The gene expression-correlation studies in Figure 1, combined with the functional genetic analysis of Clic orthologues in Drosophila (Figures 2–4) and C. elegans (Figure 5), suggested that Clic4 might influence acute behavioral sensitivity to ethanol in mammals. To directly test this possibility, we constructed AAV2 viral vectors to over-express CLIC4 protein in specific regions of mouse brain. As expected, stereotactic injection of AAV2/Clic4 virus in medial prefrontal cortex (including cingulate and secondary motor cortex) in DBA/2J mice increased Clic4 transcript abundance (not shown) and increased expression of epitope-tagged CLIC4 protein (Figure 6A) compared to control animals injected with empty vector (Figure 6B). AAV2-mediated overexpression of Clic4 in DBA/2J mice also decreased the sensitivity to high-dose ethanol (3.8 g/kg) loss-of-righting reflex (LORR) (Figure 6C; *, two-sample t-test, t(49)=2.49, p=0.016, n=26) without changing the anxiolytic or locomotor-activating responses to lower doses of ethanol (1.8 g/kg, not shown). The decreased LORR time with Clic4 overexpression is consistent with results of Fig. 1C showing that higher Clic4 expression correlates with higher blood ethanol levels being required for initiation of rotarod ataxia (decreased sensitivity). These
