Behavioral Sensitization to the Disinhibition Effect of Ethanol Requires the Dopamine/Ecdysone Receptor in Drosophila

Abstract

Male flies under the influence of ethanol display disinhibited courtship, which is augmented with repeated ethanol exposures. We have previously shown that dopamine is important for this type of ethanol-induced behavioral sensitization but the underlying mechanism is unknown. Here we report that DopEcR, an insect G-protein coupled receptor that binds to dopamine and steroid hormone ecdysone, is a major receptor mediating courtship sensitization. Upon daily ethanol administration, dumb and damb mutant males defective in D1 (dDA1/DopR1) and D5 (DAMB/DopR2) dopamine receptors, respectively, showed normal courtship sensitization; however, the DopEcR-deficient der males exhibited greatly diminished sensitization. der mutant males nevertheless developed normal tolerance to the sedative effect of ethanol, indicating a selective function of DopEcR in chronic ethanol-associated behavioral plasticity. DopEcR plays a physiological role in behavioral sensitization since courtship sensitization in der males was reinstated when DopEcR expression was induced during adulthood but not during development. When examined for the DopEcR's functional site, the der mutant's sensitization phenotype was fully rescued by restored DopEcR expression in the mushroom body (MB) αβ and γ neurons. Consistently, we observed DopEcR immunoreactivity in the MB calyx and lobes in the wild-type Canton-S brain, which was barely detectable in the der brain. Behavioral sensitization to the locomotor-stimulant effect has been serving as a model for ethanol abuse and addiction. This is the first report elucidating the mechanism underlying behavioral sensitization to another stimulant effect of ethanol.

Keywords: D1 receptors; DopEcR; behavioral sensitization; courtship disinhibition; dopamine; ethanol; mushroom body; tolerance.

Figure 1

D1 family receptors are dispensable for ethanol tolerance. The wild-type Canton-S and D1 receptor mutants were exposed to ethanol and mean sedation time (MST) was measured. (A) der mutant males defective in DopEcR showed decreased sensitivity to the sedative effect of ethanol (***p < 0.0001 by two-tailed Student’s t-test; Canton-S, n = 26; der, n = 22). (B) dumb¹and dumb² mutant males defective in dDA1 (D1) and damb defective in DAMB (D5) exhibited normal sensitivity. Analysis of variance (ANOVA): p > 0.05, n = 8; ns, not significant. (C) der males showed normal tolerance development and maintenance to the sedative effect of ethanol. (***p < 0.0001 by ANOVA and post hoc Tukey-Kramer HSD tests; Canton-S, n = 26; der, n = 22). (D) dumb and damb males displayed normal tolerance. Student t-test; **p < 0.001; ***p < 0.0001; n = 8.

Figure 2

der mutant males exhibit impaired sensitization to the disinhibition effect of ethanol. (A) der mutant males showed significantly reduced disinhibited courtship compared to the control Canton-S on the 2nd (exposure 2) and 6th (exposure 6) day of daily ethanol exposure (***p < 0.0001 by ANOVA and post hoc Tukey-Kramer HSD tests; Canton-S, n = 17; der, n = 19). ns, not significant. (B) dDA1 receptor mutants dumb¹ and dumb² as well as DAMB receptor mutant damb developed normal behavioral sensitization to the ethanol-induced disinhibition. (dumb¹, p < 0.0001, n = 7; dumb², p < 0.0001, n = 7; damb, p < 0.0001, n = 6). E, exposure.

Figure 3

DopEcR knockdown in the mushroom body (MB) αβ and γ neurons suppresses sensitization. (A) Pan-neuronal DopEcR knockdown (elav-GAL4/+;UAS-DopEcR-RNAi/+, R² = 0.50, n = 6) led to substantially reduced sensitization compared to Canton-S or the transgenic control (UAS-GFP/+;UAS-DopEcR-RNAi/+, R² = 0.91, n = 6). Different letters on the bars (i.e., a, b and c) denote significant difference when all genotypes on exposure 6 were compared (ANOVA, p < 0.0001). Normal behavioral sensitization was observed when DopEcR was knocked down in the fruitless (fru) neurons (fru-GAL4/+;UAS-DopEcR-RNAi/+, R² = 0.70, n = 5). DopEcR knockdown in the projection neurons resulted in slightly increased sensitization (NP225-GAL4/+;UAS-DopEcR-RNAi/+, R² = 0.75, n = 7) (c, p = 0.0186 compared to the transgenic control by post hoc Dunnett’s test). (B) DopEcR knockdown in the MB α, β and γ neurons (MB247-GAL4/+;UAS-DopEcR-RNAi/+, R² = 0.61, n = 7) led to significant reduction in behavioral sensitization (p < 0.0001). DopEcR knockdown in individual MB subsets (αβ, c739/+;UAS-DopEcR-RNAi/+, R² = 0.95, n = 6; α’β’, c305a/+;UAS-DopEcR-RNAi/+, R² = 0.81, n = 6; γ, NP1131/+;UAS-DopEcR-RNAi/+, R² = 0.93, n = 6) resulted in normal sensitization.

Figure 4

DopEcR is needed during adulthood to mediate sensitization. The der mutant males carrying tub-GAL80^ts, MB247-GAL4 and UAS-DopEcR cDNA were reared at 30°C before eclosion to induce DopEcR expression during development (A) or after eclosion to induce DopEcR expression during adulthood (B). (A) The der males with reinstated DopEcR expression only during development (MB247, GAL80^ts/UAS-DopEcR cDNA;der, n = 7) exhibited behavioral sensitization at the level comparable to that of the der transgenic mutant (MB247, GAL80^ts/+;der; p > 0.05, n = 6) but lower than that of the Canton-S control (***p < 0.0001, n = 6). (B) The der males with reinstated DopEcR expression only during adulthood (MB247, GAL80 ^ts/UAS-DopEcR cDNA;der; n = 7) showed behavioral sensitization comparable to the control (ns, p > 0.05, n = 7) but higher than the der mutant (MB247, GAL80^ts/+;der; *p < 0.05, n = 4). (C) The der males carrying UAS-DopEcR-cDNA and either tub-GS-GAL4 (UAS-DopEcR cDNA/+;tub-GS-GAL4, der/der, R² = 0.8486, n = 7) or MB247-GS-GAL4 (UAS-DopEcR cDNA /+;MB247-GS-GAL4, der/der, R² = 0.9834, n = 4) displayed sensitization similar to the control (R² = 0.9113, n = 5) when treated with RU486 (ns, p > 0.05), but significantly higher than the der mutant controls (tub-GS-GAL4, der/der, n = 4; MB247-GS-GAL4, der/der, n = 4) treated with RU486 or the der mutants carrying the rescue transgenes without RU486 treatment (***p < 0.0001). The percent intermale courtship on the exposure 6 are shown. ns, not significant.

Figure 5

DopEcR expression in the MB neuropil. DopEcR immunoreactivity is evident in the calyx (A, arrowhead) and medial lobes β and γ (C, arrows) in the Canton-S brain but barely detectable in the der brain (B, arrowhead for calyx; D, arrows for medial lobes). One micron optical sections were made using a 40x (A,B) or 63x (C,D) objective in the confocal microscope and three sections were stacked in all images. Scale bar, 25 micron.