Figure 2

D1 receptor agonist SKF-81297 enhances the expression and clustering of NR2B subunit. (A) PFC neurons at 16 DIV were treated with vehicle (a, d, g, j), SKF-81297 (10 μM; b, e, h, k), or SKF in the presence of SCH (15 μM; c, f, i, l), respectively, and double labeled for endogenous NR2B (green) and PSD95 (red). Panel j, k and l are merged from respective NR2B (green) and PSD-95(red) staining. Scale bars = 10 μm. (B) Quantification of total NR2B immunofluorescence staining. White bars: NR2B puncta number; black bars: NR2B fluorescence intensity. SKF-81297 significantly increased total NR2B puncta number and immunofluorescence intensity compared with control. Results are presented as mean number and fluorescence intensity of total NR2B puncta. Statistical analysis was performed using ANOVA followed by Tukey multiple comparison test (n = 20, ** p < 0.01, *** p < 0.001). Data represent mean ± SEM. (C) Proteins were isolated at 16 DIV from cultured high-density PFC neurons treated with DMSO, D1 receptor agonist SKF-81297, or SKF-81297 + SCH-23390. The proteins were resolved on SDS-PAGE and immunoblotted for NR2B and reprobed with tubulin. (D) Quantification of NR2B protein expression in PFC neurons. SKF-81297 significantly increased NR2B expression, which was completely blocked by pretreatment with SCH-23390 (n = 4, ** p < 0.01, *** p < 0.001). Integrated intensity was measured using Image J and control protein level of NR2B was set to 100% after being normalized to tubulin. These results indicate that D1 receptor stimulation does increase NR2B expression, which may eventually increase NMDA receptor trafficking to membrane surface.