Fig. 3. Water learning requires reinforcing dopamine from specific neurons in the PAM cluster.

(a) Screening GAL4 lines that label subsets of PAM dopaminergic neurons revealed significantly impaired water learning performance in R48B04; UAS-shi^ts1 flies (P<0.0001, n≥8; ANOVA followed by post hoc Tukey’s HSD test). (b) Expression pattern of R48B04-GAL4 driven GFP in the adult fly brain. The open yellow box marks the position of dopaminergic neurons in the PAM cluster. Scale bar 40 μm. (c) R48B04-GAL4 driven GFP expression in the PAM cluster is removed by R58E02-GAL80. Scale bar 40 μm. (d) The water learning defect in R48B04; UAS-shi^ts1 flies (P<0.0001, n=8; ANOVA followed by post hoc Tukey’s HSD test) is rescued by removing expression in dopaminergic neurons with R58E02-GAL80 (P>0.4, n=8; ANOVA followed by post hoc Tukey’s HSD test). (e, f) R48B04 dopaminergic neurons innervate the γ4, γ5 and β′2 zones of the horizontal mushroom body lobes. (g, h) 0104 dopaminergic neurons also innervate the γ4, γ5 and β′2 zones. (i, j) Intersectional genetics removing 0104 neuron expression from R48B04 leaves dopaminergic neurons that innervate γ4 and γ5 and deletes expression in β′2 (dotted lines). The genotype is R48B04-LexA::P65/lexAop-rCD2::GFP; 0104-GAL4/UAS-LexA^RNAi. Scale bar 40 μm. (k) Blocking the PAM-γ4/5 neurons significantly impairs water learning (P<0.0001, n=12; ANOVA). (l, m) Pairing a 2 min odor presentation with PAM-γ4/5 neuron stimulation forms appetitive memory that is significantly greater than controls in (l) thirsty flies (P=0.01, n=11; ANOVA) but not in (m) water sated flies (P=0.8, n=8; ANOVA). (n) Subtracting R15A04 neuron expression from R48B04 completely removes labeling in neurons innervating γ5 (dotted lines). Scale bar 40 μm. (o) Removing γ5 neurons from R48B04 did not reverse the water learning defect (P<0.0001, n=8; ANOVA).