Fig. 6
RIM1 is involved in both constitutive and activity-dependent NMDAR trafficking. a RIM1 KD significantly decreases the surface level of GluN2B/NMDARs. Left: cultured hippocampal neurons were transfected with GFP-tagged control RNAi plasmid (control RNAi, n = 45 neurons from three independent cultures), GFP-tagged RIM1 shRNA plasmid (RIM1 RNAi, n = 29 neurons from three independent cultures), or GFP-tagged RIM1 shRNA-resistant plasmid (RIM1 rescue, n = 26 neurons from three independent cultures), and surface GluN2B was detected by live cell-surface staining. Scale bar, 20 µm. Right: statistical analysis of surface GluN2B intensity. One-way ANOVA with Dunnett’s post test, *p < 0.05. b RIM1 KD significantly reduces the evoked NMDAR currents in cultured cortical neurons. Left: representative recordings of NMDAR currents evoked by NMDA together with glycine. Left: representative traces of NMDA-evoked currents from neurons transfected with control RNAi (n = 11 neurons from three independent cultures), RIM1 RNAi (n = 11 neurons from three independent cultures), or RIM1 KD rescue (n = 26 neurons from three independent cultures) plasmids. Right: statistical analysis of NMDAR-evoked currents. One-way ANOVA with Dunnett’s post test, *p < 0.05. c RIM1 overexpression significantly increases the surface GluN2B/NMDARs levels. Left: representative images of surface GluN2B staining in cultured hippocampal neurons transfected with GFP-RIM1 (n = 20 neurons from three independent cultures) or pEGFP-N1 (n = 35 neurons from three independent cultures). Scale bar, 20 µm. Right: statistical analysis of surface GluN2B intensity; t-test, *p < 0.05. d Treatment with Forskolin (20 μM) and Rolipram (0.1 μM) for 30 min (FSK/Rol, n = 51 neurons from three independent cultures) significantly increases the surface localization of endogenous GluN2B in cultured hippocampal neurons transfected with control RNAi plasmid compared with DMSO (n = 27 neurons from three independent cultures). Scale bar, 20 µm; t-test, **p < 0.01. e Treatment with FSK/Rol (n = 25 neurons from three independent cultures) does not alter the surface localization of endogenous GluN2B in neurons transfected with RIM1 RNAi plasmid compared with DMSO (n = 15 neurons from three independent cultures). Scale bar, 20 µm; t-test, p > 0.05
