1516 VOLUME 15 | NUMBER 11 | NOVEMBER 2012 NATURE NEUROSCIENCE

A R T I C L E S

Sleep has been observed in many organisms. The cellular basis under-lying sleep regulation, however, remains poorly understood. Studies of cellular mechanisms for sleep regulation in mammals are hindered by their complex brain structure. Drosophila melanogaster, however, has a relatively simple brain, and is therefore suitable for studying the cellular basis that underlies a specific behavior1,2. The phyloge-netic conservation of sleep regulating molecular mechanisms between mammals and fruit flies has been established previously3–8.

Sleep regulation in Drosophila involves dopamine6,7. Dopamine is synthesized from l-3,4-dihydroxyphenylalanine (DOPA) by DOPA decarboxylase (Ddc), and DOPA is synthesized from tyrosine by tyrosine hydroxylase. Administration of 3-iodo-tyrosine, a tyrosine hydroxylase inhibitor, increases sleep duration, whereas administra-tion of psychostimulants that enhance dopamine signaling, such as cocaine or amphetamine, decreases sleep. DATfmn mutant flies show a reduction in sleep time as a result of an increase in postsynaptic dopamine signaling7,9. Increased dopamine signaling also alters the temporal characteristics of the activities, metabolic rate and tempera-ture sensitivities of these flies10,11. Similarly, in mammals, deletion of the dopamine transporter gene or transient activation of dopamine neurons results in a decrease in sleep time12,13. However, it is not known whether dopamine transmission between a specific subset of dopamine neurons and defined receiving cells regulates arousal. Alternatively, broad background dopamine levels could be respon-sible for arousal regulation. Although dopamine was recently shown to regulate arousal, in part, through a pigment dispersing factor (PDF) neuron that regulates circadian rhythm, the primary target for dopamine in sleep regulation remains unknown13,14. There are two major subfamilies of dopamine receptors in Drosophila: two D1-like receptors, DA1 and DopR2, and a D2-like receptor, D2R, have been identified15. Although it has been reported that DA1 mediates wake-fulness through caffeine and methamphetamine16, startle-induced

arousal14, and ethanol-stimulated locomotion17, its contribution to physiological sleep regulation and the cellular loci in which it func-tions remain unclear.

Dopamine also mediates other physiological functions, such as memory and attention18,19. Specific subsets of dopamine neurons that project to the mushroom bodies mediate aversive and appetitive rein-forcement in olfactory memory in Drosophila20–22. As the mushroom body has been shown to regulate sleep23,24, an important question arises as to whether the same dopamine neurons can regulate both sleep/arousal and learning. Sleep also interacts with various brain functions, including efficient memory formation25–27. Whether the functional interaction between sleep and memory is implemented in the same dopamine neuron(s) remains an open question.

We set out to map the dopamine pathway that is required for sleep and arousal regulation. To this end, we employed a combinatorial approach. We identified FSB as a target for dopamine using cell type–specific genetic manipulation of DA1, and presynaptic neurons to the FSB using the GFP reconstitution across synaptic partners (GRASP) method. We measured the physiological response of the FSB neurons to dopamine application and mapped the wake-promoting dopamine neuron to a single posterior protocerebral 3 (PPM3) neuron using mosaic analysis with a repressible cell marker (MARCM).

RESULTSDopamine mediates arousal via DA1 in dFSB neuronsTo identify the target of dopamine in sleep regulation, we employed a genetic strategy that used the dopamine transporter DATfmn mutant. Given that presynaptic DAT clears the released dopamine from the synaptic cleft, the short sleep phenotype of DATfmn flies can be attrib-uted to an increase in postsynaptic dopamine signaling in all dopamine pathways. Flies containing the hypomorphic DA1 allele dumb2 show a substantial sleep increase when under constant darkness conditions14.

1Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan. 2Max Planck Institut für Neurobiologie, Martinsried, Germany. 3Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan. 4Global Center of Excellence, Kumamoto University, Kumamoto, Japan. Correspondence should be addressed to K.K. (kkume@kumamoto-u.ac.jp).

Received 27 August; accepted 17 September; published online 14 October 2012; doi:10.1038/nn.3238

Identification of a dopamine pathway that regulates sleep and arousal in DrosophilaTaro Ueno1, Jun Tomita1, Hiromu Tanimoto2, Keita Endo3, Kei Ito3, Shoen Kume1,4 & Kazuhiko Kume1

Sleep is required to maintain physiological functions, including memory, and is regulated by monoamines across species. Enhancement of dopamine signals by a mutation in the dopamine transporter (DAT) decreases sleep, but the underlying dopamine circuit responsible for this remains unknown. We found that the D1 dopamine receptor (DA1) in the dorsal fan-shaped body (dFSB) mediates the arousal effect of dopamine in Drosophila. The short sleep phenotype of the DAT mutant was completely rescued by an additional mutation in the DA1 (also known as DopR) gene, but expression of wild-type DA1 in the dFSB restored the short sleep phenotype. We found anatomical and physiological connections between dopamine neurons and the dFSB neuron. Finally, we used mosaic analysis with a repressive marker and found that a single dopamine neuron projecting to the FSB activated arousal. These results suggest that a local dopamine pathway regulates sleep.

npg

© 2

012

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

NATURE NEUROSCIENCE VOLUME 15 | NUMBER 11 | NOVEMBER 2012 1517

A R T I C L E S

As previously reported, DATfmn flies showed a significant decrease in sleep (P < 0.0001). The short sleep phenotype of DATfmn flies was rescued by administration of a dopamine synthesis inhibitor (tyro-sine hydroxylase inhibitor) or conditional DAT expression, which suggests that the phenotype is not a result of developmental changes (Supplementary Fig. 1a,b). We also examined GAL4 expression using various drivers in DAT fmn flies, and confirmed that the expres-sion patterns were equivalent to the control (w118 flies, see Online Methods; Supplementary Fig. 1c). When DATfmn was coupled with the DA1 allele dumb2, the short sleep phenotype DATfmn flies was completely rescued by the additional DA1 mutation in both 12-h light:dark cycle (F3,124 = 55.87, t = 13.97) and constant dark (F3,124 = 97.77, t = 18.25) conditions (Fig. 1), indicating that dopamine elicits arousal through DA1. As previously reported10, the waking activity, defined as the total activity divided by the active period length, was similar even after changes in dopamine signaling (Fig. 1c,d). We then veri-fied the expression of DA1 using immunohistochemistry. Prominent

DA1 expression was observed in the mushroom body and the central complex, including the FSB and the ellipsoid body, in the wild-type brain28 that was not present in DA1dumb2 brain. On the other hand, dopamine neurons were not affected in DATfmn, DA1dumb2 or DATfmn; DA1dumb2 double mutant flies (Supplementary Fig. 2).

To investigate the brain area in which DA1 mediates the arousal effects of dopamine, we expressed DA1 in the DATfmn; DA1dumb2 double mutant. A piggyBac insertion at the first intron of the DA1dumb2 con-tained a UAS construct, enabling GAL4-dependent DA1 expression28. We used OK107-GAL4 to target DA1 expression to all of the mush-room body neurons and 104y-GAL4 and c259-GAL4 to target dFSB neurons29,30. We confirmed tissue-specific expression of DA1 with these drivers by immunohistochemistry (Fig. 2a). DA1 expression in the mushroom body of the DATfmn; DA1dumb2 double mutant did not induce any change in sleep, but DA1 expression in the dFSB converted their normal sleep phenotype to the DATfmn short sleep phenotype (F4,75 = 15.30, t = 8.54, 6.28; Fig. 2b–e). DA1 is a D1-like receptor

a

c d

b60

20

15

10

5

0

20

15

10

5

0

Control fmn fmn; dumb2dumb2

Control Controlfmn fmn fmnfmndumb2

dumb2

Control fmn fmndumb2

dumb2 Control

ns

fmn fmndumb2

dumb2 Control fmn fmndumb2

dumb2Control fmn fmndumb2

dumb2

dumb2dumb2

Control fmn fmndumb2dumb2

Control fmn fmndumb2dumb2

Control

CT0 CT12 CT0 CT12 CT0 CT12

fmn fmn; dumb2dumb2

ZT0 ZT12 ZT0 ZT12 ZT0 ZT12

Sle

ep (

min

h−1

)

Sle

ep (

h da

y−1)

Sle

ep (

h da

y–1)

Act

ivity

(co

unts

day

−1)

Wak

ing

activ

ity

Sle

ep b

out d

urat

ion

(min

)

Act

ivity

(co

unts

day

−1)

Sle

ep (

min

h−1

)

50

40

30

20

10

0

2,000 ******

*

*****

*

*

***

***

***

******

********

***

******

1,500

1,000

500

0

3 120

100

80

60

40

20

0

Sle

ep b

out d

urat

ion

(min

)

80

60

40

20

0

2

1

0

Wak

ing

activ

ity

3

2

1

0

2,000

1,500

1,000

500

0

60

50

40

30

20

10

0

Figure 1 DA1 mediates the wake-promoting effects of dopamine. (a) Sleep profiles for each genotype (n = 32 flies). Sleep over 3 d in the 12-h light:dark cycle condition is shown. Day and night are depicted by the white and black bars, respectively. The short sleep phenotype of the DATfmn mutant (shown as fmn in the figure) was rescued by the hypomorphic allele of DA1, dumb2 (DA1dumb2 is shown as dumb2 in the figure). ZT, zeitgeber time. (b) Sleep profiles for each genotype (n = 32 flies). Sleep over 3 d in constant darkness is shown. Subjective day and subjective night are depicted by the gray and black bars, respectively. CT, circadian time. (c) Summary of the sleep parameters in 12-h light:dark cycle condition. Total activity counts, total sleep amount, waking activity index and mean sleep bout duration are shown. Waking activity is defined as the total daily activity divided by the length of the daily active period. (d) Summary of the sleep parameters in constant darkness. Note that all alleles are homozygous in these flies. Data are represented as mean s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant (P = 0.38); one-way ANOVA with Tukey-Kramer HSD post hoc test.

npg

© 2

012

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

1518 VOLUME 15 | NUMBER 11 | NOVEMBER 2012 NATURE NEUROSCIENCE

A R T I C L E S

that couples to the Gs protein -subunit, G s, and increases cAMP levels31. We conditionally expressed a constitutively active form of G s (Gs*) in dFSB with 104y-GAL4 to mimic the effect of DA1 activation32. Gs* expression resulted in a significant sleep decrease (P = 0.004), whereas no significant difference (P = 0.27) in sleep levels was observed prior to the induction of Gs* expression (Supplementary Fig. 3). These results indicate that the arousal-inducing effects of dopamine are mediated by DA1 expression in the dFSB.

It has been reported that activation of the dFSB neurons induces sleep33. To confirm that the dFSB neurons function as downstream targets of dopamine neurons, we activated dFSB neurons in control flies and DATfmn flies by expressing a thermo-sensitive cation channel, TrpA1, using 104y-GAL4. Activation of the dFSB neurons by temperature elevation increased sleep levels in both control flies (F2,87 = 15.87, t = 7.96, 4.24) and DATfmn flies (F2,87 = 19.88, t = 7.51, 7.61; Fig. 3). These results suggest that the dFSB is a down-stream target of dopamine neurons.

Sleep and memory are closely linked25, and dopamine regulates both of these physiolo-gical phenomena6,7,19. Thus, we asked whether DA1 expression in the dFSB could contribute to memory formation in aversive associative learning. As previously reported28,

the DA1dumb2 mutant (both in control and DATfmn backgrounds) showed severely impaired learning. DA1 expression in the mushroom body (P = 0.01), but not in the FSB (P = 0.77), significantly rescued the memory impairment, showing the reversed pattern of rescue in arousal (Supplementary Fig. 4).

Furthermore, dopamine is known to regulate arousal, in part, through the PDF neurons that regulate circadian rhythm13,14. To study their contribution, we genetically ablated the PDF neurons by expressing the apoptotic gene hid (also known as wrinkled) using Pdf-GAL4, which exclusively labels the PDF neurons (Fig. 4a). hid expression driven by Pdf-GAL4 successfully eliminated the PDF neurons (Fig. 4b). Sleep analysis of control and DATfmn flies with and without PDF neurons revealed that the sleep decrease mediated by the excess dopamine present in DATfmn flies was independent of the PDF

nc82

a b

d e

c

DA1

fmn; dumb2

fmn

3,000 ******

ns

ns

******

ns

***

ns

20

3

fmn; dumb2;OK107

fmn 104y; dumb2

fmn; c259 dumb2

fmndumb2c259

fmndumb2

fmndumb2OK107

fmndumb2 104y

fmn fmndumb2c259

fmndumb2

fmndumb2OK107

fmndumb2 104y

fmn fmndumb2c259

fmndumb2

fmndumb2OK107

fmndumb2 104y

fmn fmndumb2c259

fmndumb2

fmndumb2OK107

fmndumb2 104y

Merge nc82 DA1 Merge2,500

2,000

1,500

Act

ivity

(co

unts

day

–1)

Wak

ing

activ

ity

Sle

ep (

h da

y–1)

Sle

ep b

out (

min

)

1,000

500

0

2

1

0

15

10

5

0

60

50

40

30

20

10

0

Mushroom body FSB

Figure 2 DA1 in the FSB, but not in the mushroom body, mediates the wake-promoting effects of dopamine. (a) DA1 expression in the brain. The brain was stained with antibodies to nc82 (purple) and DA1 (green). Left, a single slice around the mushroom body (arrowheads). Right, a slice around the FSB. In the bottom row, GAL4 expression indicates that the dopamine neurons project to the hinge region of the mushroom body (arrowheads). Scale bars represent 20 m. (b) Average total activity counts per day (n = 16 flies). (c) Average total sleep amount per day. (d) Average activity per waking minute. (e) Average sleep bout duration. Note that all alleles are homozygous in these flies. Data are represented as mean s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant (P = 0.07); one-way ANOVA with Tukey-Kramer HSD post hoc test.

a c TrpA1104y

104y; TrpA1

***

Sle

ep (

min

day

–1)

1,400

1,200

1,000

800

600

400

200

022 °C 31°C

d***

Sle

ep (

min

day

–1)

700

600

500

400

300

200

100

031°C22 °C

fmn TrpA1fmn 104yfmn 104y; TrpA1

60

Sle

ep (

min

h–1

) 50

40

30

20

10

0CT0 CT12

22 °C 31°C 22 °C

CT0 CT12 CT12CT0

TrpA1104y104y; TrpA1

b60

Sle

ep (

min

h–1

)

50

40

30

20

10

0CT0 CT12

22 °C 31 °C 22 °C

CT0 CT12 CT12CT0

fmn TrpA1fmn 104yfmn 104y; TrpA1

Figure 3 Activation of the dFSB induces sleep in control and DATfmn flies. (a) Sleep profiles for each genotype in control (n = 30 flies). Sleep over 3 d in constant darkness is shown. The behavioral assay was started at 22 °C in 12-h light:dark cycle and constant dark and shifted to 31 °C for 1 d to activate the dFSB neuron, and then placed back to 22 °C. (b) Sleep profiles for each DATfmn mutant genotype (n = 30 flies). (c) Total daily sleep amounts for each control genotype. (d) Total daily sleep amounts for each DATfmn mutant genotype. Data are represented as mean s.e.m. ***P < 0.001; one-way ANOVA with Tukey-Kramer HSD post hoc test.

npg

© 2

012

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

NATURE NEUROSCIENCE VOLUME 15 | NUMBER 11 | NOVEMBER 2012 1519

A R T I C L E S

neurons (12-h light:dark cycle: control, 917 42.6 min; DATfmn, 428 52.4 min; t = 7.24, P < 0.0001; constant dark: control, 931 40.6 min; DATfmn, 213 38.6 min; t = 12.81, P < 0.0001; Fig. 4c,d).

Dopamine neurons have connections with the dFSBTo confirm the connection between the dopamine neurons and the dFSB neurons labeled by 104y-GAL4, we employed the GRASP method34,35 under the control of two independent binary transcrip-tional systems36. By screening the expression patterns of LexA driver lines37, we found that Nv131-LexAøVP16 labeled the same dFSB neurons that were labeled by 104y-GAL4 (Fig. 5a and Supplementary Video 1). Nv131-LexAøVP16 also labeled other parts of the brain, including the mushroom body, but not the dopamine neurons (Fig. 5b and Supplementary Video 2).

We expressed CD4øspGFP1-10 and CD4øspGFP11 under the control of TH-GAL4 (ref. 38) and Nv131-LexAøVP16, respectively. GRASP signals were detected in the dFSB as well as in the mushroom body, indicating that there was close contact between dopamine neurons and Nv131-LexAøVP16–positive neurons in the dFSB and the mushroom body neurons. GRASP signals were not detected following expression of either split-GFP alone, proving the spe-cificity of the GRASP signals (Fig. 5c–e). These results indicate that dopamine neurons have connections with the dFSB neurons labeled by 104y-GAL4.

The dFSB neurons respond to dopamineTo further confirm dopamine action in the dFSB neurons, we per-formed a live imaging analysis. Given that DA1 is a D1-like receptor, which signals predominantly through cAMP31, we performed real-time cAMP measures that take advantage of the cAMP-binding properties of Epac (exchange protein directly activated by cAMP), a molecule that mediates non–protein kinase A–dependent cAMP signaling. We expressed Epac1-camps, a genetically encoded cAMP fluorescence resonance energy transfer (FRET) sensor39, in the dFSB neurons using 104y-GAL4. In response to dopamine application, the dFSB area showed a robust FRET signal change, which indicates the accumulation of cAMP (F2,18 = 45.57, t = 7.98; Fig. 6a–e), suggest-ing a physiological response of the FSB neurons to dopamine.

mCD8::GFPPDF

a c

d

b6050

Pdf > mCD8::GFP

Pdf>hid fmn Pdf>hid

Pdf > hid, mCD8::GFP

mCD8::GFPPDF

40

Sle

ep (

min

h–1

)

3020100

605040

Sle

ep (

min

h–1

)

3020100

ZT0 ZT12 ZT0 ZT12 ZT0 ZT12

CT0 CT12 CT0 CT12 CT0 CT12

Pdf>hid fmn Pdf>hid

Figure 4 Ablation of PDF neurons does not eliminate the arousal effect of dopamine. (a) PDF neurons were visualized with mCD8øGFP driven by Pdf-GAL4 (green) and the PDF antibody (purple). (b) PDF neurons were ablated through the expression of the apoptotic gene hid using Pdf-GAL4. PDF neurons were visualized with mCD8øGFP driven by Pdf-GAL4 (green) and the PDF antibody (purple). (c) Sleep profiles for each genotype (n = 16 flies). Sleep over 3 d in 12-h light:dark cycle is shown. Day and night are depicted by the white and black bars, respectively. (d) Sleep profiles for each genotype (n = 16 flies). Sleep over 3 d in constant darkness is shown. Subjective day and night are depicted by the gray and black bars, respectively. Data are represented as mean s.e.m. Scale bars represent 20 m.

a b

Nv131>CD2::GFP104y>mCD8::RFP

c

GFP TH Merge

TH>GFP1-10, Nv131>GFP11

d e

GFP GFP

TH>GFP1-10Nv131>GFP11

Nv131>CD2::GFPTH

Figure 5 The FSB neuron is anatomically connected to a dopamine neuron. (a) Nv131-LexAøVP16 was expressed in the dFSB neuron labeled by 104y-GAL4. CD2øGFP (green) was expressed by Nv131-LexAøVP16 and mCD8øRFP (purple) was expressed by 104y-GAL4. (b) Nv131-LexAøVP16 was not expressed in dopamine neurons. CD2øGFP (green) was expressed by Nv131-LexAøVP16 and the dopamine neurons were visualized with antibody to tyrosine hydroxylase (TH, purple). (c) The anatomical connection of a dopamine neuron to the FSB was revealed using the GRASP method. Brains were stained with a reconstituted GFP-specific antibody (green) and antibody to tyrosine hydroxylase (magenta). The arrowhead indicates a GFP signal detected in the FSB in the experimental fly (genotype, +/+; Nv131-LexAøVP16/LexAop-CD4øspGFP11; TH-GAL4/UAS-CD4øspGFP1-10; n = 24 flies). (d,e) GFP signal in negative control flies for GRASP analysis (d, +/+; +/LexAop-CD4øspGFP11; TH-GAL4/UAS-CD4øspGFP1-10; e, +/+; Nv131-LexAøVP16/LexAop-CD4øspGFP11; +/UAS-CD4øspGFP1-10; n = 4 flies for each genotype). Scale bars represent 20 m.

npg

© 2

012

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

1520 VOLUME 15 | NUMBER 11 | NOVEMBER 2012 NATURE NEUROSCIENCE

A R T I C L E S

Next, we applied tetrodotoxin (TTX) to block neuronal activation, thereby eliminating indirect activation of dFSB neurons by other dopamine-sensitive neurons. Dopamine still showed a robust, even larger increase in cAMP after TTX treatment (t = 13.42; Fig. 6d,e). The effect of dopamine was concentration dependent, and a signi-ficant FRET signal change in the dFSB was observed with 1 mM dopamine (P = 0.04; Fig. 6f).

We knocked down DA1 in dFSB by expression of double-stranded RNA (dsRNA) against DA1 using UAS-DA1 RNAi. The expression of the dsRNA was driven by 104y-GAL4. DA1 knockdown resulted in the elimination of the cAMP response induced by dopamine (t = 1.27, P = 0.23; Fig. 6g), although these flies did not show substan-tial sleep changes in the behavior assay (data not shown). No morpho-logical changes were observed when either Dicer-2 or DA1 RNAi was expressed. We conclude that the dFSB receives direct synaptic inputs from dopamine neurons and that dopamine increases cAMP levels in the dFSB through DA1.

Dopamine neurons for memory on sleepAs previously reported, thermo-activation of most dopamine neu-rons with TH-GAL4 significantly decreased the amount of sleep (t = 22.74, P = 0.00001; Fig. 7a)11,13. To identify the sleep-regulating dopamine pathways, we genetically activated different subsets of dopamine neurons using various GAL4 drivers (Supplementary Fig. 5). The binary GAL4-UAS-TrpA1 switch mechanism enabled us to induce transient neural activation using a temperature shift in selected populations of neurons, thereby avoiding any developmental effects resulting from transgenic gene expression. Previous studies have shown that the two types of mushroom body–projecting dopamine neurons, the MB-M3 neurons in the paired anterior medial (PAM) cluster (labeled using NP1528-GAL4 and NP5272-GAL4) and the MB-MP1 neurons in the protocerebral posterior lateral 1 (PPL1) cluster (labeled using NP2758-GAL4), can induce aversive olfactory memory20.

Activation of these mushroom body–projecting dopamine neurons did not affect the daily sleep amount (Fig. 7b–d). Activation of the MB-MVP1 pathway in the PAM cluster (labeled using NP6510-GAL4) or activation of the PPM3 dopamine neurons that project to the ellipsoid body (labeled using c346-GAL4), which contribute to an ethanol-stimulated locomotion increase17, did not change sleep levels (Fig. 7e–g). Furthermore, addition of the TH-GAL80 suppressor in TH-GAL4; UAS-TrpA1 flies inhibited sleep reduction at 29 °C (Supplementary Fig. 6).

FSB-projecting PPM3 dopamine neuron regulates arousalUsing the MARCM40 method, we targeted TrpA1 expression to a limited number of dopamine neurons using TH-GAL4 with FLP-induced recom-bination, which resulted in the clonal cells lacking GAL80, a transcrip-tional repressor of GAL4. The TrpA1-expressing dopamine neurons were simultaneously labeled with a membrane-bound GFP (mCD8-GFP). This enabled post hoc identification of manipulated neurons in individual animals after behavioral quantification of their sleep (Supplementary Fig. 7). This stochastic labeling technique also allowed us to address the importance of laterality, namely whether activation of a unilateral neuron is sufficient for arousal induction. Given that each fly generated using MARCM shows a unique pattern of recombination, and different neuron populations were labeled by TH-GAL4 (ref. 41), we first examined the amount of sleep and then GFP expression for each individual fly.

As a preliminary assay, we quantified the sleep of the progeny with or without 1-h FLP induction at 22 °C and then at 29 °C (Fig. 8a). There was no apparent difference in the amount of sleep between the two groups at 22 °C, indicating that FLP-induced TrpA1 expression alone had no substantial effect on sleep. There was no gross difference in the amount of sleep at 29 °C between the two groups of flies, but some flies with FLP induction showed a larger reduction in sleep at 29 °C (Supplementary Fig. 8a). GFP expression in these flies labeled a uni-lateral neuron in the PPM3 cluster that has arborizations in the FSB (Fig. 8b–d), whereas those with average sleep either showed GFP labe-ling in other neurons or showed no GFP expression (data not shown).

a d

f ge

b c

104y>mCD8::GFP

1.2

1.30

1.1

CF

P/Y

FP

CF

P/Y

FP

CF

P/Y

FP

104y>Epac1-camps

Water

Water

WaterDopamine

WaterDopamine

Dopamine + TTX

Dopamine

***

Dopamine Dopamine + TTX

CFP/YFP 1.0

0 100 200 300 400 500 600

1.25

1.20

1.15

1.10

1.05

1.00

0.95

0.90

1.10

ns

Dicer-2

DA1 RNAi +

Dicer-2

1.05

1.00

0.95

0.90

**

*

1.12

CF

P/Y

FP

1.10

1.08

1.06

1.04

1.02

1

Dopamine

1

µM mM

10 1100 10

Figure 6 The FSB neurons show a response to dopamine. (a) FSB neurons were visualized using UAS-mCD8øGFP under the control of 104y-GAL4. Dashes outline the region of interest for quantification of the FRET signal. (b,c) Representative images and false-color representation of responses to H2O (b) or 10 mM dopamine (c) of the FSB neurons in 104y-GAL4/UAS-Epac1-camps. Scale bars represent 20 m. (d) A time course of the average responses of the FSB (n = 7 flies). The point of drug application is indicated by the arrow. The line indicates the mean and the shaded area indicates the s.e.m. (e) A summary of the relative changes in cAMP levels. (f) Dose-response curve for dFSB cAMP responses to dopamine in 104y-GAL4/UAS-Epac1-camps flies (n = 5 flies). (g) FRET signal after dopamine application in dFSB in DA1 knockdown flies (n = 7 flies). As a control, flies expressing Dicer-2 in dFSB were used. Data are represented as mean s.e.m. *P < 0.05, ***P < 0.001; ns, not significant (P = 0.22); one-way ANOVA with Tukey-Kramer HSD post hoc test or two-sided Student’s t test.

npg

© 2

012

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

NATURE NEUROSCIENCE VOLUME 15 | NUMBER 11 | NOVEMBER 2012 1521

A R T I C L E S

To quantitatively correlate induced arousal with manipulated neu-rons, we dissected all of the flies in the second experiment. Of 312 flies examined, 197 flies were classified into groups on the basis of the labeled neuron clusters. The difference in the amount of sleep follow-ing activation was analyzed for all individuals and grouped for each dopamine cluster. Most flies had only one GFP-labeled neuron and approximately 20% had no GFP signals in their brain (Fig. 8d). The expression of the TrpA1 channel itself did not affect the amount of sleep at 22 °C (Supplementary Fig. 8b). However, thermo-activation at 29 °C resulted in a significantly larger reduction in sleep when the TrpA1 channel was expressed in PPM3 neurons, which arborize in the FSB (t = 3.30, P = 0.0008; Fig. 8d and Supplementary Fig. 8c). The PPM3 cluster is comprised of two distinct cell types, one projecting to the ellip-soid body and the other to the FSB (Supplementary Fig. 9)41. MARCM clones that labeled the PPM3 neurons projecting to the ellipsoid body did not show a significant change compared to the controls (P = 0.83), which is consistent with the result of neural activation using c346-GAL4 (Figs. 7f and 8d). As the PPM3 neurons have their arbors close to the vertical lobes of the mushroom body, we carefully examined the pro-cesses and found that the PPM3 neurons did not project to the mush-room body directly (Fig. 8e–h). This arborization in the vicinity of the mushroom body is consistent previous findings41.

DISCUSSIONThe target structure of the dopaminergic arousal effectWe found that neurons in the dFSB are involved in dopaminergic sleep regulation in Drosophila and that the PPM3-FSB dopamine pathway, which is distinct from that required for memory formation, regulates arousal (Supplementary Fig. 10). A previous study found

that the rescue of DA1 mutants outside of the mushroom body using a pan-neuronal GAL4 driver, elav-GAL4, coupled with the mush-room body suppressor MB-GAL80 in DA1dumb1 mutants can recover methamphetamine sensitivity9. This suggests that dopamine regu-lates arousal outside of the mushroom body. Previous findings have shown that DA1 in the PDF neurons (lateral ventral neurons) regu-lates sleep-wake arousal and that DA1 in the ellipsoid body regulates stress-induced arousal14.

A previous study identified PDF neurons that mediate the buffer-ing effects of light on dopamine-induced arousal13. However, our ablation experiments showed that dopamine can elicit strong arousal effects without PDF neurons. The previous report used heterozygous DA1 mutant flies in a wild-type background for most experiments. However, we used homozygous (null) DA1 mutants, as we found that the heterozygous DA1 mutants crossed with DATfmn showed an almost equivalent short sleep phenotype (data not shown). Thus, one possible explanation for the difference between the previous study and ours is that heterozygous expression of DA1 in the FSB is suffi-cient to elicit the arousal effects of dopamine, which is also regulated partly through PDF neurons. Given that DA1 expression in the dFSB neurons alone is sufficient for the majority of the wake-promoting effects of dopamine, the arousal regulating dopamine pathway appears to converge at the FSB.

Identification of the arousal-inducing dopamine neuronActivation of the dopamine neurons required for aversive memory formation20 had little effect on sleep. On the other hand, TrpA1 stimu-lation in a single FSB-projecting PPM3 dopamine neuron was able to reduce sleep. As the magnitude of the decrease in sleep by the

d

a60

Sle

ep (

min

h–1

)

5040302010

0CT0

22 °C 22 °C29 °C

CT0 CT0CT12 CT12 CT12

TH-GAL4/+ TH-GAL4/UAS-Trpa1

+/UAS-Trpa1 b60

Sle

ep (

min

h–1

)

50403020100

22 °C29 °C22 °C

CT0 CT0 CT0CT12 CT12 CT12

NP1528-GAL4/+ NP1528-GAL4/UAS-Trpa1

c60

Sle

ep (

min

h–1

)

50403020100

22 °C 22 °C29 °C

CT0 CT0 CT0CT12 CT12 CT12

NP2758-GAL4/+ NP2758-GAL4/UAS-Trpa1

22 °C29 °C22 °C

60

Sle

ep (

min

h–1

)

50403020100CT0 CT0 CT0CT12 CT12 CT12

NP5272-GAL4/+ NP5272-GAL4/UAS-Trpa1

e

22 °C29 °C22 °C

60

Sle

ep (

min

h–1

)

50403020100

CT0 CT0 CT0CT12 CT12 CT12

NP6510-GAL4/+ NP6510-GAL4/UAS-Trpa1

f

22 °C29 °C22 °C

60

Sle

ep (

min

h–1

)

50403020100

CT0 CT0 CT0CT12 CT12 CT12

c346-GAL4/+ c346-GAL4/UAS-Trpa1

g TH NP1528 NP2758 NP5272 NP6510 c346TrpA1

ns

ns

nsns

ns

***

∆ S

leep

(m

in)

0– TrpA1 – TrpA1 – TrpA1 TrpA1– – TrpA1 TrpA1–

–200

–400

–600

–800

Figure 7 The activation of dopamine neurons by TrpA1 resulted in a reduction in sleep. Averaged sleep profiles for 60-min intervals for flies expressing TrpA1 in different dopamine neurons in constant dark conditions. Behavior was monitored for 3 d at 22 °C (only the third day is shown) in constant dark and shifted to 29 °C at CT0 to activate the neurons for 1 d, and then placed back to 22 °C. Subjective day and night are depicted by the gray and black bars, respectively. (a) Transient activation with TH-GAL4 significantly decreased sleep levels ((P < 0.001) n = 32 flies). (b–d) In contrast, activation of memory- forming dopamine neurons with NP1528-GAL4 (P = 0.39), NP2758-GAL4 (P = 0.94) and NP5272-GAL4 (P = 0.22) did not show significant sleep changes (n = 32 flies). (e,f) Activation of other dopamine neurons with NP6510-GAL4 (P = 0.15) and c346-GAL4 (P = 0.26) also had little effect on sleep (n = 32 flies). (g) The average decrease in the amount of sleep was calculated by subtracting daily sleep at 29 °C from that at 22 °C (before 29 °C). Error bars represent s.e.m. ***P < 0.001; ns, not significant; two-sided Student’s t test.

npg

© 2

012

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

1522 VOLUME 15 | NUMBER 11 | NOVEMBER 2012 NATURE NEUROSCIENCE

A R T I C L E S

activation of the single PPM3 neuron was smaller than that of most dopamine neurons with TH-GAL4, it is possible that other dopamine neurons, which were not labeled in our MARCM screening, also affect arousal. For example, in the PPL1 cluster, at least five types of pro-jections to the mushroom body and FSB-projecting neurons have been described41.

However, in our MARCM experiment, many of the labeled neu-rons in the PPL1 cluster were seen to project to the alpha lobe of the mushroom body; it is possible that we did not fully determine the contribution of other PPL1 neurons. Alternatively, combinatorial activation of the FSB-projecting PPM3 neurons and other dopamine neurons may have a synergistic effect. Further MARCM experiments using various clone induction protocols may help to formulate a more comprehensive characterization of single dopamine neurons. In addi-tion, it is also possible that dopamine neurons that are not labeled by TH-GAL4 also have an arousal effect. We and others have noticed that TH-GAL4–induced GFP expression and tyrosine hydroxylase staining do not overlap completely, and some clusters, such as the PAM cluster, are covered only partially by TH-Gal4.

Sleep and memoryThe association between sleep and memory in Drosophila has recently been described in various reports. DATfmn flies have a short sleep phenotype and poor memory retention42. Other short sleep flies, hyperkinetic and calcineurin knockdown, also suffer from impaired memory26,43. Sleep deprivation has been shown to impair aversive olfactory memory27 and learned phototaxis suppression44. Conversely, memory formation increased sleep duration in normal

flies, and this has not been observed in learning-deficient mutants45. In the Drosophila brain, the expression of synaptic component proteins decreased during sleep and increased during waking, suggesting that sleep is required for the maintenance of synapses46,47. In contrast, our data imply a functional dissociation between sleep and memory circuits by different dopamine neurons. These results will provide clues to uncover a possible physiological relationship between them, for example, by the activation of only one to examine the causal relationship.

The FSB has been reported to have a role in visual memory process-ing29,30,48, but its involvement in other behaviors is not yet fully understood. A recent report showed that activation of dFSB neurons induces sleep33. This suggests that the dopamine signal regulates sleep via control of the neural properties of the dFSB neurons through DA1. In mammals, dopamine signaling via the D1-type dopamine receptor is thought to increase firing probability49. However, dopamine sig-naling via the D1-like receptor DA1 in flies results in the inhibition of neural activity50. Although the previous report suggested that the inhibition is independent of protein kinase A, activation of adenylyl cyclase with Gs* in dFSB decreased sleep levels. Taken together, these findings indicate that DA1 activation in the dFSB inhibits neural activity and results in the promotion of wakefulness. It has been reported that sleep induction through the activation of the dFSB neurons promotes memory consolidation after courtship condition-ing. However, we found that DA1 expression in the FSB itself had little effect on aversive olfactory memory. The contradiction between these findings might be a result of the difference in the memory tasks. It is also possible that functional interaction between sleep and memory

37 C 1 h(FLP induction)

22 °C 22 °C

29 C(TrpA1 activation)

DissectionSleep analysisEclosionCross

amCD8::GFP

nc82

Anterior Posterior

bmCD8::GFP

nc82

g FasllmCD8::GFP h Fasll

mCD8::GFP

e FasllmCD8::GFP

MB

FSB

f FasllmCD8::GFP

FSB

MB

d No cell PAL PAM PPL1 PPL2 PPM1

***

PPM2PPM3(FSB)

PPM3(EB)

0

200

400

600

800

1,000

Sle

ep (

min

)

6050403020100

Sle

ep(m

in h

1 )

CT0 CT12

22 °C 29 °C 22 °C

CT0 CT12 CT0 CT12

No cell PPM3 (FSB)c

Figure 8 Activation of an FSB-projecting PPM3 dopamine neuron was sufficient to decrease sleep. (a) Experimental procedure. For the chromosome recombination by FLP, animals received a heat shock at 37 °C for 1 h between 1 and 2 d of development. Adult flies were entrained to 12-h light:dark cycle for 3 d before entering constant dark. After the sleep analysis, flies were dissected and the labeled neurons were identified. (b) A representative fly brain that showed a decrease in sleep between 22 °C and 29 °C, as shown in c. The TrpA1 channel and mCD8øGFP were expressed in the PPM3 cluster neurons that had FSB and/or mushroom body arborizations. The brain was stained with antibody to nc82 to stain the neuropil (purple). Green indicates mCD8øGFP expressed by the dopamine neurons that underwent recombination. Left, anterior view; Right, posterior view of the same brain. Scale bar represents 20 m. (c) The sleep profile of the representative fly shown in b (PPM3 (FSB)) compared with a fly with no GFP-positive neurons (no cell). (d) Decrease in sleep shown by the MARCM clones for each of the cell populations indicated. Each dot represents the daily sleep change of a single fly between 22 °C and 29 °C. Red bars indicate the mean in each cell population. ***P < 0.001 compared with the no cell group, two-sided Student’s t test. (e,f) PPM3 arborizations in the vicinity of the mushroom body (stained with antibody to FasII, magenta) and the FSB. Green indicates mCD8øGFP. MB, mushroom body. (g,h) Higher magnitude images of e and f. g shows the anterior view and h shows the posterior view. Scale bars represent 20 m.

npg

© 2

012

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

NATURE NEUROSCIENCE VOLUME 15 | NUMBER 11 | NOVEMBER 2012 1523

A R T I C L E S

is implemented downstream of the FSB. Further studies are required to elucidate the causal relationship between sleep and memory.

METHODSMethods and any associated references are available in the online version of the paper.

Note: Supplementary information is available in the online version of the paper.

ACKNOWLEDGMENTSWe thank M. Wu and Q. Liu for sharing unpublished results, K. Ueno, T. Sakai and S. Sato for encouraging advice, M. Yamazaki, A. Miyoshi, Y. Kawahara and W. Honghang for technical assistance, F.W. Wolf for the kind gift of an antibody, R. Jackson and the members of Kume laboratory for discussion and critical reading of the manuscript, and P. Garrity, D. Armstrong, J. Hirsh, K. Scott, the Kyoto Drosophila Genetic Resource Center and the Bloomington Stock Center for fly stocks. This work was supported by a grant (22300132) from the Japanese Society for the Promotion of Science (JSPS). T.U. is a JSPS fellow. S.K. is a member of the Global COE Program (Cell Fate Regulation Research and Education Unit).

AUTHOR CONTRIBUTIONST.U. and K.K. designed the study. T.U. performed the experiments and data analysis. J.T., H.T., K.E. and K.I. contributed unpublished reagents. H.T., S.K. and K.K. directed the study. T.U., J.T., H.T., K.E., K.I., S.K. and K.K. wrote the manuscript.

COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.

Published online at http://www.nature.com/doifinder/10.1038/nn.3238. Reprints and permissions information is available online at http://www.nature.com/reprints/index.html.

1. Crocker, A., Shahidullah, M., Levitan, I.B. & Sehgal, A. Identification of a neural circuit that underlies the effects of octopamine on sleep:wake behavior. Neuron 65, 670–681 (2010).

2. Kohatsu, S., Koganezawa, M. & Yamamoto, D. Female contact activates male-specific interneurons that trigger stereotypic courtship behavior in Drosophila. Neuron 69, 498–508 (2011).

3. Hendricks, J.C. et al. Rest in Drosophila is a sleep-like state. Neuron 25, 129–138 (2000).

4. Shaw, P.J., Cirelli, C., Greenspan, R.J. & Tononi, G. Correlates of sleep and waking in Drosophila melanogaster. Science 287, 1834–1837 (2000).

5. Agosto, J. et al. Modulation of GABAA receptor desensitization uncouples sleep onset and maintenance in Drosophila. Nat. Neurosci. 11, 354–359 (2008).

6. Andretic, R., van Swinderen, B. & Greenspan, R.J. Dopaminergic modulation of arousal in Drosophila. Curr. Biol. 15, 1165–1175 (2005).

7. Kume, K., Kume, S., Park, S.K., Hirsh, J. & Jackson, F.R. Dopamine is a regulator of arousal in the fruit fly. J. Neurosci. 25, 7377–7384 (2005).

8. Yuan, Q., Joiner, W.J. & Sehgal, A. A sleep-promoting role for the Drosophila serotonin receptor 1A. Curr. Biol. 16, 1051–1062 (2006).

9. Kume, K.A Drosophila dopamine transporter mutant, fumin (fmn), is defective in arousal regulation. Sleep Biol. Rhythms 4, 263–273 (2006).

10. Ueno, T., Masuda, N., Kume, S. & Kume, K. Dopamine modulates the rest period length without perturbation of its power law distribution in Drosophila melanogaster. PLoS ONE 7, e32007 (2012).

11. Ueno, T., Tomita, J., Kume, S. & Kume, K. Dopamine modulates metabolic rate and temperature sensitivity in Drosophila melanogaster. PLoS ONE 7, e31513 (2012).

12. Wisor, J.P. et al. Dopaminergic role in stimulant-induced wakefulness. J. Neurosci. 21, 1787–1794 (2001).

13. Shang, Y. et al. Imaging analysis of clock neurons reveals light buffers the wake-promoting effect of dopamine. Nat. Neurosci. 14, 889–895 (2011).

14. Lebestky, T. et al. Two different forms of arousal in Drosophila are oppositely regulated by the dopamine D1 receptor ortholog DopR via distinct neural circuits. Neuron 64, 522–536 (2009).

15. Draper, I., Kurshan, P.T., McBride, E., Jackson, F.R. & Kopin, A.S. Locomotor activity is regulated by D2-like receptors in Drosophila: an anatomic and functional analysis. Dev. Neurobiol. 67, 378–393 (2007).

16. Andretic, R., Kim, Y.C., Jones, F.S., Han, K.A. & Greenspan, R.J. Drosophila D1 dopamine receptor mediates caffeine-induced arousal. Proc. Natl. Acad. Sci. USA 105, 20392–20397 (2008).

17. Kong, E.C. et al. A pair of dopamine neurons target the D1-like dopamine receptor DopR in the central complex to promote ethanol-stimulated locomotion in Drosophila. PLoS ONE 5, e9954 (2010).

18. Van Swinderen, B. & Andretic, R. Dopamine in Drosophila: setting arousal thresholds in a miniature brain. Proc. Biol. Sci. 278, 906–913 (2011).

19. Waddell, S. Dopamine reveals neural circuit mechanisms of fly memory. Trends Neurosci. 33, 457–464 (2010).

20. Aso, Y. et al. Specific dopaminergic neurons for the formation of labile aversive memory. Curr. Biol. 20, 1445–1451 (2010).

21. Claridge-Chang, A. et al. Writing memories with light-addressable reinforcement circuitry. Cell 139, 405–415 (2009).

22. Liu, C. et al. A subset of dopamine neurons signals reward for odour memory in Drosophila. Nature 488, 512–516 (2012).

23. Joiner, W.J., Crocker, A., White, B.H. & Sehgal, A. Sleep in Drosophila is regulated by adult mushroom bodies. Nature 441, 757–760 (2006).

24. Pitman, J.L., McGill, J.J., Keegan, K.P. & Allada, R. A dynamic role for the mushroom bodies in promoting sleep in Drosophila. Nature 441, 753–756 (2006).

25. Diekelmann, S. & Born, J. The memory function of sleep. Nat. Rev. Neurosci. 11, 114–126 (2010).

26. Tomita, J. et al. Pan-neuronal knockdown of calcineurin reduces sleep in the fruit fly, Drosophila melanogaster. J. Neurosci. 31, 13137–13146 (2011).

27. Li, X., Yu, F. & Guo, A. Sleep deprivation specifically impairs short-term olfactory memory in Drosophila. Sleep 32, 1417–1424 (2009).

28. Kim, Y.C., Lee, H.G. & Han, K.A. D1 dopamine receptor dDA1 is required in the mushroom body neurons for aversive and appetitive learning in Drosophila. J. Neurosci. 27, 7640–7647 (2007).

29. Young, J.M. & Armstrong, J.D. Structure of the adult central complex in Drosophila: organization of distinct neuronal subsets. J. Comp. Neurol. 518, 1500–1524 (2010).

30. Liu, G. et al. Distinct memory traces for two visual features in the Drosophila brain. Nature 439, 551–556 (2006).

31. Sugamori, K.S., Demchyshyn, L.L., McConkey, F., Forte, M.A. & Niznik, H.B. A primordial dopamine D1-like adenylyl cyclase–linked receptor from Drosophila melanogaster displaying poor affinity for benzazepines. FEBS Lett. 362, 131–138 (1995).

32. Connolly, J.B. et al. Associative learning disrupted by impaired Gs signaling in Drosophila mushroom bodies. Science 274, 2104–2107 (1996).

33. Donlea, J.M., Thimgan, M.S., Suzuki, Y., Gottschalk, L. & Shaw, P.J. Inducing sleep by remote control facilitates memory consolidation in Drosophila. Science 332, 1571–1576 (2011).

34. Feinberg, E.H. et al. GFP reconstitution across synaptic partners (GRASP) defines cell contacts and synapses in living nervous systems. Neuron 57, 353–363 (2008).

35. Gordon, M.D. & Scott, K. Motor control in a Drosophila taste circuit. Neuron 61, 373–384 (2009).

36. Lai, S.L. & Lee, T. Genetic mosaic with dual binary transcriptional systems in Drosophila. Nat. Neurosci. 9, 703–709 (2006).

37. Shinomiya, K., Matsuda, K., Oishi, T., Otsuna, H. & Ito, K. Flybrain neuron database: a comprehensive database system of the Drosophila brain neurons. J. Comp. Neurol. 519, 807–833 (2011).

38. Friggi-Grelin, F. et al. Targeted gene expression in Drosophila dopaminergic cells using regulatory sequences from tyrosine hydroxylase. J. Neurobiol. 54, 618–627 (2003).

39. Shafer, O.T. et al. Widespread receptivity to neuropeptide PDF throughout the neuronal circadian clock network of Drosophila revealed by real-time cyclic AMP imaging. Neuron 58, 223–237 (2008).

40. Lee, T. & Luo, L. Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron 22, 451–461 (1999).

41. Mao, Z. & Davis, R.L. Eight different types of dopaminergic neurons innervate the Drosophila mushroom body neuropil: anatomical and physiological heterogeneity. Front. Neural Circuits 3, 5 (2009).

42. Zhang, S., Yin, Y., Lu, H. & Guo, A. Increased dopaminergic signaling impairs aversive olfactory memory retention in Drosophila. Biochem. Biophys. Res. Commun. 370, 82–86 (2008).

43. Bushey, D., Huber, R., Tononi, G. & Cirelli, C. Drosophila Hyperkinetic mutants have reduced sleep and impaired memory. J. Neurosci. 27, 5384–5393 (2007).

44. Seugnet, L., Suzuki, Y., Vine, L., Gottschalk, L. & Shaw, P.J. D1 receptor activation in the mushroom bodies rescues sleep loss–induced learning impairments in Drosophila. Curr. Biol. 18, 1110–1117 (2008).

45. Donlea, J.M., Ramanan, N. & Shaw, P.J. Use-dependent plasticity in clock neurons regulates sleep need in Drosophila. Science 324, 105–108 (2009).

46. Gilestro, G.F., Tononi, G. & Cirelli, C. Widespread changes in synaptic markers as a function of sleep and wakefulness in Drosophila. Science 324, 109–112 (2009).

47. Bushey, D., Tononi, G. & Cirelli, C. Sleep and synaptic homeostasis: structural evidence in Drosophila. Science 332, 1576–1581 (2011).

48. Pan, Y. et al. Differential roles of the fan-shaped body and the ellipsoid body in Drosophila visual pattern memory. Learn. Mem. 16, 289–295 (2009).

49. Neve, K.A., Seamans, J.K. & Trantham-Davidson, H. Dopamine receptor signaling. J. Recept. Signal Transduct. Res. 24, 165–205 (2004).

50. Yuan, N. & Lee, D. Suppression of excitatory cholinergic synaptic transmission by Drosophila dopamine D1-like receptors. Eur. J. Neurosci. 26, 2417–2427 (2007).

npg

© 2

012

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

NATURE NEUROSCIENCE doi:10.1038/nn.3238

ONLINE METHODSFly stocks. Flies (Drosophila melanogaster) were reared on a standard corn meal, yeast and glucose agar medium at 24.5 °C under a 12-h:12-h light:dark cycle. The DATfmn mutant has been described previously6. TH-GAL4 (ref. 38) and TH-GAL80 were gifts from J. Hirsh (University of Virginia). UAS-TrpA1 (ref. 51) was a gift from P.A. Garrity (Brandeis University). UAS-CD4øspGFP1-10 and LexAop-spGFP11 were gifts from K. Scott (University of California, Berkeley). UAS-Gs* was a gift from K. Ueno (Tokyo Metropolitan Institute of Medical Science). 104y and c259 were gifts from D. Armstrong (University of Edinburgh). NP lines, hs-FLP and tub-GAL80 were obtained from the Kyoto Drosophila Genetic Resource Center, UAS-Epac1-camps and DA1dumb2 were obtained from the Bloomington Stock Center, UAS-Dicer and UAS-DA1-RNAi were from Vienna Drosophila RNAi Center. For the tissue specific dDA1 rescue, we used a DA1dumb2 mutant. The piggyBac inserted at the first intron of the DA1dumb2 gene has a UAS construct so the DA1 expression is induced by crossing it with a GAL4 driver27 (note that all alleles are homozygous in these flies). To remove possible modi-fiers and allow comparisons in a common genetic background, we outcrossed all the alleles into the w1118 or DATfmn background over at least five consecutive generations. As controls, we used w1118 or DATfmn flies.

Locomotor activity and sleep analysis. We placed 3–7-d-old flies individually in glass tubes (length, 65 mm; inside diameter, 3 mm) containing 1% agar and 5% sucrose at 24.5 °C. Male flies were used except for the MARCM analysis. They were entrained for at least 3 d to 12-h light:dark cycle before transferring to constant dark conditions. Locomotor activity was monitored by recording infrared beam crossings by individual flies in 1 min bins using the Drosophila acti-vity monitoring system (Trikinetics). On the basis of previous reports, sleep was defined as periods of inactivity lasting 5 min or longer. Daily sleep time was calcu-lated with software written in Excel or R 2.11.1. For the pharmacological manipu-lations, 3-iodo-d-tyrosine and RU486 were obtained from Sigma-Aldrich. Flies were transferred to vehicle (1% agar + 5% sucrose) with 3 mM 3-iodo-d-tyrosine, 0.5 mg ml−1 cocaine or 0.5 mM RU486.

Aversive odor memory. Olfactory conditioning with two odors (4-methyl-cyclohexanol and 3-octanol) was performed as described previously52. Briefly, approximately 100 flies were placed in a training chamber where they were exposed to odors and electric shocks. One of the aversive odors, either 3-octanol (Sigma) or 4-methylcyclohexanol (Sigma) was paired with 12 pulses of electrical shocks (60 V, direct current) for 1 min, and the other was not. Testing was per-formed for 2 min after the training by placing flies at a choice point in a T maze between the two odors. If no flies moved to either of the arms of the T maze, or if a small number of flies were trapped in the middle compartment, then the flies did not choose either odor. A performance index was calculated so that a 50:50 distribution (no memory) yielded a performance index of zero and a 0:100 dis-tribution away from the shock-paired odor yielded a performance index of 100. Individual performance indexes were always the average of two experiments in which the shock-paired odor was alternated.

Neural activation with TrpA1. The assay was started at 22 °C. After transfer-ring the flies to constant darkness, behavior was assayed for 3 d at 22 °C in con-stant dark and shifted to 29 °C at CT0 to activate the neuron for 1 d, and then placed back to 22 °C. Data were collected continuously for 5 d under constant dark conditions.

MARCM methods. We used flies with the FRT (Flp recombination target) site as well as FLP recombinase, and GAL80 was driven by a tubulin promoter on the first chromosome. The second chromosome carried the UAS-TrpA1 transgene. The third chromosome carried the TH-GAL4 and UAS-mCD8øGFP trans-genes. Parents of the final offspring were allowed to lay eggs for 1 d before being

removed from the vial; eggs and larvae were then heat pulsed at 37 °C. Animals received a heat shock at 37 °C for 1 h between 1 and 2 d of development, as described previously1.

Immunohistochemistry. Brains of adult flies were dissected in cold PBS and fixed in 4% paraformaldehyde in PBS for 20 min at 25 °C. After three 20 min washes in 0.3% Triton X-100 in PBS, samples were blocked and penetrated in 5% normal sheep serum (NSS) for 30 min at 25 °C. Samples were then incubated in a primary antibody solution in 5% NSS at 4 °C overnight. We used mouse antibody to nc82 (1:100, Developmental Studies Hybridoma Bank (DSHB), University of Iowa), mouse antibody to FasII (1:20, DSHB), mouse antibody to PDF (1:100, DSHB), chicken antibody to GFP (1:1,000, #13970, Abcam), mouse antibody to GFP (1:500, Sigma, #G6539), rabbit antibody to tyrosine hydroxylase (1:25, #40101-0, Pel-Freez), rabbit antibody to dsRed (1:500, #632496, Clontech) and rabbit antibody to DA1 (1:1,250, gift from F.W. Wolf, Ernest Gallo Clinic and Research Center) as primary antibodies. After three 20 min washes in 0.3% Triton X-100 in PBS, brains were incubated at 4 °C overnight in a secondary antibody solution in 5% NSS. We used Alexa Fluor 488–conjugated goat antibody to mouse IgG (1:200, Invitrogen), Alexa Fluor 568–conjugated goat antibody to mouse IgG (1:200, Invitrogen), Alexa Fluor 488–conjugated goat antibody to chicken IgG (1:200, Invitrogen) and Alexa Fluor 568–conjugated goat antibody to rabbit IgG (1:200, Invitrogen) as secondary antibodies. After three 20-min washes in PBS, brains were mounted using PermaFluor (Thermo Scientific) between two coverslips separated by ~200- m-thick electrical tape, so that the brain sample was not flattened. Immunolabeled adult brains were imaged using a Leica TCS SP2 AOBS confocal microscope using 20× magnification. z stack images were scanned at 2- m section intervals with a resolution of 1,024 × 1,024 pixels.

Functional imaging. Living brains expressing GAL4 driven UAS-Epac1-camps were dissected under ice cold calcium-free AHL saline containing 108 mM NaCl, 5 mM KCl, 8.2 mM MgCl2, 4 mM NaHCO3, 1 mM NaH2PO4, 5 mM trehalose, 10 mM sucrose and 5 mM HEPES. Dissected brains were held with custom-made platinum grid and placed at the bottom of a slide (Ibidi) beneath calcium- containing (2 mM CaCl2) AHL saline. All imaging was performed with a confocal microscope (Zeiss LSM 710). To minimize the effect of any z plane drift, the pin-hole was adjusted to its full open diameter (600 m). The specimen was excited using an argon laser (458 nm), and emission was detected between 473 nm and 493 nm, and between 512 nm and 532 nm (to detect emission from cyan (CFP) and yellow fluorescent protein (YFP), respectively). Time series images were collected over a period of 600 s at 0.5 Hz. After taking the baseline images, water or dopamine (final concentration of 10 mM) were applied at 60 s by bath application using a pipette. TTX was purchased from Wako and a stock solution (100 M) was prepared in H2O. The final concentration was used at 1 M. Brains were pre-incubated in 1 M TTX for 15 min before adding dopamine.

Quantification of FRET signals. A region of interest (ROI) was drawn around the target structure using ImageJ (US National Institutes of Health). The average fluorescence in this ROI was calculated at each time point. The magnitudes of cAMP transients are reported in terms of the normalized change in fluorescence (R/R0 = (CFP – CFP0)/(YFP – YFP0)). CFP0 and YFP0 represent the baseline fluorescence in the ROI averaged across the first 30 frames.

Statistical analysis. Data were analyzed as described in the figure legends using R 2.11.1.

51. Hamada, F.N. et al. An internal thermal sensor controlling temperature preference in Drosophila. Nature 454, 217–220 (2008).

52. Tully, T. & Quinn, W.G. Classical conditioning and retention in normal and mutant Drosophila melanogaster. J. Comp. Physiol. A 157, 263–277 (1985).

npg

© 2

012

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

Nature Neuroscience: doi:10.1038/nn.3238

Nature Neuroscience: doi:10.1038/nn.3238

Nature Neuroscience: doi:10.1038/nn.3238

Nature Neuroscience: doi:10.1038/nn.3238

Nature Neuroscience: doi:10.1038/nn.3238

Nature Neuroscience: doi:10.1038/nn.3238

Nature Neuroscience: doi:10.1038/nn.3238

Nature Neuroscience: doi:10.1038/nn.3238

Nature Neuroscience: doi:10.1038/nn.3238

Nature Neuroscience: doi:10.1038/nn.3238

Supplementary Figure 10. Schematic drawing of the sleep-regulating dopamine circuit in the fly brain as identified by this study. The PPM3 dopamine neuron projecting to the FSB regulates sleep.

Nature Neuroscience: doi:10.1038/nn.3238