CNQX-induced exaggerated upscaling and impaired downscaling in Ppt1^−/− cortical neurons. A, Representative images of surface immunolabeled GluA1 in DIV16 WT and Ppt1^−/− cortical neurons, treated with bicuculline (20 μm) or CNQX (1 μm) for 48 h. Vehicle controls are displayed in Figure 3. Scale bars for low-magnification images = 20 µm, for high-magnification images = 5 µm. B, Quantification of the number of surface immunolabeled GluA1 puncta over 20 µm of dendrite at a standard distance 50–100 µm from the soma. n = 2–5 cultures/group (total number of cells analyzed are listed for each group on the graph). Data are normalized to the WT vehicle control (Veh) condition. Two-way ANOVA: interaction genotype × treatment (F_(3,18) = 5.620, **p = 0.0067); main effect of genotype (F_(1,18) = 10.06, **p = 0.0053); main effect of treatment (F_(3,18) = 52.74, ****p < 0.0001). Tukey's multiple comparisons shown on graph: *p = 0.0218 WT Veh versus WT CNQX; ****p < 0.0001 Ppt1^−/− Veh versus Ppt1^−/− CNQX. C, Quantification of the percent area covered by surface immunolabeled GluA1 puncta over 20 µm of dendrite at a standard distance of 50–100 µm from the soma. n = 2–5 cultures/group (total number of cells analyzed are listed for each group on the graph). Data are normalized to the WT vehicle control condition. Two-way ANOVA: interaction genotype × treatment (F_(3,18) = 26.21, ****p < 0.0001); main effect of genotype (F_(1,18) = 51.25, ****p < 0.0001); main effect of treatment (F_(3,18) = 208.2, ****p < 0.0001). Tukey's multiple comparisons shown on graph: *p = 0.0425 WT Bic versus WT Veh; ****p < 0.0001 WT Veh versus WT CNQX; ****p < 0.0001 Ppt1^−/− Veh versus Ppt1^−/− CNQX; ***p = 0.0004 WT CNQX versus Ppt1^−/− CNQX. D, Representative immunoblot of surface GluA1 and transferrin control following surface biotinylation assay in DIV16 cortical neuron cultures. Cells were treated with bicuculline (20 μm) or vehicle (DMSO, final concentration 0.1%) for 48 h. Note that all samples displayed are from the same gel. E, Quantification of the surface GluA1 levels in WT and Ppt1^−/− treated and untreated cortical neurons, normalized to transferrin for each experiment and expressed as percent change versus vehicle condition. n = 6 independent cultures. Two-way ANOVA: interaction genotype × treatment (F_(2,10) = 7.061, *p = 0.0122); main effect of genotype (F_(1,5) = 14.21, *p = 0.0130); main effect of treatment (F_(2,10) = 34.77, ****p < 0.0001). Tukey's multiple comparison indicated on graph: *p = 0.0418 WT Bic versus WT Veh. F, Representative immunoblots of GluA1, GluA2, and β-actin loading control for input samples (lysates). n = 6 cultures. Note that all displayed immunoblot samples for a given protein are from the same gel. G, Quantification of input GluA1 levels in WT and Ppt1^−/−-treated and Ppt1^−/−-untreated cortical neurons, normalized to β-actin for each experiment and expressed as percent change versus vehicle condition. n = 6 independent cultures. H, Quantification of input GluA2 levels in WT and Ppt1^−/−-treated and Ppt1^−/−-untreated cortical neurons, normalized to β-actin for each experiment and expressed as percent change versus vehicle condition. n = 6 independent cultures. Two-way ANOVA: interaction genotype × treatment (F_(2,10) = 9.617, **p = 0.0047); no main effect of genotype (F_(1,5) = 1.699, p = 0.2492); no main effect of treatment (F_(2,10) = 3.316, p = 0.0786). Tukey's multiple comparison indicated on graph: *p = 0.0112 WT Bic versus WT Veh.