To determine if Clic genes act exclusively in initial sensitivity or if they also influence the development of acute functional tolerance to ethanol, we extended our genetic analysis to another invertebrate model, the nematode C. elegans. Worms have two Clic genes, exc-4 and exl-1 (Berry et al., 2003). We assessed the responses to acute ethanol using an established behavioral assay in which worms were exposed to the drug on agar plates and locomotor speed was assessed as a function of exposure time (Davies et al., 2004, Davies et al., 2003, Kapfhamer et al., 2008). In this assay, locomotor speed in N2 control worms is increasingly depressed in the presence of ethanol over the first 15 minutes of drug exposure (indicating initial sensitivity). Thereafter, locomotor speed increases despite the continued presence of ethanol (indicating the development of acute functional tolerance to ethanol) (Davies et al., 2004). Repeated measures two-way ANOVA indicated significant effects of time (F(10,120)=14.66, p=0.0244) and genotype (F(3,120)=11.06, p=0.0009) on locomotor behavior in the presence of ethanol with no interaction between the factors (F(10,120)=1.78, p=0.177). Worms harboring exc-4(rh133), a
