(A) Spaced olfactory memory paradigm for lasting cue-associated alcohol preference. A moderate dose of ethanol (90:60 EtOH:Air, 13.8 ± 3 mM internal concentration) was paired with either ethyl acetate or isoamyl alcohol. (B) The associative memory center mushroom body (MB) comprises intrinsic neurons (Kenyon cells) that have posteriorly positioned somas and dendritic arbors (calyx), and anteriorly positioned αβ, α’β’, γ axon bundles (blue, green, red). Representation of dopaminergic neurons innervating the MB axon bundles (purple). (C) Scabrous (Sca) knockdown in adult neurons (elav) impaired alcohol associative preference; Control (maintained at 18°C) n = 17, 13, 14; Adult knockdown (shifted after eclosion from 18→30°C) n = 27, 28, 23. (D) Sca knockdown in sca5-120-expressing cells during adulthood, but not development, impaired alcohol associative preference; Developmental Knockdown (shifted after eclosion from 30→18°C) n = 16, 13, 15; Adulthood Knockdown (shifted after eclosion from 18→30°C) n = 27, 22, 30. (E) Sca knockdown in all MB neurons (30Y or split gal4 MB152B) impaired alcohol associative preference; 30Y n = 22, 18, 23; MB152B n = 21, 20, 24. (C-E) Mean ± SEM with statistical significance evaluated by ANOVA, posthoc Bonferroni compared to experimental genotype, *p < 0.05, †Temperature insensitive split-GAL4. See also Figures S1, S2, S9.