Ethanol sedation (loss of righting, LOR) assays were performed essentially as previously described (Corl et al., 2009; Rothenfluh et al., 2006). Samples of 25 to 30 male flies were allowed to equilibrate for approximately 10 minutes to humidified air in the booz-o-mat apparatus (Wolf et al., 2002) before starting exposure to a 100:50 mixture of ethanol vapor:humidified air (100:50 E:A). Ethanol exposure commenced at 0 minutes of the assay and was continuous thereafter. At 2-minute intervals, each tube of flies (8 tubes per assay) was twirled and the number of flies that appeared unable to right themselves was scored by an experimenter blinded to sample identity. Typical assay duration was 30 minutes. To measure ethanol absorption, samples of 25 flies were exposed to 100:50 E:A for 15 minutes and were immediately snap-frozen on dry ice and processed to determine internal ethanol concentration using a kit (Genzyme Diagnostics, Framingham, MA).
