The mushroom body consists of three major classes of neurons whose axonal branches occupy distinct subsets of lobes: the αβ, α’β’, and γ neurons. We investigated the role of different mushroom body neurons in the conditioned response to ethanol by suppressing synaptic transmission using UAS-shits in combination with a series of mushroom body GAL4 drivers (Fig. 5a, Supplementary Figs 5a-f). Impairing neurotransmission during training and testing in the entire mushroom body (OK107, Supplementary Fig. 5b), in a combination of γ and αβ neurons (MB247 and 201Y, Supplementary Fig. 5c,d), or in just αβ neurons (5-66a, Supplementary Fig. 5e) disrupted both conditioned aversion (Fig. 5b) and preference (Fig. 5c). There were no differences in conditioned responses in these groups at the permissive temperature with the exception of the highly expressed OK107 driver (Supplementary Figs. 5g,h), an effect that is likely due to low levels of expression at the permissive temperature. In addition, olfactory control tests showed that manipulating mushroom body neurotransmission did not significantly impair odor attraction (Supplementary Table 1). Intriguingly, the odor acuity showed more within-group variation than conditioned preference (Supplementary Table 1), suggesting that it may be sensitive to small fluctuations in ambient humidity, temperature, and fly activity.
