Figure 6.

Phosphomimetic nNOS mutations S1412D and S847D regulate AMPAR surface expression. Wild-type and nNOS mutants were expressed in cultured hippocampal neurons (21 DIV). Cells were immunostained for surface GluR2 (red) followed by permeabilization and staining for Myc-epitope-tagged nNOS. A–F, Representative images of hippocampal neurons expressing S1412D, wild-type nNOS, or GFP (green), respectively, from Sindbis virus. Scale bars, 20 μm. To the left of each panel, a segment of primary dendrite displays the levels of surface GluR2 or GluR1. A total of 240 boxed regions containing proximal dendrites were analyzed to quantify surface GluR2. G, Results of the quantification of surface GluR2 from cells expressing S1412D, S1412A, S847D, S847A, wild-type nNOS, and GFP as a control. We quantify surface GluR1 by using the same strategy used for determining surface GluR2. H, Results of the quantification of surface GluR1 from cells expressing S1412D, S1412A, S847D, S847A, wild-type nNOS, and GFP as a control. I, Cells were immunostained for total GluR1 and GluR2 by permeabilization and staining with antibody to GluR1 and GluR2. A total of 40 boxed regions containing proximal dendrites were analyzed for each experiment to quantify GluR2 and GluR1. For GluR2 surface expression, the experiments were repeated four times (n = 4; *p ≤ 0.0068; **p ≤ 0.0002) as indicated, and for GluR1 the experiments were repeated three times (n = 3; *p ≤ 0.0001).