Early treatment with Ampakine ameliorates behavioral deficits of Mecp2 null mice A: Schematic representation of the in vivo CX546 administration protocol.Figure 5A: Schematic representation of the in vivo CX546 administration protocol. B: Kaplan Mayer test shows an improved lifespan of Mecp2 null CX546 treated animals compared to Mecp2 null control animals. Sample size: wt=11, ko=16, ko treated with CX546=11. Gehan-Breslow-Wilcoxon test was used to compare the two groups. **: p-value<0.01. C: The body weight of mice was assessed at four different time points (P30, P50, P70 and P90). Two-way ANOVA indicated a significant effect for both time (F(1,813,47,13)=421,1 and p<0,0001) and treatment (F(2,33=7,540 and p<0,0001). Tukey's multiple comparison test was used to compare within each time point the effect of the CX546 treatment on Mecp2 null mice. **: p-value<0.01; ****: p-value<0.0001. Values are represented as average ± SEM. Sample size: n=10 wt and n= 27 ko (16 treated with vehicle and 11 treated with CX546). Each dot represents a single animal. Severity scores were calculated at different time points (P55, P70, P80 in D). Sample size for D n=10 wt treated with vehicle and n= 20 ko (9 treated with vehicle and 11 treated with CX546). Data are represented as mean ± SEM. E: The score for hind limbs clasping was assessed twice a week starting from P30 (E). Sample size for E: n=10 wt treated with vehicle and n= 20 ko (9 treated with vehicle and 11 treated with CX546). Data are represented as mean ± SEM. F,G: Line graph (F) shows mice motor learning assessed on the accelerating rotarod during three consecutive trials. Two-way ANOVA indicated a significant effect of time (F(2,21)=5,490 and p=0,0121) and genotype (F(2,33)=16,33 and p<0.0001). Bonferroni multiple comparison test highlighted a significant increase in the latency to fall between trial 1 and trial 3 only for null mice treated with CX546. *: p-value<0.05; **: p-value<0.01. The scatter plot (G) shows latency to fall (in seconds) on the accelerating rotarod in the last trial. One-way ANOVA was used to compare ko mice and ko treated mice to the wt group. The Tukey's multiple comparison test highlighted a significant reduction in the latency to fall between wt and ko but not between wt and ko treated with Ampakine; **: p-value<0.01. Values are represented as average ± SEM. Sample size: n=6 wt treated with vehicle and n=14 ko (6 treated with vehicle and 8 treated with CX546). Each dot represents a single animal. H: The graph shows the assessment of the discrimination index (DI) between two different objects for each mouse during the last trial of a Novel Object Recognition test. One-way ANOVA was used to compare ko mice and ko treated mice to the wt group. The Tukey's multiple comparison test highlighted a p-value of 0,07 between wt and ko treated with vehicle but not between wt and ko treated with CX546. Data are represented as mean ± SEM. Sample size: n=8 wt treated with vehicle n= 12 ko (6 treated with vehicle and 6 treated with CX546). Each dot represents a single animal. I,J: The graphs show the expression level of Bdnf (I) and Kcc2 (J) in cortical samples obtained from P45 animals. One-way ANOVA was used to assess difference in the expression of the two selected genes between wt, ko e CX546 treated ko animals. For Bdnf expression (I) Tukey's multiple comparison test highlighted significant differences between both wt and ko and ko and ko treated with CX546 but no difference between wt and ko treated with CX546 (*: p-value<0.05; **: p-value<0.01). For Kcc2 expression (J) Tukey's multiple comparison test highlighted significant differences between wt and ko but no difference between wt and ko treated (*: p-value<0.05). Data are represented as values ± SEM. Sample size for I: n=10 wt, n=9 ko and n=6 ko treated with CX546. Sample size for J: n=11 wt, n=12 ko and n=7 ko treated with CX546. Each dot represents a single animal.