Fly internal ethanol concentrations were determined from whole-fly homogenates of 50 flies per sample. Flies were exposed to vaporized ethanol or air as outlined in training protocol. Flies were frozen immediately in liquid nitrogen and stored at -80°C. Flies were homogenized in 500 μL of cold 50 mM Tris-HCl (pH 7.5, Sigma, St. Louise, MO) and the homogenate was centrifuged at 14 g for 20 minutes at 4°C. Ethanol concentrations in supernatants were measured using an alcohol dehydrogenase-based spectrophotometric assay (Ethanol Assay Kit#229-29, Diagnostic Chemicals Limited, P.E.I., Canada). To calculate fly internal ethanol concentration, the volume of 1 fly was estimated to be ~2 μL43. Ethanol concentration control data is presented in Supplementary Table 2.
