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Title: Probing spatial and subunit-dependent signalling by the NMDA receptor
Author: McKay, Sean
ISNI:       0000 0004 5921 1048
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2015
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NMDARs are ligand-gated cation channels which are activated by the neurotransmitter glutamate. NMDARs are essential in coupling electrical activity to biochemical signalling as a consequence of their high Ca2+ permeability. This Ca2+ influx acts as a secondary messenger to mediate neurodevelopment, synaptic plasticity, neuroprotection and neurodegeneration. The biological outcome of NMDAR activation is determined by a complicated interrelationship between the concentration of Ca2+ influx, NMDAR location (synaptic vs. extrasynaptic) as well as the subtype of the GluN2 subunit. Despite the recognition that NMDAR mediated physiology is multifaceted, tools used to study subunit and location dependent signalling are poorly characterized and in other cases, non-existent. Therefore, the aim of this thesis is to address this issue. Firstly, I assessed the current pharmacological approach used to selectively activate extrasynaptic NMDARs. Here, synaptic NMDARs are first blocked with MK-801 during phasic activation and then extrasynaptic NMDARs are tonically activated. This approach relies on the continual irreversible blockade of synaptic NMDARs by MK-801 yet contrary to the current dogma, I demonstrate this blockade is unstable during tonic agonist exposure and even more so when physiologically relevant concentrations of Mg2+ are present. This confines a temporal limit in which selective activation of extrasynaptic NMDARs can occur with significant consequences for studying synaptic vs. extrasynaptic NMDAR signalling. Dissecting subunit-dependent signalling mediated by the two major GluN2 subunits in the forebrain, GluN2A and GluN2B, has been advanced significantly by selective GluN2B antagonism yet a reciprocal GluN2A selective antagonist has been lacking. Utilizing novel GluN2A-specific antagonists, I demonstrate a developmental upregulation of GluN2A-mediated NMDA currents which concurrently dilutes the contribution of GluN2B-mediated currents. Moreover, I tested the hypothesis that the Cterminus of GluN2A and GluN2B are essential in controlling the developmental switch of GluN2 subunits utilizing knock-in mice whereby the C-terminus of GluN2A is replaced with that of GluN2B. Surprisingly, the exchange of the C-terminus does not impede the developmental switch in subunits nor the proportion of NMDARs at synaptic vs extrasynaptic sites. However, replacing the C-terminus of GluN2A with that of GluN2B induces a greater neuronal vulnerability to NMDA-dependent excitotoxicity. Collectively, this work enhances our understanding of the complex physiology mediated by the NMDAR by determining how pharmacological tools are best utilized to study the roles of NMDAR location and subunit composition in addition to revealing the importance of the GluN2 C-terminus in development and excitotoxicity.
Supervisor: Hardingham, Giles ; Wyllie, David Sponsor: Biotechnology and Biological Sciences Research Council (BBSRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: NMDA Receptor ; pharmacology ; excitotoxicity ; neuroprotection ; GluN2A ; GluN2B