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DATE DUE DATE DUE DATE DUE MSU le An Nflrmetlve Action/Equal Opportunity InetItmIon m ms __—-—_-._—— HISTAMINERGIC REGULATION OF CENTRAL CATECHOLAMINERGIC AND 5-HYDROXYTRYPTAMINERGIC NEURONAL ACTIVITY IN THE RAT BY Annette Elizabeth Fleckcnstcin 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 1994 ABSTRACT HISTAMINERGIC REGULATION OF CENTRAL CATECHOLAMINERGIC AND 5-HYDROXYTRYPTAMINERGIC NEURONAL ACTIVTTY IN THE RAT By Annette Elizabeth Fleckenstein The influence of histaminergic neurons on activity of selected catecholaminergic and 5-hydroxytryptaminergic neuronal systems, and on circulating levels of prolactin and a-melanocyte-stimulating hormone (aMSH) whose secretion these systems control, was investigated in male rats. Procedures were employed which mimic (i.e., intracerebroventricular administration of histamine), facilitate (i.e. , blockade of histamine metabolism and/or H3 autoreceptors), increase (i.e. , exposure to stress of physical restraint) or decrease (i.e. , disruption of histamine synthesis, blockade of post-synaptic histaminergic receptors, stimulation of H, autoreceptors) histaminergic transmission. Dopaminergic, noradrenergic and 5-hydroxytryptaminergic neuronal activity was estimated by measuring concentrations of neurotransmitters, their precursors and/or metabolites in brain regions containing perikarya and/or terminals of these neurons. Results are summarized below. Intracerebroventricular histamine administration increases mesolimbic dopaminergic neuronal activity via an action at HI receptors, but is without effect on nigrostriatal, tuberoinfundibular, periventricular-hypophysial, or incertohypothalamic dopaminergic neuronal activity. Intracerebroventricular histamine also increases activity of dopaminergic neurons terminating in the suprachiasmatic, caudal periventricular and paraventricular hypothalamic nuclei via Hl receptor activation. In contrast, blockade of histamine metabolism and/or H, receptors does not affect dopaminergic neuronal activity in systems under study. Restraint stress decreases perivcntricular-hypophysial, increases mesolimbic, and is without effect on tuberoinfundibular or nigrostriatal dopaminergic neuronal activity. Histaminergic neurons mediate stress-induced changes in periventricular-hypophysial, but not mesolimbic dopaminergic neuronal activity via an action at H, receptors. In the absence of stressful stimuli, histaminergic neurons do not affect dopaminergic neurons. Intracerebroventricular histamine administration increases activity of both noradrenergic and 5-hydroxytryptaminergic neurons projecting to the hypothalamus via an action at H, receptors. Restraint stress likewise increases activity of these neurons; histaminergic neurons mediate these increases by activating H, receptors. Blockade of histamine metabolism and/or H, receptors affects neither hypothalamic noradrenergic nor 5-hydroxytryptaminergic neuronal activity. In the absence of stressful stimuli, histaminergic neurons do not affect these systems. Pharmacological manipulations which affect central histamine concentrations or transmission do not affect aMSH secretion. Intracerebroventricular histamine administration increases prolactin secretion through a mechanism independent of changes in tuberoinfundibular dopaminergic neuronal activity. Restraint stress increases both prolactin and aMSH secretion; neither effect is mediated via histamine. To my mother and father, Sandra and John Fleclmnstein, and my brother, John, forrmfailingloveandmanywordsofwisdom and toDr.KennethE. Moore fortheopportunitytoseetheocean iv fat 01h ACKNOWLEDGMENTS I wish to express profound appreciation to my mentor, Kenneth Moore, for his encouragement and support, both professional and personal, throughout my course of study. His enthusiasm for research and teaching, commitment to excellence, and love of learning never fail to inspire. I am grateful and honored to have been his student. Appreciation is expressed to Keith Lookingland for valuable scientific discussion. I thank him and the other members of my thesis committee, Philip Von Voigtlander, Gregory Pink and Ralph Pax and for their interest, insights and guidance. I thank the Pharmaceutical Manufacturers Association Foundation for financial support. I would not have attempted graduate school were it not for the encouragement of Lynne Wathen and Leonard Bcuving, nor would I have completed it without the support and friendship of Sarah Smith, Misty Eaton, Margarita Contreras and Jorge Manzanares; to each I express heartfelt gratitude. I thank also Nelda Carpenter, Marty Burns, Mickie Vanderlip, Diane Hummel, Christine Stevens, Carol Loviska, Derek Foster, Michael Woolhiser, Thomas Toney and Robert Kranich for fellowship and many acts of kindness. Deep appreciation is expressed to my father and mother, John Sr. and Sandra Fleckenstein, for their example, guidance and unfailing love. In particular, I thank my father for teaching me that "it’s not the critic that counts. . . " and my mother for countless other words of wisdom. I thank my parents also for the many sacrifices that they made to secure for me the Catholic education that formed the foundation for future success. V I thank my brother, John Fleclmnstein Jr., whose enthusiasm and brilliance have been andwillcontinuetobemyinspirationandchallenge. Finally,IthankElirabeth Fleckenstein, MaryJeanneJuzwiak, FrancesPowersand therestofmy familyfortheir prayersandconcern. Nowordscanexpressadequately myrespectforandgratitudeto my family. Most of all, I thank Almighty God, who has blessed me with wonderful family and friends, and by whose grace this dissertation was completed. AnnetteElizabethFleckenstein TABLE OF CONTENTS LIST OF TABLES ..................................................................... xiii LIST OF FIGURES .................................................................... xv LIST OF ABBREVIATIONS ........................................................ xix I. INTRODDCI'ION .................................................................. 1 A. Statement of Purpose ..................................................... l B. Histaminergic Neuronal System ......................................... l l. Histamine as a Neurotransmitter .............................. 1 2. Anatomy of Histaminergic Neuronal System ............... 4 3. Biochemistry of Histaminergic Neurons ...................... 8 4. Afferent Regulation of Histaminergic Neurons ............. 11 5. Histaminergic Receptors: Localization, Agonists and Antagonists ................................................... 13 a. H, Receptors ............................................. 13 b. H, Receptors ............................................. 14 c. H, Receptors ............................................. 15 6. Strategies for Assessing Role of Histaminergic Neurons. 16 a. Pharmacological Strategies for Mimicking or Facilitating Histaminergic Neuronal Transmission . 17 b. Pharmacological Strategies for Disrupting Histaminergic Neuronal Transmission ............... 19 c. Stress as a Physiological Strategy for Increasing Histaminergic Neuronal Activity ...................... 20 vii C. Non-Neuronal Stores of Histamine in Brain ......................... D. Catecholaminergic Neuronal Systems ................................. 1. Anatomy of Catecholaminergic Neuronal Systems ......... a. Anatomy of Dopaminergic Neuronal Systems ..... b. Anatomy of Noradrenergic Neuronal Systems ..... 2. Biochemistry of Catecholaminergic Neurons ................ a. Biochemistry of Dopaminergic Neurons ............ b. Biochemistry of Noradrenergic Neurons ............ 3. Neurochemical Techniques to Estimate Catecholaminergic Neuronal Activity ......................... ' a. DOPA Accumulation as an Index of Dopaminergic Neuronal Activity ..................... b. Concentrations of DOPAC as Indices of Dopaminergic and Noradrenergic Neuronal Activity ........................................ c. Concentrations of MHPG as Indices of Noradrenergic Neuronal Activity ...................... 4. Dopaminergic Regulation of Prolactin and aMSH Secretion .................................................. a. Prolactin .................................................. b. a-MSH .................................................... E. 5-Hydroxytryptaminergic Neuronal Systems ........................ 1. Anatomy of 5-Hydroxytryptaminergic Neuronal Systems . 2. Biochemistry of 5-Hydroxytryptaminergic Neurons ........ 3. Neurochemical Techniques to Estimate 5-Hydroxytryptaminergic Neuronal Activity ................ II. MATERIALS AND METHODS ............................................... A. Animals .................................................................... B. Drugs ....................................................................... viii 20 21 21 21 23 24 24 27 29 29 30 31 31 31 31 32 32 34 36 37 37 38 C. Intracerebroventricular Injections ....................................... D. Neurochernical Lesion of the Ventral Noradrenergic Bundle . . . . E. Restraint Stress ............................................................ F. Tissue Dissection and Neurochemical Assays ....................... G. Acid Hydrolysis Procedure ............................................. H. Radioimmunoassays for aMSH and Prolactin ......................... 1. Statistical Analyses ........................................................ III. I-IISTAMINERGIC REGULATION OF CATECHOLAMINERGIC NEURONS ...................................... A. Exogenous Histamine Increases the Activity of Mesolimbic, but Not Nigrostriatal Dopaminergic Neurons ........................... 1. Introduction ....................................................... 2. Results ............................................................. 3. Discussion ......................................................... B. Exogenous Histamine Differentially Affects the Activity of Hypothalamic Dopaminergic Neurons ................................ 1. Introduction ...................... 2. Results ............................................................. 3. Discussion ......................................................... C. Exogenous Histamine Increases the Activity of Noradrenergic Neurons Projecting to the Diencephalon ................................. 1. Introduction ....................................................... 2. Results ............................................................. 3. Discussion ......................................................... ix 38 39 39 42 42 43 46 51 53 53 55 74 D. Differential Role of Histamine in Mediating Stress-Induced Changes in Dopaminergic Neuronal Activity ............................ 1. Introduction ....................................................... 2. Results ............................................................. 3. Discussion ......................................................... E. Histaminergic Neurons Mediate Restraint Stress-Induced Increases in the Activity of Noradrenergic Neurons Projecting to the Hypothalarnus ............................................ 1. Introduction ....................................................... 2. Results ............................................................. 3. Discussion ......................................................... F. H, Receptor Ligands Do Not Affect the Activity of Catecholaminergic Neurons ................................................. 1. Introduction ....................................................... 2. Results ............................................................. 3. Discussion ......................................................... G. Histaminergic Neurons Do Not Regulate Basal Catecholaminergic Neuronal Activity .......................................................... H. Summary and Conclusions .............................................. IV. HIST AMINERGIC REGULATION OF 5-HYDROXYTRYPTAMINERGIC NEURONS ............................ A. Effects of Histamine on 5-Hydroxytryptarninergic Neuronal Activity in the Rat Hypothalamus ......................................... 1. Introduction ....................................................... 2. Results ............................................................. 78 78 87 91 98 98 100 106 109 111 111 111 112 3. Discussion .' ........................................................ B. Histamine Mediates Restraint Stress-Induced Increases in the Activity of S-Hydroxytryptaminergic Neurons ..................... 1. Introduction ....................................................... 2. Results ............................................................. 3. Discussion ......................................................... C. Histaminergic Neurons Do Not Regulate Basal 5-Hydroxytryptaminergic Neuronal Activity ............................ D. Summary and Conclusions ............................................. V. HISTAMINERGIC REGULATION OF PROLACTIN AND aMSH SECRETION ...................................................... A. Histamine-Stimulated Prolactin Secretion Is Not Mediated by an Inhibition of Tuberoinfundibular Dopaminergic Neurons ......... 1. Introduction ....................................................... 2. Results ............................................................. 3. Discussion ......................................................... B. Pharmacological Manipulations Which Alter Histaminergic Transmission May Not Affect Prolactin Secretion ..................... C. Central Histamine Does Not Mediate aMSH Secretion ........... D. Histamine Does Not Mediate Restraint Stress-Induced Increases in Prolactin or aMSH Secretion ............................... 1. Introduction ....................................................... 2. Results ............................................................. 3. Discussion ......................................................... 115 126 126 127 128 134 137 138 138 138 139 141 144 147 148 148 148 151 E. Summary and Conclusions .............................................. 151 VI. CONCLUDING DISCUSSION .............................................. 153 VII. LIST OF REFERENCES ...................................................... 159 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.1 LIST OF TABLES Dose response effects of histamine on DOPAC and dopamine concentrations in selected brain nuclei of male rats ..................... Time course effects of histamine on DOPAC and dopamine concentrations in the intermediate lobe of the pituitary inmale'rats .................................................................... Effects of ventral noradrenergic bundle-lesion on norepinephrine concentrations in selected brain nuclei of male rats .................... Time course effects of thioperamide on DOPAC and dopamine concentrations in various brain nuclei of male rats .................... Time course effects of thioperamide on MHPG concentrations intheMZIandMPNofmalerats ........................................ Effects of thioperamide, metoprine and combined thioperamide! metoprine treatment on DOPAC and dopamine concentrations in various brain nuclei of male rats ....................................... Effects of R-a-methylhistamine on DOPAC and dopamine concentrations in various brain nuclei of male rats .................... Effects of BP 2.94 on DOPAC and dopamine concentrations in various brain nuclei of male rats ....................................... Time course effects of aFMH on DOPAC and dopamine concentrations in various brain nuclei of male rats .................... Dose-response effects of histamine on SHT and SHIAA concentrations in various hypothalamic nuclei in male rats .......... 56 57 61 101 102 103 104 105 108 113 4.2 4.3 4.4 4.5 4.6 5.1 5.2 5.3 5.4 6.1 6.2 6.3 Time course effects of thioperamide on SHT and SHIAA concentrations in various hypothalamic nuclei in male rats .......... Effects of thioperamide, metoprine and combined thiOperamide/ metoprine on SHT and SI-IIAA concentrations in various hypothalamic nuclei of male rats .......................................... Effects of R-a-methylhistamine on SHIAA and SHT concentrations in various hypothalamic nuclei of male rats .......... Effects of BP 2.94 on SI-IIAA and SHT concentrations in various hypothalamic nuclei of male rats ............................ Effects of aFMH on SHIAA and SI-IT concentrations in various hypothalamic nuclei of male rats .......................................... Dose response effects of thioperamide on plasma prolactin and median eminence DOPAC and dopamine concentrations in male rats ................................................. Dose response effects of metoprine on plasma prolactin and median eminence DOPAC and dopamine concentrations in male rats ................................................. Dose response effects of R-a-methylhistamine and BP 2.94 on plasma prolactin concentrations in male rats ............................ Dose response effects of R-a-methylhistamine and BP 2.94 on plasma aMSH concentrations in male rats ............................... Summary of effects of i.c.v. histamine administration on the activity of selected central dopaminergic neuronal systems in the rat Summary of effects of i.c.v. histamine administration on the activity of hypothalamic S-hydroxytryptaminergic and noradrenergic neuronal systems in the rat ............................... Summary of role of histaminergic neurons in effecting stress-induced changes in the activity of selected central catecholaminergic and S-hydroxytryptaminergic neuronal systems in the rat ....................................................................... xiv 120 121 135 136 136 145 145 146 147 154 154 155 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 3.1 LIST OF FIGURES Frontal sections through posterior hypothalamus of the rat illustrating the topical localization of histaminergic perikarya and the subgroups E,-E, ......................................... Schematic representation of the distribution of histaminergic nerve fibers in rat brain ..................................................... Major synthetic and metabolic pathway of histamine in the brain. . . . Schematic representation of the sites of action of the histidine decarboxylase inhibitor aFMH, the H, agonist R-a-methylhistamine, the H, antagonist mepyramine, the histamine-N-methyltransferase inhibitor metoprine, the H, antagonist thioperamide and the H, antagonist zolantidine ................................................... Schematic representation of the distribution of catecholamine- containing perikarya in rat brain .......................................... Schematic representation of the neurochemical events occurring within nigrostriatal and tuberoinfundibular dopaminergic neurons .. Schematic representation of the neurochemical events occurring within noradrenergic neurons ............................................... Schematic representation of the distribution of SHT-containing perikarya in rat brain ........................................................ Schematic representation of the neurochemical events occurring within S-hydroxytryptaminergic neurons ................................. Dose-response effects of histamine on DOPA accumulation and on concentrations of DOPAC and dopamine in the nucleus accumbens and striatum of male rats ...................................... XV 18 22 25 28 33 35 47 3.2 Time course effects ofhistamineon concentrations of DOPAC and dopamine in the nucleus accumbens and striatum ofmalerats .................................................................... 3.3 Dose-response effects of mepyramine on DOPAC concentrations in the nucleus accumbens of histaminetreated male rats .............. 3.4 Effects of mepyramine and zolantidine on DOPAC concentrations in the nucleus accumbens of histamine- and vehicle-treated male rats ........................................................................ 3.5 Time course effects of histamine on DOPAC and dopamine concentrations in the DMN and M21 of male rats ..................... 3.6 Time-course effects of histamine on DOPAC and dopamine concentrations in the MPN and rPeVN of male rats ................... 3.7 Effects of histamine on DOPAC and dopamine concentrations in the DMN and M21 of sham and ventral noradrenergic bundle-lesioned male rats ................................................... 3. 8 Effects of histamine on DOPAC and dopamine concentrations in the MPN and rPeVN of sham and ventral noradrenergic bundle-lesioned male rats ................................................... 3.9 Time course effects of histamine on DOPAC and dopamine concentrations in the SCN, cPeVN and PVN of male rats ........... 3.10 Effects of histamine on DOPAC concentrations in the SCN, cPeVN and PVN of sham and ventral noradrenergic bundle- lesioned male rats ............................................................ 3.11 Effects of histamine on DOPAC concentrations in the SCN, cPeVN and PVN of saline- and mepyrarnine-treated male rats ...... 3.12 Effects of histamine on DOPAC concentrations in the SCN, cPeVN and PVN of saline- and zolantidine-treated male rats ........ 3.13 Dose response effects of histamine on MHPG concentrations in the DMN, MZI, MPN and rPeVN of male rats ..................... xvi 3.14 3.15 3.16 3.17 3.18 3.19 3.20 3.21 3.22 3.23 3.24 4.1 TimecourseeffectsofhistamineonMHPGandnor-epinephrine concentrationsintheDLm,MZI,MPNandrPeVNofmalerats.. Effects of mepyramine and zolantidine on MHPG concentrations in the DMN, MZI, MPN and rPeVN of histamine- and vehicle- treated male rats .............................................................. Tune course effects of physical restraint on DOPAC and dopamine concentrations in the nucleus accumbens, intermediate lobe of the pituitary, striatum, and median eminence of male rats ............ Effects of aFMI-I on basal and restraint stress-affected DOPAC concentrations in the nucleus accumbens and intermediate lobe of the pituitary of male rats .......................................... Effects of mepyramine on basal and restraint stress-affected DOPAC concentrations in the nucleus accumbens and intermediate lobe of the pituitary of male rats .......................... Effects of zolantidine on basal and restraint stress-affected DOPAC concentrations in the nucleus accumbens and intermediate lobe of the pituitary of male rats .......................... Effects of aFMH, mepyramine and zolantidine on basal and restraint stress-affected aMSH concentrations in the plasma of male rats .......................................................... Time course effects of physical restraint on MHPG and norepinephrine concentrations in the PVN and MPN of male rats .. Effects of aFMH on basal and restraint stress-increased MHPG concentrations in the PVN and MPN of male rats ..................... Effects of mepyramine on basal and restraint stress-increased MHPG concentrations in the PVN and MPN of male rats ............ Effects of zolantidine on basal and restraint stress-increased MHPG concentrations in the PVN and MPN of male rats ............ Time course effects of histamine on concentrations of SHT and SHIAA in the PVN of male rats ..................................... xvii 76 82 83 85 86 93 94 95 96 116 4.2 4.3 4.4 4.5 4.6 4.7 4.8 5.1 5.2 5.3 5.4 Effects of fluoxetine on SHT and SHIAA concentrations in thePVNofhistamine-andvehicle—treatedmalerats ................. Effects of mepyramine on SHT and SI-IIAA concentrations in the PVN of histamine- and vehicle- treated male rats ....................... Effects of zolantidine on SHT and SI-IIAA concentrations in the PVN of histamine- and vehicle- treated male rats ...................... Time course effects of physical restraint on SHIAA and SHT concentrations in the nucleus accumbens and SCN of male rats ..... Effects of aFMH on basal and restraint stress-increased SHIAA concentrations in the nucleus accumbens and SCN of male rats .................................................................... Effects of mepyramine on basal and restraint stress- increased SHIAA concentrations in the nucleus accumbens and SCN of male rats ........................................................ Effects of zolantidine on basal and restraint stress- increased SHIAA concentrations in the nucleus accumbens and SCN of male rats ........................................................ Dose-response effects of histamine on concentrations of prolactin in the plasma and DOPA, DOPAC and dopamine in the median eminence of male rats ......................................... Time course effects of histamine on concentrations of prolactin in the plasma and DOPAC in the median eminence of male rats ....................................................... Time course effects of physical restraint on prolactin and oMSH concentrations in the plasma of male rats ...................... Effects of aFMH, mepyramine and zolantidine on basal and restraint stress-affected proIactin concentrations in the plasma of male rats ....................................................... xviii 117 118 119 129 130 131 132 140 142 149 150 CG Cer CX DOPAC DOPA DOPEG DA DMN Ext. capsule aFMI-I GABA LIST OF ABBREVIATIONS amino acid decarboxylase anterior hypothalamic area arcuate nucleus catecholamine-O—methyltransferase caudal periventricular nucleus central gray cerebellum corpus callosum cortex 3,4-dihydroxypheny1acetic acid 3,4-dihydroxyphenyla1anine’ 3,4-dihydroxyphenylglycol dorsal hypothalamic area dorsomedial nucleus external capsule a-fluoromethylhistidine gamma aminobutyric acid xix i.c. i.c.v. i.p. oMSH 3MT t-MeHA hour 6-hydroxydopamine S-hydroxyindoleacetic acid S-hydroxytr'yptarnine 5-hydroxytryptopban intracerebral intracerebroventricular intraperitoneal lateral hypothalamic nucleus lateral mammillary nucleus lateral septum medial forebrain bundle medial longitudinal fasciculus medial mammillary nucleus medial preoptic nucleus medial zona incerta mediodorsal thalamus a-melanocyte-stimulating hormone 3-methoxy-4-hydroxyphenylethylenglycol 3-methoxytyramine t-methylhistamine t-MeIAA norepi. N. caudate OB PVN PeVN p.o. PM rPeVN s Sol SEM stria. term. ST S.C. SCN SuM 3V TRP-H t-methylimidazole acetic acid minute norepinephrine nucleus caudate olfactory bulb paraventricular nucleus periventricular nucleus per os pontine nuclei premammillary nucleus rostral periventricular nucleus second solitary tract nucleus standard error of the mean stria terminalis striatum subcutaneous suprachiasmatic nucleus supramammillary nucleus supraoptic decussation third ventricle thyrotropin releasing hormone tryptophan hydroxylase xxi TYR-H tyrodne hydroxylase VMH ventral medial hypothalamic nucleus VBD vertical limb of the diagonal band I. INTRODUCTION A. Statement of Purpose Histamine (2-(4-imidazolyl)ethylamine) has long been recognized as an important mediator in cell-to—cell communication. Whereas peripheral actions of the amine such as effecting. smooth muscle contraction, vasodilation, or gastric acid secretion are well characterized, relatively little is known regarding the role of histamine as a neurotransmitter in the mammalian central nervous system. The aim of this research was to characterize in the rat the influence of histaminergic neurons on the activity of selected central catecholaminergic and S-hydroxyu'yptaminergic neuronal systems, and on the secretion of pituitary hormones (prolactin, a-melanocyte-stimulating hormone (aMSH)) whose secretion these neuronal systems control. As an introduction to these studies, synopses of the anatomy, biochemistry and techniques applied to the study of histaminergic, catecholaminergic and S-hydroxytryptaminergic neuronal systems are provided in the discussion below. B. Histaminergic Neuronal System I . Histamine as a Neurotransmitter Strong evidence supports the view that histamine is a neurotransmitter in the mammalian central nervous system, the criteria for which have been reviewed (Erulkar, 2 1989; Bloom, 1990). In order to be classified as a transmitter, a substance must be present in nerve terminals, usually in association with enzymes required for its synthesis. Lesion of presynaptic neurons or their tracts should cause a disappearance of the transmitter and/or its synthetic enzyme. These criteria have been fulfilled for histamine. Subcellular fractionalization studies have demonstrated the presence of histamine within both mitochondrial (P,) and microsomal (P,) fractions, indicating its presence within nerve endings (Kataoka and DeRobertis, 1967; Snyder et al., 1974; see also Schwartz et al., 1991). The presence of histamine within neurons has been further demonstrated immunohistochemically using antibodies against the amine (Panula et al., 1984; Wilcox and Seybold, 1982). The regional distribution of histidine decarboxylase, the enzyme responsible for histamine synthesis (Schayer, 1966), closely parallels that of histamine (Schwartz et al., 1970; Hough, 1988). Both electrolytic lesion and physical transection of histaminergic neurons cause the disappearance of histidine decarboxylase (Garbarg et al., 1974). A second criterion for classification as a neurotransmitter is that the putative transmitter should be released from neurons concomitantly with presynaptic nerve activity and in a pharmacologically identifiable form. Again, histamine fulfills this criterion; the amine can be released by depolarization in vitro from brain slices (Atack and Carlsson, 1972; Verdiere et al., 1975) in a Ca’ ’-dependent manner (Verdiere et al., 1975). Both electrical stimulation and perfusion with high l(+ concentrations increase histamine release in vivo as measured in microdialysates from rat hypothalamus (Mochizuki etal., 1991; Itoh et al., 1991). This histamine is of neuronal origin; the only other plausible histamine source, mast cells (see this Chapter, Section C), does not contribute to Sti ch H. ml its HI re sy st. and jm'e 3 stimulated histamine release since mast cells do not possess voltage-dependent Ca+ * channels and therefore do not release histamine on depolarization (Schwartz et al. , 1991). A third criterion for classification as a neurotransmitter is that its turnover be rapid and a mechanism for terminating its action be demonstrated. The turnover of histamine is indeed rapid and can be modified almost instantaneously (for reviews, see Hough, 1988; Schwartz etal., 1991). Central histamine is metabolized by histamine-N- methyltransferase (for review, see Schwartz et al., 1991) and the regional distribution of its metabolites is similar to the distribution of histamine (Hough and Domino, 1979; Hough, 1988). I Finally, in order to be classified as a neurotransmitter, a substance must act upon post-synaptic neurons and "reproduce in every way the specific effects of transmission resulting from stimulation of the presynaptic neurons (Erulkar, 1989)." The post- synaptic effects must be obtained at concentrations as low as are present after nerve stimulation and be blocked by competitive antagonists in a dose-dependent manner similar to that observed after neuronal transmission. An ability of histamine, like many other putative transmitters, to satisfy this requirement has not been demonstrated. For example, electrophysiological studies have revealed increased hypothalamic neuronal activity following microapplication of histamine (Haas, 1974; Haas and Wolf, 1977; Stehle, 1991); these effects often persist in low Ca“ media thereby indicating post- synaptic sites of action (Stehle, 1991). Furthermore, histamine-induced excitation of hypothalamic neurons can be blocked by histaminergic (H,) receptor antagonists (Haas and Wolf, 1977; Stehle, 1991). Correlations between this effect of exogenous histamine and the effects of stimulating histaminergic neurons have not, however, been investigated. 4 2. Anatomy of Histaminergic Neuronal System In 1974, Garbarg et a1. first suggested the existence of histaminergic neurons in the mammalian central nervous system. More specifically, the activity of the enzyme responsible for histamine synthesis, histidine decarboxylase, was decreased in several brain regions following lesion of the lateral hypothalamus and thereby a histaminergic neuronal system with diffuse projections was suggested. Subsequently, histamine- containing perikarya have been localized immunohistochemically in the posterior hypothalamus; specifically in the tuberal and caudal magnocellular nuclei (Watanabe et al., 1984) and the postmammillary caudal magnocellular nucleus (Takeda etal., 1984). Collectively, these nuclei have been referred to as the tuberomammillary nucleus (Kohler et al., 1985). Five distinct subgroups of histaminergic perikarya have been demonstrated in the tuberomammillary nucleus; these subgroups have been designated E,-E, (Inagaki et al., 1990; Figure 1.1). The E, and E, subgroups are located on the lateral surface of the mammillary body. The E, subgroup is found ventral to the mammillary body and the E4 subgroup is located dorsolateral to the mammillary recess. The E, subgroup consists of neurons scattered between the E, and E, subgroups. Retrograde tracing studies indicate that individual subgroups lack distinct projection fields (Ericson et al., 1987). Instead, individual histamine-containing brain regions receive input from several or all perikarya subgroups (Ericson et al., 1987; Panula et al., 1989; Schwartz et al., 1991). Accordingly, the E,-E, subgroups are commonly considered to be a single functional group (Staines et al., 1987; Inagaki et al., 1990; Tohyama et al., 1991). tc Vt Ve Rt Figure 1.1 Frontal sections through posterior hypothalamus of the rat illustrating the topographic localization of histaminergic perikarya and the subgroups E,-E,. 3V, third ventricle; DA, dorsal hypothalamic area; LH, lateral hypothalamic nucleus; VMH, ventromedial hypothalamic nucleus; Arc, arcuate nucleus; MM, medial mammillary nucleus; PM, premammillary nucleus; SuM, supramammillary nucleus; LM, lateral mammillary nucleus (modified from Wada et al., 1991). 6 From the tuberomammillary nucleus, two major ascending and one descending histaminergic pathways have been demonstrated (Panula et al., 1989; Figure 1.2). The first ascending pathway courses along the ventral and ventrolateral hypothalamus towards the horizontal limb of the diagonal band; some of these fibers continue dorsally towards the vertical limb of the diagonal band and medial septal nucleus while others continue rostrally towards the olfactory nuclei. The second ascending pathway courses along the lateral side of the third ventricle and branches towards rostral forebrain regions and the thalamus. Fibers of the descending histaminergic pathway project to the caudal medullary nuclei and spinal cord. The distribution of histaminergic fibers in the rat brain has been reviewed extensively (Inagaki et al., 1988a; Panula et al., 1989; Tohyama, et al., 1991). The cerebral cortex, caudate putamen, globus pallidus, amygdala, and nucleus accumbens are among the telencephalic brain regions to which histaminergic neurons project. The thalamus and hypothalamus are diencephalic regions which likewise contain histaminergic fibers. The mesencephalon contains few histaminergic fibers except in the central gray and trigeminal nucleus wherein moderate to high densities are found. The rhombencephalon similarly contains few histaminergic fibers. The anterior and intermediate lobes of the pituitary and the cerebellum are relatively devoid of histaminergic innervation. The hypothalamus contains the highest density of histaminergic fibers of any brain region (Inagaki et al., 1988a; Panula et al., 1989). In particular, the supraoptic, suprachiasmatic (SCN), paraventricular (PVN), arcuate (ARC) and ventromedial (VMH) Figure 1.2 Schematic representation of the distribution of histaminergic nerve fibers in rat brain. AH, anterior hypothalamic area; cc, corpus callosum; Cer, cerebellum; CG, central gray; CX, cortex; Hip, hippocampus; LS, lateral septum; MD, mediodorsal thalamus; OB, olfactory bulb; Pn, pontine nuclei; Sol, nucleus of solitary tract; sox, supraoptic decussation; VBD, nucleus of vertical limb of diagonal band; VMH, ventromedial hypothalamic nucleus (modified from Schwartz et al., 1991). 8 nuclei contain dense concentrations of histaminergic fibers (Panula et al., 1984; Panula eta1.,1989;Tohyamaetal.,1991). Histaminergicfiberscanbetraced from theinternal hyerofthemedianeminarcedrmughmeinfundibularsmlkmtheponenmpiunmry (Inagaki et al., 1988b). The periventricular (PeVN), dorsomedial (DMN) and medial preoptic nuclei (MPN) contain moderate to high densities of histaminergic fibers (Tohyama et al., 1991). Histaminergic neurons have 2-4 dendrites and often a few dendritic spines (I-Iayashi et al., 1984). In addition to histidine demrboxylase, histaminergic neurons purportedly contain other enzymes including glutamate decarboxylase and adenosine deaminase (Senba et a1. , 1985). Immunoreactivity against met-enkephalyl-Arg‘-Phe7 heptapeptide (Kohler et al. , 1985), galanin (thler et al., 1986) and substance P (Kohler et al., 1985) has been observed in the tuberomammillary nucleus and these substances may colocalize with histidine decarboxylase (Kahler et a1. 1986; Wooterlood and Groenewegen, 1985). 3. Biochemistry of Histaminergic Neurons Histamine does not readily cross the blood brain barrier, so any histamine found within the brain must be synthesized there. Synthesis of the amine involves two steps: transport of the amino acid precursor L-histidine into histaminergic neurons and decarboxylation of this amino acid by histidine decarboxylase (Schayer, 1966) to form histamine (Figure 1.3). Information pertaining to histidine transport is lacking; lesion of central histaminergic neurons does not alter histidine uptake (Hegstrand and Simon, 1985) and hence a specific histidine transport system in histaminergic neurons has not as yet been identified. A saturable, energy-dependent histidine uptake has, however, been demonstrated in synaptosomes (Chudomelka and Murrin, 1983; Hegstrand and HIS \ H3 (- l N I-tletldtne Decerboxyleee HIS é a HA——-> . HA Histamine-N-Methyltraneteraee-u» t-MeHA Monoamine Oxideae 8 RV Aldehyde Dehydrooeneee+ t-MelAA Figure 1. 3 Major synthetic and metabolic pathway of histamine in the brain; HA, histamine; HIS, L-histidine; t-MeHA, t-methylhistamine; t-MeIAA, t-methylimidazole acetic acid. , 10 Simon, 1985) and brain slices (Verdiere et al., 1975). In contrast, the physical properties of histidine decarboxylase have been studied extensively (for review, see Schwartz et a1. , 1991). Like other mammalian decarboxylases, histidine decarboxylase is a pyridoxyl-S ’-phosphate requiring enzyme found predominantly in the cytoplasm. The enzyme displays a high substrate specificity for histidine, with the only other naturally occurring substrate being 3-methylhistidine. 3-Methylhistidine is converted by histidine decarboxylase into t-methylhistamine (t-MeHA), a substance with little biological activity. Several studies suggest that histamine is packaged within synaptic vesicles (Kataoka and De Robertis, 1967; Snyder etal., 1974) which can be released in response to the arrival of nerve action potentials. H, receptors, autoreceptors located on histaminergic nerve terminals, modulate histamine release, at least in part, by modulating Ca” influx into nerve terminals (Arrang et al., 1985a; Arrang et al., 1985b). Heteroreceptors may also play a minor role in this regulation in that az-adrenergic agonists (Prast et al., 1991), muscarinic (Oishi et al., 1990a) agonists, x-opioid (Gulat- Mamay et al., 1990) agonists, and galanin (Arrang et al., 1991) have inhibitory effects on neuronal histamine release (see this Section, Part 4). The major metabolic pathway of central histamine is depicted in Figure 1.3. Histamine is metabolized in the brain by either of two pathways: chiefly by transmethylation to t-MeHA by histamine-N-methyltransferase or less commonly by oxidative deamination to imidazole acetaldehyde by diamine oxidase. t-MeHA is oxidized by monoamine oxidase (MAO) to t-methylimidazole acetaldehyde which, in turn, is further oxidized by aldehyde dehydrogenase to t—methylimidazole acetic acid (I- MeIAA). The metabolites of histamine have no reported physiological significance; 11 although the imidazole acetaldehyde is purportedly a GABA, agonist and when histamine-N-methyltransferase is inhibited may be of significance (Prell et al., 1991). Themeanhflf-fifeofneumnflhiMneinrflbrainisappmximatelmethhwam et al., 1991), although values of up to 50 min in the hypothalamus have been reported (Oishi et al., 1984). 4. Afl'erent Regulation of Histaminergic Neurons Whereas ' efferent projections of histaminergic neurons have been well documented, fewer studies have investigated afferent projections to brain regions containing these neurons. Noradrenergic and 5¢hydroxytryptaminergic projections to the tuberomammillary nucleus originating in the A,-A, and B,-B, cell groups (Dahlstrbm and Fuxe, 1964; see also this Chapter, Sections D and E), respectively, have been documented (Ericson et a1. , 1989). The former may influence histaminergic transmission since superfusion of the posterior hypothalamus of conscious, freely moving rats with either norepinephrine or the az-adrenergic agonist clonidine decreases histamine release (Prast et al., 1991). S-hydroxytryptaminergic neurons may likewise affect histaminergic neurons since histamine turnover in the whole mouse brain is inhibited by the S-HT,A agonists 8-hydroxy-2-(di-n-propylamino)tetralin and buspirone (Oishi etal., 1992). Histaminergic neurons in the hypothalamus receive synaptic contacts from substance P- (Tamiya et al., 1990) and neuropeptide Y- (Tamiya et al., 1989) containing neurons. Dopaminergic neurons likewise innervate the hypothalamus (see below) and may make synaptic contact with histaminergic neurons. Since neuronal histamine release 12 is stimulated and inhibited, respectively, by D, and D, receptor agonists (Prast et al., 1993), an interaction among dopaminergic and histaminergic neuronal systems is implied. Galanin, a peptide colocalized with histamine in the tuberomammillary nucleus (thler et al., 1986; Staines et al., 1986), inhibits potassium-evoked histamine release from rat hypothalamic slices (Arrang et al., 1991). M,-muscarinic (Oishi et al., 1990a) agonists and x-opioid (Gulat-Marnay et a1. , 1990) agonists inhibit histamine turnover and release, respectively. Effects of endogenous galanin, cholinergic, and opioids on histaminergic-neuronal activity can therefore be postulated. Using dual-labeling immunocytochemistry, fibers originating in the infralimbic region of the prefrontal cortex form varicosities, presumed to be axon terminals (Wouterlood and Groenewegen, 1985), are found in close approximation to histidine decarboxylase immunoreactive neurons in the posterior hypothalamus (Wouterlood et a1. , 1987). Projections from the septum-diagonal band region, MPN, olfactory tubercle and dorsal tegmental area to the posterior hypothalamus have likewise been identified (Wouterlood and Steinbusch, 1991). Because histaminergic neurons project to brain regions containing the cell bodies of these afferent projections, neuronal feedback loops can be postulated. The identities of neurotransmitter(s) contained within these afferent projections neurons have not, however, been established nor have neuronal feedback loops involving histamine been demonstrated. 13 5. Histaminergic Receptors: Localization, Agonists and Amaganists a. H, Receptors Histaminergic receptors have been classified into three major classes: H,, H, and H,. H, receptors, post-synaptic receptors long recognized for mediating smooth muscle contraction and capillary permeability, are distributed widely throughout the brain. A detailed autoradiographic mapping of H, receptors in the rat brain using the radiolabeled H, antagonist [3H]mepyramine has been described (Palacios et al., 1981). Highest concentrations of H, receptors are found in the bed nucleus of the stria terminalis, the nucleus tractus solitarii, and the pontine nuclei. The hypothalamus likewise contains high concentrations of H, receptors, particularly in the supraoptic, SCN, VMH and premammillary nuclei. The MPN, PeVN and PVN contain moderate, whereas the ARC and zona incerta contain low levels of H, receptors. Other brain regions containing moderate concentrations of H, receptors include the hippocampus, amygdala, periventricular nucleus of the thalamus, raphe dorsalis and locus coeruleus. Most brain regions which contain significant concentrations of H, receptors also receive histaminergic projections. There are, however, exceptions to this rule. For example, the hippocampal formation, although populated with H, receptors, is relatively devoid of histaminergic innervation as evidenced by a lack of histidine decarboxylase activity (Palacios et al., 1981). ”Mismatching" among neurotransmitter and receptor localization such as this has been demonstrated for other systems including the catecholaminergic, Sohydroxytryptaminergic and cholinergic neuronal systems (Herkenham, 1987). There are no highly selective agonists for the H, receptor. In contrast, numerous 14 H, antagonists havebeendeveloped, manyforuseinthetreatmentofallergiesormotion sickness. Many of these antagonists, including the 'classic antihistamines“ diphenhydramine, promethazine, and chlorpheniramine, possess marked anticholinergic and, at high concentrations, local anesthetic properties. Mepyramine (also known as pyrilamine; Ash and Schild, 1966; Haley, 1983) is the most common H, antagonist employed in experimental studies of cenual histamine since it readily crosses the blood brain barrier and is relatively devoid of anticholinergic and membrane stabilizing properties (Garrison, 1990). Mepyramine is highly selective and possesses a high affinity (K, 0.8 nM) for the H, receptor (for review, see Haaksma et al., 1990; Hill et al., 1990; Hill et al., 1992). b. H, Receptors H, receptors are post-synaptic receptors and are distributed heterogeneously throughout the brain. The cerebral cortex, caudate putamen, nucleus accumbens and hippocampus are among brain regions most populated with H, receptors. The substantia nigra and superior colliculus also contain H, receptors. H, receptors are few in numbers in most other brain regions including the hypothalamus and thalamus. The pituitary is nearly devoid of H, receptors (Schwartz et al., 1991). Several H, receptor agonists have been developed; these include dimaprit and 4- methylhistamine (for review, see Haaksma et al., 1990; Hill et al., 1990). These agents have received little attention as they have little therapeutic value. In contrast, considerable attention has been directed towards developing H, antagonists since these agents are useful in treatment of peptic ulcers. Ranitidine and cimetidine are among the 15 mostpotcntandcommonlyemployedEantagonists. Thesecommundsare, however, offimiwdvalueinmrdyingcarnaIMmminagicsynemsnncendmawmpoundreadfly paretrates the blood brain barrier. Zolantidinc is aunique and important compound in matitismeonlysdecdveandpomntmantagonistwhichreadilycrossesthebloodbrain barrier (Calcuttetal., 1988; HoughetaL, 1988). c. H, Receptors H, receptors are distributed heterogeneously among brain regions known to receive histaminergic projections. A detailed autoradiographic mapping of H, receptors in the rat brain using the radiolabeled H, agonist [’H](R)a-methylhistamine (Arrang et al., 1987a; Pollard et al., 1993) reveals that H, receptors are densely concentrated in the cerebral cortex, nucleus accumbens, globus pallidus and striatum. Moderate densities ofH,receptorsarefoundinthetha1amusandsubstantianigra. FewH,receptorsare present in the cerebellum, pons or medulla. H, receptors are found in moderate densities in the hypothalamus, the region in which highest densities of histaminergic fibers are found. Slightly higher concentrations are found in the anterior and medial regions than in the- remainder of the hypothalamus. H, receptors are located near the tuberomammillary nucleus and hence may affect histaminergic perikarya (Pollard et al., 1993). Regional differences between the distribution of H, receptors and histaminergic fibers have been demonstrated. Pollard et a1. (1993), for example, reported ”a significant decreasing rostral-caudal gradient of H, receptor density from the frontal and insular cortex, to the occipital and entorhinal cortex whereas L-histidine decarboxylase 3F 16 activity, a marker of histaminergic axons, was distributed with an opposite increasing gradient in these areas.” Moreover, the density ’of H, fibers in the thalamus is high relativetothedensityofhistaminergicfibers. DatasuchastheseindieatethatH, receptors may not only affect histamine, but also other types of neurons. A role for H, receptors as presynaptic heteroreceptors affecting norepinephrine and $- hydroxytryptamine (SHT) release in vitro has been postulated (Schlicker et a1. , 1989; Fink et a1. 1990). The effects of H, agonists and antagonists on noradrenergic and 5- hydroxytryptaminergic neuronal activity will be discussed in Chapters III and IV. R-a—methylhistamine (Anang et al., 1987a) is only H, agonist commonly employed in the study of histaminergic neurons, although other H, agonists including BP 2.94 (BIOPROJET, Paris) are currently being evaluated. Burimamide, developed originally as an H, antagonist, is a highly potent H, antagonist. Thioperamide (Arrang et a1. , 1987a) is, however, the H, antagonist employed most frequently because of its high selectivity and affinity (K, 4 nM) for the H, receptor. Both R-a-methylhistamine and thioperamide readily cross the blood brain barrier (for review, see Haaksma et al., 1990; Hill et al., 1990; Hill et al., 1992). 6. Strategies for Assessing Role of Histaminergic Neurons Although the presence of histaminergic neurons in the brain has been recognized since the 1970’s (Garbarg et al., 1974), the effects in viva of these neurons on the activity of other central aminergic neuronal systems has remained unknown. This lack of information was originally attributable to a lack of pharmacological agents which specifically affect histaminergic transmission. The recent development of 17 l l'lagents 1., '5 fly ",fi'l'tateorl' 1' . . uannnissionhasmadedresurdyoftherespmseofaminergicnanonalsystemsm histamine now possible; the use of some of these agents is included in the discussion below. a. Pharmacological Strategies for Mimicking or Facilitating Histaminergic Neuronal Transmission Three pharmacological strategies for mimicking or facilitating histaminergic transmission include administration of histamine per se, the H, receptor antagonist thioperamide (Arrang et al., 1987a), or the histamine-N-methyltransferase inhibitor metoprine (Duch et al., 1978; Hough et al., 1986). Of these, central administration of histamine has been the most commonly employed; since histamine does not readily cross the blood brain barrier, it must be administered via intracerebral (i.c.) or intracerebroventricular (i.c.v.) injection. Administration of the H, receptor antagonist thioperamide has been used to increase central histamine synthesis (Arrang et al. , 1987b) and release (Arrang et al., 1985a). Finally, administration of metoprine has been used to block histamine metabolism and thereby effect long lasting increases in central histamine concentrations (Duch et al., 1978; Hough et al., 1986). The sites of action of thioperamide and metoprine are depicted in Figure 1.4; effects of these agents will be described in greater detail in Chapters III, IV and V. 18 - R-ot-Methylhistamlne - H3 Thio eramide “M HIS X > HA——> /. HA - Metoprine >< Mepyramine Zolantidinc V __>.—4 \V/ t-MQHA H1 H2 Figure 1.4 Schematic representation of the sites of action of the histidine decarboxylase inhibitor aFMH, the H, agonist R-a-methylhistamine, the H, antagonist mepyramine, the histamine-N-methyltransferase inhibitor metoprine, the H, antagonist thioperamide and the H, antagonist zolantidine. 19 b. Pharmacological Strategies for Disrupting Histaminergic Neuronal Transmission Pharmacological strategies for disrupting histaminergic transmission include: administration of the irreversible histidine decarboxylase inhibitor a-fluoromethylhistidine (aFMH; Kollonitsch et al., 1978; Garbarg et al., 1980), the H, agonist R-a- methylhistamine (Arrang et al., 1987a), the H, antagonist mepyramine (Ash and Schild, 1966; Haley, 1983), and the H, antagonist zolantidine (Calcutt et al., 1988; Hough et al., 1988). Administration of aFMH blocks neuronal histamine synthesis and thereby decreases histamine release (Cumming et al., 1991; Itoh et al., 1991; Mochizuki et al., 1991). Administration of R-a-methylhistamine acts, via presynaptic H, receptors, to decrease histamine synthesis (Arrang et al., 1987b) and release (Arrang et al., 1985b; 1987b). The actions of aFMH and R-a-methylhistamine, and of the post-synaptic antagonists mepyramine and zolantidine are schematically depicted in Figure 1.4; effects of these agents will be described in greater detail in Chapters III, IV and V. c. Stress as 0 Physiological Strategy for Increasing Histaminergic Neuronal Activity An array of stressful procedures of short duration increase central histamine con- centrations (Campos and Jurupe, 1970; Mazurkiewicz-Kwilecki and Taub, 1978; Mazur- kiewicz-Kwilecki, 1980; Mazurkiewicz-Kwilecki and Bielkiewicz, 1982; Gillich and Meaney, 1992). Moreover, histamine has been implicated in several endocrine responses to stress including increased secretion of prolactin (Alvarez, 1982; Knigge et al., 1988a; soc-Jensen, 1993), aMSH (Knigge et al., 1991) and renin (Matzen et al., 1990). Stress- induced antinociception (Oluyomi et al., 1993) likewise may be mediated by central histamine. The importance of stress-induced increases in histaminergic neuronal activity 20 will be discussed in detail in Chapters III, IV and V. C. Non-Neuronal Stores of Histamine in Brain Not all histamine in the central nervous system is neural in origin. Mast cells, connective tissue elements which are characterized by metachromatic staining, histamine content, and sensitivity to histamine releasers, can be found centrally (for review, see Ibrahim, 1974; Dropp, 1979; Hough, 1988; Schwartz et al., 1991). Outside of the central nervous system, these cells represent major sites of histamine storage and release; exposure to antigens, drugs, or less commonly, neurotransmitters/neuromodulators (catecholamines, adenosine, substance P) increase histamine release. Within the central nervous system, the physiological significance of mast cells is unknown. Until the 1970’s, the brain was believed to be devoid of these cells. Mast cells are scarce, except in areas lacking the blood brain barrier, in several species including humans. In the rat, central mast cells have been identified primarily within the thalamus, and to a lesser degree, in the cortex, basal ganglia and median eminence (Goldschmidt et al., 1984; Hough et al., 1985; Pollard et al.. 1976). No function for mast cell histamine has been demonstrated. In addition to mast cells. cerebrovascular endothelial cells contain histamine and may contribute to the non~neuronal central histamine pool (Robinson-White and Beaven, 1982). Histamine has been found within vascular channels in the brain; the cellular origin of this histamine is unknown (Hough, 1988). 21 D. Catecholaminergic Neuronal Systems 1. Anatomy of Catecholaminergic Neuronal systems Catecholaminergic neurons have been classified according to the alpha-numeric system of Dahlstrdm and Fuxe (1964; Figure 1.5). Noradrenergic neurons located caudally in the medulla and pans are identified as A,-A-,; these neurons project diffusely throughout the brain and spinal cord. Dopaminergic neurons located more rostrally are identified as A,-A,,; these neurons are organized into several discrete anatomical systems. a. Anatomy quDaparninergic Neuronal Systems Dopaminergic neurons originating in the pars compacta of the substantia nigra (A, and A,) and in the ventral tegmental area (A,,) give rise to the mesotelencephalic system. This major ascending dapaminergic system has been subdivided into three systems: nigrostriatal, mesolimbic and mesocortical. Nigrostriatal dopaminergic neurons originate in the substantia nigra and project to the caudate putamen. Mesolimbic dopaminergic neurons originate in the ventral tegmental area and project to a number of subcortical regions including the nucleus accumbens, olfactory tubercle and septum. Mesocortical dopaminergic neurons project to the cingulate, entorhinal, prefrontal and pyriform cerebral cortices. The diencephalon contains four distinct groups of dopaminergic cell bodies (A,,, A,,, A,, and A,,) from which project dopaminergic neurons including those of the tuberoinfundibular, periventricular-hypophysial, and incertohypothalamic systems. Tubcroinfundibular dopaminergic neurons, with cell bodies (the A,, cell group) in the ARC, project to the external layer of the median eminence (Bjorklund et a1. , 1973); these Cerebellum ; Dopamine : Figure 1.5 Schematic representation of the distribution of catecholamine-containing perikarya in rat brain. CC, corpus callosum; HIP, hippocampus; ST, striatum; Norepi., norepinephrine (from Moore, 1987). 23 neurons tonically inhibit the secretion of prolactin from the anterior pituitary (see this Chapter, Part 4). Periventricular-hypophysial dopaminergic neurons have cell bodies (the A,, cell group) in the caudal periventricular nucleus (cPeVN) and terminate in the intermediate lobe of the pituitary (Goudreau et a1. , 1992); these neurons tonically inhibit the secretion pro—opiomelanocortin-derived hormones such as aMSH (see this Section, Part 4); Millington and Chronwall, 1988). Incertohypothalamic dopaminergic neurons originate in the A,, and A,4 cell groups (Bjdrklund et al., 1975). The caudal portion of this system has perikarya (the A,, cell group), dendrites and terminals in the medial zona incerta (M21) and DMN (Bjorklund and Nobin, 1973). Rostral incertohypothalamic neurons originate in the A,, cell group and project to several brain regions including the PeVN, MPN and suprachiasmatic preoptic nucleus. In addition to the tuberoinfundibular, periventricular-hypophysial and incertohypothalamic systems, the hypothalamus contains many dopaminergic neurons arising from or terminating in the PeVN, MPN, SCN, and PVN whose origin and projections are as yet undefined (Palkovits, 1981). b. Anatomy of Noradrenergic Neuronal Systems As mentioned above, noradrenergic neurons located in the medulla and pons are identified as A,-A,. These neurons project diffusely throughout the brain and spinal cord. The A, noradrenergic group is found in the lateral reticular nucleus of the medulla oblongata. The A,, A, and A, groups are found in or near the nucleus of the solitary tract, the pans and the mesencephalic reticular formation, respectively. The locus coeruleus contains noradrenergic cell bodies of the A, group. dI 24 The projections of noradrenergic neurons in the brain have been reviewed extensively (Moore and Bloom, 1979; Palkovits, 1981). Of particular importance to this dissertation (Chapter III) are noradrenergic neurons projecting to the hypothalamus; neurons originating primarily in the brainstem catecholaminergic cell groups of the lateral tegmentum. The ventral noradrenergic bundle, originating primarily in the A, cell group, constitutes the major noradrenergic input to the hypothalamus. Noradrenergic neurons of the periventricular bundles, originating in the locus coeruleus, provide relatively minor. inputs to the PVN, DMN and PeVN (Moore and Bloom, 1979; Palkovits, 1981). 2. Biochemistry of Catecholaminergic Neurons 0. Biochemistry of Dopaminergic Neurons Information pertaining to the synthesis, storage, release and metabolism of dopamine were obtained originally from studies of the nigrostriatal dopaminergic neuronal system. The neurochemical events occurring within nigrostriatal dopaminergic nerve terminals are depicted in the upper portion of Figure 1.6. The amino acid precursor L-tyrosine is actively transported into dopaminergic nerve terminals and then converted by tyrosine hydroxylase (TYR-H) to 3,4-dihydroxyphenylalanine (DOPA). This conversion by TYR-H is the rate-limiting step in dopamine synthesis. TYR-H is regulated, at least in part, by end product inhibition such that increases in cytoplasmic dopamine coneentrations decrease dopamine synthesis. DOPA is rapidly decarboxylated by L-aromatic amino acid decarboxylase (AAD) to form dopamine. Newly synthesized dopamine can then be stored within synaptic vesicles for subsequent release. / """"" ‘~ - Posrsmrnc ceu. TYROSINE '-¢L.+’DOPA —' DA] 9007 on oeuoam: l “ rrnosms Dom ' CK ..:\ l I """" “‘d erooo rraosma —‘=I— DOPA ——-DA DA 0‘] Wrfi Figure 1.6 Schematic representation of the neurochemical events occurring within nigrostriatal (upper panel) and tuberoinfundibular dopaminergic neurons (lower panel). COMT, catecholamine-Gmethyltransferase; DOPA,- 3,4-dihydroxyphenyla1anine; DOPAC, 3,4-dihydroxyphenylacetic acid; DA, dopamine; HVA, homovanillic acid; 3- MT, 3—methyltyrosine; MAO, monoamine oxidase (from Moore, 1987). d: 26 Dopamine that has been newly released from nigrostriatal dopaminergic neurons can: 1) activate dopaminergic receptors on post—synaptic neurons, 2) activate presynaptic autoreceptors and thereby inhibit further dopamine synthesis and release, or 3) be removed from the synaptic cleft chiefly by a high affinity active transport uptake mechanism located on the dopaminergic nerve terminal. Once inside the nerve terminal, dopamine can be repackaged within vesicles for re-release or oxidatively deaminated by mitochondrial MAO to form 3,4—dihydroxyphenylacetic acid (DOPAC). DOPAC diffuses from the neuron and metabolized by catecholamine-O-methylnansferase (COMT) to form homovanillic acid (HVA). A relatively small proportion of extraneuronal dopamine escapes reuptake and does not affect pre- or post-synaptic receptors but instead is oxidatively deaminated by extraneuronal MAO to form DOPAC or directly methylated by COMT to form 3-methoxytyramine (3MT). 3MT is oxidized to form HVA. The neurochemical events described above for nigrostriatal dopaminergic neurons presumably occur in most dopaminergic neuronal systems including those comprising the mesolimbic and incertohypothalamic systems. Neurochemical events associated with periventricular-hypophysial dopaminergic neurons likewise resemble those of the nigrostriatal system except that the uptake system on nerve terminals in the posterior pituitary has a lower affinity for dopamine than does that in the striatum (Demarest and Moore, 1979a). Tuberoinfundibular dopaminergic neurons likewise differ from nigrostriatal dopaminergic neurons. As depicted in the lower portion of Figure 1.6, tuberoinfundibular dopaminergic neurons lack autoreceptors for regulating dopamine synthesis (Demarest and Moore, 1979b). Furthermore, the uptake system on tuberoinfundibular dopaminergic nerve terminals has a lower affinity for dopamine than does that of nigrostriatal dopaminergic neurons (Demarest and Moore, 1979a; Annunziato 27 et al., 1980). Finally, tuberoinfundibular dopaminergic neurons do not form classical synapses but instead release dopamine into capillaries of the hypophysial portal blood system; dopamine released into this system tonically inhibits the secretion of prolactin from the anterior lobe of the pituitary. b. Biochemistry of Noradrenergic Neurons Neurochemical events occurring within noradrenergic nerve terminals are similar to those occurring within dopaminergic neurons (Figure 1.7). The amino acid precursor L-tyrosine in taken up by the neurons and converted to dopamine in reactions catalyzed by TYR-H and AAD. Once formed, dopamine is transported into synaptic vesicles and converted by dopamine-B-hydroxylase into norepinephrine. Upon the arrival of an action potential, newly synthesized norepinephrine can be released. Like dopamine, norepinephrine released into the synaptic cleft can: 1) activate post-synaptic receptors, 2) activate presynaptic autoreceptors and thereby inhibit its own release, or 3) be removed from the synaptic cleft chiefly by a high affinity active transport uptake mechanism located on the noradrenergic nerve terminal. Once inside the nerve terminal, norepinephrine can be repackaged within vesicles for re-release or oxidatively deaminated by MAO to form 3,4odihydroxyphenylglycol (DOPEG). DOPEG diffuses from synapses and is subsequently converted by COMT to form 3-methoxy-4- hydroxyphenylethyleneglycol (MHPG). A small proportion of extraneuronal norepinephrine escapes reuptake and is methylated by COMT to form normetanephrine, which is further oxidized by MAO to form MHPG. In the rat, MHPG is present in the brain primarily as a sulfonated conjugate (Schanberg et al., 1968). TYF Figure noradre ”Ethyl: acetic hydroxl TYR, t} 28 f" TYR-H AAD DBH TYFl —> DOPAéDA-fi NE NE \uao - V J com DOPAC cou\ ftao MHPG Figure 1.7 Schematic representation of the neurochemical events occurring within noradrenergic neurons. AAD. amino acid decarboxylase; COMT, catecholamineO- methyltransferase; DOPA, 3,4.dihydroxyphenylalanine; DOPAC, 3,4-dihydroxyphenyl- acetic acid; DA, dopamine; DBH, dopamine-B-hydroxylase; 3-methoxy-4- hydroxyphenylethyleneglycol, MHPG; MAO, monoamine oxidase; NE norepinephrine; TYR, tyrosine; TYR-H, tyrosine hydroxylase 29 When noradrenergic neurons are activated, transport of dopamine into the synaptic vesicle and/or the conversion of dopamine to norepinephrine by dopamine-B-hydroxylase become rate-limiting steps so that dopamine can accumulate within the nerve terminals. This unpackaged and therefore unprotected dopamine can be converted by MAO to form DOPAC. The significance of this phenomena is described below (Part 3b). 3. Neurochemical Techniques to Estimate Catecholaminergic Neuronal Acn‘vity Neurochemical estimates of catecholaminergic neuronal activity are based upon the observation that within axon terminals of catecholaminergic neurons, steady state levels of neurotransmitter are maintained during varying rates of neuronal activity by a tight coupling among dopamine synthesis, release and metabolism. Hence, change in either synthesis, release or metabolism can be quantified and used to estimate neuronal activity. Techniques based upon this principle have been employed in this dissertation and are described below. a. DOPA Accumulation as an Index of Dopaminergic Neuronal Activity As mentioned above, the rate of dopamine synthesis is regulated by TYR-H. The activity of this enzyme can be estimated indirectly by measuring the rate of accumulation of DOPA (the product of the reaction catalyzed by TYR-l-l) following administration of an inhibitor of AAD, such as 3-hydroxybenzylhydrazine (NSD 1015 ; Carlsson et al., 1972). Procedures that increase or decrease the activity of dopaminergic neurons produce corresponding changes in the accumulation of DOPA which has been used frequently to estimate dopaminergic neuronal activity. One limitation of this technique ne no Del do] esti ”Or; 30 is that DOPA is a precursor for both dopamine and norepinephrine so that upon inhibition of AAD, DOPA accumulates in both dopaminergic and noradrenergic nerve terminals. Hence DOPA accumulation is a selective index of dopaminergic neuronal activity only in brain regions where the density of dopaminergic neurons exceeds that of noradrenergic neurons. b. Concentrations of DOPAC as Indices of Dopaminergic and Noradrenergic Neuronal Activity _ Procedures which increase or decrease the activity of dopaminergic neurons produce corresponding changes in dopamine metabolism. Hence DOPAC concentrations are useful indices of dopaminergic neuronal activity in brain regions containing perikarya or terminals of these neurons (Roth et al., 1976; Lookingland et al., 1985; Moore, 1987). As mentioned above, many brain regions are innervated by both dapaminergic and noradrenergic neurons. Procedures which increase the activity of noradrenergic neurons increase DOPAC concentrations since dopamine is a precursor for norepinephrine and can be metabolized to DOPAC in noradrenergic neurons (Curet et al., 1985; Scatton et al., 1984). Hence, the contribution of DOPAC from noradrenergic neurons is a major consideration when utilizing DOPAC concentrations to estimate dopaminergic neuronal activity in norepinephrine-rich nuclei (Tian et al. , 1991). The contribution of dopaminergic neurons to changes in DOPAC concentrations can be estimated by measuring concentrations of DOPAC in brain regions to which noradrenergic innervation has been destroyed by neurotoxin lesion (see Chapter III). nc Cl 00 31 c. Concentrations of MHPG as Indices of Noradrenergic Neuronal Activity Experimental manipulations which increase or decrease the activity of noradrenergic neurons produce corresponding changes in MHPG concentrations, and this measure has been shown to be a reliable index of noradrenergic neuronal activity (Artigas et al., 1986; Lookingland et al., 1991b). 4. Dopaminergic Regulation of Prolactin and 0211133 Secretion a. Prolactin . . Tuberoinfundibular dopaminergic neurons, which originate in the A,, cell body group and project ventrally to terminate in the external layer of the median eminence, regulate the secretion of prolactin. Dopamine released from tuberoinfundibular dopaminergic neurons is transported via the hypophysial portal blood system to the anterior pituitary where it tonically inhibits the secretion of prolactin from lactotrophs. Other neurotransmitters including histamine and SHT have been implicated in the regulation of prolactin secretion; these will be discussed in Chapter V. b. a-MSH Periventricular-hypophysial dopaminergic neurons, with perikarya (the A“ cell group; Dahlstrém and Fuxe, 1964) in the caudal periventricular hypothalamic nucleus and terminals in the intermediate lobe of the pituitary (Goudreau et al., 1992), tonically inhibit the secretion of aMSH (Millington and Chronwall, 1988). Periventricular- hypophysial dopaminergic neurons are not, however, the sole regulators of aMSH secretion; epinephrine, released from the adrenal medulla, activates melanotroph B,- E. S-E 1. Ant numcr (Class: these: ObSCL t0 {ht mesa and' Don: addi desc few 32 adrenergicreceptorsintheintermediatelobethereby stimulatingaMSHsecretion(Cote et al., 1982; Kvetnansky et al., 1987; Lindley et al, 1990). Other neurotransmitters including histamine (Knigge et al., 1991) and SHT (Goudreau et al., 1993) have been implicated in the regulation of MR secretion; the role of histamine in regulating aMSHsecretionwillbediscussedinChaptersIIIandV. E. S-Hydroxytryptaminergic Neuronal Systems 1. Anatomy of 5-Hydroxytryptaminergic Neuronal Systems S-Hydroxytryptaminergic neurons have been classified according to the alpha- numeric system of Dahlstrém and Fuxe (1964; Figure 1.8). Perikarya of these neurons (classified B,-B,) are found predominantly in the raphe nuclei of the medulla, pans and mesencephalon. The B, and B, nuclei, located near the raphe pallidus and raphe obscurus, are found within the medulla. The B, and B, nuclei, corresponding roughly to the raphe magnus and raphe pontis, are found in or just caudal to the pans. The mesencephalon contains the B, and B. cell groups; the raphe dorsalis corresponds to B, and the raphe medianus and linearis correspond to B.. The 3., B. and B, nuclei are relatively small nuclei found outside of the raphe, B. and B. nuclei are found immediately ventral to the 4th ventricle in the medulla and pons, while the B, nucleus is found in the mesencephalon near the medial lemniscus. In addition, SHT-containing perikarya in the dorsomedial hypothalamic nucleus have been described (Frankfurt and Azmitia, 1983). The projections of S-hydroxytryptaminergic neurons in the brain have been reviewed extensively (Azmitia and Segal, 1978; Steinbush, 1984). Of particular 1:lgure El bra tannin lass: 33 Cingulum Cerebral cortex Cerebellum ‘3 0 av l. :3.- EX‘. 1 e ‘\ capsule ' $~. ’(//{ ‘g‘: \\ N. caudate .N A j' ’88 ill 36 34. \~ Format Sula. 89 BS 33 a: W "F3 9,0\ 81 o.\ , ,' We Med. long fasc. Figure 1.8 Schematic representation of the distribution of SI-lT-containing perikarya in rat brain. Ext. capsule, external capsule; N. caudate, nucleus caudate; stria. term. , stria terminalis; MFB, medial forebrain bundle; med. long fasc. , medial longitudinal fasciculus (from Cooper et al., 1991). hnpo. Incdkr pane: nigra, llydro region: bodies hydror will b: 2. Bio are d trans; by 1h: is um enZyr Itaso brain thfi Si 34 impomncemthisdissemfionareprojecfimuodginafinginmemphedorsahsand medianis (B, and B.). S-Hydroxytryptaminergic neurons originating in the raphe dorsalis project to numerous brain regions including the striatum, globus pallidus, substantia nigra, septum, hippocampus, amygdala, nucleus accumbens and hypothalamus. 5- Hydroxytryptarninergic neurons originating in the raphe medialis project to several brain regions including the olfactory bulb, septum, hippocampus, thalamus, mammillary bodies, frontal cortex and hypothalamus. The influence of histamine on 5- hydroxytryptaminergic neurons projecting to the nucleus accumbens and hypothalamus will be discussed in Chapter IV. 2. Biochemistry of 5 -Hydroxytryptaminergic Neurons Neurochemical events occurring within S-hydroxytryptaminergic nerve terminals are depicted in Figure 1.9. The amino acid precursor L-tryptophan is actively transported into the neurons and subsequently converted to 5-hydroxytryptophan (5111?) by the rate-limiting enzyme tryptophan hydroxylase (TRP-I-I). Tryptophan hydroxylase is unique in that, unlike many amino acid hydrolases (i.e., TYR-I-I), the activity of this enzyme is regulated not by end product inhibition but by substrate availability. For this reason, parenteral administration of L-tryptophan can increase SHT concentrations in the brain (Femstrom and Wurtman, 1971). SHTP is rapidly decarboxylated by AAD to form SHT. Newly synthesized SHT can then be stored within synaptic vesicles for subsequent release. Once released into the synaptic cleft, SHT can: 1) activate post-synaptic receptors, 2) activate presynaptic TR; Figure hydrox TRP-r hydro: 35 5HIAA use v r1 TRP-95HTP-95HT @‘ 5HT rap-n AAD V Figure 1.9 Schematic representation of the neurochemical events occurring within 5- hydroxytryptaminergic neurons. AAD, amino acid decarboxylase; TRP, tryptophan; TRP—H, tryptophan hydroxylase; SHIAA, 5—hydroxyindoleacetic acid; SHTP, 5- hydroxytryptophan; SHT, S-hydroxytryptamine; MAO, monoamine oxidase LOT tap nyd Aka hox ydrc n It tow .Slil Ianno 36 autoreceptors and thereby inhibit further SHT release, or 3) be removed from the synaptic cleft by a high affinity uptake system. Intraneuronal SHT can be packaged for re-release or metabolized by mitochondrial MAO and aldehyde dehydrogenase to form S-hydroxyindoleacetic acid (SHIAA; Fernstrom, 1983). 3. Neurochemical Techniques to Estimate 5-Hydroxytryptaminergic Neuronal Activity Experimental manipulations, such as electrical stimulation of 5- hydroxytryptaminergic cell bodies in the rnidbrain raphe, which increase the activity of S-hydroxytryptaminergic neurons, decrease SHT and increase SHIAA concentrations in brain regions which contain terminals of these neurons (Aghajanian et al., 1967; Kostowski et al., 1969; Sheard and Aghajanian, 1968). Hence, concentrations of SHT and SHIAA are frequently used as indices of 5-hydroxytryptaminergic neuronal activity (Shannon et al., 1986; Lookingland et al., 1988). A. An “why“ Rum Lilly ; 1013- 11. MATERIALS AND METHODS A. Animals Male Long-Evans rats (200-225 g) were obtained from Harlan Breeding Laboratories (Indianapolis, IN) and maintained under conditions of controlled temperature (21 j; 1°C).and lighting (illumination 0.600—20.00 h) with food (Purina Rodent Chow) and water provided ad libitum. B. Drugs Histamine dihydrochloride (Sigma Chemical Co., St. Louis, MO), a-fluoro- methylhistidine (kindly provided by Dr. Michael H. Fisher, Merck, Sharp and Dohme, Rahway, NJ), fluoxetine hydrochloride (kindly provided by Dr. William R. Fields, Eli Lilly and Company, Indianapolis, IN), 3-hydroxybenzylhydrazine dihydrochloride (NSD 1015; Sigma Chemical Co.), mepyramine maleate (Research Biochemicals Inc., Natick, MA), thioperamide maleate (Research Biochemicals Inc.), and zolantidine dimaleate (kindly provided by Dr. Derek Hills, SmithKline & Beecham, Welwyn, England) were dissolved in 0.9% saline. R-a-methylhistamine (ldndly provided by Drs. LC. Schwartz and Anai's Krikorian, BIOPROJ ET, Paris, France) was dissolved in distilled water. Metoprine (kindly provided by Dr. Anthony Dren, Burroughs Wellcome Research Laboratories, Research Triangle Park, NC) and BP 2.94 (kindly provided by Drs. LC. 37 9.72 g' 443 m 120 mi distiller C. Inlr i'P-l am the inci: Siainlesg this Can Cement. dihydro‘ aVOIUm steel inj. VCnmcle diSSCCiin‘ ventricle 38 Schwartz and Anais Krikorian, BIOPROJET) were suspended in 1% sodium carboxymethylcellulose (Sigma Chemical Co.). 6-Hydroxydopamine hydrobromide (6- OHDA; Sigma) was dissolved in 0.9% saline containing 0.1% ascorbic acid and kept on ice. All compounds were administered as indicated in the legends of appropriate figures. The anesthetic Equithesin was prepared by combining 42.51 g chloral hydrate, 9.72 g sodium pentobarbital (Sigma Chemical Co.) and 21.26 g magnesium sulfate in 443 ml warm propylene glycol. After these compounds were dissolved completely, 120 ml of 95 % ethanol was added and the total volume was brought to l L with double distilled water. C. Intracerebroventricular Iniectiom Animals receiving i.c.v. injections were anesthetized with Equithesin (3 ml/kg, i.p.) and positioned in a stereotaxic frame (David Kopf Instruments, Tujunga, CA) with the incisor bar set 2.4 mm below the horizontal plane. A 10 mm length of 23 gauge stainless-steel tubing was implanted 1.4 mm lateral to bregma and 3.2 mm below dura; this cannula guide was anchored to the skull with stainless steel screws and dental cement. Four to 7 days following implantation of cannula guides, histamine dihydrochloride or its saline vehicle was injected into conscious, freely moving rats in a volume of 3 ul from a 10 ul Hamilton microsyringe connected to a 30 gauge stainless- steel injector which protruded 1 mm beyond the tip of the cannula guide into a lateral ventricle. Cannula placement was verified postmortem in frozen brain sections using a dissecting microscope and only those animals with cannula tracts ending in the lateral ventricle were included in the study. D.N ancst'"; the in" String from U Klippe (0.3 pl for an tract. 39 D. Neurochemical Lesion of the Ventral Noradrenergic Bundle Animals receiving i.c. injections of 6—OHDA or its ascorbic acid vehicle were anesthetized with Equithesin (3 nil/kg, i.p.) and positioned in a stereotaxic frame with the incisor bar set 3.0 mm below the horizontal plane. The needle of a 5 ul Hamilton syringe was inserted into the ventral noradrenergic bundle at coordinates A: 0.0 mm from the intraural line, L: :1: 1.3 mm from midline, V: -7.0 mm from dura (KOnig and Klippel, 1963) and bilateral injections of either 6-OHDA (8 pg free base/side) or vehicle (0.3 ullside) weremadeoveras minperiod. Thesyringeneedleremainedin thebrain for an additional 10 min after injection to reduce reflux of neurotoxin into the needle tract. E. Restraint Stress Physical restraint within acrylic cylindrical tubes (64 mm diameter) was employed in experiments designed to examine the role of histaminergic neurons in effecting stress- induced changes in aminergic neuronal activity and pituitary hormone secretion. Rats were housed 2 per cage. Rats subjected to restraint stress were removed from their home cage environment, transferred to an adjacent room and placed within acrylic cylindrical tubes (64 mm diameter) for 10, 20 or 30 min prior to decapitation. Nonstressed controls were removed from their home cage environment, transferred to an adjacent room, and decapitated immediately. F. T" remm some t and so was co section Prepare these 5: Paikovit describe 30-100 stored 3 C811 D11 PTOIEin 40 F. Tissue Dissection and Neurochemical Assays Following appropriate treatments, animals were decapitated and their brains removed from the skull and frozen on aluminum foil placed directly over dry ice. In some experiments, trunk blood was collected in glass tubes containing bacitracin (150 pg) and sodium heparin (143 USP units). After centrifugation (2000 rpm; 20 min), plasma was collected and stored at -20"C until assayed for prolactin and aMSH. Frontal brain sections (600 pm) beginning at approximately A9220 (Kt‘mig and Klippel, 1963) were prepared in a cryostat (-10°C) and the appropriate brain regions were dissected from these sections using a modification (Lookingland and Moore, 1984) of the method of Palkovits (1973). The intermediate lobe was dissected from frozen pituitaries as described previously (Lookingland et al. , 1985). Tissue samples were then placed in 30-100 pl of 0.1 M phosphate-citrate buffer (pH 2.5) containing 15% methanol and stored at -20°C until assayed. On the day of the assay, tissue samples were thawed, sonicated for 3 s (Sonicator Cell Disrupter, Heat Systems-Ultrasonic, Plainview, NY), and centrifuged for 30 sin a Beckman 152 Microfuge. Tissue pellets were dissolved in l N NaOH and assayed for protein (Lowry et al., 1951). Supernatants were analyzed for DOPAC, dopamine, DOPA, SHIAA, 5H’l‘, norepinephrine and/or MHPG using high performance liquid chromatography coupled with electrochemical detection as described previously (Chapin et al., 1986; Lookingland et al., 1991b). Briefly, 50 pl of the supernatant was injected onto a C-18 reverse-phase analytical column (5 pm spheres, 250 x 4.6 mm; Biophase ODS; Bioanalytical Systems, West Lafayette, IN) that was preceded by a precolumn cartridge filter (5 pm spheres, 30 x 4.6 mm). DOPAC, DOPA, dopamine, SHIAA extra equipt a.Ag norepi daenr deeun Bedhn' +tl40 aspect of0.1] aehL 0 mecol Ofme hugh1 hon] dopar SHT ”Ore; 41 and/or 5HT content in the nucleus accumbens, striatum and median eminence tissue extracts was detected using an electrochemical detector (LC4A; Bioanalytical Systems) equipped withaTL-S glassycarbonelectrodesetatapotentialof +0.75 Vrelativeto a Ag/AgCl reference electrode. DOPAC, dopamine, SHIAA, 5HT, MHPG and/or norepinephrine content in pituitary and remaining hypothalamic tissue extracts was determined using a more sensitive detection system consisting of a single coulometric electrode set in series with dual electrode analytical cells (models #5021 and 5011; ESA, Bedford, MA). The conditioning cell electrode potential for this system was set at +0.40 V, while the analytical electrodes were set at + 0.12 V and - 0.40 V, respectively, relative to an internal Ag reference electrode. The mobile phase consisted of 0.1 M phosphate/citrate buffer (pH 2.7) containing 0.1 mM ethylenediaminetetraacetic acid, 0.03% sodium octylsulfate and 10-25% methanol. Depending on the condition of the column, components of the mobile phase were adjusted slightly to maintain separation of the compounds of interest and to minimize total retention times. The content of dopamine and DOPAC was determined by comparing sample peak heights (recorded by a Hewlett Packard Integrator, Model 3393A) with those obtained from standards run the same day. The lower limit of sensitivity for DOPAC and dopamine was approximately 2-5 pg/sample. The limit of sensitivity for SHIAA and 5HT was approximately 10-20 pg/sample. The limit of sensitivity for MHPG and norepinephrine was approximately 0.5 pg/ sample. G. At I electrc sulfon MHPC perchh pm 1986; I H. Rad radioim by Petti Mueller1 antigen oMSH Using a 42 G. Acid Hydrolysis Procedure Intheratbrain, thenorepinephrinemetabolitehflIPchistspredominantlyasa sulfonated conjugate (Schanberg et al. , 1968) and as such cannot be quantified using electrochemical methodology (Warnhoff, 1984). Accordingly, supernatants in which MHPGconcentrationsaretobequantifiedareheatedat94°C for5minin0.15M perchloric acid and then frozen immediately upon dry ice. This acid hydrolysis procedure liberates free MHPG which can be detected electrochemically (Artigas et al., 1986; Lookingland et al, 1991b). H. Radioirnrmrnoassays for aMSH and Prolactin Plasma concentrations of aMSH were determined using a double antibody radioimmunoassay modified (Lindley et al. , 1990) from a procedure originally described by Pettibone and Mueller (1984). Antisera to aMSH were kindly supplied by Dr. G. Mueller, Uniformed Services University for the Health Sciences, Bethesda, MD. These antisera cross-react on an equimolar basis with des-acetyl-aMSH and diacetyl-aMSH, but do not detect up to 30 ng/tube of deaminated aMSH, BMSH, adrenocorticotropin (ACTH), Acrrr,.,,, ACTHm, Acrn,,,,, B-lipotropin .. B-endorphin peptides. The aMSH used as a standard was obtained from Peninsula Laboratories (Belmont, CA). Using an aliquot of 200 pl, the lower limit of sensitivity of this assay was approximately 12 pg/tube and the intra-assay variability was approximately 16%. Plasma concentrations of prolactin were measured by a double radioimmunoassay utilizing reagents and procedures generously provided by Drs. S. Raiti and AD. Parlow, NIDDK National Hormone and Pituitary Program. NIDDK rat prolactin RP-3 was used 43 as the standard. Using an aliquot of 100 pl, the lower limit of sensitivity of this assay was approximately 1 ng/tube and the intra.assay variability was approximately 10% . 1. Statistical Analyses Statistical analyses between 2 groups were conducted using a two-tailed Student’s t-test. Analyses among 3 or more groups were conducted using analysis of variance followed by the Least Significant Differences test (Steel and Torrie, 1960). Differences among groups were considered significant if the probability of error was less than 5 % . III. HISTAMINERGIC REGULATION OF CATECHOLALflNERGIC NEURONS A. Exogenous Histamine Increases the Activity of Mesolimbic, but Not Nigrostriatal Daparninergic Neurons I. Introduction , Central histamine affects psychomotor activity in a variety of species (for review, see Itowi and Yamatodani, 1991). The i.c.v. administration of histamine to rats effects transitory hypoactivity followed by a prolonged increase in spontaneous motor activity (Kalivas, 1982). Administration to mice of agents which increase neuronal histamine release (i.e., thioperamide) or block histamine metabolism (i.e., metoprine) likewise increases locomotor activity (Sakai et al., 1991; 1992). Although the influence of histamine on behavioral arousal is well established, the site and mechanism of this action remain uncertain. Histamine activates adenylate cyclase in the nucleus accumbens (Chronister et al., 1982), a brain region important to motor control (Mogenson et al., 1980). Inna-accumbens injection of histamine effects biphasic changes in motor activity (Bristow and Bennett, 1988a). These findings suggest that centrally administered histamine may cause behavioral arousal by inducing neurochemical changes in the nucleus accumbens. As detailed in Chapter I, the nucleus accumbens contains terminals of mesolimbic 45 dopaminergic neurons (Ungerstedt, 1971). Procedures that mimic increased mesolimbic dopaminergic neuronal activity such as inn-accumbens injection of dopamine increase locomotor activity (Pijnenberg et al., 1976). Destruction of mesolimbic dopaminergic neurons by intra-accumbens injection of 6-OHDA attenuates the locomotor stimulation resulting from systemically administered amphetamine or cocaine (Kelly et al., 1975; Kelly and Iversen, 1976). These data suggest an important role for mesolimbic dopaminergic neurons in the regulation of motor activity. Behavioral arousal is seen in rats following administration of substances which activate mesolimbic dopaminergic neurons projecting to the accumbens (Miyamoto and Nigawa, 1977; Kalivas et al., 1983; Elliott et al., 1990; Hagan et al., 1990; Spanagel et al., 1991). It is not unreasonable, therefore, that the ability of histamine to effect behavioral arousal could be mediated by activation of mesolimbic dopaminergic neurons. The purpose of this study was to investigate the effect of i.c.v. administration of histamine on mesolimbic dopaminergic neuronal activity as estimated by neurochemical measurements of dopamine synthesis and metabolism in the nucleus accumbens. For comparison, the effects of histamine on nigrostriatal dopaminergic neurons was examined by measuring dopamine synthesis and metabolism in thc striatum. The effects of the H, antagonist mepyramine and the H, antagonist zolantidine on both basal and histamine- stimulated mesolimbic dopaminergic neuronal activity were also determined. The results reveal that central histamine administration has a stimulatory effect on mesolimbic dopaminergic neurons through an action at the H1 receptor, but has no effect on the activity of nigrostriatal dopaminergic neurons. 46 2. Results Results presented in Figure 3.1 demonstrate that the i.c.v. injection of histamine dihydrochloride 30 min prior to decapitation increased the accumulation of DOPA in the nucleus accumbens. The i.c.v. injection of either 15 o. 30 pg histamine increased whereas injection of 3.8 pg histamine did not affect DOPA accumulation in the nucleus accumbens. Similarly, i.c.v. injection of histamine increased DOPAC concentrations, but was without effect on dopamine concentrations in the nucleus accumbens. The histamine-induced increase in DOPAC concentrations following i.c.v. injection of histamine (30 pg/rat) occurred within 10 min and persisted for at least 120 min (Figure 3.2). In contrast, i.c.v. injection of histamine had no effect on the accumulation of DOPA nor on the concentrations of DOPAC or dopamine in the striatum (Figures 3.1 and 3.2). H, receptor blockade by mepyramine prevented the histamine-induced increase in DOPAC concentrations in the nucleus accumbens (Figure 3.3). The lowest dose of mepyramine necessary to antagonize the effects of histamine on mesolimbic dopaminergic activity was 5 mg/kg. Whereas H, receptor blockade by mepyramine prevented histamine-induced increases in DOPAC concentrations in the nucleus accumbens, H2 receptor blockade by zolantidine did not affect the ability of histamine to increase DOPAC levels (Figure 3.4). Neither mepyramine nor zolantidine affected basal DOPAC concentrations in the nucleus accumbens. 30 25 15 IO ng/mg protein 30 25 15 10 ng/mg protein Figure 3.1 Dose-response effects of histamine on DOPA accumulation (a) and on concentrations of DOPAC (O) and dopamine (D) in the nucleus accumbens and striatum of male rats. Histamine dihydrochloride (3.8, 15, or 30 pg/rat; i.c.v.) or its vehicle (3 pl/rat; i.c.v.) was injected 30 min prior to decapitation. determined in rats injected with the decarboxylase inhibitor NSD 1015 (100 mg/kg; i.p.) 30 min prior to decapitation. Symbols represent means and vertical lines 1 SEM of determinations in 7-9 vehicle- or histamine-treated rats. Where vertical lines are not depicted, 1 SEM is less than the radius of the symbol. Solid symbols represent values for histamine-treated rats that are significantly different from vehicle-treated rats (p<0.05). 47 NUCLEUS ACCUMBENS p b b - /A——§ é—é T 100 80 '- i i 00 - A “ " ‘°/ : so W h A DOPA 20 r- E] DOPAMINE O DOPAC - 10 '- l l 4 e l o r i . r . . J ‘0 15 30 o 15 3o STRIATUM 140 120 ‘00} ”40—0/0 30 h L 20 10 0 Fl 1 J l l L I HISTAMINE (pg/rot) DOPA accumulation was 48 NUCLEUS ACCUMBENS STRIATUM 100 140 : :gfii—H Cl 33% ——D ’6 ’° / n DOPAMINE T; / "6 50’ o DOPAC ’ ‘5. O) E \ O) c r- 20 J-s-n-l-n-u-n-s-La-a-l-a-a-Ln-i-La-n-La-a-J 2° .WMAJ 0 3° '0 ”01201501130210240 0 so so so rzorsoraozrozso MIN AFTER HISTAMINE Figure 3.2 Time course effects of histamine on concentrations of DOPAC (0) and dopamine (D) in the nucleus accumbens and striatum of male rats. Rats were decapitated at various times ( 10-240 min) after injection of histamine dihydrochloride (30 pg/rat; i.c.v.), or 10 min after injection of its vehicle (3 Wm; i.c.v.; zero time values). Symbols represent means and vertical lines 1 SEM of determinations in 8 vehicle- or histamine-treated rats. Where vertical lines are not depicted, 1 SEM is less than the radius of the symbol. Solid symbols represent values for histamine-treated rats that are significantly different from vehicle-treated rats (p<0.05). 49 50- 30- DOPAC (ng/mg protein) o o 1.2 2.5 5.0 MEPYRAMINE (mg/kg) Figure 3.3 Dose-response effects of mepyramine on DOPAC concentrations in the nucleus accumbens of histamine-treated male rats. Histamine dihydrochloride (30 pg/rat; i.c.v.) or its vehicle (3 pl/rat; i.c.v.) was injected. 30 min prior to decapitation. Histamine-treated rats (solid columns) were pretreated with mepyramine maleate (0, 1.2, 2.5 or 5 mg/kg; i.p.) whereas vehicle-treated rats (open column) were pretreated with saline (lml/kg; i.p.) 60 min prior to decapitation. Columns represent the means and vertical lines 1 SEM of determinations in 7-8 vehicle- or histamine-treated rats. *, values for histamine-treated rats that are significantly different from vehicle-treated controls (p<0.05). 50 ? 5° F [:3 vsmcts 5° ' -; * - HISTAMINE a. g .. O 30 r- 30 - ,1. e :— -"- :— 2 20 P 20 .. V 2 10 - 10 e a. o SALINE MEPYRAMINE SALINE ZOLANTIDINE Figure 3.4 Effects of mepyramine and zolantidine on DOPAC concentrations in the nucleus accumbens of histamine- and vehicle- treated male rats. Mepyramine maleate (5 mg/kg; i.p.) or saline (1 ml/kg; i.p.) was administered 60 min and histamine dihydrochloride (30 pg/rat; i.c.v.) or vehicle (3 pl/rat; i.c.v.) 30 min prior to decapitation (left panel). Zolantidinc dimaleate (25 mg/kg; s.c.) or saline (1 ml/kg; s.c.) was administered 60 min and histamine dihydrochloride (30 pg/rat; i.c.v.) or vehicle (3 pl/rat; i.c.v.) 30 min prior to decapitation (right panel). Columns represent the means, and vertical lines 1 SEM of determinations in 6-8 vehicle (open columns) or histamine- treated (solid columns) rats. ‘ values for histamine-treated rats that are significantly different from saline/vehicle-treated rats (p<0.05). 51 3. Discussion Central histamine administration (30 pg histanrine dihydrochloride or 18 pg histamine free base/rat) causes a rapid increase in both DOPA accumulation and DOPAC concentrations in the nucleus accumbens, but is without effect in the striatum. These results indicate that central administration of histamine increases the activity of mesolimbic but not nigrostriatal dopaminergic neurons. Previous studies have demonstrated a biphasic behavioral response following either i.c.v. histamine (IO-25 pg free baselrat) or bilateral intra-accumbens histamine injection (24 pg free base/rat). This biphasic response, transitory (15-20 min) hypoactivity followed by prolonged hyperactivity (Kalivas, 1982; Bristow and Bennett, 1988a), occurs during the same 120 min period throughout which, in the present study, increased mesolimbic dopaminergic neuronal activity is observed. A mechanism whereby histamine elicits either hypo- or hyperactivity has yet to be established. Increased mesolimbic dopaminergic neuronal activity is associated with increased psychomotor activity. Although histamine-induced hypoactivity does not correlate with the presently reported early increase in mesolimbic dopaminergic neuronal activity, histamine-induced hyperactivity may be related to the ability of the amine to increase the activity of these neurons. Pretreatment with the H, antagonist mepyramine prevents histamine-stimulated increases in DOPAC concentrations in the nucleus accumbens. H, antagonists, including mepyramine, can exhibit both anticholinergic and local anesthetic properties (Garrison, 1990). Mepyramine was chosen for these studies because it is among the least likely of the H, antagonists to block muscarinic receptors (Garrison, 1990). Further, the 52 concentration of mepyramine required to exhibit local anesthetic effects is several orders of magnitude greater than that required to antagonize the effects of histamine (Garrison, 1990). The lowest dose necessary to block the effects of i.c.v. histamine was determined in the present study, and the effects of this dose (5 mg/kg) were presumably the result of a selective H, receptor blockade. The ability of mepyramine to block histamine-induced increases in DOPAC concentrations in the nucleus accumbens indicates that central administration of histamine stimulates mesolimbic d0paminergic neurons through an action at the H, receptor. H, receptor-mediated effects have been noted previously in the nucleus accumbens; mepyramine pretreatment markedly attenuates intra-accumbens histamine-induced hyperactivity (Bristow and Bennett, 1988a). H, receptor blockade attenuates i.c.v. histamine-induwd hyperactivity as well (Kalivas, 1982). In both of these studies, H, receptor blockade did not prevent histamine-induwd behavioral arousal. In the present the H, receptor antagonist zolantidine, administered at a dose (25 mg/kg; i.c. , 5- to 2500- fold greater than that required to antagonize H, receptors (Gogas and Hough, 1988; Gogas et al., 1989; Koch et al., 1992) regarded as both selective and sufficient to block H2 receptors (Hough, personal communication; Houghket al., 1988), likewise failed to prevent histamine-induced increases in DOPAC concentrations. Increased motor activity induced by histamine may be related, therefore, to an H, but not H, receptor-mediated activation of mesolimbic dopaminergic neurons. Recent evidence suggests that thyrotropin-releasing hormone (TRH) modulates histamine-induced arousal (Bristow and Bennett, 1988b; Bristow and Bennett, 1989). Interestingly, TRH administration increases spontaneous motor activity, perhaps by 53 increasing mesolimbic dopamine release (Miyamotoand Nagawa, 1977; Heal and Green, 1979). Further, TRH-stimulated locomotor behavior is blocked by 6—OHDA lesions of mesolimbic dopaminergic nerve terminals and by haloperidol blockade of dopamine receptors (Heal and Green, 1979). Since histamine-induced hyperactivity is modulated by TRH, and TRH-induced hyperactivity is mediated by dopaminergic mechanisms, an interaction among histamine, TRH and mesolimbic dopaminergic neurons is implied. Additional investigations are necessary so as to establish the relationship between mesolimbic dopaminergic neurons, TRH and histamine-induced changes in spontaneous motor activity. In summary, the results demonstrate that exogenously administered histamine, through an action at H, but not H, receptors, increases the activity of mesolimbic but not nigrostriatal dopaminergic neurons. The finding that histamine stimulates mesolimbic dopaminergic neurons, which are recognized as being important for psychomotor function, supports the findings by other investigators that histamine may have an important physiological role in modulating behavioral arousal. B. Exogenous Histamine Differentially Affects the Activity of Hypothalamic Dopaminergic Neurons I. Introduction Central administration of histamine has been shown to affect both noradrenergic (Bealer, 1993; Philippu et al., 1984) and 5-hydroxytryptaminergic (Fink et al., 1990; see also Chapter IV) neuronal activity. Comparatively little information is available regarding the effects of histamine on dopaminergic neurons. Direct application of 54 histamine increases dapamine efflux both in vitro from rat brain slices (Subramanian and Mulder, 1977),andinvirofromtheposteriorhypothalamusofanesthetizedcats (Philippu et al., 1984). As described above, central administration ofhistamine increases the activity of mesolimbic, but not nigrostriatal dopaminergic neurons. Still, the mammalian brain contains several anatomically distinct dopaminergic neuronal systems (Palkovits, 1981), each regulated by different mechanisms, and the effects of histamine on these systems have yet to be established. As described in Chapter 1, Section D, the hypothalamus contains several dopaminergic neuronal systems which are important in regulating pituitary hormone secretion. For example, tuberoinfundibular and periventricular-hypophysial dopaminergic neurons tonically inhibit the secretion of prolactin and aMSH, respectively. Dopaminergic neurons of the caudal incertohypothalamic dopaminergic system reportedly influence both luteinizing hormone and prolactin secretion (Sanghera et al. , 1991). Other hypothalamic dopaminergic neurons arising from or terminating in the PeVN, MPN, SCN, and PVN (Palkovits, 1981) have functions which are as yet unclear. Central administration of histamine affects a variety of hypothalamic functions including the regulation of pituitary hormone secretion (Schwartz et al. , 1991; Knigge and Warberg, 1991), conceivably by influencing dopaminergic neuronal function. Further investigation into the mechanism whereby central histamine might affect the activity of hypothalamic dopaminergic neurons was thereby warranted. The purpose ofthis study was to characterize the effects of centrally administered histamine on the activity of hypothalamic dopaminergic neurons as estimated by measuring concentrations of dopamine and its metabolite DOPAC in brain or pituitary 55 regionscontainingcellbodiesorterrninalsoftheseneurons. Theresultsrevealthat i.c.v. administration of histamine difierentially affects the activity of the various dopaminergic neuronal systems of the rat hypothalamus. 2. Results Results presented in Table 3.1 summarize the differential effects of histamine on DOPAC and dopamine concentrations in the brain and pituitary regions selected for study. Three distinct regionally specific neurochemical responses were apparent. In the median eminence and intermediate lobe, i.c.v. administration of histamine dihydrochloride (3. 8-30 pg/rat) 30 min prior to decapitation was without effect on DOPAC and dopamine concentrations. Time course studies confirm this point; throughout the 2 h period following administration, histamine (30 pg/rat) was without effect on either DOPAC or dopamine concentrations in the median eminence (see Figure 5.2) and intermediate lobe (Table 3.2). In the DMN, MZI, MPN and rostral PeVN (rPeVN), a different pattern of response was observed (Table 3.1); histamine effected a dose-related increase in both DOPAC and dopamine concentrations, with significant increases noted in all regions at the 30 pg/rat dose. Finally, a third pattern of neurochemical response was observed in the SCN, cPeVN and PVN; 30pg histamine increased DOPAC, but was without effect on dopamine concentrations. A time course of the effects of histamine on DOPAC and dopamine concentrations in the DMN and MZI is depicted in Figure 3.5; i.c.v. administration of histamine (30 pg/rat) increased concurrently DOPAC and dopamine concentrations in these brain regions. Histamine produced a similar time course of effects in the MPN and rPeVN (Figure 3.6). Because concurrent increases in DOPAC and dopamine concentrations are 56 Table 3.1 Dose response effects of histamine on DOPAC and dopamine concentrations inselectedbrainnucleiofmalerats Dose of Histamine (pg/rat) 3.8 9.1 :t 0.9 DOPAC 4.5 00 11 7.9 36 35 00 it 0.2 36 23 00 11 0.3 35 mm. 11 7.4. 36 24. 00 11 0. 6 23 00 11 6. 5 62 00 11 95 35 4.4. 00 11 7.3 13 33 00 11 54" 13 J2 00 11 um Dopamine DOPAC rPeVN DOPAC MPN 27. 00 11 7.5 27 3A 00 it 57. 15 1.1. 00 it £3 15 23 11 4.5 15 II! Dopami 1.2 00 11 7.4. 1.4 16. 00 11 34. 15 1.2 00 11 33 15 SCN 46. 0.0 11 99 4.9 00 11 J6. 39 Dopamine cPeVN DOPAC 26. 00 11 95 119 2.3 00 it 97. 17 ‘, values for Histamine dihydrochloride (3.8. 15 or 30 pg/rat) or saline vehicle (3 pl/rat) was injected i.c.v. 30 min prior to decapitation. 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Been—ES... o... ... meeugeooeeo 3.882. e5 U [D n“ ' D CONTROL m - nesrmur goo . one a: see ‘ son '00 100 aMSH (pg/ml plasma) SALINE CFIIH SAUNE HEPYRAMINE SALINE ZOLANTIDINE Figure 3.20 Effects of aFMH. mepyramine and zolantidine on basal and restraint stress— affected aMSH concentrations in the plasma of male rats. Rats were decapitated immediately upon removal from their home cages (CONTROL) or placed in plastic restraining tubes for 10 min. removed from the tubes, and then decapitated (RESTRAINI'). A) aFMl-l (100 mg/kg, i.p.) or saline (1 ml/kg, i.p.) was administered 4 h prior to decapitation. B.C) Mepyramine maleate (5 mg/kg, i.p.), zolantidine dimaleate (25mg/kg. s.c.) or saline (1 ml/kg, i.p. or s.c.) was administered 1 h prior to decapitation. Columns represent means and vertical lines 1 SEM of determinations in 7-9 rats. ", values for stressed rats that are significantly different (p<0.05) from controls. 87 3. Discussion Thenemocbemiealresponsemsuessfidsdmuliiscomplexanddepardentupon anumberoffactorsincludinggenderoftheanimalandtypeofstressor. Theth study examines in male rats the efl’ect of physieal restraint on the activity of four distinct dopaminergic neuronal systems whose activities ean be affected by this or other stressors. The results reveal that physical restraint differentially affects the activities of mesolimbic, nigrostriatal, tuberoinfundibular and periventricular-hypophysial dopaminergic neurons; neurons that project to the nucleus accumbens, striatum, median eminence and intermediate lobe of the pituitary, respectively. Consistent with previous reports, stress rapidly increases mesolimbic (Tissari et al., 1979; Claustre et al., 1986; Roth et al., 1988; Abercrombie et al., 1989; Deutch and Cameron, 1992) and decreases periventricular-hypophysial (Lookingland et al., 1991a; Goudreau et al., 1993) dopaminergic neuronal activity. In contrast, restraint stress has no effect on the activity of tuberoinfundibular dopaminergic neurons. A lack of effect of restraint stress on tuberoinfundibular dopaminergic neuronal activity in male rats has been reported previously (Lookingland et al. , 1990). Finally, restraint stress has no effect on nigrostriatal dopaminergic neuronal activity. Although stress-induced increases in nigrostriatal dopaminergic activity have been described (Keller et al. , 1983), the absence of effect observed in the present study is consistent with several studies wherein a lack ofeffectofstressonnigrostriatal dopaminergic neuronshasbeenreported ('I'hierryet al., 1976; Deutch et al., 1985; Claustre et al., 1986; Roth et al., 1988). Central histamine concentrations increase in response to stress (Campos and 88 Jurupe, 1970; Mazurkiewicz—Kwilecki, 1980; Mazurkiewicz—Kwilecld and Bielkiewicz, 1982; Mazurkiewicz-Kwilecki and Prell, 1986) and histamine mediates several stress responses including antinociception (Oluyomi and Hart, 1991) and increased pituitary hormone secretion (Alvarez, 1982; Knigge etal., 1988a; Kniggeet al., 1991; S¢e~Jensen et al. , 1993). Since stress increases histaminergic neuronal activity, and both restraint stress and central administration of histamine (see this Chapter, Section A) increase the activity of dopaminergic neurons projecting to the nucleus accumbens, investigation into the possibility that central histamine might mediate stress-enhanced mesolimbic dopaminergic neuronal activity was warranted. The results presented in this section reveal, however, that neither depletion of neuronal histamine by ozFMH, nor post- synaptic histaminergic receptor blockade using the H, and H2 antagonists mepyramine and zolantidine, respectively, prevents the stress-induced increase in the activity of these neurons. These data indicate that histaminergic neurons are not major contributors to stress-induced increases in mesolimbic dopaminergic neuronal activity. Periventricular-hypophysial dopaminergic neurons, with perikarya (the A“ cell group; Dahlstrom and Fuxe, 1964) in the cPeVN and terminals in the intermediate lobe of the pituitary (Goudreau et al., 1992), tonically inhibit the secretion of aMSH. Restraint stress increases aMSl-I secretion, in part, by decreasing the activity of these neurons and thereby removing this tonic inhibition (Lookingland et al., 1991a). The periventricular-hypophysial dopaminergic neuronal system is not, however, the sole regulator of stress-induced aMSH secretion; epinephrine, released from the adrenal medulla in response to stress, can activate melanotroph B-adrenergic receptors in the 89 intermediate lobe and thereby stimulate aMSH secretion (Cote et al., 1982; Kvetnansky et al., 1987; Lindley et al., 1990). Previous studies have suggested that histaminergic neurons might affect both intermediate lobe dopaminergic neuronal activity and circulating epinephrine concentrations and thereby mediate stress-induced aMSH secretion (Knigge et al., 1989; 1991). The data, in part, support this assertion since disruption of histaminergic transmission by «M or mepyramine prevents stress- induced decreases periventricular-hypophysial dopaminergic neuronal activity. Disruption of histaminergic transmission does not, however, prevent the stress-induced increase in aMSH secretion suggesting that histaminergic neurons are not principle regulators of this response. Instead, the finding that aMSI-I secretion is increased in aFMH- or mepyramine-treated stressed rats even though periventricular-hypophysial dopaminergic neuronal activity is unaffected suggests that B—adrenergic receptor activation is primarily responsible for the stress-induced aMSH secretion. The finding that stress-induced decreases in periventricular-hypophysial dopaminergic neuronal activity are blocked by mepyramine but not zolantidine suggests that histamine mediates this stress-induced effect through an action at H,, but not B, receptors. Previous investigation into the effects of exogenous histamine administration revealed no such effect on periventricular-hypophysial dopaminergic neurons (see this Chapter, Section B), perhaps because histamine concentrations attained at the synaptic level following i.c.v. administration may be lower than those attained during stress and hence insufficient to affect dopaminergic neuronal activity. This disparity between the effects of exogenous and presumably stress-increased endogenous histamine illustrates the limitation of pharmacologieal approaches such as i.c.v. administration of histamine in 9O investigating histaminergic function and demonstrates the need for employing a variety of approaches to the study of central actions of histamine. In summary, results presented in this Section reveal that restraint stress differentially affects the activity central dopaminergic neurons. Specifically, restraint stress increases mesolimbic, decreases periventricular-hypophysial, and is without effect on nigrostriatal and tuberoinfundibular dopaminergic neuronal activity in male rats. Histaminergic neurons contribute to stress-induced decreases in periventricular- hypophysial dopaminergic neuronal activity through an action at Hl receptors, but appear not to mediate stress-induced increases in aMSH secretion or mesolimbic dopaminergic neuronal activity. E. Histaminergic Neurons Mediate Restraint Stress-Induced Increases in the Activity of N oradrenergic Neurons Projecting to the Hypothalamus I . Introduction As described in Chapter I, noradrenergic neurons projecting to the hypothalamus arise from brainstem catecholaminergic cell groups of the lateral tegmentum, and to a lesser extent, the locus coeruleus (Moore and Bloom, 1979; Palkovits, 1981). Stressful stimuli increase the activity of these neurons as reflected by increases in norepinephrine release and metabolism ('l‘anaka et al., 1989; Lookingland et al., 1991b; Yokoo et al., 1990; Pacak et a1. , 1992). Despite a wealth of evidence for stress increasing the activity of these noradrenergic neurons, the central mechanisms facilitating these effects remain largely undefined. As discussed previously, histaminergic neurons project diffusely to many brain 91 regions, especially to the diencephalon, and the activity of these neurons is apparently increased in response to stressful stimuli. Since acute stress apparently increases histaminergic neuronal activity and, as described in this Chapter, Section C, exogenous administration of histamine increases the activity of central noradrenergic neurons (see also Philippu et al. , 1984; Bealer, 1993), the possibility that histaminergic neurons might mediate stress-induced increases in noradrenergic neuronal activity warranted attention. The response of an animal to stressful stimuli is complex and dependent on the type of stressor. The purpose of the present study was to examine the role of histamine in mediating the effects of one particular type of stressor, immobilization within a restraining tube, on the activity of noradrenergic neurons projecting to two hypothalamic nuclei: the PVN and MPN. These nuclei were selected because each are densely innervated by noradrenergic neurons (Dahlstrom and Fuxe, 1964; Moore and Bloom, 1979), the activities of which are seemingly altered in response to stress (Saavedra, 1982; Lookingland et al., 1991b; Pacak et al., 1992). The activity of noradrenergic neurons was estimated by measurements of the norepinephrine metabolite MHPG in these nuclei. The results reveal that restraint stress increases the activity of noradrenergic neurons projecting to the hypothalamus and that histaminergic neurons contribute to this increase via an action at H, receptors. 2. Results As described above (this Chapter, Section D), an array of stressful procedures of short duration increase histamine levels in the rat brain. Results presented in Figure 3.21 92 demonstrate that restraint of short duration can also affect the activity of noradrenergic neurons. Specifically, 10, 20 or 30 min of physical restraint increased MHPG but was without effect on norepinephrine concentrations in the PVN and MPN. The shortest duration of restraint, 10 min, was employed in subsequent studies. Results presented in Figure 3.22 reveal that blockade of neuronal histamine synthesis with the histidine decarboxylase inhibitor aFMH (Garbarg et al., 1980), administered at a dose and time demonstrated to decrease central histamine release by 70% (Itoh et al., 1991), attenuated the stress-induced increase in MHPG concentrations in the PVN and MPN. Similarly, H, receptor blockade by mepyramine antagonized the stress-induced increase in MHPG concentrations in these brain regions (Figure 3.23). On the other hand, H, receptor blockade by zolantidine did not affect the stress-induced increases in MHPG in either the PVN or MPN (Figure 3.24). Neither aFMH, mepyramine, nor zolantidine affected basal MHPG concentrations in either brain region. 3. Discussion As described in this Chapter. Section C, several lines of evidence suggest that exogenous histamine can influence noradrenergic neuronal activity. In vitro, histamine increases norepinephrine efflux from rat brain synaptosomal (Tuomisto and Tuomisto, 1980) and hypothalamic slice preparations (Subramanian and Mulder, 1977 ; Blandina et al., 1989). In rat cortical slices, both stimulatory and inhibitory effects of histamine on norepinephrine release have been described (Schlicker et al. , 1989; Taube et al., 1977; Young et al., 1988). In vivo, superfusion with histamine increases norepinephrine 93 PVN MPN 150 60 90 20 : .__- .. .8 . B 6 I- M a o "' I- L O. 4 i- A 4’ :- / U’ E P {2 \ 0' 2 '- 2 " U NOREPINEPHRINE ‘ o MHPG o l l l _I o l l l I O 10 20 30 O 10 20 30 MIN OF RESTRAINT Figure 3.21 Time course effects of physical restraint on MHPG (0) and norepinephrine (0) concentrations in the PVN and MPN of male rats. Rats were placed in restraining tubes for 10, 20 or 30 min, removed from the tubes, and then decapitated. Control rats were decapitated immediately upon removal from their home cages (zero time values). Symbols represent means and vertical lines 1 SEM of determinations in 7—8 rats; where vertical lines are not depicted, 1 SEM is less than the radius of the symbol. Solid symbols represent values that are significantly different from controls (p <0.05). 94 3 " * [:3 CONTROL - RESTRAINT 6 3 T o a 2 T O E E 5 , 1, o l I 3 O — o _ SALINE aFMH SALINE «FMH Figure 3.22 Effects of aFMH on basal and restraint stress-increased MHPG concentrations in the PVN and MPN of male rats. Rats were decapitated immediately upon removal from their home cages (CONTROL) or placed in plastic restraining tubes for 10 min, removed from the tubes, and then decapitated (RESTRAINT). aFMH (100 mg/kg, i.p.) or saline (1 ml/kg, i.p.) was administered 4 h prior to decapitation. Columns represent means and vertical lines 1 SEM of determinations in 7-9 rats. ', values that are significantly different from controls; T, values that, although significantly different from controls, represent a response to restraint significantly attenuated by aFMH (p< 0.05). 95 PVN MPN 2 ' :1 CONTROL 3 ' * - RESTRAINT ’3 f -- 1' 3 I- s 2 - E' 1 .— \ 2' r“ E :— 0 1 '- O. I 2 r- 0 — o J SALINE MEPYRAMINE SALINE MEPYRAMINE Figure 3.23 Effects of mepyramine on basal and restraint stress-increased MHPG concentrations in the PVN and MPN of male rats. Rats were decapitated immediately upon removal from their home cages (CONTROL) or placed in plastic restraining tubes for 10 min, removed from the tubes, and then decapitated (RESTRAINT). Mepyramine maleate (5mg/kg, i.p.) or saline (1 ml/kg, i.p.) was administered 1 h prior to decapitation. Columns represent means and vertical lines 1 SEM of determinations in 79 rats. *, values that are significantly different from controls; 1', values that, although significantly different from controls, represent a response to restraint significantly attenuated by mepyramine (p< 0.05). 3 " [:3 CONTROL 3 P A _ - RESTRAINT _ * C E 8 2 _. * a, D E . \ 3' .1. V 1 o r O. I 3 D o ‘ ... sauna zoumtome emu: ZOLANTIDIINE Figure 3.24 Effects of zolantidine on basal and restraint stress-increased MHPG concentrations in the PVN and MPN of male rats. Rats were decapitated immediately upon removal from their home cages (CONTROL) or placed in plastic restraining tubes for 10 min, removed from the tubes, and then decapitated (RESTRAINT). Zolantidinc dimaleate (25mg/kg, s.c.) or saline (1 ml/kg, s.c.) was administered 1 h prior to decapitation. Columns represent means and vertical lines 1 SEM of determinations in 7-8 rats. *, values that are significantly different from controls (p<0.05). 97 release from the posterior hypothalamus of anesthetized cats (Philippu et al., 1984) and from the PVN/anterior hypothalamic region of conscious rats (Bealer et al., 1993). As demonstrated in this Chapter (see Section C), exogenous administration of histamine increases norepinephrine metabolism in the hypothalamus as well. Although these studies demonstrate clearly that exogenous histamine can increase noradrenergic neuronal activity, the possibility that endogenous histamine might effect similar neurochemical changes has remained unexplored. Since stress increases both endogenous histamine concentrations (Campos and Jurupe, 1970; Mazurkiewicz-Kwilecki, 1980; Mazurkiewicz- Kwilecki and Bielkiewicz, 1986) and noradrenergic neuronal activity (Tanaka et al., 1989; Yokoo et al., 1990; Lookingland et al., 1991b; Smith et al., 1991; Tanaka et al., 1991; Pacak et al., 1992), investigation into the possibility that histaminergic neurons might mediate stress-induced increases in noradrenergic neuronal activity was warranted. Results from the experiments presented in this Section demonstrate that restraint stress increases the activity of noradrenergic neurons projecting to the hypothalamus. These results are in agreement with numerous reports describing stress-induced increases in central norepinephrine synthesis, release and metabolism (Algeri et al., 1988; Yokoo et al., 1990; Tanaka et al., 1989; Lookingland et al., 1991b; Pacak et al., 1992), and extend these findings by demonstrating a role for histaminergic neurons in mediating stress-induced increases in noradrenergic neuronal activity. Specifically, disruption of neuronal histamine synthesis by the irreversible histidine decarboxylase inhibitor aFMH or antagonism of H, receptors by mepyramine attenuates, but does not prevent completely, stress-induced increases in MHPG concentrations. This suggests that 98 histaminergic neurons contribute to stress-induced increases in noradrenergic neuronal activity via an action at H, receptors. The finding that neither aFMH nor mepyramine completely prevents stress-induced increases in noradrenergic neuronal activity indicates that in addition to activation of histaminergic neurons, other neurochemical events contri- bute to increased noradrenergic neuronal activity; these events have yet to be defined. In summary, histaminergic neurons contribute to stress-induced increases in the activity of noradrenergic neurons projecting to the hypothalamus through an action at H, receptors. These data are consistent with previous reports of an H, receptor-mediated stimulatory effect of histamine on noradrenergic neuronal activity (Bealer et al., 1993; see also this Chapter, Section C), and further demonstrate the importance of histamine as a neurotransmitter in the mammalian brain. F. H, Receptor Ligands Do Not Affect the Activity of Catecholaminergic Neurons 1. Introducrion As described in Chapter 1. H, receptors are pre-synaptic autoreceptors which modulate histamine synthesis and release. Administration of H, agonists such as R-a- methylhistamine decrease. whereas H, antagonists such as thioperamide increase, synthesis and release of the amine (Arrang et al., 1985a; 1987b). Since exogenous administration of histamine increases the activity of certain noradrenergic and dopaminergic neuronal systems (see this Chapter, Sections A-C), it is not unreasonable to suggest that H, receptor-mediated changes in central histamine dynamics might affect catecholaminergic neuronal activity. Furthermore, a role in vitro for H, receptors as heteroreceptors that directly modulate norepinephrine release has been postulated (Schlicker et al., 1989). An examination of the effects of H, receptor ligands on catecholaminergic neuronal activity was thereby warranted. 99 mpurposeofthissmdywastoexaminetheabilityofH,receptorligandsto affect catecholaminergic neuronal activity as estimated from measurements of DOPAC, dopaminc, MHPG and norepinephrine in brain regions containing terminals of these neurons. The results reveal that H, receptor ligands do not affect the activity Of central dopaminergic or noradrenergic neurons in the systems under study. 2. Results Results presented in Table 3.4 demonstrate that the H, receptor antagonist thioperamide, administered at a dose (5 mg free base/kg, i.p.) reported in vivo to effect 2- to 3-fold increases in hypothalamic histamine release (Itoh et al., 1991; Mochizuki et al., 1991), had virtually no effect on DOPAC or dopamine concentrations in the nucleus accumbens, striatum, median eminence, intermediate lobe, DMN, PVN, cPeVN, or SCN during the 2 h period following injection. Similarly, thioperamide administration had no effect on MHPG concentrations, as shown for the MZI and MPN in Table 3.5. The histamine-N-methyltransferase inhibitor metoprine, administered at a dose sufficient to increase extracellular histamine concentrations 2-fold (Duch et al., 1978; Itoh et al., 1991), had no effect DOPAC or dopamine concentrations in any of the brain regions under study (Table 3.6). Combined administration of thioperamide with metoprine, a regimen which enhances (i.e., 2- to 4-fold; Itoh et al., 1991) the ability of thioperamide to increase extracellular histamine content presumably by blocking the metabolism of newly released histamine likewise had virtually no effect on concentrations of DOPAC or dopamine (Table 3.6). Administration of the H, agonist R-a-methylhistarnine, or its prodrug BP 2.94, 100 atadmedemonsuawdmeffectmaximaldecreasesmhiminesynmedsandrdease (Krikorian, A., and Schwartz, J.C., BIOPROJET, personal communication; Garbarg et al., 1989), had no effect on DOPAC or dopamine concentrations in any of the brain regions under study (Tables 3.7, 3.8). 3. Discussion The results presented in this Section demonstrate that blockade of the H, receptor, a pharmacological manipulation which increases extracellular histamine content, has no effect on the activity of nigrostriatal, tuberoinfundibular, or incertohypothalamic dopaminergic neurons. Combined administration of thioperamide with metoprine, a regimen which effects even greater increases in histamine content, likewise does not affect the activity of these systems. These data are not unexpected given the previous finding (see this Chapter, Sections A and B) that exogenous administration of histamine does not affect the activity of these dopaminergic neuronal systems. Thioperamide and/ or metoprine administration affects neither the activities of dopaminergic neurons projecting to the nucleus accumbens, PVN, cPeVN, or SCN, nor noradrenergic neurons projecting to the MZI or MPN. The finding that pharmacological manipulations which facilitate endogenous histaminergic transmission do not affect the activity of these neurons stands in contrast to findings that the activities of these dopaminergic and noradrenergic neuronal systems are increased following stress-induced increases in histaminergic neuronal activity and/or central administration of histanrine (see this Chapter, Sections B, C, E). 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H a... .N... 8. 8 on n. o 8.508.... .22 ...: 8.8.. as... an. 0.2: ..O 2&2 :5 .NE 0... :. “5.3.2088 9.12 :O 02582.2... ..O 800...? 8.30 08.... n... 030... 103 Table 3.6 Effects of thioperamide, metoprine, and combined thioperamide/metoprine treatment on DOPAC and dopamine concentrations in various brain nuclei of male rats vehicle thioperamide metoprine thioperamide! metoprine Nucleus DOPAC 39.2 1 1.6 44.2 1 2.1 33.8 1 0.8 33.2 1 2.2“ Accumbms, . Dopamine 78.0 1 3.4 85.4 1 4.9 74.7 1 3.6 69.0 1 6.7 Striatum DOPAC 34.6 :1: 1.7 36.7 1 1.5 34.2 1 1.0 34.8 1 1.4 Dopamine 108.1 1 0.5 110.8 1 3.9 115.6 1 4.1 120.5 1 3.7 Median DOPAC 11.3 1 1.1 12.7 :1; 0.7 10.4 1 0.6 11.9 1 1.0 Eminalce Dopamine 85.9 1 5.0 83.8 1 3.1 98.9 1 9.0 98.7 1 6.6 Intermediate DOPAC 1.0 1 0.1 1.3 1 0.1 1.3 1 0.1 1.2 1 0.1 Lobe Dopamine 16.2 1 0.4 17.5 1 1.8 16.5 1 1.2 17.7 1 1.6 MZI DOPAC 1.6 1 0.1 1.5 1 0.1 1.4 1 0.1 1.5 1 0.1 Dopamine 5.4 1 0.2 5.2 1 0.2 4.5 1 0.2 5.3 1 0.4 cPeVN DOPAC 2.3 1 0.1 2.2 1 0.1 2.1 1 0.1 2.1 1 0.1 Dopamine 6.9 1 0.3 7.5 1 0.3 6.9 1 0.4 7.6 1 0.3 SCN DOPAC 1.9 1 0.1 1.9 1 0.1 1.8 1 0.1 1.9 1 0.1 Dopamine 4.8 1 0.2 5.8 1 0.2" 5.8 1 0.3"I 5.3 1 0.2 MPN DOPAC 1.8 1 0.1 1.4 1 0.1 1.6 1 0.1 1.5 1 0.1 Dopamine 2.4 1 0.2 2.2 :t 0.1 2.4 1 0.2 2.4 1 0.2 Thioperamide maleate (5 mg free base/kg; i.p.) or saline (1 ml/kg; i.p.) was administered 30 min and metoprine (10 mglkg; i.p.) or 1% sodium carboxymethylcellulose vehicle (1 ml/kg; i.p.) 60 min prior to decapitation. Values represent means 1 1 SEM of DOPAC and dopamine concentrations (ng/mg protein) in tissue samples obtained from 79 rats. ", values that are significantly different (p<0.05) from controls. 104 Table 3.7 Effects of R-a-methylhistamine on DOPAC and dopamine concentrations in various brain nuclei of male rats Brain Region R-a-Metbylhistamine (mg/kg) 0 5 10 20 Nucleus DOPAC 30.7 1 1.2 33.5 1 1.3 33.0 1 1.6 32.0 1 1.6 Accumbens Dopamine 92.1 1 3.6 102.2 1 3.6 97.4 1 4.6 100.2 1 4.8 Stn'atum DOPAC 30.5 1 1.1 27.6 1 0.8 29.1 1 1.2 28.9 1 1.3 Dopamine 121.7 1 2.4 110.9 1 3.8 117.4 1 3.9 126.4 1 5.4 Median DOPAC 8.7 1 0.7 10.4 1 0.6 8.0 1 0.3 7.7 1 0.6 Eminence Dopamine 112.4 1 6.9 113.2 1 2.5 111.2 1 7.1 92.2 1 7.1 Intermediate DOPAC 1.8 1 0.1 1.6 1 0.2 1.8 1 0.2 1.3 1 0.1 Lobe Dopamine 17.0 1 0.7 15.1 1 1.0 17.0 1 1.6 15.5 1 1.0 DMN DOPAC 1.9 1 0.1 2.1 1 0.1 2.0 1 0.1 1.9 1 0.2 Dopamine 4.5 1 0.2 4.7 1 0.2 4.7 1 0.2 4.7 1 0.3 M21 DOPAC 1.8 1 0.2 1.7 1 0.1 1.9 1 0.2 1.9 1 0.2 Dopamine 5.9 1 0.4 5.8 1 0.3 6.7 1 0.4 5.9 1 0.3 cPeVN DOPAC 2.0 1 0.2 2.0 1 0.1 2.1 1 0.1 2.0 1 0.1 Dopamine 7.6 1 0.5 7.2 1 0.5 7.6 1 0.5 7.3 1 0.4 SCN DOPAC 1.5 1 0.1 1.5 1 0.1 1.4 1 0.2 1.3 1 0.1 Dopamine 5.6 1 0.5 4.6 1 0.2 4.6 1 0.2 4.7 1 0.1 R-a-methylhistamine (5. 10 or 20 tug/kg; p.o.) or distilled water (1 ml/kg; p.o.) was administered 2 h prior to decapitation. Values represent means 1 1 SEM of DOPAC and dopamine concentrations (ng/mg protein) in tissue samples obtained from 79 rats. 105 Table 3.8 Effects of BP 2.94 on DOPAC and dopamine concentrations in various brain nuclei of male rats Brain Region BP 2.94 (mg/kg) 0 5 10 20 Nucleus DOPAC 35.9 1 2.6 41.1 1 1.3 39.0 1 2.6 42.1 1 1.7 Aecumbm Dopamine 72.6 1 6.4 72.9 1 3.9 74.9 1 4.7 86.1 1 3.0 Striatum DOPAC 35.3 1 1.1 40.6 1 3.1 37.0 1 1.3 35.1 1 1.1 Dopamine' 129.0 1 3.7 135.1 1 8.7 135.4 1 5.3 125.0 1 1.4 Median' DOPAC 11.3 1 2.6 11.3 1 0.8 11.2 1 0.9 10.0 1 0.5 Eminence Dopamine 123.0 1 5.5 103.1 1 5.2 110.6 1 7.7 109.9 1 6.2 Intermediate DOPAC 2.1 1 0.2 2.4 1 0.2 2.5 1 0.2 2.5 1 0.1 Lobe Depamine 17.0 1 0.7 17.7 1 1.1 19.2 1 1.2 18.6 1 1.1 DMN DOPAC 1.4 1 0.1 1.3 1 0.1 1.3 1 0.1 1.3 1 0.1 Dopamine 5.0 1 0.4 5.0 1 0.2 5.0 1 0.3 5.3 1 0.3 MZI DOPAC 1.2 1 0.1 1.2 1 0.1 1.3 1 0.1 1.3 1 0.1 Dopamine 4.6 1 0.3 4.1 1 0.2 4.4 1 0.2 4.8 1 0.3 cPeVN DOPAC 2.8 1 0.2 2.8 1 0.2 3.0 1 0.1 3.0 1 0.2 Dopamine 9.1 1 0.5 8.4 1 0.4 9.7 1 0.4 9.8 1 0.5 MPN DOPAC 1.5 1 0.1 1.7 1 0.1 - 1.4 1 0.1 1.5 1 0.1 Dopamine 2.4 1 0.1 2.4 1 0.1“ 2.1 1 0.1 2.4 1 0.1 BP 2.94 (5, 10 or 20 mg/kg; p.o.) or 1% sodium carboxymethylcellulose (1 ml/kg; p.o.) was administered 2 h prior to decapitation. Values represent means 1 1 SEM of DOPAC and dopamine concentrations (ng/ mg protein) in tissue samples obtained from 7-9 rats. 106 neurons; a finding seemingly contradictory to the previous finding that stress-induced increases in histaminergic neuronal activity decrease the activity of these dopaminergic neurons (see this Chapter, Section D). These disparities may result from several factors. For instance, histamine concentrations attained at the synaptic level following thioperamide and/or metoprine treatment may be less than concentrations effected by stress or central histamine administration and consequently insufficient to affect catecholaminergic neuronal activity. Alternatively, exogenous histamine may affect the activity of these catecholaminergic neuronal systems at sites distant from sites of histamine release and metabolism. The differences among the effects of i.c.v. histamine, thioperamide, metoprine and stress again demonstrate the importance of employing a variety of approaches to the study of central histamine. Administration of either the H, agonist R—a-methylhistamine, or its prodrug BP 2.94, has no effect on the activity of the catecholaminergic neurons under study. The lack of effect of these compounds which decrease neuronal histamine synthesis and release suggests that histaminergic neurons do not tonically regulate the activity of catecholaminergic neurons. This lack of tonic regulation will be discussed further in this Chapter, Section G. The lack of effect of these agonists, coupled with the lack of effect of thioperamide, further indicates that H, receptor ligands do not affect the activity of eatecholaminergic neurons in the systems under study. G. Histaminergic Neurons Do N at Regulate Basal Catecholaminergic Neuronal Activity Sections A-F in this Chapter have focussed on the effects of pharmacological and physiological manipulations which mimic, facilitate or increase histaminergic transmission (i.e., i.c.v. histamine, thioperamide or metoprine administration, stress) on 107 the activity of central catecholaminergic neurons. Over the course of these experiments, it was demonstrated that neither the H, antagonist mepyramine nor the H, antagonist zolantidine affect basal eatecholamine synthesis or metabolism (Figures 3.4, 3.11, 3.12, 3. 15 , 3.18, 3.19, 3.23, 3.24). The finding that blockade of post-synaptic histaminergic receptors does not affect these indices suggests that histaminergic neurons do not tonically affect catecholaminergic neuronal activity in the systems under study. The inability of the H, agonists R—a-methylhistamine and BP 2.94, compounds which decrease histamine release, to affect dopaminergic neuronal activity further indicates a lack of tonic regulation (Tables 3.7, 3.8). Finally, the lack of effect on catecholaminergic neurons of the histidine decarboxylase inhibitor aFMH, examined at a time (4 h after administration) at which neuronal histamine synthesis is purportedly decreased by 70%, demonstrates a lack of tonic regulation (Figures 3.17, 3.22). Results presented in Table 3.9 confirm the lack of effect of aFMH on d0paminergic neuronal activity. The histidine decarboxylase inhibitor has virtually no effect on DOPAC or dopamine concentrations in the nucleus accumbens, striatum, median eminence, intermediate lobe, DMN, MZI, MPN, or SCN during a 24 h period followmg injection, a period throughout which neuronal histidine decarboxylase activity is inhibited (Garbarg et al., 1980; Hough, L.B., personal communication). These data, taken together with the findings that neither blockade of histamine release with R-a- meth)’ll'tistamine or BP 2.94, nor of postsynaptic H, and H2 receptors with mepyramine and zolantidine, respectively, affect the activity of dopaminergic and/or noradrenergic neurons suggest that histaminergic neurons do not tonically regulate the activity of cateCholaminergic neurons in the systems under study. .2888 ..080....0.0...0> 8.... Angev... 808...... .8850»... 0... ...... 0.8 00.8..-E2me .0. 002.... .... 0.8 w-.. 88.. 0025.... 00888 03.... ... 8.0.8.. «8.»... 82.88088 0280...... ... 0.....8 .0 2mm . 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H ...: 0.0 H «.0. ...0 H ...: 0.. H «... 0.0 H v... 0.200 808...... 5.00: ...n H 0.0.. .... H 0.0.. .0 H .02 0... H I: .... H 0.0: 0.« H ...««. 8.5.00 «.0 H «.0. .... H ..«n 0.. H 0... 0.. H 0.«n ... H 2.. 0.. H 0.0.. 05.8 0.0.0.00 ..0 H ...... 0.. H 0.8 0.0 H 0.2. .... H 0.2. 0... H 0.... ..« H «.00 8.0.88 83608.. ¢.« H 0.00 ...« H ..«v ....« H 0.«.. 0.. H ...... 0.. H 0.2 0.. H 0.«.. 9:8 30.82 4« 0 0 « . 0 :2"... 80... a8: 8.0.... ...-.0 a... 0...... ..0 .20.... 0...... 80...... ... 80.8.0808 008.80.. ...... qum ..0 E5... .0 38.... 8.000 00...... 0.... 0.00... 109 H. Summary and Conclusiom The effects of pharmacological and physiological manipulations which affect histaminergic neuronal transmission on catecholaminergic neuronal systems in the rat brain were examined. The conclusions of these studies are summarized below. 1 . Exogenous administration of histamine differentally affects the activity of the various dopaminergic neuronal systems. Exogenous histamine increases the activity of mesolimbic dopaminergic neurons via an action at H, receptors, but is without effect on nigrostriatal, tuberoinfundibular, periventricular-hypophysial, or incertohypothalamic dopaminergic neuronal activity. Exogenous histamine likewise increases the activity of dopaminergic neurons terminating in the PVN, SCN and cPeVN via an action at H, receptors. 2. Physical restraint decreases perivenu'icular-hypophysial, increases mesolimbic, and is without effect on tuberoinfundibular or nigrostriatal dopaminergic neuronal activity. Histaminergic neurons mediate stress-induced changes in periventricular-hypophysial, but not mesolimbic dopaminergic neuronal activity via an action at H, receptors. Histaminergic neurons do not mediate restraint stress-induced increases in aMSH secl'etion. 3- Neither H, receptor ligands nor blockade of histamine metabolism affect the activity 0f Catecholaminergic neurons in the systems under study. 110 4. Histaminergic neurons do not tonically regulate basal catecholaminergic neuronal activity in the systems under study. IV. ‘TAIWINERGIC REGULATION OF S-HYDROXYTRYPTAMINERGIC NEURON S A. Effects of Histamine on S-Hydroxytryptaminergic Neuronal Activity in the Rat Hypothalamus 1. Introduction ‘ Histamine has been implicated in a wide range of hypothalamic functions including the regulation of circadian rhythms, feeding behavior, water balance, body temperature, and pituitary hormone secretion (for reviews, see Hough, 1988; Schwartz et al. , 1991). The influence of 5-hydroxytryptaminergic neurons projecting to the hypothalamus in regulating each of these homeostatic functions is well established (Hoebel et al., 1989; Morin et al, 1990; Hillegaart, 1991; Van de Kar, 1991). It is not “Mable to suggest, therefore, that the ability of histamine to affect these functions might involve an interaction with S-hydroxytryptaminergic neurons. Although hundreds of papers have characterized the behavioral and physiological effects of histamine and histaminergic agents, very few have examined in vivo the effects 0“ S-hydroxytryptaminergic neuronal activity. Pilc and Nowak (1979) reported that e"08'~'=nous histamine administration increases 5HT release from the rat hypothalamus, although the histaminergic receptor subtype mediating this effect was not established and the possibility that histamine might displace 5HT stores could not be precluded. Little 1]] 112 is known regarding the effects on S-hydroxytryptaminergie neurons of agents which enhance endogenous histaminergic activity such as the histamine H, receptor antagonist thioperamide (Arrang et al. , 1987a) and the histamine-N-methyltransferase inhibitor metoprine (Duch et al. , 1978). Further investigation into the mechanism whereby exogenous and endogenous histamine might affect the activity of S-hydroxytryptaminergic neurons was thereby warranted. The purpose of this study was to investigate in male rats the effects of exogenous histamine and _of_ agents which enhance endogenous histaminergic transmission on the activity of S-hydroxytryptaminergic neurons projecting to the hypothalamus as estimated by neurochemical measurements of 5HT and its primary metabolite SHIAA in brain regions which contain terminals of these neurons. The results reveal that exogenous histamine has a stimulatory effect on hypothalamic S-hydroxytryptaminergic neurons through an action at histamine H, receptors. In contrast, compounds such as thioperamide and metoprine, which purportedly enhance endogenous histaminergic activity, do not affect the activity of S-hydroxytryptaminergic neurons. Overall, these results reveal that pharmacological manipulations that are commonly employed to study the actions of histamine in the brain differentially affect the activity of hypothalamic 5- hYdI'oxytl'yptaminergic neurons. 2- Results Results presented in Table 4.1 demonstrate that the i.c.v. administration of hiStamine dihydrochloride 30 min prior to decapitation decreased 5HT concentrations in the PVN, PeVN, SCN, DMN and ARC, and increased SHIAA concentrations in the 113 a 282.8 38.5.0629 88.. Anodvnc 220:5 350:2? 2a 85 88 8.856585% .8 829’ .... .38 a4. 88.. .8553 83E“... 2.3. 5 £28.. MEBS 23.3588 < 62.5383 2 3.2. so. on 3.3 ..esa o 2%.? 2:3 e 3.2 ..ea: 8 .e 2 .99 oecezoeiee assess 53 3&5 so; me. . mg a 8:. 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These data indicate that in the current in vivo model, histamine-induced decreases in 5HT concentrations are not mediated via the 5HT reuptake system. Early investigations into the receptor subtype whereby histamine might affect 5— hydroxytryptaminergic neuronal function have been inconclusive. Central administration of the histamine H, receptor agonist 4.methylhistamine decreased 5HT and increased SHIAA concentrations in the rat hypothalamus (Pile and Nowak, 1979; Nowak et al., 1980). These data were, however, difficult to interpret since the histamine H, receptor antagonist metiamide did not block i.c.v. histamine-induced decrease in 5HT concentrations (Pilc and Nowak, 1979). Further, the selectivity of 4-methylhistamine for histamine H, receptors is relatively low and central administration of the histamine H, receptor agonist dimaprit had no effect on S-hydroxytryptaminergic neuronal activity (Nowak et al., 1980). In the present study, pretreatment with the histamine H, receptor antagonist mepyramine prevented the histamine-induced decrease in 5HT and increase in SHIAA concentrations in the hypothalamus. By contrast, the histamine H, receptor antagonist zolantidine did not affect the ability of histamine to decrease and increase 5HT storage and metabolism, respectively. These data suggest that exogenous histamine increases the activity of S-hydroxytryptaminergic neurons through an action at H,, but not H, receptors. An alternative to examining the effects of exogenous histamine exploits agents which effectively enhance endogenous histaminergic activity. One such agent, the 124 histamineH,receptorantagonistthioperamide(Anangetal., l987a),actsviapresynaptic autoreceptors to increase histamine release (Garbarg et al., 1989;1toh et al., 1991; Mochizuki et al., 1991) and synthesis (Arrang et al., 1987a). A second agent, the histamine-N-methyltransferase inhibitor metoprine (Duch et al., 1978; Hough et al., 1986), increases extracellular histamine concentrations by preventing its metabolism. In thepresent study, thioperamide,administeredatadosereportedinviwtoeffect2t03- fold increases in hypothalamic histamine release (Itoh et al., 1991; Mochizuki et al., 1991), had noeffect on 5-hydroxytryptaminergic neuronal activity, thereby confirming a previousireport that thioperamide has no effect on the activity of S- hydroxytryptaminergic neurons at doses which affect the activity of histaminergic neurons (Oishi et al., 1990b). Neither metoprine, administered at a dose sufficient to increase extracellular histamine concentrations 2-fold (Duch et al., 1978; Itoh et al., 1991), nor combined administration of thioperamide with metoprine, a regimen which increases extracellular histamine content 4 to 8-fold (Itoh et al., 1991), affected 5- hydroxytryptaminergic activity. Hence, in contrast to the effects of exogenous histamine, agents which enhance endogenous histaminergic activity had no effect on 5- hydroxytryptaminergic neuronal activity in the hypothalamus. The disparity among the effects of exogenous histamine and agents which modify endogenous histamine concentrations may result from several factors. For instance, histamine concentrations attained at the synaptic level following thioperamide and/or metoprine treatment may be less than concentrations effected by i.c.v. histamine administration and consequently may be insufficient to activate S-hydroxytryptaminergic neurons. Alternatively, exogenous histamine could exert its stimulatory effect on 5- 125 hydroxytryptaminergic neurons by activating histaminergic receptors at sites that are distant from sites of histamine release and metabolism by these agents. Interpretation of datapertainingtothioperamideisfurtbercomplicated since histamineH,receptordensity may not be the same on all histaminergic neurons (Arrang et al., 1987a). Finally, the effects of thioperamide may not be confined to histamine H, autoreceptors since presynaptic H, heteroreceptors affecting 5111‘ release in vitro have been reported recently (Fink et al. , 1990). The disparity among the effects of pharmacological manipulations which mimic. _or_ enhance histaminergic transmission demonstrate the importance of assessing the effects of other manipulations which affect histaminergic neuronal activity (i.e., stress) on the activity of S-hydroxytr'yptaminergic neurons (see this Chapter, Section B). In conclusion, data presented in this Section reveal that central administration of histamine decreases 5HT and increases SHIAA concentrau'ons in rat hypothalamic nuclei. The histamine—induced reduction of 5HT concentrations is not associated with a 5HT reuptake system-mediated displacement of 5HT stores; instead, histamine increases the activity of S-hydroxytryptaminergic neurons through an action at histamine H, receptors. In contrast, neither the histamine H, receptor antagonist thioperamide, the histamine-N- methyltransferase inhibitor metoprine, nor combined thioperamide-metoprine treatment affected concentrations of 5HT or SHIAA suggesting that these agents, which effectively enhance endogenous histaminergic transmission, do not affect the activity of 5- hydroxytryptaminergic neurons. Overall, the results indicate that pharmacological manipulations such as administration of histamine, thioperamide or metoprine exert different effects on the activity of S-hydroxytryptaminergic neurons in the rat 126 hypothalamus. B. Histamine Mediates Restraint Stress-Induced Increases in the Activity of 5- Hydroxytryptaminergic Neurons I. Introduction Considerable attention has been directed towards examining the effects of stress on central S-hydroxytryptaminergic neuronal activity. Stressful stimuli increase the activity of these neurons as reflected by increases in 5HT synthesis, release and metabolism (i.e., Johnston et al., 1984; Adell et al., 1988; Pei et al., 1990; Inoue et al., 1993). Despite a wealth of evidence for stress increasing the activity of S-hydroxytrypt- aminergic neurons, central mechanisms facilitating these effects remain largely undefined. As discussed previously (Chapter III, Sections D and E), the activity of histaminergic neurons is increased in response to stressful stimuli. Since stress increases central histamine concentrations and, as described in this Chapter, Section A, exogenous administration of histamine increases the activity of central 5-hydroxytryptaminergic neurons, investigation into the possibility that histaminergic neurons might mediate stress- induced increases in S-hydroxytryptaminergic neuronal activity was warranted. As mentioned previously, the response of an animal to stress is dependent on the type of stressor. The purpose of the present study was to examine the role of histamine in mediating the effects of one particular stress, immobilization within a restraining tube, on the activity of S-hydroxytryptaminergic neurons projecting to the nucleus accumbens and SCN. These nuclei were selected because each is innervated by 5- hydroxytryptaminergic neurons (Azmitia and Segal, 1978; Steinbush, 1984), the activities 127 of which are increased in response to stress (Johnston et al., 1984; Johnston et al., 1985; Inoue et al. , 1993). The activity of S-hydroxytryptaminergic neurons was estimated by neurochemical measurements of the SET metabolite SHIAA in these nuclei. The results reveal that restraint stress increases the activity of central S-hydroxytryptaminergic neurons and that histaminergic neurons contribute to this increase via an action at H, receptors. 2. Results As discussed in Chapter III, a variety of stressful procedures of short duration increase histamine levels in the rat brain. Results presented in Figure 4.5 demonstrate that restraint of short duration can also affect the activity of S-hydroxytryptaminergic neurons. Specifically, 10, 20 or 30 min of physical restraint increased SHIAA concentrations but was without effect on 5HT concentrations in the nucleus accumbens and SCN. The shortest duration of restraint, 10 min, was employed in subsequent studies. Results presented in Figure 4.6 reveal that blockade of neuronal histamine synthesis with the histidine decarboxylase inhibitor aFMH (Garbarg et al., 1980), administered at a dose and time demonstrated to decrease central histamine release by 70% (Itoh et al. 1991), blocked the stress-induced increase in SHIAA concentrations in the nucleus accumbens and SCN. Similarly, H, receptor blockade by mepyramine antagonized the stress-induced increase in SHIAA concentrations in these brain regions (Figure 4.7). On the other hand, H, receptor blockade by zolantidine did not prevent the 128 stress-induced alterations in SHIAA in either the nucleus accumbens or SCN (Figure 4.8). Neither aFMH, mepyramine, nor zolantidine affected basal SHIAA concentrations in either the nucleus accumbens or SCN. 3. Discussion As discussed previously, several lines of evidence suggest that exogenous histamine can influence 5-hydroxytryptaminergic neuronal activity. In vitro, direct application of histamine increases 5HT release from rat brain synaptosomal (Tuomisto and Tuomisto, 1980) and cortical slice (Young et al., 1988) preparations. In vivo, exogenous administration of histamine or its agonists increases 5HT metabolism as well (File and Nowak, 1979; Nowak et al., 1980; see also this Chapter, Section A). Although these studies indicate that exogenous histamine can increase 5-hydroxytryptamincrgic neuronal activity, a role for endogenous histamine in affecting the activity of these neurons has yet to be established. To the contrary, pharmacological manipulations that affect endogenous histaminergic transmission by increasing histamine release (i.e., administration of the II, receptor antagonist thioperamide) or blocking histamine metabolism (i.e., administration of the histamine-N-methyltransferase inhibitor metoprine) have no effect on S-hydroxytryptaminergic neuronal activity (Oishi et al., 1990b; see also this Chapter, Section A). Since stress increases both histaminergic (sec Chapters 1 and Ill) and S-hydroxytryptaminergic neuronal activity, the possibility that endogenous histamine might mediate stress-induced increases in S-hydroxytryptaminergic 129 NUCLEUS ACCUMBENS SCN 8 8 ..eé -5 6 E z 4 4 .. E o 5HIAA g 2 _ a 5HT 2 _ o l l l J O L l J J 0 10 20 30 o 10 20 so MIN OF RESTRAINT Figure 4.5 Time course effects of physical restraint on SHIAA (O) and 5HT (0) concentrations in the nucleus accumbens and SCN of male rats. Rats were placed in restraining tubes for 10, 20 or 30 min, removed fromthe tubes, and then decapitated. Control rats were decapitated immediately upon removal from their home cages (zero time values). Symbols represent means and vertical lines 1 SEM of determinations in 6-8 rats; where vertical lines are not depicted, 1 SEM is less than the radius of the symbol. Solid symbols represent values that are significantly different from controls (p < 0.05). 130 NUCLEUS ACCUMBENS SCN '2 " [:3 contract. ‘0 " * ’e‘ ,3 - scanner '3 to - 8 _ E J. 2 a P s - ‘7 E '- J7 =1 ‘ _ a 5 4 - 3 z - 2 " :2 l0 0 o —S-A_L|NE :t-‘MH ETLINE and“ Figure 4.6 Effects of aFMH on basal and restraint stress-increased SHIAA concentrations in the nucleus accumbens and SCN of male rats. Rats were dceapitated immediately upon removal from their home eages (CONTROL) or placed in plastic restraining tubes for 10 min, removed from the tubes, and then decapitated (RESTRAINT). aFMH (100 mg/kg, i.p.) or saline (1 ml/kg, i.p.) was administered 4 h prior to decapitation. Columns represent means and vertical lines 1 SEM of determinations in 7-9 rats. ‘, values that are significantly different from controls (p<0.05). 131 NUCLEUS ACCUMBENS 12- ’c‘ .310- ...... '2 o. l- E 3.. \ O 5 4.- 1 E =- In °—— * SALINE [:3 CONTROL - arsrnmr HEPYRAMINE SCN EALINE MEPYRAHINE Figure 4.7 Effects of mepyramine on basal and restraint stress-increased SHIAA concentrations in the nucleus accumbens and SCN of male rats. Rats were decapitated immediately upon removal from their home cages (CONTROL) or placed in plastic restraining tubes for 10 min, removed from the tubes, and then dceapitated (RESTRAINT). Mepyramine maleate (Smg/kg, i.p.) or saline (1 ml/kg, i.p.) was administered 1 h prior to decapitation. Columns represent means and vertical lines 1 SEM of determinations in 8-9 rats. controls (p< 0.05). r, values that are significantly different from 132 NUCLEUS ACCUMBENS SCN '2 ’ C3 coarser. ‘° ’ - essraum * at: A as C 10 .- 3." * a - .o‘ ' 3. ‘ *- -T— .1. '7‘ a F" ‘ * \ ‘ ' 3‘ 4 _ V ‘ __ < S 2 a; 2 - ° 0 SALINE ZOLANTIDINE SAUNE ZOLANTIDINE Figure 4.8 Effects of zolantidine on basal and restraint stress-increased SHIAA concentrations in the nucleus accumbens and SCN of male rats. Rats were decapitated immediately upon removal from their home eages (CONTROL) or placed in plastic restraining tubes for 10 min, removed from the tubes, and then decapitated (RESTRAINT). Zolantidinc dimaleate (25 mg/kg, s.c.) or saline (1 ml/kg, s.c.) was administered 1 h prior to decapitation. Columns represent means and vertical lines 1 SEM of determinations in 7-8 rats. *, values that are significantly different from controls (p < 0.05). 133 neuronal activity warranted further attention. Results presented in this Section demonstrate that restraint stress increases the activity of central S-hydroxytryptaminergic neurons. These results are in agreement with numerous reports describing stress-induced increases in central 5HT synthesis, release and metabolism (Johnston et al., 1984; Adell et al., 1988; Pei et al., 1990; Inoue et al., 1993), and extend these findings by demonstrating a role for histaminergic neurons in mediating stress-induced increases in S-hydroxytryptaminergic neuronal activity. Specifically, both disruption of neuronal histamine synthesis by the irreversible histidine decarboxylase inhibitor aFMH and antagonism of H, receptors by mepyramine prevent stress-induced increases in SHIAA concentrations. These data indicate that histaminergic neurons mediate stress-induced increases in 5-hydroxytryptaminergic neuronal activity via an action at H, receptors. The finding that histaminergic neurons mediate stress-induced increases in 5- hydroxytryptaminergic neuronal activity is consistent with previous data (this Chapter, Section A) that exogenous administration of histamine increases the activity of these neurons. These data stand in contrast, however, to the lack of effect of pharmacological manipulations such as administration of thioperamide or metoprine which purportedly increase extracellular levels of histamine. As mentioned previously, the disparity among the effects of these various experimental manipulations may result from several factors. For instance, histamine concentrations attained at the synaptic level following thiOperamide and/or metoprine treatment may be less than concentrations effected by stress or i.c.v. histamine administration and consequently may be insufficient to activate S-hydroxytryptaminergic neurons. Moreover, the effects of thioperamide may not be 134 confined to histamine H, autoreceptors since presynaptic H, heteroreceptors affecting SHTreleaseinthhavebeenreportedrecently (Finketal., 1990). Thedisparity among the effects of exogenous histamine, stress, and pharmacological manipulations which purportedly facilitate histaminergic transmission demonstrate the need for emmoying a variety of approaches to the study of the central actions of histamine. In summary, histaminergic neurons mediate stress-induced increases in the activity of central S-hydroxytrytpaminergic neurons through an action at H, receptors. These data are consistent with previous reports of a H, receptor-mediated stimulatory effect of exogenous histamine on S-hydroxytryptaminergic neuronal activity (see this Chapter, section A), and demonstrate the importance of histaminergic neurons in the physiological response to stress. C. Histaminergic Neurons Do Not Regulate Basal S-Hydroxytryptaminergic Neuronal Activity Sections A and B in this Chapter have focussed on the effects of pharmacological and physiological manipulations which mimic, facilitate or increase histaminergic transmission (i.e., i.c.v. histamine, thioperamide or metoprine administration, stress) on the activity of central S-hydroxytryptaminergic neurons. Over the course of these experiments, it was demonstrated that neither the H, antagonist mepyramine nor the H, antagonist zolantidine affect basal S-hydroxytryptaminergic neuronal activity (Figures 4.3, 4.4, 4.7, 4.8) suggesting that histaminergic neurons do not tonically affect 5- hydroxytryptaminergic neuronal activity in the systems under study. The H, agonists R-a-methylhistamine and BP 2.94, compounds which decrease histamine release, have 135 virtually no'efl'ect on S-hydroxytryptaminergic neuronal activity (Tables 4.4 and 4.5) therebyfurtherindicatingalackoftonicregulation. Finally, thelackofeffectons- hydroxytypnmmagicnanmsofmehisfidinedecarboxyhsemhibimrmmigum 4.6, Table 4.6), examined at a time (4 h after administration) at which neuronal histamine synthesis is purportedly decreased by 70% (Itoh et al., 1991), suggests that histaminergic neurons do not tonically affect the activity of S-hydroxytryptaminergic neurons in the systems under study. Table 4.4 Effects of R-a-methylhistamine on SHIAA and 5111‘ concentrations in various hypothalamic nuclei of male rats Brain Region R-a-Methylhinmine (mg/kg) 0 5 10 20 PVN SHIAA 7.4 1 0.4 8.6 1 0.9 7.6 1 0.4 7.2 1 0.5 5HT 7.4 1 0.5 8.4 1 1.3 7.3 1 0.4 6.9 1 0.5 cPeVN SHIAA 7.0 :t: 0.4 5.9 1 0.3“ 5.4 :t 0.4“ 5.8 :t: 0.2‘I 5HT 6.8 1 0.2 6.1 1 0.3 5.9 1 0.3 6.4 1 0.1 ARC SI-IIAA 3.5 1 0.4 2.8 1 0.3 3.2 1 0.3 3.7 1 0.3 5HT 4.4 1 0.4 4.4 1 0.3 4.4 1 0.3 4.8 1 0.2 DMN SHIAA 6.2 1 0.3 5.7 1 0.3 5.9 1 0.3 5.3 1 0.4 5HT 6.1 1 0.2 5.7 :t 0.2 5.9 1 0.2 5.8 :t 0.4 R-a-methylhistamine (5, 10 or 20 mg/kg; p.o.) or distilled water (1 ml/kg; p.o.) was administered 2 h prior to decapitation. Values represent means 1 1 SEM of SHIAA and 5HT concentrations (rig/mg protein) in tissue samples obtained from 7-9 rats. ‘, values that are significantly different from controls (p< 0.05). 136 Table 4.5 Effects of BP 2.94 on SHIAA and 5HT concentrations in various hypothalamic nuclei ofmalerats Brain Regiai BP 2.94 (mg/kg) 0 5 10 20 PVN SHIAA 8.6 1 0.5 7.9 1 0.6 7.7 1 0.3 7.5 1 0.2 5HT 6.2 1 6.2 5.4 1 0.1 5.9 1 0.4 6.5 1 0.4 cPeVN SHIAA 7.7 1 0.2 9.1 1 0.4 7.8 1 0.5 8.4 1 0.5 5HT 9.0 1 0.4 9.1 1 0.4 10.0 1 0.3 10.6 1 0.6‘I SCN SHIAA 5.9 1 0.1 6.2 1 0.4 6.4 1 0.3 5.7 1 0.2 5111‘ 5.2 1 0.2 5.4 1 0.3 5.8 1 0.2 5.8 1 0.2 DMN - SHIAA 7.3 1 0.4 7.1 1 0.6 7.2 1 0.5 6.5 1 0.3 5HT 6.8 1 0.4 6.2 1 0.3 6.8 1 0.3 7.0 1 0.3 BP 2.94 (5, 10 or 20 mg/kg; p.o.) or 1% sodium carboxymethylcellulose (1 mllkg; p.o.) was administered 2 h prior to decapitation. Values represent means 1 1 SEM of SHIAA and 5HT concentrations (ng/mg protein) in tissue samples obtained from 7-9 rats. ‘, values that are significantly different from controls (p< 0.05). Table 4.6 Effects of aFMH on SHIAA and 5HT concentrations in various hypothalamic nuclei of male rats Brain Region saline aFMH (i.p.) aFMH (s.c.) PVN SHIAA 8.0 1 0.4 8.7 1 0.6 9.0 1 0.5 5HT 7.1 1 0.3 7.4 1 0.2 7.7 1 0.3 cPeVN SHIAA 7.7 1 0.2 7.2 1 0.4 6.7 1 0.3 5HT 7.8 1 0.4 7.5 1 0.3 7.1 1 0.1 SCN SHIAA 6.7 1 0.3 6.0 1 0.2 5.9 1 0.2 5HT 7.3 1 0.3 6.9 1 0.4 6.9 1 0.4 DMN SHIAA 9.0 1 0.6 8.0 1 0.3 7.5 1 0.5 5111' 9.6 1 0.6 8.0 1 0.3“ 7.7 1 0.2"l aFMI-I (100 mglkg, i.p. or s.c.) or saline (1 ml/kg; i.p.) was administered 4 h prior to decapitation. Values represent means 1 1 SEM of SHIAA and 5HT concentrations (rig/mg protein) in tissue samples obtained from 7-9 rats. *, values that are significantly different from controls (p< 0.05). 137 D. Summary and Conclusions The efiects of pharmacological and physiological manipulations which affect histaminergic neuronal transmission on S-hydroxytryptaminergic neuronal systems were examined. The conclusions of these studies are summarized below. 1. Exogenous administration of histamine increases the activity of 5- hydroxytryptarninergic neurons through an action at histamine H,, but not H,, receptors. 2. Neither H, receptor ligands nor blockade of histamine metabolism affect the activity of S-hydroxytryptaminergic neurons in the systems under study. 3. Physical restraint increases S-hydroxytryptaminergic neuronal activity; histaminergic neurons mediate these increases via an action at H, receptors. CHAPTER V: HISTAMINERGIC REGULATION OF PROLACTIN AND ME SECRETION A. Hktarnine-Stimulated Prolactin Secretion 3 Not Mediated by an Inhibition of Tuberoinfundibular Dopaminergic Neurons I. Introduction Histamine dose-dependently stimulates prolactin secretion when administered to human males (Knigge et al., 1986b) or rats of either gender (Gibbs et al., 1979; Knigge et al., 1986a). The amine has no direct effect upon the anterior pituitary (Libertun and McCann, 1976; Rivier and Vale, 1977) but instead acts indirectly at the hypothalamic level to stimulate prolactin release (Alvarez and Donoso, 1981; Tuominen et al., 1991). The mechanism whereby histamine exerts this effect is complex and controversial (for review, see Schwartz et al., 1991); several transmitters including 5HT (Knigge et al., 1988c), arginine vasopressin (Kjatr et al., 1991) and dopamine (Knigge et al., 1986b; Knigge et al. , 1988b) reportedly mediate histamine-induced prolactin secretion. Considerable attention has focussed on the role of tuberoinfundibular dopaminergic neurons in histamine-stimulated prolactin secretion (Knigge et al., 1986b; Gibbs et al., 1979; Knigge et al., 1988b). Tuberoinfundibular dopaminergic neurons terminating in the median eminence tonically inhibit prolactin secretion from lactotrophs of the anterior pituitary (Gudelsky, 1981). Pharmacological agents which inhibit dopamine synthesis and release (i.e. a-methyltyrosine or NSD 1015) or block dopamine 138 139 receptors on pituitary lactotrophs (i.e. the dopaminergic receptor antagonist haloperidol) increase circulating levels of prolactin (for review, see Ben-Jonathan et al., 1989, Moore, 1987). Since blockade of dopamine receptors inhibits the prolactin-releasing effect of histamine (Knigge et al., 1988b) and histamine reduces dopamine release into the pituitary portal blood (Gibbs et al., 1979; Knigge et al., 1988b), the prolactin-releasing effect of histamine could be mediated by removal of the tonic inhibitory action of tuberoinfundibular dopaminergic neurons on pituitary lactotrophs. The purpose of this study was to determine if the stimulatory effect of i.c.v. histamine injection on prolactin secretion involves an inhibition of tuberoinfundibular dopaminergic neuronal activity. The activity of these neurons was estimated by measuring the rate of dopamine synthesis [the accumulation of DOPA after administration of NSD 1015] and metabolism [concentrations of DOPAC] in the median eminence of male rats. The results reveal that central histamine administration stimulates prolactin secretion by a mechanism independent any change in the activity of tuberoinfundibular dopaminergic neurons. 2. Results Results presented in Figure 5.1 demonstrate that i.c.v. injection of histamine dihydrochloride 30 min prior to decapitation caused a dose-related increase in plasma prolactin concentrations in male rats with the highest dose (30 rig/rat) causing an 8-fold increase. In contrast, even the highest dose of histamine was without effect on the accumulation of DOPA or the concentrations of dopamine or DOPAC in the median eminence (Table 3.1 and Figure 5.1). 140 A B 120 ’° " new A PROLACTIN en E is - 5 '2 O 8 *5 to E 10 - 2 ' E E ° F u DOPAMINE E 5 . g , _ o DOPAC r: */ 2 __ O l l l 1 1 J O l l l 1 L l O 15 30 O 15 30 HISTAMINE (pg/rot) 1a- 14- .512- D ‘5 tdfiM L Q .- o i 0" oDOPA as ‘_ C 2!— o A A l L L_] 0 15 30 HISTAMINE (pg/rot) Figure 5.1 Dose-response effects of histamine on concentrations of prolactin in the plasma and DOPA. DOPAC and dopamine in the median eminence of male rats. Histamine dihydrochloride (3.8. 15, or 30 ug/rat) or its saline vehicle (3 ill/rat) was injected i.c.v. 30 min prior to decapitation. DOPA accumulation was determined in rats injected with NSD 1015 (100 mg/kg; i.p.) 30 min prior to decapitation. Symbols represent means and vertical lines 1 SEM of determinations in 7-8 rats. Solid symbols represent values for histamine-treated rats that are significantly different from vehicle- treated rats (p<0.05). 141 ‘I'hetimecourseoftheeffectsofasinglei.c.vinjectionofhistamine(30pg/rat) on plasma concentrations of prolactin and DOPAC concentrations in the median eminence is depicted in Figure 5.2. Histamine caused a rapid (within 15 min) and short-lasting (less than 60 min) increase in circulating levels of prolactin. Throughout this period, histamine had no effect on DOPAC concentrations in the median eminence. The results ofthesedose-andtime-coursesmdiessuggestthathistamineincreases thesecretionof prolactin through a mechanism that is independent of any change in tuberoinfundibular dopaminergic neuronal activity. 3. Discussion Central histamine infusion causes a rapid and short-lasting increase in circulating prolactin levels. In the present study, the dose used (30 uglrat) is comparable and the time required to reach maximal prolactin levels (less than 15 min) is identical to doses and intervals reported previously (Libertun and McCann, 1976; Knigge et al., 1986a; Seltzer and Donoso, 1986; Knigge et al., 1988b). Consistent with previous findings, the duration of enhanced prolactin secretion is short. Although plasma prolactin levels have been reported to be elevated as long as 90 min following histamine administration (Knigge et al., 1986a), in the present and in previous studies (Gibbs et al., 1979; Seltzer and Donoso, 1986), increased circulating prolactin levels do not persist beyond 60 min. Although numerous studies have demonstrated that central administration of histamine stimulates prolactin secretion, the mechanism whereby histamine exerts this effect remains uncertain. Prolactin secretion is controlled predominantly, in a tonic 142 140 40 - ‘28 o A PROLACTIN '80 c 10 ’ of 8 I E. a D §\§/§——§ _ 2° '- 0! I r- E E , __ c1 DOPAMINE E ,0 g o DOPAC C 2 .— 0 J l l l l l l l l l 14 o l A! L l l l L l l l J 0 so so so 120 0 so so so 120 MIN AFTER HISTAMINE Figure 5.2 Time course effects of histamine on concentrations of prolactin in the plasma and DOPAC and dopamine in the median eminence of male rats. Rats were decapitated 15 , 30, 60 or 120 min after i.c.v. injection of histamine dihydrochloride (30 ug/rat), or 15 min after i.c.v. injection of saline vehicle (3 ul/rat; zero time values). Symbols represent means and vertical lines 1 SEM of determinations in 7-8 rats. Solid symbols represent values for histamine-treated rats that are significantly different from vehicle- treated rats (p<0.05). 01 M in hi: CO 143 inhibitory fashion, by dopamine released from tuberoinfundibular dopaminergic neurons into the hypophysial portal blood (Gudelsky, 1981). It has been suggested that histamine might stimulate prolactin secretion through an inhibition of these neurons (Knigge et al. , 1986b; Knigge et al., 1988b). The present results indicate, however, that the increase in plasma prolactin levels following i.c.v. histamine is not accompanied by a decrease in tuberoinfundibular dopaminergic neuronal activity; neither the metabolism (DOPAC concentration) nor the synthesis (DOPA accumulation) of dopamine in the median eminence is altered following i.c.v. histamine. Although central administration of histamine is reported to decrease dopamine levels in the portal blood (Gibbs et al. , 1979; Knigge et al., 1988b), these decreases are insufficient to account for the large increases in prolactin secretion following i.c.v. histamine (Gibbs et al., 1979). Furthermore, previous studies have demonstrated that central administration of histamine at a dose comparable to that used in the present study did not affect dopamine turnover in the median eminence (Seltzer and Donoso, 1986). These results are in agreement with the conclusion of the present study that histamine-stimulated prolactin secretion is not mediated by changes in tuberoinfundibular dopaminergic neuronal activity. Since histamine has no effect on either tuberoinfundibular dapaminergic neurons or the anterior pituitary, it is likely that histamine-stimulated prolactin secretion is mediated through the release of one or more prolactin releasing factors (Samson and Mogg, 1989). Histamine effects prolactin secretion through the activation of nerve tracts in the rostral hypothalamus since anterolateral hypothalamic deafferentation inhibits histamine-induced prolactin secretion (Tuominen et al., 1991). These nerve tracts may consist of or be regulated by 5HT neurons since 5HT receptor antagonists partially block Not of F nCU' SlllC or 1 San thlt Unl to hi: do of 144 histamine—stirnulated prolactin secretion (Knigge et al., 1988c). Arginine vasopressin maydwbemvolvedsincehismminesfimmatesvasopresnnrdeaseinmtsmogtaom et al., 1976) and antibodies against vasopressin attenuate histamine-induced prolactin secretion (chr et al., 1991). The mechanism by which histamine regulates prolactin secretion is undoubtedly complicated, but it can be concluded from the present study that the complex mechanism whereby centrally administered histamine stimulates prolactin secretion does not involve an inhibition of tuberoinfundibular dopaminergic neurons. B. Pharmacological Manipulations Which Alter Histaminergic Transmission May Not Affect Prolactin Secretion IneachofthestudiesdescribedinChaptersIIIanlepertainingtotheeffects of pharmacological manipulations which alter histaminergic transmission on aminergic neuronal activity, effects on prolactin secretion were examined concurrently. In several studies, manipulations which facilitate histaminergic transmission such as thioperamide or metoprine administration had no effect on prolactin secretion; in other studies, these same compounds increased prolactin secretion. The reason behind the inability of thioperamide and metoprine, respectively, to consistently increase prolactin secretion is unknown, but may result from extracellular histamine concentrations afforded by these two compounds being less than those effected by i.c.v. histamine and hence insufficient to consistently stimulate prolactin secretion. Consistent with the finding that i.c.v. histamine—induced increases in prolactin secretion are not mediated by tuberoinfundibular dopaminergic neurons (see this Chapter, Section A), any thioperamide- or metoprine- induced increases in prolactin secretion were not accompanied by decreases in the activity of tuberoinfundibular dopaminergic neurons (Tables 5.1 and 5.2). .. hhdam 145 Table5.1Doserespmseeffectsofthioperamideonplasmapmlacdnandmedian eminenceDOPACanddopamineconcentrationsinmalerats. Thioperamide (mg/kg) 0 2.5 s 10 prolactin 8.7 1 1.4 9.4 1 1.4 18.9 1 315* 8.0 1 0.9 DOPAC 12.2 1 0.3 14.0 1 0.8 12.4 1 0.6 9.8 1 0.4-1 dopamine 93.8 1 5.3 99.3 1 5.8 90.5 1 4.7 91.9 1 4.8 Thioperamide maleate (2.5, 5 or 10 mg free base/kg, i.p.) or saline (1 ml/kg, i.p.) was ft '1'- administered _1_ h prior to decapitation. Values represent means 11SEM of determinations in 7-9 rats. *, values for thioperamide-treated rats that are significantly different from controls (p<0.05). , .. Table 5 .2 Dose response effects of metoprine on plasma prolactin and median eminence DOPAC and dopamine concentrations in male rats. Metoprine (mg/kg) 0 1 ~ ' 5 10 prolactin 3.1 1 0.8 3.9 1 1.1 5.7 1 2.0 12.0 1 3.2“ DOPAC 9.6 1 1.2 11.0 1 0.7 9.5 1 0.6 11.7 1 1.0 dopamine 112.8 1 6.7 83.0 1 5.6 105.5 1 12.3 110.2 1 6.5 Metoprine(1, 5 or 10 mglkg, i.p.) or 1% sodium carboxymethylcellulose (1 ml/kg, i.p.) was administered 1 h prior to decapitation. Values represent means 1 1 SEM of determinations in 7-8 rats. ', values for metoprine-treated rats that are significantly different from controls (p<0.05). E53. Tabl: prolz 146 Procedures which disrupt histaminergic transmission such as administration of «M, mepyramine, zolantidine, BP 2.94 or R-a-methylhistamine, had no effect on basal prolactin secretion (Figure 5.4, Table 5.3). Table 5.3 Dose response effects of R-a—methylhistamine and BP 2.94 on plasma prolactin concentrations in male rats R-a-methylhistamine or BP 2.94 (mg/kg) 0 5 10 20 Roar-methylhistamine 13.5 :1: 4.3 16.4 1 6.6 18.1 1 6.4 14.6 1 4.6 BP 2.94 14.5 1 5.1 24.0 1 5.1 19.0 1 3.7 18.8 1 6.6 R-a-methylhistamine (5, 10 or 20 mglkg, p.o.), BP 2.94 (5, 10 or 20 mglkg, p.o) or vehicle (distilled water or 1% sodium carboxymethylcellulose, 1 ml/kg, p.o.) was administered 2 h prior to decapitation. Values represent means 1 1 SEM of prolactin concentrations (ng/ml plasma) in samples obtained from 8-9 rats. 147 C.CentanistamineDouNotMediateaMSHSeu'etion IneachofdrestudiesdeseibedinChaptesHIandIVpertainingtodieeffects of histamine on catecholaminergic and 5-hydroxytryptaminergic neuronal activity, effects on aMSH secretion wee examined concurrently. Neither ceitral administration of histamine, blockade of histamine metabolism with metoprine, nor ethancement of histamine release with thioperamide affected aMSH secretion in these studies. Procedures which disrupted histaminergic transmission such as administration of aFMI-I, mepyramine, zolantidine, or R-a-methylhistamine, similarly did not affect the secretion of this hormone (Figure 3.20, Table 5.4). Histaminergic neurons do not mediate stress- induced increases in aMSH secretion (Chapter III, section G). In summary, no evidence forhistamineasaregulatorofaMSH secretionwasfound. Table 5.4 Dose response effects of R-a-methylhistamine and BP 2.94 on plasma aMSH conceitrations in male rats R-a-methylhistamine or BP 2.94 (mg/kg) 0 5 10 20 R-a-rnethylhistamine 161.6 1 10.9 154.9 1 12.2 163.7 1 12.2 167.8 1 15.3 HP 2.94 96.7 1 8.5 100.9 1 8.57 107.7 1 16.1 108.4 1 9.2 R-a-methylhistamine (5, 10 or 20 mglkg, p.o.), BP 2.94 (5, 10 or 20 mglkg, p.o) or vehicle (distilled water or 1% sodium carboxymethylcellulose, 1 ml/kg, p.o.) was administered 2 h prior to decapitation. Values represent means 1 1 SEM of aMSH concentrations (ng ml plasma) in samples obtained from 9 rats. 148 D.HktamineDoesNotMedhteReshaintSfiw-InducedlnereasesinProlactinor aMSH Secretion 1. Introduction A variety of stressful stimuli increase the secretion of prolactin and aMSH, respectively. Histaminergic neurons reportedly mediate these increases when resulting from thestress ofether exposureorS min supinerestraint (Alvarez, 1982; Kniggeetal., 1988a; Knigge et al., 1991; See-Jersei et al., 1993). The role of histamine in mediating pituitary hormonal responses to other stressors has not beer well defined. The purpose of this experimeit was to examine the role of histamine in mediating the effects of the stress of immobilization within a restraining tube on the secretion of prolactin and aMSH, respectively. The results reveal that histaminergic neurons do not mediate restraint stress-induced increases in prolactin or aMSH secretion. 2. Results Results presented in Figure 5 .3 revesl that 10, 20 or 30 min of physical restraint increased plasma prolactin and aMSH concentrations, respectively. As was shown in Figure 3.20, pretreatment with either aFMH, mepyramine or zolantidine affected neither basal aMSH concentrations nor prevented the stress-induced increase in aMSH secretion. Similarly, stress-induced increases in prolactin concentrations were not prevented by either aFMI-I, mepyramine or zolantidine treatmeit (Figure 5.4). 1 ( l ‘ c <\ (‘M f 149 PROLACTIN aMSH 70 - 500 60 - o.." 'I‘ O 400 S 50 ~ g 2 4o— 2 300 n. _ Q E 3°: E 200 E 20 - E C 10: ‘1 too o P l l #J O l l l J O 10 20 30 o 10 20 :50 MIN OF RESTRAINT Figure 5.3 Time course effects of physical restraint on prolactin and aMSH concentrations in the plasma of male rats. Rats were placed in restraining tubes for 10, 20 or 30 min, removed from the tubes, and then decapitated. Control rats were decapitated immediately upon removal from their home cages (zero time values). Symbols represent the means and vertical lines 1 SEM of determinations in 6-8 rats. Solid symbols represent values for stressed rats that are significantly different (p<0.05) from control rats. 21> prolactin (ng/ml plasma) 150 > U) 0 " ' comet murmur * prolactin (rig/ml plasma) ‘ 0 SALINE INN “LINE “WINE SALINE ZOLANTIDINE Figure 5.4 Effects of aFMH, mepyramine and zolantidine on basal and restraint stress- affected prolactin concentrations in the plasma of male rats. Rats were decapitated immediately upon removal from their home cages (CONTROL) or placed within plastic restraining tubes for 10 min, removed from the tubes, and then decapitated (RESTRAINT). A) aFMH (100 mglkg, i.p.) or saline (1 mllkg, i.p.) was administered 4 h prior to decapitation. B,C) Mepyramine malmte (5 mglkg, i.p.), zolantidine dimaleate (25 mglkg, s.c.) or saline (1 mllkg, i.p. or s.c.) was administered 1 h prior to decapitation. Columns represent means and vertical lines 1 SEM of determinations in 7-9 rats. ', values for stressed rats that are significantly different (p<0.05) from controls. 3. D1 nor 1 SCCII that Sinc I651 19% C01 1121 HE di 151 3. Discussion Results from the present study reveal that neither depletion of neuronal histamine nor blockade of post-synaptic histaminergic receptors prevents the increased prolactin secretion associated with the presently employed model of stress. These data indicate that histaminergic neurons do not mediate restraint stress-induced secretion of prolactin. Since tuberoinfundibular dopaminergic neuronal activity is not altered by physical restraint (Figure 3.16; Lookingland et al., 1990), it is likely that the increased prolactin secretion is mediated by one or more prolactin releasing factors (Samson and Mogg, 1989). The identity of this factor is unknown. Its release, however, is probably not controlled by S-hydroxytryptaminergic neurons, since disruption of histaminergic transmission prevents restraint stress-induced increases in S-hydroxytryptaminergic neuronal activity (Chapter IV). As discussed previously (Chapter 111, Section E), the results presented in this dissertation reveal that histaminergic neurons do not mediate the stress-induced increase in aMSH secretion associated with 10 min tube restraint; it is likely that B-adrenergic receptor activation mediates this increase. E. Summary and Conclusions The effects of pharmacological and physiological manipulations which affect histaminergic neuronal transmission were examined. The conclusions of these studies are summarized below. 1. Exogenous administration of histamine increases prolactin secretion, an effect that is not mediated by an inhibition of tuberoinfundibular dopaminergic neurons. Other 152 pharmacological manipulations which erhance or disrupt histaminergic transmission have inconsistent effects on prolactin secretion. 2. Pharmacological manipulations that affect central histamine concentrations or transmission do not affect aMSH secretion. 3. Histaminergic neurons do not mediate restraint stress-induced increases in prolactin or arMSH secretion. VI. CONCLUDING DISCUSSION The effects of histamine on the activities of selected catecholaminergic and 5- hydroxytryptaminergic neuronal systems have beer evaluated. As summarized in Tables 6.1 and 6.2, the studies within this dissertation have demonstrated that histamine can increase the'activity of noradreiergic, 5-hydroxytryptaminergic, and certain dopaminergic neuronal systems, as evidenced by the finding that exogeious administration of histamine increases the synthesis and/or metabolism of norepinephrine, dopamine and 5HT, respectively, in brain regions containing these neurons. Of greater significance, however, is the finding that effects of histamine on catecholaminergic and 5- hydroxytryptaminergic neurons are apparent not only after the pharmacological manipulation of exogenous histamine administration, but also as a component of the physiological response to stress. Specifically, histaminergic neurons mediate stress- induced decreases in periventricular-hypophysial dopaminergic neuronal activity and increases in noradrenergic and S-hydroxytryptaminergic neuronal activity; these findings are summarized in Table 6.3. Striking similarities exist between effects of histamine following exogetous administration and its mediatory role in the stress-response. In both cases, histamine increases noradrenergic and 5-hydroxyuyptaminergic neuronal activity via an action at H, receptors. Since histaminergic, noradrenergic and 5-hydroxytryptaminergic neurons 153 154 Table 6.1 Summary of effects of i.c.v. histamine administration on the activity of selectedceitraldopaminergicneuronalsystemsintherat Dopaminergic Effect of i.c.v. histamine administration‘ ‘ neuronal system ; , mesolimbic 1 activity, H, 1 ‘ nigrostriatal no effect ‘ [tuberoinfundibular no effect I periventricular-hypophysial no effect I incertohypothalamic (caudal) no effect ’ undefined“ 1 activity, H, L (PVN, SCN, cPeN) ' see text, Chapter III, for details Table 6.2 Summary of effects of i.c.v. histamine administration on the activity of hypothalamic 5-hydroxytryptaminergic and noradrenergic neuronal systems in the rat ' Aminergic Effect of i.c.v. histamine administration' neuronal system 5-hydroxytryptaminergic ° see text, Chapters III and IV, for details 155 23% .8 .>_ c5 E 6.95 is 8» . .3, .8535 55.8 « 03:05:.5eb3xe6bmn .: 6.5—00.: .9550: e . 0530:0505: .: .853:— 553 a fi_m>.._ec§:-:a_:0§:0>t0e 05: 55.00 6 035.82: 59055850 , .2950: 590555 .«c 0.3— .305 .5830: we sootm 80?? .223: 5905:}. .E 05 5 ”E03? 3:050: 590585eb§x215n 3: 59055220200 35:00 300.8 Co 5.60: 05 5 8m:£0 “000505-305... 9200...? 5 82:0: 03:05.:sz ..e 0.2 ..c SE85 «.0 2%.: haw 156 have been implicated in regulating a variety of functions ranging from neuroendocrine secretion to sleep-wakefulness (see Chapters III and IV), an appreciation of histamine- regulated noradrenergic and 5-hydroxytryptaminergic neuronal activity is of importance. On the other hand, significant differences are observed between effects of exogenous histamine and its role in stress-affected dopaminergic neuronal activity. As mentioned in Chapter III, i.c.v. histamine increases mesolimbic dopaminergic neuronal activity whereas disruption of histaminergic transmission does not prevent stress-induced increases in the activity of these neurons. These data suggest that either histaminergic neurons do- not effect stress-induced increases in mesolimbic dopaminergic neuronal activity or that other factors, in addition to histamine, cause these increases such that disruption of histaminergic neuronal activity per se is not sufficient to prevent increased mesolimbic dopaminergic neuronal activity. Furthermore, i.c.v. histamine does not affect periventricular-hypophysial dopaminergic neuronal activity whereas histaminergic neurons mediate stress-induced decreases in the activity of these neurons. As eluded to in Chapter 111, this discrepancy may be related to differences in histamine levels effected by i.c.v. administration versus stress. The role of histaminergic neurons in mediating stress-induced decreases in periventricular dopaminergic neuronal activity is of interest in light of recent data pertaining to afferent regulation of the latter. Exposure to stressful stimuli increases 5- hydroxytryptaminergic neuronal activity (Table 6.3). S-Hydroxytryptaminergic neurons are involved in stress-induced decreases in periventricular-hypophysial dopaminergic neuronal activity as evidenced by the finding that either destruction of 5- hydroxytryptaminergic neurons with 5 ,7-dihydroxytryptamine, blockade of post-synaptic 157 SET, receptors, or activation of 5HT autoreceptors with 8-hydroxy-2-(di-n-propylamino)— tetralin prevents this decrease (Goudreau et al., 1993). GABA inhibitory intemeurons may mediate this “stress - S-hydroxytryptaminergic - periventricular-hypophysial dopaminergic” pathway, since administration of the GABA, antagonist 2-hydroxysaclofen blocks both stress- and 5HT, receptor agonist-induced decreases in periventricular dopaminergic neuronal activity (Goudreau, Wagner, Lookingland and Moore, unpublished). Since disruption of histaminergic neuronal transmission (i.e., with the H, receptor antagonist mepyramine or the histidine decarboxylase inhibitor aFMl-I) prevents both stress-induced increases in S-hydroxytryptaminergic and decreases in periventricular- hypophysial dopaminergic neuronal activity, it is not unreasonable to suggest that histaminergic, S-hydroxytryptaminergic, GABAergic and periventricular hypophysial- dopaminergic neurons may be arranged in series. More specifically, stress may increase the activity of histaminergic neurons thereby increasing S-hydroxytryptaminergic neuronal activity; this latter event might cause an activation of inhibitory GABAergic neurons resulting in a decrease in periventricular-hypophysial dopaminergic neuronal activity. Alternatively, histaminergic, S-hydroxytryptaminergic and GABAergic neurons might act independently to decrease periventricular-hypophysial. dopaminergic neuronal activity. Further investigation is necessary to better define the role of histaminergic neurons in the afferent regulation of periventricular-hypophysial dopaminergic neuronal activity. In conclusion, data presented within this dissertation indicate that histaminergic neurons can affect the activity of central catecholaminergic and S-hydroxytryptaminergic neurons. 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