To determine when each subset of mushroom body neurons was required for memories of ethanol reward, we blocked transmission specifically during acquisition, consolidation, or retrieval of conditioned preference. We found that silencing a combination of γ and αβ neurons (OK107, MB247, 201Y) during acquisition blocked conditioned preference (Fig. 6a). Because acquisition was not affected by silencing of αβ neurons alone (5-66a), we infer that the γ neurons rather than αβ neurons are important for acquisition (Fig. 6a, Supplementary Fig. 5a-e). In contrast, silencing the α’β’ neurons (OK107, 4-59) during consolidation or the αβ neurons (5-66a) during testing blocked conditioned preference (Figs. 6b,c). Thus, the function of different subsets of mushroom body neurons is needed for the distinct phases involved in forming conditioned preference. Our results thus reveal sequential use of the γ, α’β’, and then αβ neurons. Since our data show that neurotransmission of both dopaminergic and αβ neurons specifically affected conditioned preference expression, we hypothesize that the manifestation of ethanol reward memory may be mediated by dopaminergic innervation of the αβ neurons. This is in line with recent studies
