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��Drosophila D1 dopamine receptor mediates caffeine-induced arousal Rozi Andretica.b, Young-Cho Kimc, Frederick S. Jonesa, Kyung-An Hanc, and Ralph J. Greenspana,1 a b c The Neurosciences Institute, San Diego, CA 92121; Department of Psychology, University of Rijeka, 51000 Rijeka, Croatia; and Department of Biology, Pennsylvania State University, University Park, PA 16802 Edited by Jeffrey C. Hall, University of Maine, Orono, ME, and approved November 10, 2008 (received for review July 14, 2008) The arousing and motor-activating effects of psychostimulants are receptor (dDA1) and the DAT. We show that the wake- mediated by multiple systems. In Drosophila, dopaminergic trans- promoting action of CAFF engages both adenosine and dopa- mission is involved in mediating the arousing effects of metham- mine receptors and that CAFF leads to modulation of D1-like phetamine, although the neuronal mechanisms of caffeine (CAFF)- receptors. Wake-promoting actions of CAFF, in particular, induced wakefulness remain unexplored. Here, we show that in require an area of the fly brain that has been linked to both sleep Drosophila, as in mammals, the wake-promoting effect of CAFF regulation and learning and memory, namely, the mushroom involves both the adenosinergic and dopaminergic systems. By bodies (MBs). Our findings emphasize the conservation of measuring behavioral responses in mutant and transgenic flies neural mechanisms regulating the wake-promoting actions of exposed to different drug-feeding regimens, we show that CAFF- psychostimulants between mammals and invertebrates and pro- induced wakefulness requires the Drosophila D1 dopamine recep- vide a model for the neural and molecular basis of behaviors tor (dDA1) in the mushroom bodies. In WT flies, CAFF exposure modulated by arousal. leads to downregulation of dDA1 expression, whereas the trans- Results genic overexpression of dDA1 leads to CAFF resistance. The wake- promoting effects of methamphetamine require a functional do- Adenosinergic and Dopaminergic Systems Mediate Wake-Promoting pamine transporter as well as the dDA1, and they engage brain Effects of CAFF in Drosophila. In Drosophila, CAFF, an adenosine areas in addition to the mushroom bodies. receptor antagonist, decreases sleep and cycloxyladenosine, a specific A1 receptor agonist, promotes sleep; however, the mutants sleep adenosinergic methamphetamine mushroom bodies neural mechanism for this action is not known (13, 14). We exposed WT flies to increasing concentrations of CAFF admin- istered through regular fly food either (i) during a 12-h period ptimal behavioral performance in humans and animals of lights off short-term exposure (STE) or (ii) continuously Odepends on an adequate arousal level, which often involves during a 96-h period of long-term exposure (LTE). Both STE diffuse afferent inputs from the dopaminergic system. Caffeine and LTE reduced sleep dose dependently, mirrored by a dose- (CAFF) displays strong arousing properties and is the most dependent increase in locomotor activity, indicating CAFF s consumed psychoactive drug in the world. CAFF competitively arousing and motor-activating effects in Drosophila [Fig. 1A and inhibits adenosine A1 and A2 receptors, antagonizing the effects supporting information (SI) Fig. S1 A]. Although CAFF signif- of the sleep-promoting neuromodulator adenosine that accu- icantly increases activity, such activity remains lower than the mulates during waking (1). CAFF also leads to increased dopa- activity of unexposed active flies during the day (Fig. S1 A), minergic and glutamatergic transmission in different striatal similar to that of METH-exposed WT flies (Fig. S1B). CAFF- subcompartments, which has been linked to its activating and induced sleep loss is mimicked by the specific A1 (Adenosine 1) reinforcing effects (2 4). receptor antagonist, 8-Cyclopentyll-1,3-dimethlxanthin (CPT) Although CAFF-induced wakefulness has been related to and the A2 (Adenosine 2) receptor antagonist, 3,7-Dimethyl- modulation of cholinergic and histaminergic arousal systems (5, 1 2-propynylxanthine (Fig. 1B). Because both specific and non- 6), CAFF s induction of increased dopaminergic transmission specific adenosine antagonists led to similar behavioral conse- and its effect on wakefulness have not been adequately exam- quences, this suggests that the wake-promoting effects of CAFF ined. Animals with increased dopaminergic transmission, such as are mediated by adenosine receptors. Because a single adenosine dopamine transporter (DAT) mutant mice, have decreased receptor (AdoR), a likely counterpart of the A2B receptor in non-rapid eye movement sleep and increased sensitivity to the mammals, has been described in Drosophila (15), the arousing wake-promoting action of CAFF (7). Dopaminergic action on effects of adenosine receptor antagonists and CAFF are most both the D1 and D2 receptors contributes to the alert waking likely mediated by the same AdoR. state, based on the action of centrally administered D1 and D2 D1-like receptors in mammals mediate the wake-promoting agonists in rodents (8). Molecularly, CAFF modulates D2 tran- and motor-activating properties of psychostimulants such as scription in vitro and in vivo (9). Motor-activating effects of cocaine and amphetamines (8, 16 18). CAFF leads to increased CAFF are diminished in D2R mutant mice (10, 11); however, the dopaminergic transmission by antagonizing A1 receptors on role of D2R in the arousing effect of CAFF remains unknown, presynaptic dopaminergic neurons (19). To determine if dDA1 and functional tests of the brain regions mediating these effects are lacking in mammals. We have shown previously that the wake-promoting effects of Author contributions: R.A., F.S.J., K.-A.H., and R.J.G. designed research; R.A. performed methamphetamine (METH) in Drosophila are mediated research; Y.-C.K. contributed new reagents/analytic tools; R.A., F.S.J., K.-A.H., and R.J.G. through dopaminergic transmission, indicating some evolution- analyzed data; and R.A., K.-A.H., and R.J.G. wrote the paper. ary conservation in the behavioral and neurochemical effects of The authors declare no conflict of interest. psychostimulants (12). This article is a PNAS Direct Submission. Treatment of flies with CAFF induces wakefulness; however, 1 To whom correspondence should be addressed at: The Neurosciences Institute, 10640 John the mechanism underlying this activity is currently unknown (13, Jay Hopkins Drive, San Diego, CA 92121. E-mail: greenspan@nsi.edu. 14). In the present study, we investigate the role of dopamine This article contains supporting information online at www.pnas.org/cgi/content/full/ 0806776105/DCSupplemental. signaling in the wake-inducing properties of CAFF and METH, with emphasis on the role of the Drosophila D1 dopamine � 2008 by The National Academy of Sciences of the USA 20392 20397 PNAS December 23, 2008 vol. 105 no. 51 www.pnas.org cgi doi 10.1073 pnas.0806776105 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-propynylxan- thine (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. mediates the arousing effects of CAFF, we measured sleep in the 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 CAFF-exposed dDA1 mutant dumb1 (20). The dumb1 mutants concentrations of CAFF in dumb1 female flies (dDA1 mutants, n 14 31 showed pronounced resistance to the wake-promoting effect of flies/concentration; ANOVA; F(5, 150) 2.6, P 0.3) and fmn female flies (DAT CAFF and lost a small amount of sleep only at the highest CAFF mutants, n 14 28 flies/concentration; ANOVA; F(5, 96) 7.9, P 3.1 6). doses (see Fig. 2A and Fig. S2 for additional details). The *Significant difference by Student s t test (P 0.05) compared with 0-mg/ml behavioral consequences of CAFF exposure were similar during CAFF group. (B) Percent change in amount of sleep during the 96-h LTE to STE and LTE (Fig. 2 A and B), indicating that the mutant s increasing concentrations of CAFF in dumb1 flies (n 25 30 flies/concentra- resistance cannot be overcome by long-term cumulative effects tion; ANOVA; F(2, 71) 1.6, P 0.21) and fmn flies (n 20 30 flies/ of the drug. We also observed that lethality in dumb1 flies caused 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, by LTE occurred at the same concentrations as in WT flies, that only when flies survived until day 4. Concentrations greater than 1 mg/ml CAFF is, at concentrations higher than 1 mg/ml (Figs. 1 A and 2B). in dumb1 flies and 0.5 mg/ml in fmn flies led to lethality. *Significant differ- Similar mortality in WT and dumb1 flies, but distinct wake- ence by Student s t test (P 0.05) compared with 0-mg/ml CAFF group. (C) promoting effects, suggest that the wake-promoting effects occur dumb1 Mutant flies are resistant to the wake-promoting effect of specific via a different mechanism. adenosine receptor antagonist. Percent change in amount of sleep during 12 h To determine if increased dopaminergic signaling influences on CPT in WT (n 16), dumb1 (n 20), and fmn (n 25) flies. *Significant CAFF responsiveness, we measured the amount of sleep in difference by Student s t test (P 0.001). CAFF-exposed DAT mutant flies, fumin ( fmn) (21). Increased dopaminergic signaling in fmn leads to increased arousal (less because high doses commonly suppress motor activity (24). The sleep) and hyperactivity, as in DAT mutant mice (21 23). motor depressant effect of CAFF was evident at lower concen- Compared with WT flies, fmn flies show significant sleep loss trations than in the WT flies. The fmn flies were also more and a trend toward greater sensitivity at lower concentrations of sensitive to the lethal effects of CAFF, evident as lethality during CAFF. At 0.25 mg/ml, fmn lost 18.4 6.5% (P 0.05 compared LTE at doses higher than 0.5 mg/ml (Fig. 2B). with their baseline) versus WT loss of 11.1 5.6% (P 0.05 compared with their baseline); at 0.5 mg/ml, fmn lost 31.5 To determine whether the effectiveness of CAFF in reducing 5.4% versus WT loss of 24.1 2.9% (both P 0.05 compared sleep requires a functional dopaminergic system, we exposed with their respective baselines). At high concentrations, fmn flies dumb1 and fmn flies to 2.5 mg/ml CPT. As shown in Fig. 2C, lost more sleep at 2.5 mg/ml (40.1 10%) than at 5 mg/ml CAFF treatment of these mutants with CPT mimicked the action of (27.3 5%) (Figs. 1 A and 2 A). This was likely attributable to CAFF. The dumb1 flies were resistant to the wake-promoting a motor depressant effect of high CAFF dose in fmn flies, effects of CPT, whereas fmn flies lost more sleep than WT flies Andretic et al. PNAS December 23, 2008 vol. 105 no. 51 20393 GENETICS plays an important role in olfactory associative learning (20). Therefore, we expressed a functional copy of dDA1 in the MBs of otherwise mutant flies in an attempt to rescue the resistance of dumb1 flies to CAFF. As shown in Fig. 3A, C747, a MB driver, fully restored the wake-promoting effects of CAFF in dumb1 mutant flies. The dDA1/C747; dumb1 flies on CAFF lost 40.7% of their usual amount of sleep, whereas control flies with only one of the two components, DA1/ ; dumb1 or C747/ ; dumb1, showed no significant sleep loss (Fig. 3A). We attained similar results using another MB driver, MB247, suggesting a major role for MBs in mediating the wake-promoting effects of CAFF (Fig. 3A). Expression of dDA1 in the entire brain, using the elav driver, produced greater CAFF-induced sleep loss compared with restricted MB expression (dDA1/elav; dumb1 56.5 6.5, dDA1/C747; dumb1 40.7 5.5, dDA1/MB247; dumb1 31.1 5.4). A subtraction experiment, expressing dDA1 every- where except MBs, confirmed that MB expression was necessary for rescue of the CAFF response (Fig. S3). Altogether, these results suggest that expression of dDA1 in MBs is sufficient to permit the arousing properties of CAFF and that expression of dDA1 in brain areas other than MBs may further augment CAFF responsiveness only if dDA1 is simultaneously expressed in MBs. In mammals, CAFF induces the expression of D2 receptors in vivo and in vitro (9); however, it is unknown if the D1 receptor is under similar transcriptional regulation. To deter- mine if changes in expression of dDA1 are correlated with wake-inducing properties of CAFF in Drosophila, we analyzed Fig. 3. dDA1 expression in the MBs mediates the arousing effects of CAFF. the expression of dDA1 mRNA in the heads of WT flies (A) Functional rescue of the arousing effect of CAFF by expressing dDA1 following STE and LTE to CAFF. Samples were collected transgene in the whole brain or MBs of dumb1 mutant flies. Percent change in from flies that lost more than 30% of their baseline amount of amount of sleep during the STE to 2.5 mg/ml CAFF compared with the baseline sleep (64.1 2.5% during STE and 50.5 5.7% during LTE) night (n 13 38 flies; ANOVA; F(6, 133) 20, P 1.4 16). *Significance level by compared with sham-treated flies that changed their amount 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 resis- of sleep by less than 10%. Fig. 3B (Inset) shows that CAFF led tance to the arousing effect of CAFF. Percent change in the amount of sleep to significant downregulation of dDA1 receptor expression during the STE to 2.5 mg/ml CAFF (n 20 58 flies; ANOVA; F(6, 230) 2.13, P after STE and LTE. 4.7 18). *Significance level of P 0.01 by Student s t test between transgenic To determine if the downregulation is functionally related to flies on the left and their respective controls on the right. (B, Inset) CAFF CAFF-induced sleep loss, as opposed to being a consequence of exposure decreases expression of dDA1 transcript in the heads of WT flies. CAFF exposure unrelated to sleep loss, we overexpressed dDA1 Percent change in the expression of the dDA1 transcript was measured by in the brains of WT flies with the aim of offsetting the modu- quantitative PCR assay in the whole heads of WT female flies. Animals were lation of the dDA1 expression. Fig. 3B shows that transgenic flies selected and frozen after either 12 h (STE) or 96 h (LTE). Only flies that lost with dDA1 overexpressed in the entire brain, dDA1/elav flies, more than 30% of their baseline amount of sleep when exposed to 2.5 mg/ml maintained their CAFF sensitivity and displayed loss of sleep. 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 This loss was similar to elav/ and dDA1/ control lines, 0.02) between the drug-treated strains and their sham-treated siblings. indicating that dDA1 overexpression in the entire brain does not have a functional consequence for the arousal effect of CAFF. However, flies with dDA1 overexpression restricted to the MBs, (Canton-S 30.5 4.8, fmn 48.4 11.3). Resistance to dDA1/C747, behaved significantly differently from their control the wake-promoting effects of either the nonspecific adenosine siblings, DA1/ and C747/ . The dDA1/C747 flies were CAFF receptor antagonist CAFF or the specific A1 antagonist CPT in resistant (Fig. 3B). Similar results were obtained using another dDA1 mutant flies indicates a functional relation between the MB driver, MB247 (Fig. 3B). The possibility that this disparity adenosine and dopamine receptors in mediating the wake- merely reflects inadequacy of MB expression in the elav GAL4 promoting effects of adenosine receptor antagonists. Significant strain is contradicted by experiments showing full rescue of CPT-induced sleep loss in fmn flies further shows that functional CAFF sensitivity with, and no rescue without, the MB contri- DAT is not necessary for the wake-inducing effect of adenosin- bution of elav (Fig. 3A and Fig. S3). Thus, absence of a functional ergic antagonists. dDA1 receptor in MBs, as in dumb1 mutant flies, or overex- pression of the receptor only in the MBs, as in C747/dDA1 Wake-Promoting Effects of CAFF Involve Modulation of dDA1 Recep- transgenic flies, both had the same outcome: resistance to the tor in MBs. To identify brain areas in which the dDA1 receptor arousing effects of CAFF. These findings suggest that dDA1 mediates the action of CAFF, we expressed a WT copy of dDA1 receptor downregulation in the MBs is functionally important for in the brains of dumb1 mutant flies, using the UAS/GAL4 binary eliciting the arousing effects of CAFF. expression system. We first expressed dDA1 in all the neurons of dumb1 mutant flies, using the elav promoter. The dDA1/elav; METH-Induced Wakefulness Does Not Involve Modulation of dDA1 dumb1 flies showed significant sleep loss at 2.5 mg/ml CAFF, Receptor. Acute METH exposure in mammals induces wakeful- indicating successful rescue of CAFF responsiveness (Fig. 3A). ness attributable, in part, to increased dopaminergic signaling (8, Control flies that do not express a functional dDA1 receptor, 18). We have shown previously that METH-induced arousal dDA1/ ; dumb1 and elav/ ; dumb1, remained resistant to CAFF correlates with increased dopaminergic signaling in Drosophila (Fig. 3A). (12). To determine if the arousing effects of CAFF and METH In WT flies, dDA1 is strongly expressed in the MBs, where it are mediated by the same receptor and transporter, we exposed 20394 www.pnas.org cgi doi 10.1073 pnas.0806776105 Andretic et al. 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 concen- trations of METH in WT (n 15 62 flies/concentration; ANOVA; F(5, 247) 13.5, P Fig. 5. dDA1 expression in MB mediates the arousing effect of METH but 1.2 11), dumb (n 16 32 flies/concentration; ANOVA; F(5, 161) 0.05, P 0.99), does not lead to the modulation of dDA1 transcript in the whole heads. (A) and fmn (n 12 15 flies/concentration; ANOVA; F(5, 76) 1.08, P 0.4) flies. Functional rescue of the arousing effect of METH by expressing dDA1 trans- (B) Percent change in amount of sleep for four nights of LTE to increasing gene in the MBs of dumb1 mutant flies. Percent change in amount of sleep concentrations of METH in WT (n 13 15 flies/concentration; ANOVA; F(3, 53) during the STE to 2.5 mg/ml METH (n 12 21 flies; ANOVA; F(6, 99) 6.8, P 5.5, P 0.002), dumb (n 25 30 flies/concentration; ANOVA; F(3, 50) 3.11, P 4.4 6). All comparisons are experimental flies vs. controls. *Significance level 0.03), and fmn (n 20 30 flies/concentration; ANOVA; F(3, 57) 1.27, P 0.29) of P 0.001 by Student s t test between transgenic flies on the left and their flies. *Significance level by Student s t test (P 0.01) between the drug-treated respective controls on the right. #Sleep loss in dDA1/elav; dumb1 flies is strains and their sham-treated siblings. #Significance level by Student s t test significantly different only in respect to one control line elav/ ; dumb1 (P (P 0.05) between the transgenic flies, C747/dDA1, and one of the control 0.047) and is not significant compared with dDA1/ ; dumb1. (B) Flies overex- lines, dDA1/ . pressing 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 dumb1 and fmn flies to increasing doses of METH. In contrast between transgenic flies dDA1/elav and dDA1/C747 and the control strain to WT flies, which lost sleep during the STE and LTE to METH, dDA1/ . (B, Inset) METH exposure does not change the expression of dDA1 fmn and dumb1 flies were resistant (Fig. 4). This result agrees 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 well with findings from D1R and DAT mutant mice, which are the dDA1 transcript was measured by a quantitative PCR assay in the whole likewise resistant to the psychostimulant effect of cocaine and heads of WT female flies. Animals were selected and frozen after either 12 h METH (7, 25). Extended METH exposure in the fly mutants did (STE) or 96 h (LTE). Only flies that lost more than 30% of their baseline amount not increase METH sensitivity; instead, it tended to increase of sleep when exposed to 2.5 mg/ml METH and their sham-treated siblings sleep in fmn flies (Fig. 4B). A similar sleep-promoting effect of with less than a 10% change in amount of sleep were used for the analysis. METH has also been observed in DAT mutant mice (7). Thus, in Drosophila, the arousing effects of METH and CAFF involve partially overlapping components of the dopaminergic system: dDA1, C747/dDA1, and MB247/dDA1 transgenic flies all showed the dDA1 receptor is involved in the behavioral effects of both substantial sleep loss similar to or greater than the control strains drugs, whereas DAT is required only for the arousing effects of (Fig. 5B). This finding suggests that although downregulation of METH. dDA1 in MBs may be required for CAFF-induced wakefulness, Knowing that the expression of dDA1 in MBs is required for METH-induced wakefulness does not involve downregulation of the wake-inducing properties of CAFF, we asked if dDA1 in MBs dDA1. Because sleep loss in C747/dDA1 flies is somewhat mediates METH-induced wakefulness. The dumb1 mutant trans- greater than in elav/dDA1 flies, it is possible that METH-induced genic flies that expressed dDA1 either in the MBs alone (DA1/ wakefulness involves differential regulation of dDA1 expression C747; dumb1 or DA1/MB247; dumb1) or in the entire brain in different brain areas: more in MBs and less in other brain (DA1/elav; dumb1) both showed sleep loss on METH (Fig. 5A). areas. Such a scenario agrees well with our finding that the dDA1 The amount of sleep lost was similar with either whole-brain or transcript does not change in the samples extracted from whole MB expression (DA1/elav; dumb1 16 6.1% sleep loss and heads of METH-exposed flies. DA1/C747; dumb1 20.3 2.9% sleep loss), indicating that although the expression of dDA1 in MBs mediates METH- dDA1 Receptor Does Not Regulate Baseline Sleep. Although dDA1 is induced wakefulness, the expression of dDA1 in areas outside of strongly expressed in MBs, where most genes affecting baseline the MBs does not have a significant additive effect. Thus, dDA1 sleep in Drosophila are expressed (26 31), the average amount expression in MB mediates the arousing effects of METH, of sleep and activity during waking were indistinguishable be- although not as completely as it does CAFF (see also Fig. S3). tween WT and dumb1 flies (Fig. S4 A and B), in contrast to fmn Unlike CAFF, STE to METH did not lead to downregulation flies, which have lowered baseline amounts of sleep and in- of dDA1 transcripts in WT flies (Fig. 5B, Inset); thus, we speculated that overexpression of dDA1 in WT flies would not creased locomotor activity during waking (ref. 21; Fig. S4 A and lead to resistance to METH. Indeed, METH-exposed elav/ B; see SI Text for additional details.) Andretic et al. PNAS December 23, 2008 vol. 105 no. 51 20395 GENETICS Discussion dDA1 in WT flies after CAFF exposure support a model in which the adenosinergic system acts as a neuromodulator of dopaminergic Wake-Inducing Properties of Psychostimulants Are Mediated by dDA1. signaling. CAFF acting through AdoR on dopaminergic neurons In Drosophila, the wake-promoting action of the adenosinergic could stimulate dopamine synthesis or release through protein antagonist CAFF is mediated through the dDA1 receptor. Genetic manipulations of the dDA1 receptor, as in dumb1 kinase A dependent mechanisms similar to the A2A receptor in mammals (4). Postsynaptically, dDA1 receptors located on MB mutants, or overexpression of dDA1 in the MBs of transgenic neurons respond homeostatically by downregulating their expres- flies both lead to resistance to the arousing effects of CAFF. sion, a common adaptive mechanism in response to excessive These apparently paradoxical findings can be reconciled if the stimulation. A related mechanism involving A1-D1 receptor inter- CAFF response requires downregulation of the dDA1 receptor action was observed in the rodent brain and implicated in the in the MBs within a certain range. In support of this model (Fig. psychostimulant properties of CAFF (36). Furthermore, a recent S5), the dDA1 mRNA transcript in WT flies is downregulated in Drosophila report shows increased dopaminergic content concom- response to either STE or LTE to CAFF (see Results), the dDA1 itant with decreased dDA1 expression in the brains of sleep- product is already reduced to a negligible level in the MBs (and deprived flies (37). most other regions) of the dumb mutant (20), and excess expression of the dDA1 receptor in the MBs produces CAFF Role of MBs in Psychostimulant Effects on Sleep. Although the resistance (see Results), suggesting that levels in these flies function of sleep still remains a mystery, one line of evidence cannot be sufficiently downregulated. suggests that synaptic plasticity underlying memory consolidation A role for the MBs in the control of arousal has been proposed might occur during sleep (e.g., ref. 38). That such a conserved in the past (32). MBs have an inhibitory effect on locomotor function of sleep might be present in Drosophila has been sparked activity but a stimulatory effect toward sleep (33, 34). Genetic by a number of recent reports showing overlap between genes and transgenic manipulations of MBs, which lead to decreasing [dunce, rutabaga, Clock, Shaker, 5HT1A, and GABA(A)] and ana- amounts of sleep, are often accompanied by a shortening of tomical regions (MBs), which regulate sleep as well as learning and sleep episodes, and can thus be explained by a premature memory (26, 27, 29, 30, 31, 33, 34). Our findings show that dDA1, arousing signal (21, 26, 29, 31, 33, 34). a receptor with a role in neuronal plasticity in MB-dependent Our observation that the doses of CAFF that decrease sleep learning tasks, has only a moderate role in regulation of baseline also increase motor activity is similar to the effect of CAFF in sleep, although it is important in conditions of elevated arousal, vertebrates. In mammals, the antagonistic effect of CAFF on such as those induced by stimulants. adenosine receptors located on dopaminergic neurons leads to Optimal behavioral performance, such as learning, is depen- increased release (2, 3, 19). A similar mechanism might be dent on adequate levels of arousal (39, 40). Although psycho- operating in flies, based on the correlation that we have shown stimulant exposure increases dopaminergic transmission and between CAFF responsiveness and functional dDA1 receptors in increases general arousal, it also influences specific functions MBs as well as on the motor-activating effects of dopamine (21). related to reward (40). These multiple roles are preserved in Although CAFF and METH lead to similar wake-promoting Drosophila, in which mechanisms for arousal and learning con- and motor-activating effects, the neuronal mechanisms under- verge on the dDA1 receptor, thus ensuring that learning asso- lying responses to these drugs are only partially overlapping. ciated with survival occurs in an attentive and awake organism. Both responses require a functional dDA1 receptor, particularly CAFF and METH effects on dDA1 receptors in MBs could be in the MBs, but METH does not lead to uniform downregulation mimicking, albeit at an elevated level, the increased dopaminer- of dDA1 in the brain, although it is conceivable that downregu- gic signaling that otherwise occurs during learning and memory, lation might occur in a limited area of the brain outside of the reflecting the role that dDA1 receptors play in that process. MB. Although CAFF-induced wakefulness involves dDA1 down- regulation in MBs, METH-induced wakefulness could involve a Methods selective increase of dDA1 in MBs, whereas dDA1 expression Animals. Flies were housed at 25 �C, 60% humidity, and a 12-h light/dark cycle might be unchanged or even decreased in other brain areas. Such on standard agar- and yeast-based food (12). Canton-S was our standard an interpretation is supported by the lack of significant modu- background for all strains: dumb1, fmn, UAS-dDA1; dumb1/TM3, Tb, C747; lation of dDA1 transcript in samples obtained from the entire dumb1, elavGAL4/CyO; dumb1, MB247/CyO; and dumb1, UAS-dDA1, and elav- brain of METH-fed flies as well as weaker rescue of METH Gal4, C747. response when dDA1 was expressed in the entire brain vs. the MBs (Fig. 5A). When dDA1 expression is restricted only to areas Sleep Measurements. Sleep was measured using the Drosophila Activity Mon- outside of the MBs (Fig. S3), METH response is at least as great itoring System (TriKinetics), with a data collection interval set at 5 min as as in panneural (elav) expression, further suggesting the possi- described previously (41), with ad libitum food. bility of antagonism between MBs and other areas for this effect. Pharmacological Treatment. Flies were exposed to CAFF or METH during 12 h Another DA receptor, damb, which is specific to the MBs (35), of lights off or for 96 h starting at lights on. Water-based solutions of drugs is not relevant to these responses. It does not show altered were mixed into the food. Drug effects during STE were calculated by com- regulation in response to CAFF or METH in WT or dumb paring the amount of sleep during the baseline night (without drug) with that mutants, and dDA1 expression alone or in combination with during the treatment night. The sham control for manipulation was a group CAFF or METH is not altered in damb mutants (R.A., Y.-C.K., of flies transferred to food without drug. K.-A.H., R.J.G., unpublished data). Altogether, these findings suggest a model in which the arousing Quantitative PCR. Total RNA extraction, reverse transcription, and quadrupli- and motor-activating effects of CAFF are a consequence of its cate RT quantitative PCR assays were performed as described elsewhere (28). neuromodulatory action on dopaminergic signaling (Fig. S5). This Primers were as follows: dumb forward 5 -CCGTCGTGTCCAGCTGTA-3 and is based on similar behavioral responses to CAFF and CPT in reverse 5 ATAGCAGTATAGCCGACAGTAGATG-3 and RP49 forward 5 - TGGAGGTCCTGCTCATGCA-3 and reverse 5 -GGCATCTCGCGCAGTAAAC-3 . Drosophila, which implies that the arousing properties of CAFF involve close interaction between the adenosine and dopamine ACKNOWLEDGMENTS. We thank H. Dierick, J. Gally, and C. Hughes for helpful systems, as they do in mammals. Presynaptically, CAFF can increase comments and Jen�e Wagner for expert technical assistance. This work was dopamine release by antagonizing adenosine receptors on dopa- supported by National Science Foundation (NSF) grant 052326 (to R.J.G.), by minergic neurons (2, 3). Resistance to the wake-promoting effect of the Neurosciences Research Foundation, and by grants from the National the A1R antagonist in dumb1 mutants and decreased expression of Institute of Child Health and Human Development and NSF (to K.-A. Han). 20396 www.pnas.org cgi doi 10.1073 pnas.0806776105 Andretic et al. Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2006 For Evaluation Only. 1. Porkka-Heiskanen T, Alanko L, Kalinchuk A, Stenberg D (2002) Adenosine and sleep. Sleep 22. Spielewoy C, et al. (2000) Behavioural disturbances associated with hyperdopaminergia in Med Rev 6:321 332. dopamine-transporter knockout mice. Behav Pharmacol 11:279 290. 2. Solinas M, et al. (2002) Caffeine induces dopamine and glutamate release in the shell of 23. Zhuang X, et al. 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