Drosophila D1 dopamine receptor mediates caffeine-induced arousal

Abstract

The arousing and motor-activating effects of psychostimulants are mediated by multiple systems. In Drosophila, dopaminergic transmission is involved in mediating the arousing effects of methamphetamine, although the neuronal mechanisms of caffeine (CAFF)-induced wakefulness remain unexplored. Here, we show that in Drosophila, as in mammals, the wake-promoting effect of CAFF involves both the adenosinergic and dopaminergic systems. By measuring behavioral responses in mutant and transgenic flies exposed to different drug-feeding regimens, we show that CAFF-induced wakefulness requires the Drosophila D1 dopamine receptor (dDA1) in the mushroom bodies. In WT flies, CAFF exposure leads to downregulation of dDA1 expression, whereas the transgenic overexpression of dDA1 leads to CAFF resistance. The wake-promoting effects of methamphetamine require a functional dopamine transporter as well as the dDA1, and they engage brain areas in addition to the mushroom bodies.

Fig. 1.

Adenosine receptor antagonists decrease sleep in WT Drosophila. (A) CAFF leads to significant sleep loss in WT flies. Percent change in amount of sleep during the 12 h of lights off (STE) to increasing concentrations of CAFF mixed in food in WT CantonS female flies (n = 16–31 flies/concentration; ANOVA; F_{(5, 90)} = 18.7, P = 1.1⁻¹²) and during continuous 96 h of LTE (n = 26–30 flies/concentration; ANOVA; F_{(2, 80)} = 24.6, P = 4.8⁻⁹). LTE values represent average amount of sleep loss during the night, for four nights of the exposure, only for flies that survived until day 4. LTE to CAFF concentrations greater than 1 mg/ml led to lethality. More than 90% of flies survived until day 4 on 1 mg/ml CAFF, similar to the sham-treated group. At 2.5 mg/ml, survival to day 4 was variable (∼50% of flies). (B) Specific adenosine receptor antagonists lead to sleep loss in WT flies. Percent change in amount of sleep during the 12 h of lights off on 2.5 mg/ml nonspecific adenosine antagonist CAFF (n = 14), A1R antagonist CPT (n = 16), and A2R antagonist 3,7-Dimethyl-1–2-propynylxanthine (DMPX) (n = 28) compared with baseline night in WT female flies. *Significant difference by Student's t test (P < 0.05) compared with 0-mg/ml CAFF group.

Fig. 2.

dDA1 receptor, but not DAT, mediates the arousing effects of CAFF. (A) Percent change in amount of sleep during the 12-h STE to increasing concentrations of CAFF in dumb¹ female flies (dDA1 mutants, n = 14–31 flies/concentration; ANOVA; F_{(5, 150)} = 2.6, P = 0.3) and fmn female flies (DAT mutants, n = 14–28 flies/concentration; ANOVA; F_{(5, 96)} = 7.9, P = 3.1⁻⁶). *Significant difference by Student's t test (P < 0.05) compared with 0-mg/ml CAFF group. (B) Percent change in amount of sleep during the 96-h LTE to increasing concentrations of CAFF in dumb¹ flies (n = 25–30 flies/concentration; ANOVA; F_{(2, 71)} = 1.6, P = 0.21) and fmn flies (n = 20–30 flies/concentration; ANOVA; F_{(2, 81)} = 12.7, P = 1.6⁻⁵). LTE values represent the average amount of sleep loss during the night, for four nights of the exposure, only when flies survived until day 4. Concentrations greater than 1 mg/ml CAFF in dumb¹ flies and 0.5 mg/ml in fmn flies led to lethality. *Significant difference by Student's t test (P < 0.05) compared with 0-mg/ml CAFF group. (C) dumb¹ Mutant flies are resistant to the wake-promoting effect of specific adenosine receptor antagonist. Percent change in amount of sleep during 12 h on CPT in WT (n = 16), dumb¹ (n = 20), and fmn (n = 25) flies. *Significant difference by Student's t test (P < 0.001).

Fig. 3.

dDA1 expression in the MBs mediates the arousing effects of CAFF. (A) Functional rescue of the arousing effect of CAFF by expressing dDA1 transgene in the whole brain or MBs of dumb¹ mutant flies. Percent change in amount of sleep during the STE to 2.5 mg/ml CAFF compared with the baseline night (n = 13–38 flies; ANOVA; F_{(6, 133)} = 20, P = 1.4⁻¹⁶). *Significance level by Student's t test (P < 0.001) between the drug-treated group and the sham-treated control strain. (B) Overexpression of dDA1 in the MBs leads to resistance to the arousing effect of CAFF. Percent change in the amount of sleep during the STE to 2.5 mg/ml CAFF (n = 20–58 flies; ANOVA; F_{(6, 230)} = 2.13, P = 4.7⁻¹⁸). *Significance level of P < 0.01 by Student's t test between transgenic flies on the left and their respective controls on the right. (B, Inset) CAFF exposure decreases expression of dDA1 transcript in the heads of WT flies. Percent change in the expression of the dDA1 transcript was measured by quantitative PCR assay in the whole heads of WT female flies. Animals were selected and frozen after either 12 h (STE) or 96 h (LTE). Only flies that lost more than 30% of their baseline amount of sleep when exposed to 2.5 mg/ml CAFF and their sham-treated siblings with less than 10% change in amount of sleep were used for the analysis. *Significance level by Student's t test (P < 0.02) between the drug-treated strains and their sham-treated siblings.

Fig. 4.

Resistance to the arousing effect of METH in dDA1 and DAT mutant flies. (A) Percent change in amount of sleep during the 12-h STE to increasing concentrations of METH in WT (n = 15–62 flies/concentration; ANOVA; F_{(5, 247)} = 13.5, P = 1.2⁻¹¹), dumb (n = 16–32 flies/concentration; ANOVA; F_{(5, 161)} = 0.05, P = 0.99), and fmn (n = 12–15 flies/concentration; ANOVA; F_{(5, 76)} = 1.08, P = 0.4) flies. (B) Percent change in amount of sleep for four nights of LTE to increasing concentrations of METH in WT (n = 13–15 flies/concentration; ANOVA; F_{(3, 53)} = 5.5, P = 0.002), dumb (n = 25–30 flies/concentration; ANOVA; F_{(3, 50)} = 3.11, P = 0.03), and fmn (n = 20–30 flies/concentration; ANOVA; F_{(3, 57)} = 1.27, P = 0.29) flies. *Significance level by Student's t test (P < 0.01) between the drug-treated strains and their sham-treated siblings. #Significance level by Student's t test (P < 0.05) between the transgenic flies, C747/dDA1, and one of the control lines, dDA1/+.

Fig. 5.

dDA1 expression in MB mediates the arousing effect of METH but does not lead to the modulation of dDA1 transcript in the whole heads. (A) Functional rescue of the arousing effect of METH by expressing dDA1 transgene in the MBs of dumb¹ mutant flies. Percent change in amount of sleep during the STE to 2.5 mg/ml METH (n = 12–21 flies; ANOVA; F_{(6, 99)} = 6.8, P = 4.4⁻⁶). All comparisons are experimental flies vs. controls. *Significance level of P < 0.001 by Student's t test between transgenic flies on the left and their respective controls on the right. #Sleep loss in dDA1/elav; dumb¹ flies is significantly different only in respect to one control line elav/+; dumb¹ (P = 0.047) and is not significant compared with dDA1/+; dumb¹. (B) Flies overexpressing dDA1 are responsive to METH similar to control flies. Percent change in amount of sleep during the STE to 2.5 mg/ml METH (n = 21–49 flies; ANOVA; F_{(6, 218)} = 2.14, P = 8.4⁻⁵). #Significance level of P < 0.01 by Student's t test between transgenic flies dDA1/elav and dDA1/C747 and the control strain dDA1/+. (B, Inset) METH exposure does not change the expression of dDA1 transcript in the heads of WT flies (P > 0.1 by Student's t test between the drug-treated strains and their sham-treated siblings). Percentage change in the dDA1 transcript was measured by a quantitative PCR assay in the whole heads of WT female flies. Animals were selected and frozen after either 12 h (STE) or 96 h (LTE). Only flies that lost more than 30% of their baseline amount of sleep when exposed to 2.5 mg/ml METH and their sham-treated siblings with less than a 10% change in amount of sleep were used for the analysis.