(A and B) Immunostaining for GluR1 (A) and GluR2 (B) in cultured neurons (9 DIV) from the cerebral cortex of E17.5 Rng105+/+ and Rng105−/− littermates. The neurons were cultured with (+) or without (-) TTX and APV prior to the staining. GluR1 and GluR2 staining before permeabilization (green, surface proteins), after permeabilization (magenta, intracellular and residual surface proteins), and merged images (total proteins) are shown. GluR1 and GluR2 are distributed in a punctate manner both in the soma and dendrites. The insets show magnified images of boxed areas. Arrowheads denote representative GluR1 and GluR2 puncta which were stained both before and after permeabilization (white), only before permeabilization (yellow) and only after permeabilization (blue). Scale bars, 10 µm. (C and D) Quantitative analysis of GluR1 and GluR2 surface expression in dendrites. C, the number of surface GluR1 puncta in dendrites normalized by the number of total GluR1 puncta (left), and fluorescence intensity of surface GluR1 puncta in dendrites normalized by GluR1 fluorescence intensity after permeabilization and in the soma (right). D, the same quantification for GluR2. Data are represented as the mean ± s.e.m. In C, n = 31 (Rng105+/+, −), 35 (Rng105+/+, +), 34 (Rng105−/−, −), and 33 (Rng105−/−, +) neurons from 4 experiments. In D, n = 39 (Rng105+/+, −), 40 (Rng105+/+, +), 39 (Rng105−/−, −), and 38 (Rng105−/−, +) neurons from 4 experiments. ***p<0.005, ****p<0.001 using two-way ANOVA followed by post-hoc Student's t-test. See also Figure 10—figure supplement 1.