Mecp2 null neuronal network responsiveness is rescued by early treatment with CX546 A: Representative images of DIV14 wt and Mecp2 null primary cortical neurons loaded with Fluo-4 before and after NMDA 100 µM exposure.Figure 4A: Representative images of DIV14 wt and Mecp2 null primary cortical neurons loaded with Fluo-4 before and after NMDA 100 µM exposure. High fluorescent signals correspond to high calcium levels. Dotted circles are representative of the Region Of Interest (ROI) used during the analysis. Scale bars: 20 µm. B-C: The graphs represent the binned distribution of the magnitude of NMDA induced calcium transients in wt (in blue), Mecp2 null (in red) and treated Mecp2 null (light and dark green for early and late treatment respectively). The distribution of responses on X-axis are expressed as percentage compared to wt (100%), values on Y-axis refer to percentage of cells over total (100%). Two-way ANOVA followed by Bonferroni post hoc multiple comparison test was used to compare calcium responses between wt, Mecp2 null and Mecp2 null early (B) and late treated (C) neurons. *: P-value<0.05 ***: p-value<0.005. Red *: comparison between wt and ko; green *: comparison between wt and treated ko. Inserts b' and c' represent the protocols of CX546 exposure; dotted lines indicate the wash out of the drug. Values are represented as mean ± SEM. Sample size: n= 11 and 10 animals for wt and Mecp2 null control groups (for a total of 710 and 670 cells measured respectively) and n= 4 and 6 animals for early and late CX546 treated groups (for a total of 270 and 360 cells measured respectively). D: Representative images of DIV6 and DIV14 wt and Mecp2 null primary cortical neurons loaded with MQAE dye. High levels of fluorescence correspond to low level of intracellular chloride. Dotted circles are representative of the Region Of Interest (ROI) used during the analysis. Scale bars: 20 µm. E: Line graphs represent intracellular chloride concentration at different consecutive time points in wt and Mecp2 null neurons. Two-way ANOVA indicated a significant effect for both time (F(2,1541)=840,9 and p<0,0001) and genotype (F(1,1541)=155,7 and p<0.0001). Bonferroni post hoc test revealed a statistically significant reduction of Mecp2 null MQAE intensity at all the analysed time points. **: p-value<0.01; #: p<0.0001. Values of MQAE intensity are represented as mean ± SEM. Sample size: n≥200 cells from three different embryos per genotype. F,G: Violin plots represent the average values of MQAE intensity for early (F) or late (G) treated null neurons and their relative controls. One-way ANOVA followed by Tukey's multiple comparison test was used to compare null control neurons and null treated neurons to wt neurons. ***: p<0.001; ****: p <0.0001. The central line of the violin plot is the median while the two dotted lines represent the upper and lower quartiles. Sample size: n≥300 cells from five different embryos per genotype. H: Schematic representation of CX546 and Nifedipine administration; dotted line indicates drug wash out. I,J: In I One-way ANOVA was used to compare the expression of 9 neuronal genes between Mecp2 null primary neurons (set to 100), null neurons treated with CX546 from DIV1 to DIV4 and null neurons treated with CX546 and Nifedipine from DIV1 to DIV4. * refers to differences between Mecp2 null untreated neurons and Mecp2 null neurons treated with CX546 (*: p-value<0.05; **: p-value<0.01; ***: p-value<0.001); # refers to differences between Mecp2 null neurons treated with CX546 and null neurons treated with both CX546 and Nifedipine (#: p-value<0.05; ##: p-value<0.01). Panel J shows the ratio between the expression level of Kcc2 and Nkcc1 (expressed as 2-∆ct). Box plot shows the median (central band), the lower and upper quartiles (boxes) and the max and min values (whiskers) from at least 3 wells obtained from four different embryos. One-way ANOVA was used to compare the effects of the different treatments on null neurons compared to the untreated ones. **: p-value<0.01. Sample size: n≥3 wells from four different embryos.