A Drosophila model for alcohol reward.

Ethanol is both aversive and rewarding to flies(a) Flies were trained by three spaced training sessions of a 10-min exposure to one odor followed by a 10-min exposure to the second odor paired with 53% ethanol vapor. During the test flies were given the choice of the two odors, and a preference index was calculated by subtracting the number of flies entering the Odor- vial from the Odor+ vial and dividing this number by the total number of flies. Conditioned preference index (CPI) was calculated by averaging the preference indexes of the two reciprocal groups. All values are reported as mean ± s.e.m. (b) Flies showed conditioned aversion when tested 30 min after training (N=8, p=0.006) and conditioned preference 24 hrs after training (N=8, p=0.02) compared to an unpaired control (Wilcoxon 2-sample). (c) Conditioned preference lasted for up to seven days if flies were left undisturbed (Wilcoxon 1-way, N=8, p=0.007 on day 7). (d) Compared to flies that received either odor or ethanol alone, flies conditioned with ethanol or sucrose (Dunnett’s, N=11/group, p=0.05 and p=0.006 respectively) walked over a 2 cm, 100V electric grid to attain the conditioned odor, whereas only flies conditioned with ethanol walked over a 120V electric grid to attain the conditioned odor (p=0.0004). Exposures to either odor alone or ethanol alone (p=0.99 and p=0.87 respectively) did not affect the likelihood to walk over an electric grid.

Pharmacological properties of ethanol induce preference(a) Flies absorbed significant amounts of ethanol during conditioning (Student’s-t post-hoc, N=11/group, p=0.04, p=0.02, p=0.004 for trials 1, 2, and 3 respectively) and recovered within 30 min (p=0.54). (b) Ethanol absorbed during training induced a significant increase in locomotor activity characteristic of acute intoxication (Repeated Measures ANOVA, N=11/group, p=0.002). (c) An odor presented prior to ethanol resulted in significant conditioned aversion (Wilcoxon 1-way, N=8/group, p=0.007) but not conditioned preference (p=1.00) suggesting that an odor can predict onset of the aversive effects of ethanol. (d) An odor presented directly after ethanol resulted in significant conditioned aversion and significant conditioned preference (Wilcoxon 1-way, N=8, p=0.007 for both behaviors) suggesting that ethanol intoxication is required for conditioned preference to form. All data are shown as mean ± s.e.m.

Dopamine is required for conditioned preference(a) Blocking synaptic transmission in dopaminergic neurons during both training and testing did not affect conditioned aversion tested 30 min after training (Kruskal-Wallis, N=8/group, p=0.84), but (b) blocked the formation of preference in both TH− and Ddc–expressing neurons tested 24 hrs later (Wilcoxon 1-way, N=8, p=0.0003 and p=0.007, respectively). (c) Conditioned aversion was not affected by decreasing serotonin levels in the brain using αMTP or dopamine levels using 3IY (Kruskal-Wallis, N=8/group, p=0.07). (d) Conditioned preference was not affected by αMTP (Student’s-t post-hoc, N=8/group, p=0.21) but was blocked by decreasing dopamine levels in the brain using 3IY (p=0.0002). All data are shown as mean ± s.e.m.

Dopamine is required for expression of ethanol rewardTransiently blocking neurotransmission of TH-expressing cells during (a) acquisition or (b) consolidation did not affect conditioned preference (Kruskal-Wallis N=8/group, p=0.06 and p=0.27, respectively). (c) Activity of TH-expressing cells was required for the retrieval or expression of conditioned preference (Kruskal-Wallis N=8/group, p=0.0005). All data are shown as mean ± s.e.m.

The mushroom body is required for aversion and preference(a) Schematic of the subsets of mushroom body neurons: yellow = γ neurons, blue = αβ neurons, red = α’β’ neurons. (b) We transiently inactivated neurotransmission in selected sets of mushroom body neurons using the GAL4 drivers OK107, 201Y, MB247, 5-66a and 4-59. Blocking synaptic transmission of specific mushroom body neurons using the GAL4 drivers OK107 (Student’s-t post-hoc, N=8/group, p<0.0001), 201Y (p=0.003), MB247 (p<0.0001) and 5-66a (p=0.01) during training and testing disrupted conditioned aversion tested 30 min after training. Colored circles represent mushroom body neurons in which GAL4 drivers are expressed as defined above. (c) Inactivation of mushroom body using OK107 (p<0.0001), 201Y (p=0.003), MB247 (p<0.0001) and 5-66a (p=0.01) during both training and test disrupted conditioned preference 24 hrs after training. All data are shown as mean ± s.e.m.

Sequential use of mushroom body neurons(a) Inactivation using drivers OK107 (Student’s-t post-hoc, N=8/group p<0.0001), 201Y (p<0.0001) and MB247 (p=0.006) but not 5-66a during training disrupted acquisition, implicating the γ neurons in acquisition of conditioned preference. (b) Inactivation using drivers OK107 (p=0.004), and 4-59 (p=0.02) disrupted stabilization, implicating the α’β’ neurons in consolidation of conditioned preference. (c) Inactivation using drivers OK107 (p<0.0001), 201Y (p=0.003), MB247 (p<0.0001) and 5-66a (p<0.0001) disrupted retrieval, implicating the αβ neurons in retrieval or expression of conditioned preference. All data are shown as mean ± s.e.m.

scabrous (sca) affects memories for ethanol reward(a) A screen for conditioned ethanol preference of 160 P{GawB}-containing strains with known expression in the mushroom body identified 3 mutations in which conditioned aversion persisted 24 hrs after training (magenta), 54 mutations in which conditioned preference was not expressed (orange), and 3 mutations in which conditioned preference was enhanced (green). Values represent mean (N=8 per strain). (b) In the 5-120 mutant, the P{GawB} element was inserted 125 bp 5’ of exon 1 of scabrous. (c) qPCR showed that the 5-120 mutation decreased scabrous mRNA expression to 55% that of wild-type controls (mean ± s.e.m., N=6 independent samples). (d) – (g) CPI values represent mean ± s.e.m. (d) sca5-120 does not affect conditioned aversion for ethanol 30 min after training. (e) sca5-120 affects conditioned preference for ethanol 24 hrs after training. (f) Complementation analysis of conditioned preference 30 min after training with two independent sca alleles confirms that sca does not affect conditioned aversion. (g) sca5-120 fails to complement the sca1 and scaBP2 alleles for 24 hr conditioned preference. (h) The 5-120 GAL4 expression pattern suggests that sca is expressed in the mushroom body αβ and γ neurons, the antennal lobe (AL), eye, and a number of cell bodies near the ventrolateral protocerebrum and subesophageal ganglia (See also Supplementary Figure 7).