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. Author manuscript; available in PMC: 2014 Sep 1.
Published in final edited form as: Mol Cell Neurosci. 2013 Jul 31;56:10.1016/j.mcn.2013.07.010. doi: 10.1016/j.mcn.2013.07.010

Figure 4. Knockdown of GRIP occludes the increased constitutive GluA2 cycling induced by the absence of activity.

Figure 4

A. Constitutive AMPAR endocytosis was measured in RGCs maintained for >8 hours in the presence or absence of synaptic activity (−Activity or +Activity). Surface GluA2 receptors were immunolabeled in live RGCs that were subsequently fixed at multiple time points. GluA2 endocytosis was also measured in RGCs 72 hours following knockdown of GRIP expression by siRNA. Endocytosis of GluA2 AMPARs was monitored in siRNA transfected (Control and GRIP) cells both maintained in the presence of activity, a condition under which GRIP levels are normally high, and in the absence of activity. Images depict remaining labeled surface GluA2 receptors at 0 and 45 minutes after initial labeling (scale bar = 5μm). B. Quantitation of constitutive GluA2 endocytosis in cultured retinal ganglion cells maintained for >8 hours in the absence or presence of synaptic activity (−Activity or +Activity). The reduction in surface GluA2 labeling is higher in the absence of activity at 30 minutes consistent with an increase in the cycling of the receptors (n=9, * p<.05). C. Images depict expression of GRIP following siRNA treatment (scale bar=10μm). D. GRIP and surface GluA2 levels were measured immunocytochemically in control and GRIP siRNA-treated neurons, 72 hours after infection. Knockdown was measured in cultures maintained for >8 hours in the presence of activity (+Activity), when GRIP levels are normally higher. While GRIP levels were significantly decreased in the siRNA treated cells (n=5, ** p= 0.01), there was little effect on surface GluA2 levels (n=6). E. Quantitation of constitutive AMPAR endocytosis in control and GRIP siRNA treated RGCs. Reduced GRIP expression in cultures maintained in activity (+Activity) resulted in enhanced AMPAR endocytosis comparable to that seen in the absence of activity (−Activity). Knockdown of GRIP had no further effect on AMPAR endocytosis in cultures maintained in the absence of activity, suggesting the effect on receptor trafficking is occluded (n=9, * p< 0.05).