Figure 4.

ARN23746 Rescues Social Deficits and Repetitive Behaviors in the Valproic Acid Mouse Model of Autism, with No Diuretic Effect

(A) Schematic cartoon of the experimental protocol for the treatment of control (CTR) and VPA mice with bumetanide or ARN23746 to the assess the diuretic effect.

(B) Quantification of mean ± SEM and single animal cases of the urine volume collection for 120 min after mice were treated with the indicated drugs (two-way ANOVA on Ranks, F_{treatment (2,63)} = 11.635, p < 0.001, Tukey’s post hoc test, ∗p < 0.05, ∗∗p < 0.01)

(C) Schematic cartoon of the experimental protocol for the treatment of young adult WT and VPA mice with ARN23746 for in vivo efficacy assessment of improved sociability and repetitive behaviors in ASD mice.

(D) Top, schematic representation of the three-chamber test. Bottom left, quantification of the mean ± SEM and single animal cases of sociability index in mice treated with the indicated drugs (two-way ANOVA, F_{condition (1,42)} = 9.575, p = 0.003, Tukey’s post hoc test, ∗∗p < 0.01; ∗∗∗p < 0.001). Bottom right, quantification of the mean ± SEM and single animal cases of social novelty index in mice treated with the indicated drugs (two-way ANOVA, F_{condition (1,42)}= 8.195, p = 0.007, Tukey’s post hoc test, ∗p < 0.05, ∗∗p < 0.01).

(E) Top, schematic representation of the male-female interaction test. Bottom quantification of the mean ± SEM and single animal cases of the interaction time in mice treated with the indicated drugs (two-way ANOVA, F_{treatment (1,42)} = 6.351, p = 0.016, Tukey’s post hoc test, ∗∗p < 0.01).

(F) Top, schematic representation of the marble burying test. Bottom, quantification of the mean ± SEM and single animal cases of the number of marbles buried by mice treated with the indicated drugs (two-way ANOVA, F_{condition (1,56)} = 6.727, p = 0.012, Tukey’s post hoc test, ∗p < 0.05, ∗∗p < 0.01).

(G) Top, schematic representation of the grooming test. Bottom quantification of the mean ± SEM and single animal cases of the grooming time for mice treated with the indicated drugs (two-way ANOVA on Ranks, F_{interaction (1,59)}= 5.019, p = 0.029, Tukey’s post hoc test, ∗p < 0.05, ∗∗p < 0.01).

(H) Top, representative immunoblots for NKCC1 and KCC2 on extracts of membrane-enriched protein fractions from prefrontal cortices of individuals diagnosed with ASD and controls. Bottom left, quantification of KCC2 in samples from individuals with ASD in comparison to age- and sex-matched controls (two-tailed t test, t = 2.128, p = 0.0492). Bottom right, quantification of the NKCC1/KCC2 expression ratio in samples from individuals with ASD in comparison to age- and sex-matched controls (Mann-Whitney rank sum test, p = <0,001). See also Figures S3 and S4 and Tables S4–S6.