Studying GABAA Receptors using AII Amacrine Cells in the Rat Retina By Tuan Van Trinh MASTER THESIS IN PHARMACY Department of Biomedicine/ Centre for Pharmacy University of Bergen May 2018 The picture of AII amacrine cells in front page is adapted from Zhou et al., 2016. 2 ACKNOWLEDGEMENTS This study was carried out at the department of Biomedicine, University of Bergen, during the period August 2012 to April 2013. Due to a serious illness, the project was interrupted, and continued again in April 2018 to May 2018. I would like to thank several people for their support during this project. First I would like to express my sincere gratitude to my supervisor prof. Ph.d Margaret Lin Veruki and co-supervisor prof. dr. med. Espen Hartveit for valuable advice and much appreciated guidance during the period. Ph.d. Yifan Zhou is thanked for helping me with collecting the data, and of course thanks to Marte Nørve Årvik, Lise Skålvik Amble and all my co-workers and lab personnel that have helped me during this period. To my family and my friends thank you for supporting me during this hard period of life. Bergen, May 2018 3 TABLE OF CONTENTS ACKNOWLEDGEMENTS……………………………………………………..3 TABLE OF CONTENTS………………………………………………………..4 ABBREVIATIONS……...………………………………………………………8 AIMS……………………...…………………………………………………....11 SUMMARY……………………...…………………………………………….13 1.0 INTRODUCTION AND THEORY……………………………………………16 1.1 Nerve cell and signal communication ………………………………16 1.1.1 Cell membrane……………………………..……………………...17 1.1.2 The membrane potential………….…………………….……….……..18 1.1.3 The membrane receptors……………………………….……….……..19 1.1.3.1 Enzyme-linked receptors..…………………….……….………...19 1.1.3.2 G-protein-coupled receptors....………………….………...20 1.1.3.3 Ligand-gate ion channels, with examples...……...……….21 The tetrameric superfamily of ionotropic glutamate receptors……………………………………………………22 The trimeric superfamily of LGIC…………….….………22 The pentameric super family of receptors……….…………22 1.2 GABA A receptors……..………………………………………………….23 1.2.1 The GABA receptors…………………………………….…….…..23 1.2.2 Function and structure of GABA A-receptor….…………….……...26 1.2.3 GABA A receptors: drugs, ligands, subunits and binding sites …....27 1.2.3.1 GABA AR subunits expression in brain region……………28 1.2.3.2 The GABA binding site…………………………………...29 1.2.3.3 The benzodiazepine binding site………………………….30 1.2.3.4 The picrotoxin sites……………………………………….32 4 1.2.3.5 The general anesthetic sites….……………………………33 1.3 Studying the GABA A receptor using patch clamp electrophysiology……...34 1.3.1 GABA A receptor and patch clamp technique……………………...34 1.3.1.1 Patch clamp configurations…………………………………...…35 Cell-attached…………...…………………………………………36 Whole-Cell patch…..……………………………...........................36 Outside-out patch…………………………………………………37 Inside-out patch……………………………………………...……37 Perforated-patch…………………………………………………..38 Nucleated patch……………………………………………….…..38 1.3.2 Electric theory of a neuron………………………………………...39 1.3.2.1 Nernst equations and Goldman –Hodgkin-Katz equation…….………..39 1.3.2.2 Neuron as electric circuits with capacitance and series resistance……………..41 1.3.2.3 Voltage clamp and amplifier…………………………………….43 1.4 The retina ……...…………………………………………………………45 1.4.1 Function of the retina………………………………………...........45 1.4.2 The rod and cone pathway ……….……………………………….46 1.5 AII Amacrine cells……...…………………………………………….….47 1.5.1 AII-amacrine cells………………………………………………....47 1.5.2 AII amacrine classification and localisation…………...………….48 2.0 MATERIALS…………...………………………………………………….51 2.1 Solutions………………………………………………………………….51 2.1.1 Extracellular solution, EC 1000………………….………………...51 2.1.2 Extracellular solution, EC 3000…………………….………...……51 2.1.3 Ames solution……………………………………………………...51 2.1.4 Intracellular solution, IC 6302………………………..……………52 2.2 Chemicals and drugs……………………………………………………...52 5 2.2.1 GABA ……………………………………………………….…….52 2.2.2 Isoflurane…………………………………………………….…….52 2.3 Dissection equipment…………………………………...………………..53 2.4 Technical equipment……………………………………………….……54 3.0 METHODS…………………...………………………………………….…55 3.1 Methods involving handling the rat and dissection of the rat’s retina…...55 3.1.1 Anaesthesia and enucleation the rat…………………….…………55 3.1.2 Cutting the retina into four quadrants…………………….….…….55 3.1.3 Retinal slices……………………………………………………….56 3.2 Patch clamping set-up…………………………….…………………….…56 3.2.1 Set-up, microscope and visualization……………………………...56 3.2.2 Perfusion chamber setup…………………………………………..58 3.3 The pipettes…………….………………………………………………...58 3.3.1 The recording pipettes……………………………………………..58 3.3.1.1 Filling……………………………..…………………………59 3.3.1.2 Connecting the pipette to the set-up………………………...59 3.3.2 The application pipettes…………………………………….……...60 3.3.2.1 The multi-barrel pipette………………………….………….60 3.4 The recording configurations used for the experiments………………….61 3.4.1 Whole-cell and nucleated patch…………………………………...61 3.5 Drug application.…………………………………………….…………...62 3.6 Recording software……….………………………………….…………...63 3.7 Recording parameters….…….…………………………….……………..63 3.8 The application protocol…..………………………………….……….….63 3.9 Measurements and analyses………..…………………………….……....64 4.0 RESULTS…………...……………………………………………….……..66 4.1 Identification of the AII amacrine cells…………………………….…….66 6 4.2 The GABA-response current……………………………………….….....67 4.2.1 Desensitization and run down …………………………………….69 4.2 The results and current-voltage curves (I-V-curves)………………….….69 5.0 DISCUSSION………...………………………………………………….…72 5.1 The application of GABA………….…………………………………….72 5.1.1 Variations in concentration of GABA reaching the GABA A receptors…………………………………………………………..72 5.2 Desensitization and rundown…………………………………………….73 5.3 Sensitivity of patch clamp methods and technical challenging………..…74 5.3.1 Dissection of the retina slices……………………………………...74 5.3.2 Identification of the AII amacrine cells……………………………74 5.3.3 Vibration isolation table…………………………………………...74 5.3.4 Electrical noise…………………………………………………….75 5.4 Data which, were not included…………………..………………………75 5.5 Patch clamp in drug-discovery…………………………………………..75 5.5.1 Basic research…………………………..………………………….76 5.5.2 Primary screening………………………………………………….76 5.5.3 Secondary screening……………………………………………….77 5.5.4 Safety screening………………………………...…………………77 6.0 FUTURE WORK……………………...…………………………………...78 7.0 REFERENCES……...……………………………………………….……..79 7 ABBREVIATIONS 5-HT3R Serotonin receptors 7TM Seven-(pass)-transmembrane domain AChBP Acetylcholine binding protein AMPAR α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors ATP Adenosine triphosphate BBB Blood–brain barrier BZs Benzodiazepines BZR benzodiazepine receptor CNS Central nervous system CX36 Connexin36 DZ Diazepam EC Extracellular solution ECD Extracellular domain EGTA Ethylene glycol tetraacetic acid Eq Equation GABA γ-amino butyric acid GABA AR γ-amino butyric acid receptors type A GAD Glutamic acid decarboxylase GCL Ganglion cell layer GluR Ionotropic glutamate receptors (iGluR for ionotropic) GlyR Glycine receptors GPCRs G-protein-coupled receptors GTP Guanosine Triphosphate HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HERG The human Ether-à-go-go-Related Gene HTS High throughput screening IC Intracellular solution ICD Intracellular domain INL Inner nuclear layer IPSP Inhibitory postsynaptic potential IPL Inner plexiform layer ILM Inner limiting membrane 8 LGICs ligand-gated ion channels LJP Liquid junction potential ms millisecond nAChR nicotinic acetylcholine receptors NAM Negative allosteric modulation NMDAR N-methyl D-aspartate receptors OFF-GC OFF-ganglion cells OFF-CBP OFF-cone bipolar cells more common to use CBC OLM Outer limiting membrane ON-CBP ON-cone bipolar cell more common to use CBC ON-GC ON-ganglion cells ONL Outer nuclear layer ON-RBP ON-rod bipolar cell more common to use RBC PAM Positive allosteric modulation PLGICs Pentameric ligand-gated ion channels Pro Propofol Pyr Pyrazoloquinoliines RPE Retinal pigment epithelium TEA-CL Tetramethylammonium-Chloride THIP (4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol), Gaboxadol TM Transmembrane TMD Transmembrane domain VGICs Voltage-gated ion channels Ions Ca 2+ Calcium ion Cl - Chloride ion Cs + Cesium ion - HCO 3 Hydrogen carbonate ion K+ Potassium ion Na + Sodium ion Mg 2+ Magnesium ion 9 Electrical parameters Cm Membrane capacitance Cp Pipette capacitance Gm Membrane conductance Ic Capacitive current Im Membrane currents Rm Membrane resistance Rp Pipette resistance Rs Series resistance 10 AIMS GABAergic drugs or compounds that directly and indirectly activate the GABA receptors (GABA AR) in CNS typically have anti-anxiety, muscle relaxant, amnesic, sedative, hypnotic, euphoriant and anti-convulsive effects. (Chapouthier & Venault, 2001; Foster & Kemp, 2006; Olsen & Sieghart, 2008; Uusi-Oukari & Korpi, 2010; Lorenz-Guertin & Jacob, 2017). There are many severe problems that are related to the long-term therapeutic use of drugs that affect GABAergic system (Uusi-Oukari & Korpi, 2010; Lorenz-Guertin & Jacob, 2017; Olsen, 2018). GABA AR play a very important role in medicine treatment (Johnston, 1996; Chapouthier & Venault, 2001; Möhler et al., 2004; Foster & Kemp, 2006; Santhakumar et al., 2007; Jacob et al., 2008; Lager et al., 2008; Olsen & Sieghart, 2008; Uusi-Oukari & Korpi, 2010; Lorenz-Guertin & Jacob, 2017). The active site of the GABA AR is the binding site for GABA and several drugs (Johnston, 1996; Santhakumar et al., 2007; Mrunmayee et al., 2010, Olsen, 2018). Drugs that act via GABA AR often affect the regulation of various GABAAR subunits. Different subunit compositions give the receptor different