To assess ethanol behavior in Clic mutant flies, we used an ethanol Rapid Iterative Negative Geotaxis (eRING) assay developed in one of our component laboratories (Bhandari et al., 2009). Female ClicG0472/+ and ClicEY04209/+ mutants maintained their negative geotaxis behavior longer than did the w1118 female controls during exposure to ethanol (Supplemental Figure S2; two-way ANOVA; time, F(7,216)=249.75, p<0.0001; genotype, F(2,216)=40.36, p<0.0001; n=5), suggesting that Clic mutants have reduced sensitivity to the sedative effects of ethanol. Data from several experiments confirmed the blunted ethanol sensitivity in female ClicG0472/+ and ClicEY04209/+ flies (Figure 3A and B, respectively; *, t tests; ClicG0472/+, t(26)=6.31, p<0.0001; ClicEY04209/+, t(28)=6.87, p<0.0001, n=13–15). Additionally, ClicG0472 and ClicEY04209 hemizygous males and homozygous females (reared at 20°C to improve viability) had diminished ethanol sensitivity (Supplemental Figure S3; two-sample t tests; ClicG0472 males, t(6)=3.55, p=0.012; ClicG0472 females, t(6)=9.39, p<0.0001; ClicEY04209 males, t(7)=5.62, p=0.0008; ClicEY04209 females, t(8)=13.04, p<0.0001, n=3–5). (Note that data in Figure 3 and Supplemental Figure S3 are T50 values that reflect the time required for ethanol to inhibit negative geotaxis by 50%. Higher values indicate blunted ethanol sensitivity; i.e.
