LOCALISATION AND MODULATION OF GABA b AND 5-HT3 RECEPTORS IN RODENT BRAIN: AN AUTORADIOGRAPHIC STUDY Gerard David Pratt B.Sc. A thesis submitted to the University of London in part fulfilment of the requirements for the degree of Doctor of Philosophy Department of Pharmacology, The School of Pharmacy, 29/39, Brunswick Square, LONDON, WC1N 1AX. May 1991 ProQuest Number: U050912 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest U050912 Published by ProQuest LLC(2017). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 2 ABSTRACT Autoradiographical visualisation of GABA b binding sites in sections of rat brain has extended the findings of previous reports in that within the frontal cortex, the distribution of such sites is clearly laminated into four distinct regions. Furthermore, GABA b binding sites have also been identified in the dorsal vagal complex of the brainstem, especially within the nucleus tractus solitarius (NTS). In parallel with these GABAergic medullary receptors, detailed analysis of the autoradiography of the 5-HT3 receptor radioligand, [3H]BRL43694 (as well as [3H]zacopride, [3H]GR65630 and [3H]quipazine) has also been investigated within this region. Using histological markers in conjunction with autoradiography, 5-HT3 binding sites were localised in the area postrema, dorsal motor vagal nucleus and the nucleus of the spinal tract of the trigeminal nerve. However, by far the highest concentration of sites was observed in the NTS. As the NTS/dorsal vagal complex provides the site of termination for the majority of vagal afferent fibres, 5-HT3, GABA b and GABA a receptor autoradiography was examined 10 days after vagal lesioning achieved via chronic unilateral nodose ganglionectomy. Since all three binding site categories were reduced ipsilaterally in the dorsal vagal complex, this suggested that such sites are likely to be located presynaptically on vagal afferent fibres that terminate in this region. Conflicting and inconsistent reports regarding GABA b receptor modulation by chronic antidepressant treatment prompted the use of receptor autoradiography (restricted to the sub-laminal regions of the frontal cortex) in an attempt to resolve this issue. Administration of imipramine (14 days) either orally or via subcutaneously implanted osmotic minipumps failed to increase GABA b receptor numbers in this region. In contrast, chronic oral and i.p. administration (21 days) of desipramine 3 significantly up-regulated GABA b receptors in lamina I of the frontal cortex with a concomitant beta-adrenoceptor down-regulation. Repeated adminstration of amitriptyline, paroxetine or the centrally-active GABA b receptor antagonist, CGP 35348 was ineffective at modulating GABA b receptors. Conversely, another putative GABA b receptor antagonist, designated as Compound X, like desipramine, also increased GABA b receptor densities in the frontal cortex, but again only in lamina I. Despite the increase in GABA b receptor numbers produced by despramine, this drug, like the majority of the above treatment regimes, failed to increase the sensitivities of the GABA b receptor-modulation of forskolin- and noradrenaline-stimulated adenylyl cyclase activity. However, treatment with Compound X produced an apparent enhancement of the GABA b receptor-mediated inhibition of forskolin-stimulated adenylyl cyclase. The phosphonic acid derivative of GABA, 3-aminopropylphosphinic acid (3-APA), has been characterised as a GABA b receptor agonist. Whilst it is 10 times more potent than (-)-baclofen at displacing [3H]GABA from GABA b binding sites, it is equipotent with (-)-baclofen at inhibiting forskolin-stimulated adenylyl cyclase. Conversely, whilst (-)-baclofen is a full agonist with respect to the noradrenaline-stimulated system, 3-APA appeared to act as a partial agonist in this response. These contrasting findings may be indices of the possible existence of GABA b receptor subtypes. 4 CONTENTS ABSTRACT 2 List of figures 10 List of tables 13 Abbreviations 15 Publications arising from this thesis 18 Acknowledgements 19 CHAPTER 1 General Introduction 20 GABA as a neurotransmitter - historical perspective 21 Synthesis and distribution 21 Release 22 Uptake, inactivation and metabolism 22 GABA as an inhibitory neurotransmitter 23 The heterogeneity of GABA receptors 24 GABA a receptors 25 GABA b receptors 27 Autoradiographical receptor localisation 29 Characteristics of GABA b receptor activation 31 Electrophysiological studies 31 a) Calcium current modulation by GABA b receptors 31 b) Postsynaptic hippocampal actions 33 c) Presynaptic hippocampal actions 35 Second messenger systems 35 Evidence in favour of possible GABA b receptor heterogeneity 38 GABA b receptor antagonists 40 Aspects of the pharmacology of antidepressant drugs 45 Functional changes in beta-adrenoceptors following chronic antidepressant administration 46 5 Functional changes in serotonergic receptors following chronic antidepressant administration 47 The involvement of GABAergic mechanisms in depression 49 a) Clinical investigations 49 b) The role of GABA in animal models of depression 49 c) GABA b receptor-modulation following chronic antidepressant administration 50 Gastric motility and GABA b receptors 51 The evolution of 5-HT3 receptor antagonists from gastric motility stimulants 55 5-HT3 receptor antagonists 57 5-HT3 receptors and the central nervous system 59 Aims of the thesis 64 CHAPTER 2 General Methodology 66 Autoradiographical procedures 67 Tissue preparation 67 Localisation of GABA a and GABA b binding sites 67 a) Saturation analysis 68 b) Analysis of binding data 68 Localisation of beta-adrenoceptor binding sites 70 Localisation of 5-HT3 binding sites 70 Autoradiographical analysis 71 Membrane binding procedures 72 Preparation of whole brain synaptic membranes 72 Determination of GABA a and GABA b binding sites 72 Expression of specific radioligand binding 73 6 Adenylyl cyclase studies 73 Tissue preparation and adenylyl cyclase activation 73 Determination of cAMP concentrations 74 a) Preparation of cAMP binding protein 74 b) cAMP radioimmunoassay 75 Protein concentration determination 75 Radioligands, drugs and chemicals 76 Sources of radioligands 76 Sources of drugs and chemicals 77 CHAPTER 3 Localisation of central GABA receptors, beta-adrenoceptors and 5-HT3 receptors using receptor autoradiography 78 Introduction 79 Results 79 Autoradiographical localisation of GABA receptors 79 Characterisation of GABA b receptor agonists and antagonists in brain tissue 80 Characterisation of (-)-[125I]iodopindolol binding to beta- adrenoceptors in rat cortex 81 Colocalisation of GABA b and beta-adrenoceptor binding sites in rat frontal cortex 83 Localisation of [3H]BRL 43694 binding sites in rat hindbrain 83 Saturation analysis of [3H]BRL 43694 binding in the NTS 85 Localisation of [3H]zacopride, [3H]GR65630 and [3H]quipazine binding sites in rat hindbrain 84 Drug inhibition profiles for the displacement of [3H]BRL 43694 7 binding in the NTS 86 Colocalisation of GABA b and 5-HT3 receptor sites in rat hindbrain 86 Discussion 125 Structure activity comparisons of GABA b receptor agonists and antagonists in brain tissue 127 Characterisation of central beta-adrenoceptors 128 5-HT3 receptor distribution in mammalian hindbrain 128 Colocalisation of 5-HT3 binding sites with those for GABA b receptors in rat dorsal vagal complex 132 CHAPTER 4 Similarity of the distribution of GABA b and 5-HT3 receptors in the rat hindbrain 134 Introduction 135 Methods (surgical procedures) 136 Nodose ganglionectomy 136 Hemisection of the nucleus tractus solitarius 137 Results 137 Denervation studies with 5-HT3 receptors 137 Denervation studies with GABA b and GABA a receptors 138 Discussion 153 5-HT3 receptor association with vagal afferent terminals 153 Serotonergic mechanisms in cisplatin-induced emesis 155 Fine microstructure of the dorsal vagal complex 157 The association of GABA receptors with central vagal terminals 160 Gastrointestinal responses to central GABAergic receptor activation 161 Cardiovascular responses to central GABAergic receptor activation 163 Central respiratory control by GABA b receptors 165 8 CHAPTER 5 Autoradiographical analysis of GABA b receptors in rat frontal cortex following repeated antidepressant administration 167 Introduction 168 Methods Drug treatment protocols and GABA b receptor binding conditions 173 First Investigation 173 Second Investigation 173 Third Investigation 174 Fourth Investigation 174 Results 175 First Investigation 175 Second Investigation 175 Third Investigation 177 a) GABA b receptor-modulation by antidepressants 177 b) Beta-adrenoceptor-modulation by antidepressants 178 Fourth Investigation 179 a) Drug-induced modulation of GABA b receptor binding 179 b) Drug-induced modulation of beta-adrenoceptor binding 180 Discussion 211 Chronic antidepressant administration via osmotic minipumps 214 Discrete GABA b receptor up-regulation by desipramine 215 Beta-adrenoceptor modulation by antidepressants 216 Selective GABA b receptor-modulation