Moreover, cotransfection of siRNA-GluN2B along with the CaMKII co

Moreover, cotransfection of siRNA-GluN2B along with the CaMKII constitutively active mutant CaMKII T286D (Fong et al., 1989), but not WT CaMKII or the nonphosphorylatable mutant T286A, was able to rescue GluN2B loss of function (Figures 7E and 7F). These data suggest that both proper localization and activation of CaMKII downstream of GluN2B are critical for maintaining appropriate levels of AMPARs at developing cortical synapses. The decrease in levels of phosphorylated CaMKII was not

due to a decrease in total CaMKII protein, because this was actually enhanced in the dendrites of siRNA-GluN2B-expressing neurons (Figure 7D). Another important protein effector of NMDAR function is the synaptically localized GTPase activating protein, SynGAP. SynGAP has been shown to interact preferentially with GluN2B-containing NMDARs, and the phenotype of the SynGAP knockout animal is strikingly similar to the GluN2B knockout (Kim et al., 2003, Vazquez et al., 2004, Kim et al., 2005 and Kutsuwada et al., 1996). We examined SynGAP expression and function in the 2B→2A mouse, hypothesizing that it could be a major effector of GluN2B signaling at glutamatergic synapses. Consistent with previous reports, we observed a significant decrease in mean mEPSC

amplitudes in neurons transfected with WT full-length SynGAP (Figures S6A and S6B). From this we inferred that if SynGAP-mediated regulation of AMPAR trafficking acted downstream of GluN2B, coexpression of SynGAP would rescue GluN2B loss of function. However, overexpression of SynGAP did not rescue mEPSC amplitudes recorded in GluN2B-siRNA-expressing neurons (Figure S6B). Furthermore, we tested the requirement for SynGAP activation in this system by cotransfecting neurons with 2BsiRNA + CaMKII T286D and siRNA against SynGAP. Fossariinae SynGAP siRNA did not block the rescue induced by CaMKII T286D, and in neurons expressing 2BsiRNA and SynGAP siRNA, we observed an additive increase in mEPSC amplitudes (Figure S6D).

Together, these data suggest that although SynGAP can regulate AMPAR content at developing synapses, it is not a strong candidate for effecting GluN2B signaling and regulating homeostatic synaptic plasticity. NMDARs are critical for proper circuit development, and suppression of NMDAR function during development can be genetically induced via decreased expression of the obligatory GluN1 subunit (GluN1 hypomorph) (Mohn et al., 1999). This manipulation results in a behavioral phenotype marked by hyperlocomotion and decreased sociability. Due to the strong synaptic phenotype we observed in the 2B→2A mice, we wondered whether the changes observed in the GluN1 hypomorph animal might be attributable to specific loss of GluN2B function during development. In support of this hypothesis, 2B→2A animals exhibited increased spontaneous locomotion in a familiar cage setting when measured P15–P21 (cage transect counts per 3 min) (Figure 8C).

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