IMAGING AND PROPERTIES OF EXTRASYNAPTIC GABAA RECEPTORS IN THE BRAIN Saku Sinkkonen Institute of Biomedicine, Pharmacology University of Helsinki, Finland and Turku Graduate School of Biomedical Sciences and Department of Pharmacology and Clinical Pharmacology University of Turku, Finland ACADEMIC DISSERTATION To be presented, with the permission of the Medical Faculty of the University of Helsinki, for public examination in the Small Lecture Hall of Haartman Institute, Haartmaninkatu 3, on November 5th, 2004, at 12 o’clock. Helsinki 2004 Supervised by Professor Esa R. Korpi, M.D. Institute of Biomedicine, Pharmacology Faculty of Medicine University of Helsinki, Finland Reviewed by Associate Professor Bryndis Birnir, Ph.D. Department of Physiological Sciences Faculty of Medicine University of Lund, Sweden and Docent Petteri Piepponen, Ph.D. (Pharm.) Department of Pharmacology and Toxicology Faculty of Pharmacy University of Helsinki, Finland Dissertation opponent Professor Emeritus Simo S. Oja, M.D., Ph.D., M.LL. Brain Research Center Medical School University of Tampere, Finland ISBN 952-91-7825-5 (paperback) ISBN 952-10-2092-X (PDF http://ethesis.helsinki.fi) University Printing House Helsinki 2004 To Wera 4 TABLE OF CONTENTS ABSTRACT.............................................................................................................................. 7 ORIGINAL COMMUNICATIONS ......................................................................................... 8 ABBREVIATIONS...................................................................................................................9 1. INTRODUCTION............................................................................................................... 11 2. REVIEW OF THE LITERATURE..................................................................................... 14 2.1. GABA as neurotransmitter......................................................................................... 14 2.2. GABA receptors......................................................................................................... 14 2.3. GABAA receptor structure.......................................................................................... 15 2.3.1. Subunit genes ................................................................................................. 15 2.3.2. Subunit proteins.............................................................................................. 16 2.3.3. Receptor assembly.......................................................................................... 16 2.3.4. Heterologous receptor expression .................................................................. 17 2.3.5. Receptor subtypes in the native brain ............................................................ 19 2.4. GABAA receptor function .......................................................................................... 19 2.4.1. Phasic inhibition............................................................................................. 21 2.4.2. Tonic inhibition.............................................................................................. 22 2.4.3. Inhibition in the cerebellar granule cell layer................................................. 24 2.4.4. Inhibition in the hippocampus........................................................................ 26 2.5. Molecular pharmacology of GABAA receptor binding sites...................................... 27 2.5.1. GABA............................................................................................................. 28 2.5.2. Picrotoxinin/TBPS ......................................................................................... 29 2.5.3. Benzodiazepines............................................................................................. 31 2.5.4. DMCM ........................................................................................................... 32 2.5.5. Furosemide..................................................................................................... 33 2.6. Tiagabine.................................................................................................................... 34 2.7. GABAA receptors in disease ...................................................................................... 34 2.7.1. Anxiety........................................................................................................... 34 2.7.2. Epilepsy.......................................................................................................... 35 2.8. GABAAergic drug therapy and its problems.............................................................. 38 3. AIMS OF THE STUDY...................................................................................................... 39 4. MATERIALS AND METHODS........................................................................................ 40 4.1. Experimental animals................................................................................................. 40 4.2. Ligand autoradiographic assays (I, II, III, IV, V) ...................................................... 40 4.2.1. Preparation of brain sections (I, II, III, IV, V) ............................................... 40 4.2.2. [35S]TBPS binding (I, II, III, IV, V)............................................................... 41 4.2.3. [3H]Ro 15-4513 binding (I, III, IV)................................................................ 41 4.2.4. [3H]Muscimol binding (I, III)......................................................................... 42 4.3. Localization of GABAA receptor subunits by in situ hybridization (II) .................... 42 4.4. Quantitative autoradiographic film analysis (I, II, III, IV, V).................................... 43 4.5. Recombinant GABAA receptors in HEK 293 cells (III) ............................................ 43 4.5.1. Receptor expression ....................................................................................... 43 4.5.2. [35S]TBPS binding assay................................................................................ 44 4.6. Electrophysiological recordings from acutely isolated mouse hippocampal CA1 pyramidal cells (IV) ................................................................................................... 44 5 4.7. Determination of receptor subunit composition (IV)................................................. 44 4.7.1. Preparation of receptor extracts...................................................................... 44 4.7.2. Immunoaffinity chromatography ................................................................... 44 4.7.3. Quantification of GABAA receptor subtypes by immunoprecipitation and [3H]muscimol binding assays......................................................................... 45 4.8. Mouse behavioral studies (IV) ................................................................................... 45 4.8.1. Observational functional analysis by the SHIRPA protocol.......................... 45 4.8.2. Light-dark choice test..................................................................................... 46 4.8.3. T-maze test ..................................................................................................... 46 4.8.4. Convulsion tests ............................................................................................. 47 4.9. Recombinant GABAA receptors in Xenopus oocytes (V).......................................... 47 4.9.1. Receptor expression ....................................................................................... 47 4.9.2. Electrophysiological recordings..................................................................... 47 4.10. Statistics (I, II, III, IV, V)......................................................................................... 48 5. RESULTS AND DISCUSSION ......................................................................................... 49 5.1. Development of a receptor autoradiographic method to reveal GABA-insensitive [35S]TBPS binding (I).................................................................. 49 5.2. Localization and prevalence of GABA-insensitive [35S]TBPS binding in the brain (I, II)........................................................................................................ 52 5.3. Roles of different GABAA receptor subunits in GABA-insensitive [35S]TBPS binding...................................................................................................... 53 5.3.1. Correlation with receptor subunit mRNA localization by in situ hybridization................................................................................................... 53 5.3.1.1. Adult rat (II) ....................................................................................... 53 5.3.1.2. Developing rat.................................................................................... 54 5.3.2. GABA-insensitive [35S]TBPS binding in genetically engineered mouse lines..................................................................................................... 54 5.3.2.1. The β3 subunit knockout mice (β3-/-) (II) ......................................... 54 5.3.2.2. The δ subunit knockout mice (δ-/-) (II) ............................................. 56 5.3.2.3. The α6 subunit knockout mice (α6-/-) (II) ........................................ 56 5.3.2.4. Mice with ectopic α6 subunit expression (Thy1α6) (IV) .................