Synaptic upscaling of GluA1 is exaggerated in primary cortical Ppt1^−/− neurons. A, Representative images of surface immunolabeled GluA2 in DIV16 WT and Ppt1^−/− cortical neurons, treated with vehicle control (DMSO, final concentration 0.1%) or TTX (1 μm) for 48 h. Scale bars for low-magnification images = 20 µm, for high-magnification images = 5 µm. B, Quantification of the number of surface immunolabeled GluA2 puncta over 20 µm of dendrite at a standard distance 50–100 µm from the soma. n = 16–23 cells/group across 2 independent cultures. Data are normalized to the WT vehicle control (Veh) condition. Two-way ANOVA: interaction genotype × treatment (F_(1,4) = 5.008, ns); main effect of treatment (F_(1,4) = 199, ***p = 0.0001); main effect of genotype (F_(1,4) = 30.99, **p = 0.0051). Tukey's multiple comparison indicated on graph: **p = 0.0038 WT Veh versus WT TTX; *p = 0.0179 WT Veh versus Ppt1^−/− Veh; **p = 0.0011 Ppt1^−/− Veh versus Ppt1^−/− TTX. C, Quantification of the percent area covered by surface immunolabeled GluA2 puncta over 20 µm of dendrite at a standard distance 50–100 µm from the soma. n = 16–23 cells/group across 2 independent cultures. Two-way ANOVA: interaction genotype × treatment (F_(1,4) = 0.1787, ns); main effect of treatment (F_(1,4) = 85.69, ***p = 0.0008); main effect of genotype (F_(1,4) = 20.03, *p = 0.0110). Tukey's multiple comparison indicated on graph: **p = 0.0082 WT Veh versus WT TTX; *p = 0.0115 Ppt1^−/− Veh versus Ppt1^−/− TTX. D, Representative images of surface immunolabeled GluA1 in DIV16 WT and Ppt1^−/− cortical neurons treated with vehicle control (DMSO, final concentration 0.1%) or TTX (1 μm) for 48 h. Scale bars for low-magnification images = 20 µm, for high-magnification images = 5 µm. E, 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 = 3–5 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 comparison indicated on graph: **p = 0.0097 WT Veh versus WT TTX; ****p < 0.0001 Ppt1^−/− Veh versus Ppt1^−/− TTX; *p = 0.0218 WT TTX versus Ppt1^−/− TTX. Data represent mean ± SEM. F, 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 = 3–5 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 comparison indicated on graph: ****p < 0.0001 WT Veh versus WT TTX; ****p < 0.0001 Ppt1^−/− Veh versus Ppt1^−/− TTX; ****p < 0.0001 WT TTX versus Ppt1^−/− TTX. G, Representative immunoblot of surface GluA1 and transferrin control following surface biotinylation assay in DIV16 cortical neuron cultures. Cells were treated with vehicle (DMSO, final concentration 0.1%), or TTX (1 μm) for 48 h. Note that all displayed immunoblot samples are from the same gel. H, Quantification of the surface GluA1 levels in WT and Ppt1^−/−-treated and Ppt1^−/−-untreated cortical neurons, normalized to transferrin for each experiment and expressed as percent change versus vehicle condition. n = 6 independent cultures. Note that all samples displayed are from the same gel. 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 = 0.0240 WT Veh versus WT TTX; ***p = 0.0002 Ppt1^−/− Veh versus Ppt1^−/− TTX; **p = 0.0038 Ppt1^−/− TTX versus WT TTX. Data represent mean ± SEM. I, Representative immunoblots of GluA1, GluA2, and β-actin loading control for input samples (lysates). n = 6 cultures. Note that all displayed immunoblot samples are in fact from the same gel. J, 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. Two-way ANOVA: no interaction genotype × treatment (F_(2,10) = 2.902, p = 0.1014); no main effect of genotype (F_(1,5) = 2.432e-005, p = 0.9963); main effect of treatment (F_(2,10) = 14.81, **p = 0.0010). Tukey's multiple comparison indicated on graph: *p = 0.0106 WT Veh versus WT TTX; ***p = 0.0001 Ppt1^−/− Veh versus Ppt1^−/− TTX. Data represent mean ± SEM. K, 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). Data represent mean ± SEM. ns = non significant.