To determine whether conditioned odor preference could be attributed to the pharmacological effects of ethanol, we first measured internal ethanol levels in conditioned flies. Indeed, extracts of conditioned flies contained significant levels of ethanol (Fig 2a, Supplementary Fig. 3c), which were sufficient to stimulate locomotion (Fig. 2b). We next asked if flies needed to be “under the influence” in order to form associations with odors by delivering the odor either immediately before (Fig. 2c) or after (Figs. 2d) the ethanol exposure. We found that conditioned preference was elicited only when the odor was presented immediately after the ethanol exposure, when flies still contained significant levels of ethanol (Fig. 2d). Thus, the formation of conditioned preference required the temporal coincidence of internal ethanol levels and the odor cue.
