To establish whether flies experience ethanol intoxication as rewarding, we exposed flies to two attractive odor cues (isoamyl alcohol and an ethyl acetate mixture), one of which was associated with a moderately intoxicating dose of ethanol vapor. We then determined which odor the flies preferred to move towards in a Y-maze (Supplementary Fig. 1a). Although naïve flies had no significant preference for the odors used (Supplementary Fig. 1b), a reciprocal training procedure was used to control for any inherent odor preferences (Fig. 1a). Preference for the ethanol-associated odor was expressed as a conditioned preference index (CPI), where a positive CPI indicates attraction and a negative CPI implies repulsion to the ethanol-paired odor. Control odor tests, ethanol absorption assays performed for each fly strain, and detailed statistical analyses are listed in Supplementary Tables 1-5.
