ROLE OF 5-HT6 RECEPTOR IN CONDITIONED LEARNING AND MEMORY Susie Woods, BSc. THESIS SUBMITTED TO THE UNIVERSITY OF NOTTINGHAM FOR THE DEGREE OF DOCTOR OF PHILOSOPHY School of Biomedical Sciences July 2011 Abstract The recently discovered 5-HT6 receptor has generated interest due to increasing evidence for its role in feeding, obesity, anxiety, depression and cognition. Initial studies utilising selective 5-HT6 receptor antagonists found pro-cognitive effects in various cognitive paradigms. In the last three years selective 5-HT6 receptor agonists have been developed and initial reports suggested they impaired cognition as predicted, but more recent reports have found paradoxical pro- cognitive effects in learning and memory tasks. The main aim of the current thesis was to determine the role of the 5-HT6 receptor in a conditioned emotion response (CER) task in rats. Both the effects of 5-HT6 receptor antagonists and agonists given alone, and their abilities to reverse a cholinergic- or glutamatergic- induced memory impairment were analysed. Secondly, to analyse the intracellular mechanisms involved in the behavioural effects exerted following treatment with 5-HT6 receptor ligands by examining changes in hippocampal protein expression. Pre-treatment with either the muscarinic receptor antagonist, scopolamine, or the NMDA receptor antagonist, MK-801, induced memory impairment in the 24 hour retention trial. Post-training administration of 5-HT6 receptor antagonist, SB-271046, and agonists, EMD 386088 and E-6801, had little effect on CER- induced behaviour when given alone, but both reversed the cholinergic- and glutamatergic-induced deficits. Western blot analysis revealed no significant difference between hippocampal BDNF and 5-HT6 receptor protein levels following any drug or shock treatment, i but some interesting trends were observed. CER slightly increased BDNF expression, this was reduced by scopolamine and MK-801 which in turn was reversed with SB-271046 and EMD 386088. CER decreased 5-HT6 receptor expression, scopolamine caused further reduction, SB-271046 and EMD 386088 increased the expression following scopolamine. MK-801 increased 5-HT6 receptor expression, whilst SB-271046 further enhanced this expression, EMD 386088 reduced it. No significant results were observed in the proteomic studies. These findings provide further evidence for the exciting potential therapeutic use of 5-HT6 receptor compounds in the treatment of cognitive dysfunction. ii Publications Poster Woods, S. W., Clarke, N. N., Topham, I. A., Layfield, R., Fone, K. C. F. (2008). Do 5-HT6 receptor antagonists alter emotional learning in a contextual fear conditioning paradigm? Journal of Psychopharmacology S22, 5, A14 Woods, S. W., Clarke, N. N., Topham, I. A., Layfield, R., Fone, K. C. F. (2008). Effects of 5-HT6 receptor antagonist, SB-271046, on fear motivated learning and memory. Serotonin Club Meeting, SCP020. Woods, S. W., Clarke, N. N., Layfield, R., Fone, K. C. F. (2009). Can a 5-HT6 receptor agonist, EMD 386088, ameliorate a scopolamine-induced memory deficit in contextual fear conditioning? British Neuroscience Association Abstract., 20, P39.05 Oral Woods, S. W., Clarke, N. N., Layfield, R., Fone, K. C. F. (2009). Can 5-HT6 receptor antagonists and agonists reverse impairments in hippocampal-dependent memory? Short Oral Presentation, BAP Summer Meeting, Oxford, UK iii Acknowledgments I would like to thank my supervisors Kevin Fone and Robert Layfield for their encouragement and support throughout my PhD. I would also like to thank the University of Nottingham and the Medical Research Council for their financial support. I am very grateful to GSK and Esteve for the kind gifts of the pharmacological compounds which made this work so interesting. As I knew very little on arrival I have been taught a lot along the way at Nottingham, therefore I would like to thank friends in the lab, Liaque for Western blots (and the discussions), Ian K, Maddy, Dan and Dave for their advice on the models, and Ian T for his advice and help in every study. During first year I would have been a bit lost without Nola, so thanks to her for keeping me afloat. A huge thanks to the girls, Irish, Warner and Welshy I would have gone insane a long time ago if it was not for you three and the good old gossips over tea, also for the advice and support you gave me, thank you. A bit further away, a big thanks to Daniella and Sam for always being on the other end of the phone when I needed you. My biggest thanks has to go to my family, Mum, Dad, Lucy, Tom and Guy, for their love and support throughout my studies at Nottingham. Finally to my husband John, who has always been there at the end of the day, some longer than others! I want to thank you so much for listening to my rants (work and non- work related), cheering me up and having a constant supply of sweets, helping with my presentations and abstracts, basically for believing in me and always being there! For their constant love and support I would like to dedicate my thesis to my family and John. iv Table of Contents Abstract .............................................................................................................. i Publications...................................................................................................... iii Acknowledgments..............................................................................................iv Table of Contents ...............................................................................................v Table of Figures and Tables............................................................................. ix Abbreviations and Pharmacological compounds ............................................. xi 1 General Introduction ................................................................................. 1 1.1 Cognition .........................................................................................................1 1.2 Conditioned Emotion Response.....................................................................5 1.3 5-Hydroxytryptamine.....................................................................................6 1.3.1 Discovery of 5-HT ...................................................................................................6 1.3.2 Synthesis, storage, release and metabolism of 5-HT................................................7 1.3.3 Anatomy of serotonergic system..............................................................................9 1.3.4 Classification of 5-HT receptors ............................................................................11 1.3.5 5-HT6 receptors ......................................................................................................15 1.3.5.1 Molecular biology.........................................................................................15 1.3.5.2 Ligand Binding sites .....................................................................................18 1.3.5.3 Signal Transduction ......................................................................................19 1.3.5.4 Distribution of 5-HT6 receptors ....................................................................20 1.3.5.5 Pharmacology of the 5-HT6 receptor ............................................................23 1.3.5.6 5-HT6 receptor antagonists ...........................................................................24 1.3.5.7 5-HT6 receptor agonists ................................................................................30 1.3.5.8 Functional Roles of 5-HT6 receptors ............................................................33 1.3.5.9 Behavioural syndrome ..................................................................................34 1.3.5.10 Depression ....................................................................................................34 1.3.5.11 Anxiety .........................................................................................................36 1.3.5.12 Nociception...................................................................................................37 1.3.5.13 Seizure threshold ..........................................................................................38 1.3.5.14 Feeding .........................................................................................................39 1.3.5.15 Schizophrenia ...............................................................................................39 1.3.5.16 Cognition ......................................................................................................42 1.4 Aims ...............................................................................................................52 1.5 Hypotheses.....................................................................................................52 2 Optimisation of a fear motivated learning paradigm in rodents: Conditioned Emotion Response........................................................................54 2.1 Introduction...................................................................................................54 2.1.1 Learning and memory ............................................................................................55 2.1.2 Conditioned Emotion Response .............................................................................59 2.1.3 Neuroanatomical and Neurochemical substrates of CER.......................................60 2.1.4 Aims .......................................................................................................................65