Drosophila D1 dopamine receptor mediates
caffeine-induced arousal

Rozi Andretic

a.b

, Young-Cho Kim

c

, Frederick S. Jones

a

, Kyung-An Han

c

, and Ralph J. Greenspan

a,1

a

The Neurosciences Institute, San Diego, CA 92121;

b

Department of Psychology, University of Rijeka, 51000 Rijeka, Croatia; and

c

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
mediated by multiple systems. In Drosophila, dopaminergic trans-
mission is involved in mediating the arousing effects of metham-
phetamine, 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 recep-
tor (dDA1) in the mushroom bodies. In WT flies, CAFF exposure
leads to downregulation of dDA1 expression, whereas the trans-
genic overexpression of dDA1 leads to CAFF resistance. The wake-
promoting effects of methamphetamine require a functional do-
pamine transporter as well as the dDA1, and they engage brain
areas in addition to the mushroom bodies.

mutants

兩 sleep 兩 adenosinergic 兩 methamphetamine 兩 mushroom bodies

O

ptimal behavioral performance in humans and animals
depends on an adequate arousal level, which often involves

diffuse afferent inputs from the dopaminergic system. Caffeine
(CAFF) displays strong arousing properties and is the most
consumed psychoactive drug in the world. CAFF competitively
inhibits adenosine A1 and A2 receptors, antagonizing the effects
of the sleep-promoting neuromodulator adenosine that accu-
mulates during waking (1). CAFF also leads to increased dopa-
minergic and glutamatergic transmission in different striatal
subcompartments, which has been linked to its activating and
reinforcing effects (2–4).

Although CAFF-induced wakefulness has been related to

modulation of cholinergic and histaminergic arousal systems (5,
6), CAFF’s induction of increased dopaminergic transmission
and its effect on wakefulness have not been adequately exam-
ined. Animals with increased dopaminergic transmission, such as
dopamine transporter (DAT) mutant mice, have decreased
non-rapid eye movement sleep and increased sensitivity to the
wake-promoting action of CAFF (7). Dopaminergic action on
both the D1 and D2 receptors contributes to the alert waking
state, based on the action of centrally administered D1 and D2
agonists in rodents (8). Molecularly, CAFF modulates D2 tran-
scription in vitro and in vivo (9). Motor-activating effects of
CAFF are diminished in D2R mutant mice (10, 11); however, the
role of D2R in the arousing effect of CAFF remains unknown,
and functional tests of the brain regions mediating these effects
are lacking in mammals.

We have shown previously that the wake-promoting effects of

methamphetamine (METH) in Drosophila are mediated
through dopaminergic transmission, indicating some evolution-
ary conservation in the behavioral and neurochemical effects of
psychostimulants (12).

Treatment of flies with CAFF induces wakefulness; however,

the mechanism underlying this activity is currently unknown (13,
14). In the present study, we investigate the role of dopamine
signaling in the wake-inducing properties of CAFF and METH,
with emphasis on the role of the Drosophila D1 dopamine

receptor (dDA1) and the DAT. We show that the wake-
promoting action of CAFF engages both adenosine and dopa-
mine receptors and that CAFF leads to modulation of D1-like
receptors. Wake-promoting actions of CAFF, in particular,
require an area of the fly brain that has been linked to both sleep
regulation and learning and memory, namely, the mushroom
bodies (MBs). Our findings emphasize the conservation of
neural mechanisms regulating the wake-promoting actions of
psychostimulants between mammals and invertebrates and pro-
vide a model for the neural and molecular basis of behaviors
modulated by arousal.

Results

Adenosinergic and Dopaminergic Systems Mediate Wake-Promoting
Effects of CAFF in

Drosophila.

In Drosophila, CAFF, an adenosine

receptor antagonist, decreases sleep and cycloxyladenosine, a
specific A1 receptor agonist, promotes sleep; however, the
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
of lights off short-term exposure (STE) or (ii) continuously
during a 96-h period of long-term exposure (LTE). Both STE
and LTE reduced sleep dose dependently, mirrored by a dose-
dependent increase in locomotor activity, indicating CAFF’s
arousing and motor-activating effects in Drosophila [Fig. 1A and

supporting information (SI) Fig. S1 A

]. Although CAFF signif-

icantly increases activity, such activity remains lower than the
activity of unexposed active flies during the day (

Fig. S1 A

),

similar to that of METH-exposed WT flies (

Fig. S1B

). CAFF-

induced sleep loss is mimicked by the specific A1 (Adenosine 1)
receptor antagonist, 8-Cyclopentyll-1,3-dimethlxanthin (CPT)
and the A2 (Adenosine 2) receptor antagonist, 3,7-Dimethyl-
1–2-propynylxanthine (Fig. 1B). Because both specific and non-
specific adenosine antagonists led to similar behavioral conse-
quences, this suggests that the wake-promoting effects of CAFF
are mediated by adenosine receptors. Because a single adenosine
receptor (AdoR), a likely counterpart of the A2B receptor in
mammals, has been described in Drosophila (15), the arousing
effects of adenosine receptor antagonists and CAFF are most
likely mediated by the same AdoR.

D1-like receptors in mammals mediate the wake-promoting

and motor-activating properties of psychostimulants such as
cocaine and amphetamines (8, 16–18). CAFF leads to increased
dopaminergic transmission by antagonizing A1 receptors on
presynaptic dopaminergic neurons (19). To determine if dDA1

Author contributions: R.A., F.S.J., K.-A.H., and R.J.G. designed research; R.A. performed
research; Y.-C.K. contributed new reagents/analytic tools; R.A., F.S.J., K.-A.H., and R.J.G.
analyzed data; and R.A., K.-A.H., and R.J.G. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1

To whom correspondence should be addressed at: The Neurosciences Institute, 10640 John

Jay Hopkins Drive, San Diego, CA 92121. E-mail: [e-mail].

This article contains supporting information online at

www.pnas.org/cgi/content/full/

0806776105/DCSupplemental

.

© 2008 by The National Academy of Sciences of the USA

20392–20397

兩 PNAS 兩 December 23, 2008 兩 vol. 105 兩 no. 51

www.pnas.org

兾cgi兾doi兾10.1073兾pnas.0806776105

mediates the arousing effects of CAFF, we measured sleep in the
CAFF-exposed dDA1 mutant dumb

1

(20). The dumb

1

mutants

showed pronounced resistance to the wake-promoting effect of
CAFF and lost a small amount of sleep only at the highest CAFF
doses (see Fig. 2A and

Fig. S2

for additional details). The

behavioral consequences of CAFF exposure were similar during
STE and LTE (Fig. 2 A and B), indicating that the mutant’s
resistance cannot be overcome by long-term cumulative effects
of the drug. We also observed that lethality in dumb

1

flies caused

by LTE occurred at the same concentrations as in WT flies, that
is, at concentrations higher than 1 mg/ml (Figs. 1 A and 2B).
Similar mortality in WT and dumb

1

flies, but distinct wake-

promoting effects, suggest that the wake-promoting effects occur
via a different mechanism.

To determine if increased dopaminergic signaling influences

CAFF responsiveness, we measured the amount of sleep in
CAFF-exposed DAT mutant flies, fumin ( fmn) (21). Increased
dopaminergic signaling in fmn leads to increased arousal (less
sleep) and hyperactivity, as in DAT mutant mice (21–23).
Compared with WT flies, fmn flies show significant sleep loss
and a trend toward greater sensitivity at lower concentrations of
CAFF. At 0.25 mg/ml, fmn lost 18.4

⫾ 6.5% (P ⬍ 0.05 compared

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

⫾

5.4% versus WT loss of 24.1

⫾ 2.9% (both P ⬍ 0.05 compared

with their respective baselines). At high concentrations, fmn flies
lost more sleep at 2.5 mg/ml (40.1

⫾ 10%) than at 5 mg/ml CAFF

(27.3

⫾ 5%) (Figs. 1A and 2A). This was likely attributable to

a motor depressant effect of high CAFF dose in fmn flies,

because high doses commonly suppress motor activity (24). The
motor depressant effect of CAFF was evident at lower concen-
trations than in the WT flies. The fmn flies were also more
sensitive to the lethal effects of CAFF, evident as lethality during
LTE at doses higher than 0.5 mg/ml (Fig. 2B).

To determine whether the effectiveness of CAFF in reducing

sleep requires a functional dopaminergic system, we exposed
dumb

1

and fmn flies to 2.5 mg/ml CPT. As shown in Fig. 2C,

treatment of these mutants with CPT mimicked the action of
CAFF. The dumb

1

flies were resistant to the wake-promoting

effects of CPT, whereas fmn flies lost more sleep than WT flies

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.

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 dumb

1

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 dumb

1

flies (n

⫽ 25–30 flies/concentra-

tion; 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 dumb

1

flies and 0.5 mg/ml in fmn flies led to lethality. *Significant differ-

ence by Student’s t test (P

⬍ 0.05) compared with 0-mg/ml CAFF group. (C)

dumb

1

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), dumb

1

(n

⫽ 20), and fmn (n ⫽ 25) flies. *Significant

difference by Student’s t test (P

⬍ 0.001).

Andretic et al.

PNAS

兩 December 23, 2008 兩 vol. 105 兩 no. 51 兩 20393

GENETICS

(Canton-S

⫽ ⫺30.5 ⫾ 4.8, fmn ⫽ ⫺48.4 ⫾ 11.3). Resistance to

the wake-promoting effects of either the nonspecific adenosine
receptor antagonist CAFF or the specific A1 antagonist CPT in
dDA1 mutant flies indicates a functional relation between the
adenosine and dopamine receptors in mediating the wake-
promoting effects of adenosine receptor antagonists. Significant
CPT-induced sleep loss in fmn flies further shows that functional
DAT is not necessary for the wake-inducing effect of adenosin-
ergic antagonists.

Wake-Promoting Effects of CAFF Involve Modulation of dDA1 Recep-
tor in MBs.

To identify brain areas in which the dDA1 receptor

mediates the action of CAFF, we expressed a WT copy of dDA1
in the brains of dumb

1

mutant flies, using the UAS/GAL4 binary

expression system. We first expressed dDA1 in all the neurons of
dumb

1

mutant flies, using the elav promoter. The dDA1/elav;

dumb

1

flies showed significant sleep loss at 2.5 mg/ml CAFF,

indicating successful rescue of CAFF responsiveness (Fig. 3A).
Control flies that do not express a functional dDA1 receptor,
dDA1/

⫹; dumb

1

and elav/

⫹; dumb

1

, remained resistant to CAFF

(Fig. 3A).

In WT flies, dDA1 is strongly expressed in the MBs, where it

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 dumb

1

flies to CAFF. As shown in Fig. 3A, C747, a MB driver,

fully restored the wake-promoting effects of CAFF in dumb

1

mutant flies. The dDA1/C747; dumb

1

flies on CAFF lost 40.7%

of their usual amount of sleep, whereas control flies with only
one of the two components, DA1/

⫹; dumb

1

or C747/

⫹; dumb

1

,

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; dumb

1

⫽ ⫺56.5 ⫾ 6.5,

dDA1/C747; dumb

1

⫽ ⫺40.7 ⫾ 5.5, dDA1/MB247; dumb

1

⫽

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
the expression of dDA1 mRNA in the heads of WT f lies
following STE and LTE to CAFF. Samples were collected
from f lies that lost more than 30% of their baseline amount of
sleep (64.1

⫾ 2.5% during STE and 50.5 ⫾ 5.7% during LTE)

compared with sham-treated f lies that changed their amount
of sleep by less than 10%. Fig. 3B (Inset) shows that CAFF led
to significant downregulation of dDA1 receptor expression
after STE and LTE.

To determine if the downregulation is functionally related to

CAFF-induced sleep loss, as opposed to being a consequence of
CAFF exposure unrelated to sleep loss, we overexpressed dDA1
in the brains of WT flies with the aim of offsetting the modu-
lation of the dDA1 expression. Fig. 3B shows that transgenic flies
with dDA1 overexpressed in the entire brain, dDA1/elav flies,
maintained their CAFF sensitivity and displayed loss of sleep.
This loss was similar to elav/

⫹ and dDA1/⫹ control lines,

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,
dDA1/C747, behaved significantly differently from their control
siblings, DA1/

⫹ and C747/⫹. The dDA1/C747 flies were CAFF

resistant (Fig. 3B). Similar results were obtained using another
MB driver, MB247 (Fig. 3B). The possibility that this disparity
merely reflects inadequacy of MB expression in the elav GAL4
strain is contradicted by experiments showing full rescue of
CAFF sensitivity with, and no rescue without, the MB contri-
bution of elav (Fig. 3A and

Fig. S3

). Thus, absence of a functional

dDA1 receptor in MBs, as in dumb

1

mutant flies, or overex-

pression of the receptor only in the MBs, as in C747/dDA1
transgenic flies, both had the same outcome: resistance to the
arousing effects of CAFF. These findings suggest that dDA1
receptor downregulation in the MBs is functionally important for
eliciting the arousing effects of CAFF.

METH-Induced Wakefulness Does Not Involve Modulation of dDA1
Receptor.

Acute METH exposure in mammals induces wakeful-

ness attributable, in part, to increased dopaminergic signaling (8,
18). We have shown previously that METH-induced arousal
correlates with increased dopaminergic signaling in Drosophila
(12). To determine if the arousing effects of CAFF and METH
are mediated by the same receptor and transporter, we exposed

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 dumb

1

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 resis-
tance 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.

20394

兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0806776105

Andretic et al.

dumb

1

and fmn flies to increasing doses of METH. In contrast

to WT flies, which lost sleep during the STE and LTE to METH,
fmn and dumb

1

flies were resistant (Fig. 4). This result agrees

well with findings from D1R and DAT mutant mice, which are
likewise resistant to the psychostimulant effect of cocaine and
METH (7, 25). Extended METH exposure in the fly mutants did
not increase METH sensitivity; instead, it tended to increase
sleep in fmn flies (Fig. 4B). A similar sleep-promoting effect of
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:
the dDA1 receptor is involved in the behavioral effects of both
drugs, whereas DAT is required only for the arousing effects of
METH.

Knowing that the expression of dDA1 in MBs is required for

the wake-inducing properties of CAFF, we asked if dDA1 in MBs
mediates METH-induced wakefulness. The dumb

1

mutant trans-

genic flies that expressed dDA1 either in the MBs alone (DA1/
C747; dumb

1

or DA1/MB247; dumb

1

) or in the entire brain

(DA1/elav; dumb

1

) both showed sleep loss on METH (Fig. 5A).

The amount of sleep lost was similar with either whole-brain or
MB expression (DA1/elav; dumb

1

⫽ ⫺16 ⫾ 6.1% sleep loss and

DA1/C747; dumb

1

⫽ ⫺20.3 ⫾ 2.9% sleep loss), indicating that

although the expression of dDA1 in MBs mediates METH-
induced wakefulness, the expression of dDA1 in areas outside of
the MBs does not have a significant additive effect. Thus, dDA1
expression in MB mediates the arousing effects of METH,
although not as completely as it does CAFF (see also

Fig. S3

).

Unlike CAFF, STE to METH did not lead to downregulation

of dDA1 transcripts in WT flies (Fig. 5B, Inset); thus, we
speculated that overexpression of dDA1 in WT flies would not
lead to resistance to METH. Indeed, METH-exposed elav/

dDA1, C747/dDA1, and MB247/dDA1 transgenic flies all showed
substantial sleep loss similar to or greater than the control strains
(Fig. 5B). This finding suggests that although downregulation of
dDA1 in MBs may be required for CAFF-induced wakefulness,
METH-induced wakefulness does not involve downregulation of
dDA1. Because sleep loss in C747/dDA1 flies is somewhat
greater than in elav/dDA1 flies, it is possible that METH-induced
wakefulness involves differential regulation of dDA1 expression
in different brain areas: more in MBs and less in other brain
areas. Such a scenario agrees well with our finding that the dDA1
transcript does not change in the samples extracted from whole
heads of METH-exposed flies.

dDA1 Receptor Does Not Regulate Baseline Sleep.

Although dDA1 is

strongly expressed in MBs, where most genes affecting baseline
sleep in Drosophila are expressed (26–31), the average amount
of sleep and activity during waking were indistinguishable be-
tween WT and dumb

1

flies (

Fig. S4 A and B

), in contrast to fmn

flies, which have lowered baseline amounts of sleep and in-
creased locomotor activity during waking (ref. 21;

Fig. S4 A and

B

; see

SI Text

for additional details.)

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

⫽15–62flies/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 trans-
gene in the MBs of dumb

1

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; dumb

1

flies is

significantly different only in respect to one control line elav/

⫹; dumb

1

(P

⫽

0.047) and is not significant compared with dDA1/

⫹; dumb

1

. (B) Flies overex-

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

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.

Andretic et al.

PNAS

兩 December 23, 2008 兩 vol. 105 兩 no. 51 兩 20395

GENETICS

Discussion

Wake-Inducing Properties of Psychostimulants Are Mediated by dDA1.

In Drosophila, the wake-promoting action of the adenosinergic
antagonist CAFF is mediated through the dDA1 receptor.
Genetic manipulations of the dDA1 receptor, as in dumb

1

mutants, or overexpression of dDA1 in the MBs of transgenic
flies both lead to resistance to the arousing effects of CAFF.
These apparently paradoxical findings can be reconciled if the
CAFF response requires downregulation of the dDA1 receptor
in the MBs within a certain range. In support of this model (

Fig.

S5

), the dDA1 mRNA transcript in WT flies is downregulated in

response to either STE or LTE to CAFF (see Results), the dDA1
product is already reduced to a negligible level in the MBs (and
most other regions) of the dumb mutant (20), and excess
expression of the dDA1 receptor in the MBs produces CAFF
resistance (see Results), suggesting that levels in these flies
cannot be sufficiently downregulated.

A role for the MBs in the control of arousal has been proposed

in the past (32). MBs have an inhibitory effect on locomotor
activity but a stimulatory effect toward sleep (33, 34). Genetic
and transgenic manipulations of MBs, which lead to decreasing
amounts of sleep, are often accompanied by a shortening of
sleep episodes, and can thus be explained by a premature
arousing signal (21, 26, 29, 31, 33, 34).

Our observation that the doses of CAFF that decrease sleep

also increase motor activity is similar to the effect of CAFF in
vertebrates. In mammals, the antagonistic effect of CAFF on
adenosine receptors located on dopaminergic neurons leads to
increased release (2, 3, 19). A similar mechanism might be
operating in flies, based on the correlation that we have shown
between CAFF responsiveness and functional dDA1 receptors in
MBs as well as on the motor-activating effects of dopamine (21).

Although CAFF and METH lead to similar wake-promoting

and motor-activating effects, the neuronal mechanisms under-
lying responses to these drugs are only partially overlapping.
Both responses require a functional dDA1 receptor, particularly
in the MBs, but METH does not lead to uniform downregulation
of dDA1 in the brain, although it is conceivable that downregu-
lation might occur in a limited area of the brain outside of the
MB. Although CAFF-induced wakefulness involves dDA1 down-
regulation in MBs, METH-induced wakefulness could involve a
selective increase of dDA1 in MBs, whereas dDA1 expression
might be unchanged or even decreased in other brain areas. Such
an interpretation is supported by the lack of significant modu-
lation of dDA1 transcript in samples obtained from the entire
brain of METH-fed flies as well as weaker rescue of METH
response when dDA1 was expressed in the entire brain vs. the
MBs (Fig. 5A). When dDA1 expression is restricted only to areas
outside of the MBs (

Fig. S3

), METH response is at least as great

as in panneural (elav) expression, further suggesting the possi-
bility of antagonism between MBs and other areas for this effect.
Another DA receptor, damb, which is specific to the MBs (35),
is not relevant to these responses. It does not show altered
regulation in response to CAFF or METH in WT or dumb
mutants, and dDA1 expression alone or in combination with
CAFF or METH is not altered in damb mutants (R.A., Y.-C.K.,
K.-A.H., R.J.G., unpublished data).

Altogether, these findings suggest a model in which the arousing

and motor-activating effects of CAFF are a consequence of its
neuromodulatory action on dopaminergic signaling (

Fig. S5

). This

is based on similar behavioral responses to CAFF and CPT in
Drosophila, which implies that the arousing properties of CAFF
involve close interaction between the adenosine and dopamine
systems, as they do in mammals. Presynaptically, CAFF can increase
dopamine release by antagonizing adenosine receptors on dopa-
minergic neurons (2, 3). Resistance to the wake-promoting effect of
the A1R antagonist in dumb

1

mutants and decreased expression of

dDA1 in WT flies after CAFF exposure support a model in which
the adenosinergic system acts as a neuromodulator of dopaminergic
signaling. CAFF acting through AdoR on dopaminergic neurons
could stimulate dopamine synthesis or release through protein
kinase A dependent mechanisms similar to the A2A receptor in
mammals (4). Postsynaptically, dDA1 receptors located on MB
neurons respond homeostatically by downregulating their expres-
sion, a common adaptive mechanism in response to excessive
stimulation. A related mechanism involving A1-D1 receptor inter-
action was observed in the rodent brain and implicated in the
psychostimulant properties of CAFF (36). Furthermore, a recent
Drosophila report shows increased dopaminergic content concom-
itant with decreased dDA1 expression in the brains of sleep-
deprived flies (37).

Role of MBs in Psychostimulant Effects on Sleep.

Although the

function of sleep still remains a mystery, one line of evidence
suggests that synaptic plasticity underlying memory consolidation
might occur during sleep (e.g., ref. 38). That such a conserved
function of sleep might be present in Drosophila has been sparked
by a number of recent reports showing overlap between genes
[dunce, rutabaga, Clock, Shaker, 5HT1A, and GABA(A)] and ana-
tomical regions (MBs), which regulate sleep as well as learning and
memory (26, 27, 29, 30, 31, 33, 34). Our findings show that dDA1,
a receptor with a role in neuronal plasticity in MB-dependent
learning tasks, has only a moderate role in regulation of baseline
sleep, although it is important in conditions of elevated arousal,
such as those induced by stimulants.

Optimal behavioral performance, such as learning, is depen-

dent on adequate levels of arousal (39, 40). Although psycho-
stimulant exposure increases dopaminergic transmission and
increases general arousal, it also influences specific functions
related to reward (40). These multiple roles are preserved in
Drosophila, in which mechanisms for arousal and learning con-
verge on the dDA1 receptor, thus ensuring that learning asso-
ciated with survival occurs in an attentive and awake organism.
CAFF and METH effects on dDA1 receptors in MBs could be
mimicking, albeit at an elevated level, the increased dopaminer-
gic signaling that otherwise occurs during learning and memory,
reflecting the role that dDA1 receptors play in that process.

Methods

Animals. Flies were housed at 25 °C, 60% humidity, and a 12-h light/dark cycle
on standard agar- and yeast-based food (12). Canton-S was our standard
background for all strains: dumb

1

, fmn, UAS-dDA1; dumb

1

/TM3, Tb, C747;

dumb

1

, elavGAL4/CyO; dumb

1

, MB247/CyO; and dumb

1

, UAS-dDA1, and elav-

Gal4, C747.

Sleep Measurements. Sleep was measured using the Drosophila Activity Mon-
itoring System (TriKinetics), with a data collection interval set at 5 min as
described previously (41), with ad libitum food.

Pharmacological Treatment. Flies were exposed to CAFF or METH during 12 h
of lights off or for 96 h starting at lights on. Water-based solutions of drugs
were mixed into the food. Drug effects during STE were calculated by com-
paring the amount of sleep during the baseline night (without drug) with that
during the treatment night. The ‘‘sham’’ control for manipulation was a group
of flies transferred to food without drug.

Quantitative PCR. Total RNA extraction, reverse transcription, and quadrupli-
cate RT– quantitative PCR assays were performed as described elsewhere (28).
Primers were as follows: dumb forward 5

⬘-CCGTCGTGTCCAGCTGTA-3⬘ and

reverse 5

⬘ATAGCAGTATAGCCGACAGTAGATG-3⬘ and RP49 forward 5⬘-

TGGAGGTCCTGCTCATGCA-3

⬘ and reverse 5⬘-GGCATCTCGCGCAGTAAAC-3⬘.

ACKNOWLEDGMENTS. We thank H. Dierick, J. Gally, and C. Hughes for helpful
comments and Jene´e Wagner for expert technical assistance. This work was
supported by National Science Foundation (NSF) grant 052326 (to R.J.G.), by
the Neurosciences Research Foundation, and by grants from the National
Institute of Child Health and Human Development and NSF (to K.-A. Han).

20396

兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0806776105

Andretic et al.

1. Porkka-Heiskanen T, Alanko L, Kalinchuk A, Stenberg D (2002) Adenosine and sleep. Sleep

Med Rev 6:321–332.

2. Solinas M, et al. (2002) Caffeine induces dopamine and glutamate release in the shell of

the nucleus accumbens. J Neurosci 22:6321– 6324.

3. Quarta D, et al. (2004) Adenosine receptor-mediated modulation of dopamine release in

the nucleus accumbens depends on glutamate neurotransmission and N-methyl-D-
aspartate receptor stimulation. J Neurochem 91:873– 880.

4. Ferre S (2008) An update on the mechanisms of the psychostimulant effects of caffeine.

J Neurochem 105:1067–1079.

5. Basheer R, Strecker RE, Thakkar MM, McCarley RW (2004) Adenosine and sleep-wake

regulation. Prog Neurobiol 73:379 –396.

6. Ferre S, Diamond I, Goldberg SR, Yao L, Hourani SM, et al. (2007) Adenosine A2A receptors

in ventral striatum, hypothalamus and nociceptive circuitry implications for drug addic-
tion, sleep and pain. Prog Neurobiol 83:332–347.

7. Wisor JP, et al. (2001) Dopaminergic role in stimulant-induced wakefulness. J Neurosci

21:1787–1794.

8. Isaac SO, Berridge CW (2003) Wake-promoting actions of dopamine D1 and D2 receptor

stimulation. J Pharmacol Exp Ther 307:386 –394.

9. Stonehouse AH, Adachi M, Walcott EC, Jones FS (2003) Caffeine regulates neuronal

expression of the dopamine 2 receptor gene. Mol Pharmacol 64:1463–1473.

10. Zahniser NR (1998) Regulation of the dopamine transporter and cocaine sensitization.

Cocaine and a Changing Brain, ed Pilotte NS (National Institute on Drug Abuse, Rockville,
MD) pp. 23–29.

11. Chen JF, et al. (2001) The role of the D(2) dopamine receptor (D(2)R) in A(2A) adenosine

receptor (A(2A)R)-mediated behavioral and cellular responses as revealed by A(2A) and
D(2) receptor knockout mice. Proc Natl Acad Sci USA 98:1970 –1975.

12. Andretic R, van Swinderen B, Greenspan RJ (2005) Dopaminergic modulation of arousal in

Drosophila. Curr Biol 15:1165–1175.

13. Shaw PJ, Cirelli C, Greenspan RJ, Tononi G (2000) Correlates of sleep and waking in

Drosophila melanogaster. Science 287:1834 –1837.

14. Hendricks JC, et al. (2000) Rest in Drosophila is a sleep-like state. Neuron 25:129 –138.
15. Dolezelova E, Nothacker HP, Civelli O, Bryant PJ, Zurovec M (2007) A Drosophila adenosine

receptor activates cAMP and calcium signaling. Insect Biochem Mol Biol 37:318 –329.

16. Zhang D, et al. (2005) Repeated cocaine administration induces gene expression changes

through the dopamine D1 receptors. Neuropsychopharmacology 30:1443–1454.

17. Vezina P (1996) D1 dopamine receptor activation is necessary for the induction of sensi-

tization by amphetamine in the ventral tegmental area. J Neurosci 16:2411–2420.

18. Berridge CW (2006) Neural substrates of psychostimulant-induced arousal. Neuropsycho-

pharmacology 31:2332–2340.

19. Okada M, Mizuno K, Kaneko S (1996) Adenosine A1 and A2 receptors modulate extracel-

lular dopamine levels in rat striatum. Neurosci Lett 212:53–56.

20. Kim YC, Lee HG, Han KA (2007) D1 dopamine receptor dDA1 is required in the mushroom

body neurons for aversive and appetitive learning in Drosophila. J Neurosci 27:7640 –7647.

21. Kume K, Kume S, Park SK, Hirsh J, Jackson FR (2005) Dopamine is a regulator of arousal in

the fruit fly. J Neurosci 25:7377–7384.

22. Spielewoy C, et al. (2000) Behavioural disturbances associated with hyperdopaminergia in

dopamine-transporter knockout mice. Behav Pharmacol 11:279 –290.

23. Zhuang X, et al. (2001) Hyperactivity and impaired response habituation in hyperdopam-

inergic mice. Proc Natl Acad Sci USA 98:1982–1987.

24. Svenningsson P, Nomikos GG, Fredholm BB (1995) Biphasic changes in locomotor behavior

and in expression of mRNA for NGFI-A and NGFI-B in rat striatum following acute caffeine
administration. J Neurosci 15:7612–7624.

25. Xu M, et al. (1994) Elimination of cocaine-induced hyperactivity and dopamine-mediated

neurophysiological effects in dopamine D1 receptor mutant mice. Cell 79:945–955.

26. Yuan Q, Joiner WJ, Sehgal A (2006) A sleep-promoting role for the Drosophila serotonin

receptor 1A. Curr Biol 16:1051–1062.

27. Hendricks JC, et al. (2003) Gender dimorphism in the role of cycle (BMAL1) in rest, rest

regulation, and longevity in Drosophila melanogaster. J Biol Rhythms 18:12–25.

28. Shaw PJ, Tononi G, Greenspan RJ, Robinson DF (2002) Stress response genes protect

against lethal effects of sleep deprivation in Drosophila. Nature 417:287–291.

29. Hendricks JC, et al. (2001) A non-circadian role for cAMP signaling and CREB activity in

Drosophila rest homeostasis. Nat Neurosci 4:1108 –1115.

30. Agosto J, et al. (2008) Modulation of GABA(A) receptor desensitization uncouples sleep

onset and maintenance in Drosophila. Nat Neurosci 11:354 –359.

31. Cirelli C, et al. (2005) Reduced sleep in Drosophila Shaker mutants. Nature 434:1087–1092.
32. Martin JR, Ernst R, Heisenberg M (1998) Mushroom bodies suppress locomotor activity in

Drosophila melanogaster. Learn Mem 5:179 –191.

33. Joiner WJ, Crocker A, White BH, Sehgal A (2006) Sleep in Drosophila is regulated by adult

mushroom bodies. Nature 441:757–760.

34. Pitman JL, McGill JJ, Keegan KP, Allada R (2006) A dynamic role for the mushroom bodies

in promoting sleep in Drosophila. Nature 441:753–756.

35. Han KA, Millar NS, Grotewiel MS, Davis RL (1996) DAMB, a novel dopamine receptor

expressed specifically in Drosophila mushroom bodies. Neuron 16:1127–1135.

36. Ferre S, Popoli P, Tinner-Staines B, Fuxe K (1996) Adenosine A1 receptor-dopamine D1

receptor interaction in the rat limbic system: Modulation of dopamine D1 receptor
antagonist binding sites. Neurosci Lett 208:109 –112.

37. Seugnet L, Suzuki Y, Vine L, Gottschalk L, Shaw PJ (2008) D1 receptor activation in the

mushroom bodies rescues sleep-loss-induced learning impairments in Drosophila. Curr
Biol 18:1110 –1117.

38. Walker MP, Stickgold R (2006) Sleep, memory, and plasticity. Annu Rev Psychol 57:139 –

166.

39. Chudasama Y, Robbins TW (2004) Dopaminergic modulation of visual attention and

working memory in the rodent prefrontal cortex. Neuropsychopharmacology 29:1628 –
1636.

40. Schultz W (2007) Multiple dopamine functions at different time courses. Annu Rev

Neurosci 30:259 –288.

41. Andretic R, Shaw PJ (2005) Essentials of sleep recordings in Drosophila: Moving beyond

sleep time. Methods Enzymol 393:759 –772.

Andretic et al.

PNAS

兩 December 23, 2008 兩 vol. 105 兩 no. 51 兩 20397

GENETICS