Voluntary intake of psychoactive substances is regulated by the dopamine receptor Dop1R1 in Drosophila

Dysregulated motivation to consume psychoactive substances leads to addictive behaviors that often result in serious health consequences. Understanding the neuronal mechanisms that drive drug consumption is crucial for developing new therapeutic strategies. The fruit fly Drosophila melanogaster offers a unique opportunity to approach this problem with a battery of sophisticated neurogenetic tools available, but how they consume these drugs remains largely unknown. Here, we examined drug self-administration behavior of Drosophila and the underlying neuronal mechanisms. We measured the preference of flies for five different psychoactive substances using a two-choice feeding assay and monitored its long-term changes. We found that flies show acute preference for ethanol and methamphetamine, but not for cocaine, caffeine or morphine. Repeated intake of ethanol, but not methamphetamine, increased over time. Preference for methamphetamine and the long-term escalation of ethanol preference required the dopamine receptor Dop1R1 in the mushroom body. The protein level of Dop1R1 increased after repeated intake of ethanol, but not methamphetamine, which correlates with the acquired preference. Genetic overexpression of Dop1R1 enhanced ethanol preference. These results reveal a striking diversity of response to individual drugs in the fly and the role of dopamine signaling and its plastic changes in controlling voluntary intake of drugs.

Substance use disorders cause severe human health problems, including more than 350 thousand direct and many more indirect deaths per year 1 . Contribution of genetic factors to the risk of drug addiction is estimated to be around half 2 , highlighting the importance of animal models that enable investigation of the genetic underpinnings.
The fruit fly Drosophila melanogaster has been established as a useful genetic model organism for decades. These flies show many characteristic responses common in humans, especially toward ethanol. Ethanol is known to target several ion channels, protein kinase C and adenylate cyclase 3 . Exposure of the flies to vapored ethanol make them first hyperactive, uncoordinated, and eventually sedated [4][5][6] . They develop tolerance after repeated exposure 7,8 and show withdrawal-like symptoms when ethanol is suddenly withheld after chronic exposure 9 . These flies indeed like ethanol: food odor attraction is enhanced by the addition of ethanol 10,11 , and they prefer to feed or lay eggs on food containing ethanol 12,13 . When an odor cue coincides with ethanol exposure, they form associative reward memory 14,15 . Importantly, self-administration of ethanol-containing food increases after several days of drinking or acute exposure to the drug 12,[16][17][18] , capturing one of the most critical steps toward the formation of alcohol use disorder 19,20 .
Effects of other drugs have also been examined in Drosophila. X-ray crystallography showed that methamphetamine and cocaine bind to the Drosophila dopamine transporter 21 , which is the primary molecular target of these drugs in mammals 22 . Similar to humans, methamphetamine and cocaine prevent sleep and cause hyperactivity in a dopamine-dependent manner [23][24][25] . Caffeine also causes insomnia through dopamine and the cAMP pathway [26][27][28][29] . Opioid, which targets several G-protein coupled receptors known as opioid receptors in mammals, affects development and lifespan of flies [30][31][32] . An opiate antagonist is reported to suppress alcohol preference in Results Systematic investigation of drug feeding behavior of flies. To measure the drug preference and its experience-dependent changes, we performed two-choice CAFE assay 44 . A group of four wild type male flies was given a choice between 5% sucrose solution and 5% sucrose solution supplemented with five different drugs in different concentrations (ethanol, cocaine, caffeine, morphine or methamphetamine) (Fig. 1a). Daily consumption of each solution was measured for 4 days, and the drug preference index was calculated ("Methods"). Consistent with the previous report 12 , flies showed a mild preference for ethanol on the first day, which escalated on the following days (Fig. 1b). In contrast, they showed a dose-dependent aversion to caffeine, cocaine and morphine throughout the measurement (Fig. 1b). Aversion to morphine developed over time, suggesting a negative reinforcing effect of the drug (Fig. 1b). Interestingly, methamphetamine was preferred on the first day at 100 µM or 1 mM, but the preference gradually diminished on the following days (Fig. 1b). Ethanol and methamphetamine increased the total food consumption-the sum of consumed sucrose solutions irrespective of drug presence (Fig. 1c), which might be because of the hyperactivity induced by these drugs 23,25,45 . Altogether, these results revealed distinct dynamics of drug intake behavior of flies.
Repeated drug feeding underlies the preference. One of the hallmarks of drug addiction is repeated drug intake. We therefore next asked if the preference for ethanol or methamphetamine comes from more frequent feeding on drug-containing food or increased meal size on it. To dissociate these two possibilities, we estimated the number of feeding events and the average meal size of a single-housed fly by measuring the liquid level every 5 minutes (Figs. 2a and 3a). Interestingly, we found that the flies drank ethanol-or methamphetamine-containing solution more often than the control on the first day (Figs. 2b and 3b). The average size of each meal on the other hand was not significantly different (Figs. 2c and 3c). The frequency of ethanol intake, but not the average meal size, progressively increased over time (Fig. 2b,c). Conversely when methamphetamine was provided, flies became to consume the control sucrose solution more often while frequency of methamphetamine feeding stayed rather constant across the 4 days (Fig. 3b). Therefore, we conclude that the drug preference is shaped by repeated drug feeding, but not by different size of each meal. Dop1R1 signaling in the mushroom body regulates drug preference. Ethanol and methamphetamine induce dopamine release in the fly brain 21,38,[45][46][47] . Therefore, we tested the role of dopamine receptors in the preference for ethanol and methamphetamine by using mutants of the four dopamine receptors: Dop1R1, Dop1R2, Dop2R and DopEcR. We found that the mutant of Dop1R1, but not of the other receptors, failed to acquire the experience-dependent ethanol preference (Fig. 4a). The Dop1R1 mutant however showed a normal or slightly enhanced ethanol preference on the first day (Fig. 4a), suggesting that the acute ethanol preference is Dop1R1-independent. In contrast, the same mutation suppressed the preference for methamphetamine throughout the measurement (Fig. 4d). Dop1R2 mutation also suppressed the methamphetamine preference (Fig. 4d). In contrast, the DopEcR mutant showed continuously high preference for both drugs (Fig. 4a,d), suggesting its role in the drug aversion. Because sugar and ethanol reward is conveyed to the mushroom body (MB) in the context of associative learning 33,38,48,49 , we next tested the role of Dop1R1 in the MB. Genetic knock-down of Dop1R1 in the MB showed a consistent phenotype to the mutant, highlighting the importance of the MB in this behavior (Fig. 4b,e). Consistently, blockade of the reward-conveying PAM cluster dopamine neurons using shibire ts150 abolished the acquired ethanol preference (Fig. 4c). Altogether, these results highlight the importance of the Dop1R1 signaling in the MB for the drug preference but in a distinct manner: acquired preference for ethanol and basal preference for methamphetamine.
The selective requirement of Dop1R1 for acquired, but not acute, ethanol preference prompted us to hypothesize that voluntary intake of ethanol might change the Dop1R1 signaling. To test this hypothesis, we quantified the protein level of Dop1R1 and Dop2R, which mediate the opposite effects 51 , in the brain using the Venus tagged endogenous dopamine receptors 52 . Strikingly, when the flies were given an access to the ethanol-containing food for 3 days, the protein level of Dop1R1, but not Dop2R, was significantly increased in the medial lobe of the MB (Fig. 5a,b). In contrast, addition of methamphetamine did not lead to the Dop1R1 augmentation (Fig. 5c,d). We then hypothesized that the increased Dop1R1 level is sufficient to enhance ethanol preference. Indeed, we found that genetic overexpression of Dop1R1 enhanced ethanol preference (Fig. 5e). Therefore, these results www.nature.com/scientificreports/ suggest that voluntary ethanol intake increases protein level of Dop1R1, which further mediates the acquired ethanol preference (Fig. 5f).

Discussion
In this study, we systematically tested the preference and its experience-dependent changes of five different drugs using a two-choice feeding assay (Fig. 1). Flies showed a robust preference to ethanol, which escalated over time.
A previous study has shown that it occurs in a wide range of the ethanol concentration 12 . In contrast, they avoided cocaine, caffeine and morphine, all of which are plant alkaloid. They even developed an aversion to morphine (Fig. 1b). This diversity might be explained by their evolutionally background: many of the Drosophila species are naturally feeding on fruits, and melanogaster particularly prefers fermented rotten fruits 53 , which is the major source of ethanol in the wild 54 . Therefore, ethanol might function as an indicator of a good food resource, which urges them to repeatedly consume (Fig. 2). Plant alkaloids on the other hand are mainly defensive compounds against herbivore insects 55 . Indeed, cocaine and caffeine prevent the larvae of Manduca sexta from eating plant leaves and eventually kill them 56,57 . Also for flies, cocaine shortens the lifespan and disrupts oogenesis 58 , and caffeine prevents food intake 59 . Our result revealed that Drosophila can readily avoid these plant alkaloids, supporting an idea that drug intake behavior is shaped by the adaptive evolution 60,61 . www.nature.com/scientificreports/ Methamphetamine is an artificial compound and was preferred at the beginning of the measurement (Figs. 1,  3). Analysis of the mutants and the genetic knock-down suggests that the preference is dependent on Dop1R1 and Dop1R2, reminiscent of sugar-rewarded appetitive memory 33,62,63 (Fig. 4d). A recent genome wide association study consistently identified the Dop1R1 gene as a critical regulator of consumption of methamphetamine 64 . Interestingly for ethanol, the Dop1R1 mutant showed normal or slightly enhanced preference on the first day of the measurement but no significant increase on the following days (Fig. 4a). These observations clearly indicate the involvement of the dopamine system, but with different temporal dynamics among these two drugs. Because methamphetamine directly induces a robust dopamine release, primarily by induction of reverse transport through monoamine transporters 21,46,65 , it may be strong enough to stimulate Dop1R1 to promote the drug intake from the beginning. Ethanol on the other hand does not strongly activate the rewarding dopamine neurons at the first exposure but the response develops afterwards 38 . We found that self-administration of ethanol, but not methamphetamine, enhanced the Dop1R1 protein level (Fig. 5). These lines of evidence support an idea that repeated ethanol intake, but not methamphetamine, sensitizes the dopamine-Dop1R1 signaling, possibly by increasing both the dopamine release and the receptor. The sensitized reward signaling might escalate self-administration of ethanol (Fig. 5f). A recently reported switch of Dop2R isoforms in the MB after repeated ethanol exposure 40 might also contribute to the behavioral change. It would be therefore important in the future to investigate at which level, i.e. transcription, translation or protein turnover, the experience-dependent changes take place and how they are regulated.
DopEcR mutant on the other hand showed a higher preference for both ethanol and methamphetamine (Fig. 4a,d). The DopEcR mutant flies are less sensitive to ethanol sedation 66,67 , and show defects in dealing with various stressors such as starvation, heat and sexual rejection [68][69][70] . Therefore, DopEcR might mediate aversive effects of drugs, which include ethanol sedation, thereby function as a 'brake' for the further drug intake. Consistently, a prospective clinical study in humans has shown that lower sensitivity to ethanol sedation predicted a greater number of alcohol use disorders afterwards 71 . Closer look at the interaction of the accel and the brake would help understanding the neural mechanisms of the drug intake behavior.

Methods
Flies. Flies were reared in a mass culture on standard cornmeal food at 24 °C under 12-12 h light-dark cycles.
Canton-S was used as the wild-type. Following dopamine receptor mutants were used: dumb 248 , Dop1R2 attP72 , Dop2R ∆173 and DopEcR GAL466 . Following transgenic strains were used: w 1118 ;;R58E02-GAL4 33 (BDSC #41347), w 1118 ;;UAS-Shibirets1 (pJFRC100) 74 (Figs. 2, 3) was placed in a column-shaped plastic container, to which two capillaries were inserted. Female flies were used for the shibire ts1 blockade (Fig. 4c), because of the high mortality when male flies were measured at the high temperature. Capillaries with inner diameter of 0.5 mm (BF100-50-15, Sutter Instrument, CA, USA) or 0.021 mm (BR708707, BRAND GMBH, Germany) were used for grouped or single flies, respectively. Sucrose (S9378, Sigma-Aldrich, St. Louis, MO, USA), ethanol (09-0851-5, Sigma-Aldrich), caffeine (C8960, Sigma-Aldrich), cocaine hydrochloride (Takeda Pharmaceutical, Tokyo, Japan), methamphetamine hydrochloride (Sumitomo Dainippon Pharma, Osaka, Japan) and morphine hydrochloride (Daiichi-Sankyo, Tokyo, Japan) were dissolved into the evian mineral water (Danon, Paris, France) with indicated concentrations. The sucrose concentration (5%) was chosen following previous studies that measured ethanol preference 12,41,43,44 . Sulforhodamine B sodium salt (S1402, Sigma-Aldrich) was added by 0.005% (w/v, Figs. 1, 4, 5) or 0.02% (Figs. 2, 3) to stain the solution. n-Octyl Acetate (0.001%, A6042, Toyo Chemical Industry, Tokyo, Japan) and vanillin (0.0001%, H0264, Tokyo Chemical Industry) were reciprocally added to the two capillaries to give an odor cue for the flies: half of the groups were presented with the drugs flavored with n-Octyl Acetate and the other half with vanillin. Experiments were performed in a plexiglas box containing wet tissue paper to minimize evaporation in 12 h-12 h light-dark cycles. Temperature was set to 24 °C, except for the shibire ts1 blockade experiment (Fig. 4c), which was performed at 30 °C. Capillaries were pictured every day (Figs. 1, 4, 5) or every 5 min (Figs. 2, 3) using the Pentax Q-S1 camera (Ricoh, Tokyo, Japan). Evaporation was measured for each drug and dose in parallel by imaging the capillaries without flies. Daily consumption was calculated as descent of liquid level subtracted by evaporation, divided by the number of living flies. Preference index was calculated as To detect each meal event (Figs. 2, 3), a threshold was calculated for each time bin as mean evaporation + (standard deviation of evaporation) * 2 using measurements of evaporation without flies. A descent of liquid level bigger than the threshold was defined as a meal bout.

Consumption of the drug solution − Consumption of the control solution
Total consumption with 5% sucrose solution ("Sugar only") or 5% sucrose solution and 5% sucrose solution supplemented with 15% ethanol ("With ethanol") in the CAFE assay chamber for 3 days. Fly brains were then dissected in PBS, immediately fixed in 2% paraformaldehyde in PBS for 1 h at room temperature, and washed three times with PBST (0.1% Triton X-100 in PBS). The brains were then blocked with 3% goat serum in PBST for 30 min at room temperature and incubated in the primary antibody solution (rabbit anti-GFP (1:1000; catalog #A11122, Thermo Fisher Scientific, MA, USA) and 1% goat serum in PBST) and in the secondary antibody solution (Alexa Fluor 488 goat antirabbit (1:1000; catalog #A11034, Thermo Fisher Scientific) and 1% goat serum in PBST) at 4 °C for two nights respectively. Brains were then washed for three times with PBST and mounted in SeeDB2G 77 . Images were obtained using the Olympus FV1200 confocal microscope (Olympus, Tokyo, Japan) with the 20x, 0.85NA oil objective lens (UPLSAPO20XO, Olympus). Images of the two groups were acquired at the same time periods under the identical microscope settings.
Statistics. Statistical tests were performed on GraphPad Prism 8 for macOS (GraphPad Software, CA, USA).
One-sample t-test, repeated measures one-way analysis of variance (ANOVA) or ordinary one-way ANOVA followed by Dunnett's or Sidak's multiple comparison was performed when the assumption of normal distribution (D' Agostino & Pearson test) was not violated. Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli was performed to compare the consumption of the wild type flies (Fig. 1c). Otherwise nonparametric