Fig. 1: Generation of Clcn4 KO and analysis of ASD related behavior.

a To generate Clcn4 KO, exon 5 of the Clcn4 gene on chromosome 7 was deleted. Clcn4 mRNA expression mice were completely suppressed in Clcn4 KO when measured by qPCR. Clcn4 KO showed downregulation of Clcn4 but not Clcn3 or Clcn5. b The distribution of pyramidal neurons and the number of spines in the hippocampus were examined through Golgi staining. The number of dendritic spines per 10μm decreased in Clcn4 KO compared to WT. c The three-chamber test was performed at 8 weeks of age. In the social approach, WT mice spent significantly more time in the novel mouse (NM) -containing chamber than in the novel object (NO)-containing chamber (left panel), but there was no difference in the Clcn4 KO group. In social novelty, the WT spent a significantly longer time in the novel mouse (NM)-containing chamber than in the familiar mouse (FM)-containing chamber (right panel), but there was no difference in the Clcn4 KO group. d The marble burying test was performed at 8 weeks of age. Clcn4 KO showed a significant increase in the number of marbles buried, suggesting increased stereotypic behavior. All data were analyzed by t-test and are presented as the mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001).