FUNCTIONAL REGULATION OF NMDA RECEPTOR SUBTYPES AND THEIR INVOLVEMENT IN HIPPOCAMPAL PLASTICITY by Oana Cristina Vasuta BSc, University of Iasi, 2002 MSc, University of Bucharest, 2004 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Neuroscience) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2009 © Oana Cristina Vasuta 2009 ABSTRACT Regulation of NMDAR activity by desensitization is important in physiological and pathological states. We previously reported that desensitization decreases during hippocampal neuronal development, correlating with NMDAR composition, synaptic localization and association with PSD-95. To determine if PSD-95-induced changes in NMDAR desensitization occur because of direct binding to NR2 subunits or due to recruitment of regulatory proteins, we tested the effects of various PSD-95 constructs on NMDAR currents in HEK293 cells and neurons. In HEK cells, wt PSD-95 significantly reduced wt NMDAR desensitization without altering currents of NMDARs containing NR2A-S1462A, a mutation that abolishes PSD-95 binding. Moreover, PDZ1-2 domain was sufficient for this effect in neurons with low endogenous PSD-95 levels. Moreover, other PSD-95 family members with highly homologous PDZ1-2 domains significantly reduced NMDAR desensitization. In mature neurons, disruption of PSD-95/NMDAR interaction through PKC activation, or through interference peptides, increased desensitization to levels found in immature neurons. We conclude that direct binding of PSD-95 increases stability of NMDAR responses to agonist exposure. Desensitization is a property that shapes synaptic responses, and modulates the calcium signal mediated by the two predominant NMDARs subtypes in hippocampus, with possible consequences for their functioning. Further, we examined the involvement of NR2 subtypes in synaptic plasticity in hippocampal dentate gyrus of juvenile mice. Exercise was used as a means to alter expression of the NR2 subunits in this region. We compared two groups of animals: Controls, which were housed in conditions of minimal enrichment, and ii Runners, which had access to an exercise wheel. NMDAR-dependent LTP expression was significantly greater in Runners than in Controls; in the presence of NR2B subunit antagonists, it was significantly reduced in both groups. NR2A subunit antagonist blocked LTP in slices from Runners and produced a slight depression in Control animals. LTD could not be prevented by either of the NR2B specific antagonists. Strikingly, eliminating NR2A subunit-containing receptor activity prevented LTD in Runners, but not in Control animals. Overall, these results indicate that interplay between subtype, subcellular localization and size of NMDAR subpopulations accounts for their diverse role in synaptic plasticity induction, and that exercise increases the contribution of NR2A to plasticity. iii CONTENTS ABSTRACT ............................................................................................................................. ii CONTENTS ............................................................................................................................ iv LIST OF FIGURES .................................................................................................................. vii LIST OF ABBREVIATIONS ...................................................................................................... viii ACKNOWLEDGEMENTS ......................................................................................................... xi DEDICATION .........................................................................................................................xii CO-AUTHORSHIP STATEMENT .............................................................................................. xiii 1 INTRODUCTION .............................................................................................................. 1 1.1 The hippocampal NMDA receptor family ............................................................................1 1.1.1 General overview ................................................................................................................................... 1 1.1.2 Structure and assembly of NMDA receptor complexes ......................................................................... 3 1.1.2.1 Structure....................................................................................................................................... 3 1.1.2.2 Assembly ...................................................................................................................................... 5 1.1.3 Expression of hippocampal NMDA receptors ........................................................................................ 6 1.1.3.1 Regional variability of expression ............................................................................................... 10 1.1.3.2 Temporal variability of expression ............................................................................................. 13 1.1.4 Properties ............................................................................................................................................ 15 1.1.5 Cytoskeletal and signalling proteins associated with NMDARs ........................................................... 19 1.1.5.1 PSD-95 complex and other MAGUKs .......................................................................................... 19 1.1.5.2 Other interacting molecules ....................................................................................................... 22 1.1.6 NMDAR function in cellular physiology ............................................................................................... 23 1.1.6.1 Fast excitatory transmission ....................................................................................................... 23 1.1.6.2 Structural development of neural processes ............................................................................. 26 1.1.6.3 Synaptic plasticity ....................................................................................................................... 27 1.1.6.4 Neuronal survival and excitotoxicity .......................................................................................... 28 1.1.6.5 Insights on the NMDAR function from NMDAR subunit mutant mice ....................................... 30 1.2 NMDAR desensitization ................................................................................................... 31 1.2.1 Definition and classification ................................................................................................................. 31 1.2.2 Structural determinants of NMDAR desensitization ........................................................................... 34 1.2.2.1 Structural determinants at the N-terminal domain ................................................................... 35 1.2.2.2 Structural determinants at the C-terminal domain .................................................................... 36 1.2.3 Proved and hypothesised roles for NMDAR desensitization ............................................................... 37 1.3 NMDAR-dependent synaptic plasticity in hippocampus .................................................... 39 1.3.1 Definition and forms of synaptic plasticity .......................................................................................... 39 1.3.1.1 Transient and short-term forms of plasticity ............................................................................. 40 1.3.1.2 Long-term plasticity: early and late LTP/LTD ............................................................................. 42 iv 1.3.1.3 Metaplasticity ............................................................................................................................. 43 1.3.1.4 Homeostatic p lasticity ................................................................................................................ 43 1.3.2 Induction .............................................................................................................................................. 44 1.3.2.1 Classical patterns ........................................................................................................................ 45 1.3.2.2 Spike-timing protocols ............................................................................................................... 46 1.3.2.3 Calcium signalling through NMDARs in plasticity induction ...................................................... 46 1.3.3 Expression ............................................................................................................................................ 47 1.3.3.1 Presynaptic locus of expression ................................................................................................. 48 1.3.3.2 Postsynaptic locus of expression ................................................................................................ 48 1.3.4 Particularities of plasticity in hippocampal dentate gyrus ................................................................... 50 1.4 Theme and hypothesis ..................................................................................................... 53 1.5 Bibliography 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