Physiological roles of endogenous neurosteroids at α2 subunit-containing GABAA receptors Elizabeth Jane Durkin A thesis submitted to University College London for the degree of Doctor of Philosophy October 2012 Department of Neuroscience, Physiology and Pharmacology University College London Gower Street London WC1E 6BT Declaration 2 Declaration I, Elizabeth Durkin, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis Abstract 3 Abstract Neurosteroids are important endogenous modulators of the major inhibitory neurotransmitter receptor in the brain, the γ-amino-butyric acid type A (GABAA) receptor. They are involved in numerous physiological processes, and are linked to several central nervous system disorders, including depression and anxiety. The neurosteroids allopregnanolone and allo-tetrahydro-deoxy-corticosterone (THDOC) have many effects in animal models (anxiolysis, analgesia, sedation, anticonvulsion, antidepressive), suggesting they could be useful therapeutic agents, for example in anxiety, stress and mood disorders. Neurosteroids potentiate GABA-activated currents by binding to a conserved site within α subunits. Potentiation can be eliminated by hydrophobic substitution of the α1Q241 residue (or equivalent in other α isoforms). Previous studies suggest that α2 subunits are key components in neural circuits affecting anxiety and depression, and that neurosteroids are endogenous anxiolytics. It is therefore possible that this anxiolysis occurs via potentiation at α2 subunit-containing receptors. To examine this hypothesis, α2Q241M knock-in mice were generated, and used to define the roles of α2 subunits in mediating effects of endogenous and injected neurosteroids. Biochemical and imaging analyses indicated that relative expression levels and localization of GABAA receptor α1-α5 subunits were unaffected, suggesting the knock- in had not caused any compensatory effects. Electrophysiological characterization of cells in hippocampal and nucleus accumbens brain slices revealed faster-decaying inhibitory synaptic transmission in α2Q241M mice. Furthermore, the response to applied THDOC was markedly reduced compared to wild-type cells. α2 subunits therefore formed a major component of synaptic GABAA receptors in these areas. The α2Q241M knock-ins showed greater anxiety levels in two classical rodent anxiety paradigms (light-dark box and elevated plus maze), consistent with endogenous neurosteroids mediating anxiolysis via α2-type GABAA receptors. In addition, the anxiolytic response to injected THDOC is impaired by the α2Q241M mutation, which would identify the α2 isoform as an appropriate target for generating receptor subtype- selective neurosteroid therapeutics for anxiety disorders. Acknowledgements 4 Acknowledgements I will forever be indebted to the many people who have helped me along my PhD journey, especially to Prof. Trevor Smart, for allowing me to work on such a challenging and exciting project, and for his unending support, guidance and enduring positivity. This project would not have been possible without the financial support of the MRC, and helpful advice from my thesis committee, Stuart Cull-Candy, Stephen Nurrish and Antonella Riccio. Thank you to Mike Lumb, for generating the transgenic mouse strain, together with Steve Moss and GenOway. For generously providing lab space and loaning me the equipment necessary for the behavioural analyses, I am grateful to Clare Stanford and Stephen Hunt. I would also like to thank Clare, and members of her lab, especially Ruth Weir and Ewelina Grabowska, for teaching me behavioural techniques, and helping me interpret results from these experiments. Thank you also to Martin Stocker for allowing access to his microtome. Thanks to all members of the Smart lab, past and present, for creating an excellent environment to work in, and being a great source of fun and of encouragement through the inevitable experimental difficulties. I would particularly like to thank Damian and Phil, for their excellent teaching and support, and for being on hand whenever my rig misbehaved! Also, thanks to Saad for sharing his imaging expertise, and PhD pep talks. I would also like to take this opportunity to thank all of the Durkin ‘clan’ – above all, Mum, Dad, Rob, Billy and Pat – for reminding me that there is life outside of my PhD, and whose love and support has given me the drive to make it through my academic career to date. I am particularly grateful to my PhD buddies who have shared in this rollercoaster ride, especially Sinead and Kasia, who have always been there for a chat over coffee. Finally, the biggest of thanks is owed to Stuart for his patience, constant encouragement, and excellent curries! Contents 5 Contents Declaration .................................................................................................................................................. 2 Abstract ....................................................................................................................................................... 3 Acknowledgements .................................................................................................................................... 4 List of Figures ............................................................................................................................................. 8 List of Tables............................................................................................................................................... 9 List of Abbreviations ................................................................................................................................ 10 Chapter 1: Introduction ............................................................................................................................ 12 1.1. GABAA receptors ............................................................................................................................... 12 1.1.1. GABAA receptor composition and function ............................................................................................ 13 1.1.2. GABAA receptor modulation: trafficking ................................................................................................. 18 1.1.3. GABAA receptor modulation: ligands and post-translational modification .............................................. 21 1.2. Neurosteroid physiology and pharmacology .................................................................................. 27 1.2.1. Endogenous neurosteroids: synthesis and roles ................................................................................... 27 1.2.2. Neurosteroid binding to GABAA receptors ............................................................................................. 30 1.2.3. Physiological modulation of GABAA receptors by neurosteroids ........................................................... 32 1.2.4. Neurosteroids: therapeutic potential ...................................................................................................... 34 1.3. Neurosteroids and GABAA receptors in anxiety ............................................................................. 35 1.3.1. The HPA axis and neurosteroids as endogenous anxiolytics ................................................................ 35 1.3.2. Defining the GABAA receptor α subunits that are important in anxiety and anxiolysis ........................... 37 1.4. Neurosteroids and GABAA receptors in depression ...................................................................... 39 1.4.1. Roles for GABAA receptors in depression ............................................................................................. 39 1.4.2. Roles for neurosteroids as antidepressants .......................................................................................... 43 1.4.3. The nucleus accumbens in reward and depression .............................................................................. 44 1.5. Thesis Aims........................................................................................................................................ 46 Q241M 1.5.1. Generation of an α2 knock-in mouse ............................................................................................. 46 Q241M 1.5.2. Electrophysiological characterisation of α2 mice ........................................................................... 46 Q241M 1.5.3. Behavioural characterisation of α2 mice ........................................................................................ 47 1.5.4. Summary of thesis aims ........................................................................................................................ 47 Chapter 2: Materials and Methods .......................................................................................................... 49 2.1. Materials ............................................................................................................................................. 49 2.1.1. Reagents .............................................................................................................................................. 49 2.1.2. Antibodies ............................................................................................................................................. 49 2.2. Animals ..............................................................................................................................................