Table 16. Neither suckling nor lactation influenced 5-HTP accumula- tion in the ST, SCN or AN. Lactation, itself, increased 5—HT synthesis in the ME but suckling did not cause an additional effect of its own. Lactation alone did not alter 5-HTP accumulation in the MPO. However, suckling increased 5—HTP accumulation in the MPO over both the non— lactating, diestrous control and the lactating, 4 hr pup-deprived control. These results suggest an effect of lactation on 5—HT synthesis 94 TABLE 15 Effect of Acute Suckling on S-HT Metabolism in Selected Regions of the Rat Brain Region Treatment 5—HT 5—HIAA ST Diestrous Control 6.5i0.7 5.3i0.5 Non—Suckled 5.7i0.5 4.8i0.3 Suckled 5.5i0.5 4.6:0.5 ME Diestrous Control 18.4il.3 12.2il.1 Non—Suckled 22.3i2.1N 14.8il.3 Suckled 23. 4:2. 2 15.7il.5 AN Diestrous Control 20. 2:1. 9 19.9il.2 Non-Suckled 17. 7il. .4N 23.7il.9N Suckled 15.7il.5 25.3il.6 SCN Diestrous Control 23.2i2.l 18.1il.7 Non-Suckled 25.6i2.4 19.8i2.0 Suckled 21.7i2.2 17.2i1.6 MPO Diestrous Control 6.5i0.4 15.1i1. 5 Non-Suckled 7°2i0'3*N 15. 4:0. 8* Suckled 9.8i1.1 18. er. 9 Suckled animals were deprived of pups for 4 hr prior to re-establishment of suckling stimulus for 30 min. Non— suckled animals were deprived of pups for 4 hr prior to sacrifice. Values represent ng/mg protein (mean i S.E.; N17). *, sig— nificantly different from non-suckled control (p<0.01). , significantly different from diestrous, non-lactating control (p<0.01). 95 TABLE 16 Effect of Acute Suckling on 5—HTP Accumulation in Selected Regions of the Rat Brain Region Treatment 5—HTP ST Diestrous Control 4.9iO.4 Non-Suckled 3.8:0.2 Suckled 3.5:0.2 ME Diestrous Control 5.3:0.5N Non-Suckled 8.2:0.7N Suckled 10.2i1.l AN Diestrous Control 6.5i0.3 Non-Suckled 6.4i0.3 Suckled 6.4:0.4 SCN Diestrous Control 10.9i0.7 Non-Suckled 12.2i0.9 Suckled ll.0i0.6 MPO Diestrous Control 4.9:0.3 Non—Suckled 4.7i0.3*N Suckled 7.3i0.8 Suckled animals were deprived of pups for 4 hr prior to re-establishment of suckling stimulus for 30 min. Non—Suckled animals were deprived of pups for 4 hr prior to sacrifice. All animals received an injection of NSD 1015 (100 mg/kg, i.p.) 30 minutes prior to sacrifice. Values represent the rate of 5-HTP accumulation (ng/mg protein/30 min; mean i S.E.; N = 8). N, significantly different from diestrous, non— lactating control (p<0.01). *, significantly different from non-suckled control (p<0.01). 96 in the ME and that suckling increases 5-HT synthesis in the MPO. Serum prolactin concentrations in lactating rats (8.3il.2 ng/ml) was not significantly different from that in diestrous controls, but suckling elicited an increase in serum prolactin (98.4i12.3 ng/ml). These results support the large amount of evidence that certain 5—HT neuronal systems may be activated during suckling. In particular, 5-HT and 5—HIAA concentrations as well as 5—HTP accumulation are all increased in the MPO, suggesting that S-HT neuronal activity in this nucleus is greatly affected by suckling. S-HT synthesis and, to a lesser degree, 5-HT and 5-HIAA concentrations are increased in the ME during lactation whether the suckling stimulus is present or not. These results extend observations by others which indicate that 5—HT neuronal activity in the hypothalamus is stimulated during suckling (Mena e£_31,, 1976; Carr and Jimenez, 1981; Coppings and McCann, 1981). It is pos- sible that 5-HT neurons in the MPO may be part of a neuronal system that contributes in some way to the decreased DA turnover in the ME that is observed during suckling (Selmanoff and Wise, 1981). For example, 5—HT neurons could be indirectly controlling prolactin release via effects on the tuberoinfundibular DA neurons. It is also tempting to suggest that 5—HT may be involved in the inhibition of ovulation that occurs during lactation. Evidence suggests that 5—HT may inhibit gonadotropin secre— tion (O'Steen, 1965; Kordon_e£h31,, 1968; Kamberi 33.31,, 1971). In this regard, an elevation of 5—HT neuronal activity could simultaneously facilitate prolactin secretion to suckling and antagonize LH secretion; and conversely, a decrease in 5-HT neuronal activity could possibly disinhibit tuberoinfundibular DA neurons (letting them re—establish 97 their tonic inhibition on prolactin secretion) and restore normal phasic patterns of FSH and LH secretion. LHRH-containing nerve cell bodies are located in the MPO and connections between the MPO and ME are known to exist so the anatomical correlates of this latter hypothesis already exist. No causal effect between the increase in 5-HT neuronal activity in the MPO and the suckling-induced increase in prolactin secretion has been shown in the present experiment. Furthermore, suckling in the lactating rat not only produces increases in the release of prolactin but also ACTH (Gregoire, 1947; Voogt eEN31., 1969; Zarrow 31:31,, 1972), growth hormone (Grosvenor, 1964; Sar and Meites, 1969), melanocyte stimulating hormone (Taleisnik and Orias, 1966; Deis and Oriés, 1968), thyroid stimulating hormone (Blake, 1974; Grosvenor, 1964) and oxytocin (Cowie and Folley, 1961). Therefore, the possibility that activation of the 5—HT neuronal system in the MPO is involved with one of the other endocrine events occurring during suckling cannot be ruled out. B. Evidence for Involvement of 5—HT Neurons in Prolactin Secretion During Pregnancy 5—HT neurons may also be involved in the regulatory processes governing prolactin and gonadotropin secretion througout pregnancy. Following copulation or cervical stimulation during the period of estrus, two daily surges of prolactin can be observed during early pregnancy/pseudopregnancy (Butcher 33 31., 1972; Freeman 33 31., 1974). One surge of prolactin occurs at the end of the light period (diurnal surge, 1700—1900 hr) while the second daily surge occurs at the end of 98 the dark period (nocturnal surge, 300~500 hr). These surges appear to be necessary for the initiation and maintenance of progesterone secre- tion from the corpus luteum in early pregnancy (Smith egu313, 1975; Morishige and Rothchild, 1974). The daily diurnal surge of prolactin continues until day 8 of pregnancy whereas the nocturnal surge normally ends on day 10 of pregnancy (Yogev and Terkel, 1978). Presently, little is known about the neurochemical mechanisms governing the regu- lation of these surges of prolactin during pregnancy. Both surges are abolished if hypothalamic retrochiasmatic cuts are made, suggesting that information necessary for their initiation arrives at the medial hypo— thalamus from a rostral direction (Freeman 33 31., 1974). Lesions of the suprachiasmatic nuclei abolish both of the daily surges of prolactin characteristically observed in female rats during pregnancy and pseudo— pregnancy (Bethea and Neill, 1980; Yogev and Terkel, 1980). It appears that the medial preoptic area possesses neurons which inhibit the nocturnal surge and stimulate the diurnal surge of prolactin, whereas stimulation of the dorsomedial-ventromedial (DMN—VMN) areas of the hypothalamus initiates both surges of prolactin (Freeman and Banks, 1980; Gunnet 33 31,, 1981). Excitation of the DMN-VMN appears to be a definite requirement for initiation of the diurnal but not the nocturnal surge of prolactin (Gunnet_e£ 31., 1981). Although some information exists concerning the pathways and areas of the brain that are necessary for initiation and/or maintenance of these surges, experiments examining the neurochemical systems involved are few. Biphasic changes in the activity of the tuberoinfundibular DA neurons and in tyrosine hydroxy- lase activity in the ME have been reported in pregnant and pseudopregnant 99 rats that correlate inversely with prolactin secretion (McKay 33.31,, 1981; Voogt and Carr, 1981). However, few studies examining S-HT neuronal activity throughout pregnancy and especially throughout these dynamic changes in prolactin secretion have been performed. Rowland 33 31, (1978b) observed no changes in S-HT or 5—HIAA concentrations in the hypothalamus, hippocampus and cortex during selected stages of preg— nancy. Thus, it is not known if 5—HT neuronal systems are involved in the daily surges of prolactin or in the alterations in tuberoinfundi- bular DA neuronal activity. Thus, 5-HT neuronal activity in the ST, MPO, AN and SCN and serum prolactin concentrations at various times throughout day 6 of pregnancy (a day when both surges should be occur— ring) were examined. 1. Serum Prolactin and 5—HT Metabolism in Selected Regions of the Rat Brain During Dey 6 of Pregnancy The profile of serum prolactin at various times on day 6 is shown in Figure 7. The nocturnal surge peaks at 300 hr and is still present at 600 hr. The diurnal surge is much smaller than the nocturnal surge and peaks at 1800—2100 hr. 5-HT metabolism was also measured in the SCN, MPO, ST and AN at various times throughout day 6 of pregnancy, including 300 and 600 hr (times when the nocturnal surge occurs), 1200 hr (a control time point when serum prolactin is low) and 1800 and 2100 hr (times when the diurnal surge of prolactin occurs). The results of this experiment are shown on Table 17. No effects on 5-HT or S—HIAA concentrations were observed in the SCN throughout day 6 of pregnancy. In the MPO 5-HT was low at 300 hr and rose by 600 hr to levels that were maintained throughout the day whereas the concentration of 5-HIAA increased from a low at 300 hr to become significantly higher at 1200 100 udonommHm hHudmonHdem one mnouuoH udoanHHo auH3 mdem> .m.w one mnouumH udonomev nuHB mode> .n.m .hodmdwond mo 0 and .AHo.ovdV .Amo.ovmv udonomee hHudmonHdem dendv dHuomHonm ddnom .m ondem 101 m ondem A meow v Hozazummm no s nae zo mane Ban - Oo. COW. - -OON. I 00¢ r. E I l l 8 c9 .- (tm/BU) NIIOV’IOHcI mass 8 n TABLE 17 Times t Various 1118. f the Rat Bra ions 0 Selected Reg 1n 5-HT Metabolism of Day 6 of Pregnancy Time on Day 6 of Pregnancy Compound Region 600 Hr 1200 Hr 1800 Hr 2100 Hr 300 Hr .D—O NM I—II-l +|+I Nd) 5-HIAA 5-HT ST 102 GS I—II—i +l+l 00 OM“ NI—l 20.612.42’b 11.8il.8 24.5:2.2a 14.2:l.0a 5-HIAA S-HT 30.1i6.1 28.1il.8 29.6i0.9 22.8i2.2 25.2:2.6 5-HT SCN 18.7i0.8 l9.4r2.0 14.8il.4 l6.2i2.6 19.9i3.7 5-HIAA I-IO +l-H \‘TCD I-iI-‘l 12.5i1.l 13.7i1.0 8.8:l.0a 5-HIAA S—HT MPO Values represent ng/mg protein (mean i S.E., N>7). Groups without the same letter are significantly different (p<0.01). b 9 a 103 hr, decreasing again by 1800 hr. In the ST, both 5-HT and 5—HIAA concentrations were higher before 1200 hr than they were afterward. 5— HT concentrations in the AN decreased from high values at 300 hr to a low at 1200 hr before increasing back to high values at 1800—2100 hr. In contrast, 5-HIAA concentrations in the AN were highest at 1200 hr with respect to any other time point examined. 2. S-HT Synthesis in Selected Regions of the Rat Brain During Day 6 of Pregnancy 5-HTP accumulation in the ST, MPO, SCN and AN was examined at 300 hr, 600 hr, 1200 hr, 1800 hr and 2100 hr on day 6 of pregnancy. The results of this experiment are shown on Table 18. 5-HT synthesis in the ST or MPO did not change at any time on day 6 of pregnancy. S-HTP accumulation in the SCN decreased from a high at 300 hr throughout the day to a low at 2100 hr. Thus, at the time of the nocturnal surge of prolactin, 5—HT synthesis is increased in the SCN whereas at the time of the diurnal surge (1800—2100 hr) 5—HT synthesis is decreasing. In the AN, S-HTP accumulation is increased at 300 hr and at 1800 hr compared to the other time points examined on day 6 of pregnancy. 3. 5—HT Synthesis in Selected Reg1ons of the Rat Brain at 4:30 and 1200 hr on Day 8 and Day 13 of Pregnancy An experiment was performed to examine whether the changes observed in 5—HT synthesis on day 6 of pregnancy were still occurring when the prolactin surges were absent. 5—HTP accumulation in the ST, MPO, SCN and AN was examined at 430 hr (a time during the nocturnal surge of prolactin) and at 1200 hr (a time point when serum 104 .AHo.ovav udoanmHu mHudmonHdem one nouuoH mamm emu udonuHB mononon.m .Aw I z “.m.m H dmod ”mouddHa om\dHouond wa\wdv dOHudeadooo mHmIm Ho oumn msu udomondon mode> .moHHHnomm on nOHnd mondde om A.d.H .wx\wa.OOHv mHOH nmz Ho dOHuoode do vo>Hooon mHdedm HH¢ m.OHm.n m.0Hm.o m.OHm.m m.OHN.o n.0Ho.m om: £5.0Hm.m Drm.0Hm.OH DrN.HHm.OH n.mN.HHo.mH mm.HHm.qH zom 0.0HH.o Dr\..OHo.0H m.OHH.© m.0HH.H mm.HHm.qH z< N.OHo.q N.OHN.¢ m.OHN.¢ m.OHm.m H.0Hw.m Hm nm OOHN nm oowH nm oomH nm coo nm com dOmem kodwdwonm Ho o and do oBHH hodmdwonm Ho 0 >mm mo moeHH mSOHnm> um denm mom dad Ho meHmom mmuoonm dH dOHudeddoo< mHmIm wH MHmmH 105 prolactin is low) on day 8 (when the nocturnal and diurnal surges are both present) and on day 13 (When both surges of prolactin have dis— appeared) of pregnancy. The results of this experiment are shown on Table 19. No differences in 5-HTP accumulation in the ST or MPO were observed on day 8 or day 13. In the SCN the rate of S—HTP accumulation at 430 on both day-8 and day 13 is significantly greater than the 1200 hr value. On the other hand, the significant increase in 5—HTP accu- mulation observed in the AN on day 8 of pregnancy is no longer present on day 13 when the nocturnal surge in prolactin is also absent. These results suggest that 5—HT synthesis in the AN is increased during both the nocturnal and diurnal surges of prolactin and that 5-HT synthesis in the SCN is increased during the nocturnal surge and possibly decreased during the diurnal surge of prolactin. 5-HT metabolism, on the other hand, appears to be enhanced in the AN at 1200 hr and does not change in the SCN. This apparent discrepancy with the reports of Rowland et al, (1978b) that 5-HT metabolism does not change throughout pregnancy is probably explained by the fact that Rowland and his coworkers examined these concentrations at one time point on day 5, day 15 and day 21 of pregnancy. No differences would have been detected in the present study if we had examined one time point (for example, 1200 hr) on three different days. Also, Rowland gt 31. (1978b) examined effects in the whole hypothalamus. With the great variety of effects occurring in discrete areas of the hypothalamus in the present study it is not surprising that effects were not observed in the whole hypo— thalamus. 106 TABLE 19 S—HTP Accumulation in Selected Regions of the Rat Brain at 430 and 1200 hr on Day 8 and Day 13 of Pregnancy Day of Pregnancy Region Hour of Day Day 8 Day 13 ST 430 3.9i0.2 4.0i0.4 1200 4.4:0.4 3.9iO.4 AN 430 11.3i0.9 5.5i0.3 1200 6.6iO.7* 6.2i0.5 SCN 430 17.6il.5 l6.7i1.1 1200 13.1il.5* 13.0:1.1* MPO 430 6.9i1.0 4.5:0.8 1200 4.4i0.6 5.4i0.4 All animals received an injection of NSD 1015 (100 mg/kg, i.p.) 30 minutes prior to sacrifice. Values repre- sent the rate of 5—HTP accumulation (ng/mg protein/30 minutes; mean i S.E.; N = 8). *, significantly different from 430 hour value (p<0.01) on same day. 107 When 5—HTP accumulation was examined in the AN and SCN at a time when the prolactin surges have stopped, the changes in the SCN still occur; whereas the increase in 5-HT synthesis observed at 430 hr in the AN is no longer present. These results suggest that the increase in 5-HT synthesis in the AN may be correlated to the presence of the nocturnal prolactin surge whereas the increase in 5-HT synthesis in the SCN is not. Therefore, the changes in 5-HT neuronal activity in the SCN are probably not involved in the surges of prolactin observed in early pregnancy but the changes in S-HT neuronal activity in the AN may, indeed, play a role in this regulation. V. 5-Hydroxytryptamine Metabolism and Synthesis in Selected Brain Regions and Serum Prolactin Throughout Proestrus in the Female Rat 5-HT neuronal systems appear to play an important role in the regulation of neuroendocrinological processes leading to ovulation. On the afternoon of proestrus at or close to the time of ovulation there occur surges of the Secretion of luteinizing hormone (LH), follicle— stimulating hormone (FSH) and prolactin (Butcher g£_§l,, 1974). Al— though many experiments have been performed examining catecholamine content and turnover in the ME during proestrus and relating a decrease in tuberoinfundibular DA to the observed surge of prolactin (Ahren et_ al., 1971; Lofstrom, 1977; Crowley g£_§l,, 1978; Demarest §E_§l,, 1981; Rance et a1,, 1981), most work concerning the possible 5-hydroxytrypt- aminergic involvement in the neuroendocrinological events occurring on the afternoon of proestrus has been concerned with LH secretion. The marked increases in serum LH concentrations occurring on the afternoon 108 of proestrus or in estradiol-pretreated ovariectomized rats as well as ovulation can be prevented by drugs which disrupt S—HT neurotransmission (Héry gt El-a 1975, 1976, 1978; Baumgarten 23 al., 1978; Coen and Mac- Kinnon, 1979; walker, 1980; walker 25 31., 1981), and 5—HTP administra— tion can temporarily reinstate the estrogen-induced surge if it is given at certain times of the day (Héry eg al., 1976; Coen and MacKinnon, 1979). Administration of 5-HT agonists at certain times on the day of proestrus can elicit a surge in LH secretion (walker, 1980). In addi— tion, there is a correlation between the 5-HT antagonistic properties of a number of tricyclic and ergot-derivative compounds and their ability to inhibit the pre-ovulatory surges of LH (Mark6 and Flfickiger, 1980). Estradiol pretreatment in ovariectomized rats also induces a daily surge of prolactin and disrupts 5-HT neurotransmission, lesioning the SCN or interrupting frontal afferents to the mediobasal hypothalamus at the retrochiasmatic level can attenuate or abolish this surge (Caligaris and Taleisnik, 1974; Subramanian and Gala, 1976; Kawakami 25 31., 1980). walker (1980) observed an increased 5-HT turnover in the hypo— thalamus coincident with the onset of the proestrus pre—ovulatory LH and prolactin surges. Furthermore, when the LH (and probably prolactin) surge was prolonged by exposing the rats to light on the evening of proestrus, the hypothalamic S—HT turnover remained high for an extended period. Stimulation of the MPO may be at least partially responsible for the secretion of prolactin on the afternoon of proestrus (Kimura and Kawakami, 1978). Kueng g5 El: (1976) reported a decrease in 5-HT concentrations in many hypothalamic, limbic and midbrain structures at 109 1500 hr on proestrus versus 1000 hr values. The decrease was especially evident in the lateral parts of the preoptic area. The authors inter— preted this decrease as indicating a release from the 'normal' inhi— bitory effects of 5-HT on gonadotropin release mechanisms but also may have demonstrated an increased release and metabolism of 5—HT in these areas at 1500 hr before the surges of LH, FSH and prolactin occur. These results taken together suggest that the proestrus surges of FSH, LH and prolactin are accompanied by and may be at least partially dependent upon dynamic changes in 5-HT neuronal activity. Thus, S—HT neuronal activity in the ST, SCN, MPO, ME and AN and serum.prolactin were examined at various times on the day of proestrus in female rats. A. Serum Prolactin and 5-HT Metabolism in Selected Brain Regions During Proestrus in Female Rats The profile of serum prolactin at various times throughout proestrus is shown in Figure 8. Serum prolactin began to increase at 1500 hr, peaked at 1700 hr and then slowly fell throughout the rest of the evening. 5-HT~metabolism in the ST, MPO, SCN, AN and ME was also studied at 800, 1200, 1500, 1700, 1900, 2100 and 2400 hr on the day of proestrus in female Long—Evans rats. The results of this experiment are shown on Table 20. 5—HIAA concentrations did not change at any time in the ST, MPO, AN or ME but increased in the SCN from a low at 800 hr to a high at 1700-1900 hr (same time that prolactin is reaching its peak) before declining again by 2100 hr. 5—HT concentrations in the ST, MPO and AN showed small differences throughout the day of proestrus. In the ME, 5—HT concentrations remain fairly constant through 1500 hr and then drop dramatically at 1700 hr (when the surge of prolactin is peaking). llO . Go .93 aim». .E coma woo cow fioum uoouommfio hauomoHMfiome ma moam> .c .mouumooum so cfluomaouo abnom .w ouowfim 1.4 111 A Mbom V mDMHmmomm ZO mSHH 00$ Oofi 002 w ouowfim 00.». 002 OON. - Oo H . «Ho. FL (rm/Bu) NILDV'IOHa muss TABLE 20 5—HT Metabolism in Selected Regions of the Rat Brain at Various Times on Proestrus Time on Proestrus Region Compound 2400 Br 2100 Hr 1500 Hr 1700 Hr 1200 Hr 800 Hr 1900 Hr 7.2i1.0 l4.lil.3 5.4i0.9 12.8il.3 6.8il.l l3.6i0.8 6.7iO.8 12.2i0.9 6.8i0.7 13.8il.4 6.5:0.7 12.4i1.3 8.2:0.8 15.3il.7 S-HT ST 5—HIAA 14.4:2.3a’b 134452.06“b 16413.26"b 9.3:1.3a 17.8:1.9b 13.7i3.88’b 12.1:1.0a S-HT ME .5 7.4il.2 6.2i0 7.8i0.9 .5 3il.2 6.7i0.7 7.5iO.8 5.9i0 8. 5-HIAA 22.5«_~2.4a’b 23.4:2.43’b 19.6:2.0"J"b 25.412.8b 112 23.8:2.1a’b 15.0:1.6 l6.4il.5 15.3il.8 12.8il.l 13.1il.3 l4.2il.5 21.3~:2.2‘1"b 18.4:1.9a 5-HT AN b b 13’ a 23.8:2 l6.3il b ’b 26.8:2.0 3 a,b l7.lil.3 a b 24.2il.3 18.8il.1 b b a 21.3:~_2.0a’b 15.1:0.9a 5-HT SCN 20.4il.6 a,b 15.7il.l 5-HIAA 7.3%.?"b 10.1:o.3 6.9:0.5""”b 6.4i0.4b 9.6:0.4 8.4i0.4a 8.4:O.5 8.7:0.Sa 9.7:1.o 8.2:1.2“"b 10.9:2.1 7.1:o.sa’b 10.3i0.8 S-HT MPO .4 9.0i0 5-HIAA (mean i S.E.; N>7). Values represent ng/mg protein ’bGroups without the same letter are significantly different (p<0.01). a 113 By 1900 hr the 5-HT concentrations in the ME have returned to values observed at 1500 hr. S-HT concentrations in the SCN parallel those of the 5-HIAA concentrations in that they start low at 800 hr and peak at 1700-2100 hr before beginning to fall. These results suggest that 5-HT metabolism in the SCN is activated at a time when the surges in pro- lactin, LH and FSH are occurring. On the other hand, 5—HT neuronal activity in the ME is decreased at the onset of these hormonal surges. B. 5-HT Synthesis in Selected Brain Regions During Proestrus in Female Rats 5-HT synthesis in the ST, MPO, SCN, AN and ME as well as serum prolactin was examined at 800, 1200, 1500, 1700, 1900, 2100 and 2400 hr on the day of proestrus. The results of these experiments are shown on Table 21. 5-HTP accumulation was not statistically altered at any time during proestrus in the ST, MPO, SCN or AN. However, a definite trend towards an increase in 5—HT synthesis in the SCN occurs at the same time that the increases in 5-HT and S-HIAA concentrations were observed. In the ME, 5—HTP accumulation decreases at 1700 hr and 2100 hr. Results from these studies suggest that neuronal activity in the SCN is in— creased while that in the ME is decreased at the time of the peak con— centration of serum prolactin on proestrus. The results from this section support those of other investi— gators which suggest that 5-HT neuronal activity is activated in the hypothalamus during the afternoon of proestrus and may be related to the preovulatory surges of LH, prolactin or FSH (Héry 35 31., 1975, 1976, 1978; Baumgarten 35 al., 1978; Coen and MacKinnon, 1979; Marko and Flfickiger, 1980; Walker, 1980; Walker et_al,, 1981). 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