Figure 4. The light-induced increase in AMPA receptor-mediated EPSCs is specific to Y782 phosphorylation in Nlgn1.

CA1 Neurons were co-electroporated with plasmids encoding tdTomato, shRNA against endogenous Nlgn1 containing a GFP reporter, shRNA resistant AP-tagged Nlgn1-WT or -Y782F, biotin ligase (BirA^ER), and HA-tagged optoFgfr1. (A, B) Confocal images showing tdTomato (red) and GFP (green). Biotinylated Nlgn1 and optoFGFR1 were stained in different slices using streptavidin-Atto647 and anti-HA antibody, respectively (magenta). (C, D) Scatter plots of AMPAR- and NMDAR-mediated EPSCs, respectively, for electroporated versus paired non-electroporated neurons (control cell) in the indicated conditions. Representative traces (black, blue or violet) normalized to control (grey) are shown as insets. (E, F) Average of AMPAR-and NMDAR-mediated EPSCs, respectively, normalized to the control (100%) in the different conditions. Data were compared to the control condition by the Wilcoxon matched-pairs signed rank test and between themselves using one-way ANOVA followed by Tukey's multiple comparison (**p<0.01, ns: not significant).

Figure 4—figure supplement 1. The light-induced increase in spine density is specific to Y782 phosphorylation in Nlgn1.

(A) Confocal images of tdTomato in apical dendrites from CA1 neurons before (0 hr) and after (24 hr) light activation. Control neurons either did not receive light, or did not contain optoFGFR1, or were expressing the Nlgn1-Y782F mutant. Solid arrowheads point to spines which have appeared, and empty arrowheads to spines which have disappeared in the time period. (B) Normalized spine density for each condition (n = 13–33 dendrites from N = 4–10 cells). Change in spine density was assessed for each condition using the paired t-test (****p<0.0001, ns: not significant). Spine density change was compared across conditions using the Kruskal-Wallis test followed by Dunn’s multiple comparison test, paired t-test (**p<0.001).