Developmental Regulation of Glutamate Receptor Function

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DESCRIPTION (provided by applicant): The long-term objective of this proposal is to understand mechanisms of visual system plasticity. Such knowledge may prove vital to the prevention of amblyopia and the loss of stereopsis as well as to the restoration of vision following trauma or stroke. Visual system synaptic plasticity involves the NMDA subtype of glutamate receptor (NR), which is scaffolded by proteins that also interact with other glutamate receptors. A recent hypothesis is that two distinct protein complexes, consisting of NRs with different subunit compositions, NRscaffolding proteins (MAGUKs), and signaling proteins, are trafficked differently in response to visual system activity and mediate distinct glutamate-induced changes at developing synapses: an early system carries NRs enriched in the NR2B subunit and associated trafficking and signaling molecules; in response to light and eye- opening (EO), this system is replaced by a scaffolding complex with a different trafficking system and carrying NR2A-enriched NRs. To test this hypothesis, the proposed experiments focus on the superficial visual layers of the superior colliculus, which allow electrophysiological studies of changes in glutamate receptor function and synaptic refinement as well as biochemical studies of changes in synaptic protein complexes that may underlie these physiological changes. Specific Aim I is to use coimmunoprecipitation experiments to determine if the postulated receptor-MAGUK-trafficking complexes exist in vivo when they are proposed to act in synaptic competition and refinement. Specific Aim II is to determine if specific responses to EO are tightly linked to high dendritic levels of PSD-95. PSD-95 is the MAGUK protein that scaffolds the mature NR complex and that translocates to synaptic regions in response to EO. Anatomical studies will determine if new currents induced by EO are a consequence of dendritic sprouting, which has been associated with PSD-95-bound proteins. Whole-cell patch-clamping will test for a tight correlation between dendritic PSD-95 and refinement by asking if refinement is lost once PSD-95 is reduced to pre-EO levels after eye re-closure. Specific Aim III is to use NR2A knock-out and PSD-95 knockout mice to attempt to directly test the hypothesis that the PSD-95/NR complex is critical to synaptic refinement and to glutamate current changes that occur in response to EO. Specific Aim IV is to construct a hybrid NR2B/NR2A NR subunit that will allow a determination of the extent to which the biological roles of PSD-95 depend on its ability to localize specific NRs at the synapse as opposed to its ability to stimulate PSD-95-mediated signal transduction.