FIGURE 5.

Effects of GluN2A and GluN2B knockdown on NMDAR-mediated excitotoxicity and CREB phosphorylation. Live neurons cotransfected with GFP and scrambled shRNA (shRNA-Sc) or shRNA-GluN2Aa or shRNA-GluN2Bm were monitored before and after the 30-min NMDA (50 μm) treatment (A), which included the application of 2 μm glycine, 5 μm nifedipine, and 20 μm CNQX. Neurons with the formation of varicosity and cell body swelling were counted and quantified (B and C). The images of the same GFP-positive neuron before and after NMDA treatment are shown (A). The multiple varicosities appeared in representative neurons following NMDA treatment (arrows). The cell body of the same neuron before and after NMDA treatment is enlarged and compared in the right panels. Cell body swelling of the representative neurons is indicated by the non-parallel diverging lines. D, transfected neurons were treated with 50 μm NMDA for 10 min and then fixed and costained for p-CREB and GFP (D). The relative intensity of the p-CREB signal (as indicated by arrows for the representative GFP-positive cell) in neurons transfected with GFP and scrambled shRNA was defined as 1 (right panel) and compared with those in neurons transfected with GFP and GluN2A or GluN2B shRNA constructs. One-way ANOVA revealed a significant difference (i.e. p < 0.05) among different treatments. The post hoc SNK analysis revealed that the values associated with distinct SNK groups (a, b, and c) are significantly different: a < b < c.