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N-methyl-D-aspartate receptors of the central nervous system : network connectivity, trafficking, and plasticity She, Kevin

Abstract

Activity through NMDA type glutamate receptors sculpts connectivity in the developing nervous system and is typically studied in the visual system in vivo where individual synapses are difficult to visualize. Here, we developed a model of NMDA-receptor dependent synaptic competition in dissociated cultured hippocampal neurons. GluN1 -/- (KO) mouse hippocampal neurons were cultured alone or in defined ratios with wild type (WT) neurons. Synapse development was assessed by immunofluorescence for PSD-95 apposed to VGlut1. Synapse density was specifically enhanced only onto minority WT neurons co-cultured with majority KO neighbour neurons and this increased synapse density was dependent on activity through NMDA receptors. This enhanced synaptic density onto NMDA receptor-competent neurons in minority co-culture represents a cell culture paradigm for studying synaptic competition. Trafficking of NMDA receptors to the cell surface is critical for proper brain function. Recent evidence suggest that surface trafficking of other ionotropic glutamate receptors requires ligand binding for exit from the endoplasmic reticulum. We show that glutamate binding is required for trafficking of NMDA receptors to the cell surface by expressing a panel of GluN2B ligand binding mutants in heterologous cells and primary rodent neurons and found that glutamate efficacy correlates with surface expression. Such a correlation was found even with inhibition of endocytosis indicating differences in forward trafficking. These results indicate that ligand binding is critical for receptor trafficking to the cell surface. NMDA receptors mediate many forms of synaptic plasticity. GluN2B is proposed to bind and recruit CaMKII to synapses to mediate multiple forms of synaptic plasticity. We find that accumulation of CFP-CaMKIIα at synapses is induced in wild-type but not in KO neurons by bath stimulation of NMDA receptors or by a chemical long-term potentiation protocol. Stimulated synaptic accumulation of CFP-CaMKIIα was rescued in KO neurons by YFP-GluN2B or chimeric GluN2A/2B tail but not by GluN2A, chimeric GluN2B/2A tail, or GluN2B with point mutations in the CaMKII binding site. Thus, activity-regulated synaptic aggregation of CaMKII is dependent on the cytoplasmic CaMKII binding site of GluN2B and not on differential permeation properties between GluN2B and GluN2A.

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