GAL4 drivers: one expressed under the control of the tyrosine hydroxylase promotor (TH-GAL4, expressed in most dopaminergic neurons18) and one under the control of dopamine decarboxylase (Ddc-GAL4, expressed in most dopaminergic and serotonergic neurons19). Perturbing neurotransmission in Ddc– or TH– expressing neurons using UAS-shits at the restrictive temperature (30°C) during both training and testing did not affect conditioned aversion (Fig. 3a), but blocked the formation of conditioned preference (Fig. 3b). No effects were observed at the permissive temperature (24°C) (Supplementary Figs. 4a,b). Note that testing flies at 29°C compared to 24°C generally increased their conditioned responses (Figs. 3a,b, Supplementary Figs. 4a,b), possibly due to enhanced locomotion or stronger perception of the training odors. These results indicate that distinct neuronal populations mediate conditioned aversion and preference for ethanol, where activity of dopaminergic neurons is required for flies to form and/or express the association between the attractive properties of ethanol and an odor, but not required for flies to form aversive memories.
