After an extensive search for ethanol doses and training procedures that produced an optimal conditioned response, we chose a protocol in which flies were given three training sessions spaced by one hour. Each training session consisted of a 10-min exposure to odor 1, followed by a 10-min exposure to odor 2 together with 53% ethanol vapor (and vice-versa; Fig. 1a). We observed conditioned aversion when flies were tested 30 min after training, but conditioned preference 24 hrs later (Fig. 1b). The transition from aversion to preference occurred between 12 and 15 hrs post-training (Supplementary Fig 1c). Remarkably, if the flies were left undisturbed after training, conditioned preference was maintained for at least 7 days (Fig. 1c). These results show that ethanol intoxication, while initially aversive, is associated with a long-lasting expression of reward.
