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Sleep in Drosophila is regulated by adult mushroombodiesWilliam J. Joiner1, Amanda Crocker1, Benjamin H. White3 & Amita Sehgal1,2

Sleep is one of the few major whole-organ phenomena for whichno function and no underlying mechanism have been conclusivelydemonstrated. Sleep could result from global changes in the brainduring wakefulness or it could be regulated by specific loci thatrecruit the rest of the brain into the electrical and metabolic statescharacteristic of sleep. Here we address this issue by exploiting thegenetic tractability of the fruitfly, Drosophila melanogaster, whichexhibits the hallmarks of vertebrate sleep1–4. We show that largechanges in sleep are achieved by spatial and temporal enhance-ment of cyclic-AMP-dependent protein kinase (PKA) activityspecifically in the adult mushroom bodies of Drosophila. Othermanipulations of the mushroom bodies, such as electrical silenc-ing, increasing excitation or ablation, also alter sleep. These resultslink sleep regulation to an anatomical locus known to be involvedin learning and memory.Sleep duration is inversely related to PKA activity in flies5. To

determine whether specific brain loci regulate sleep, we used theGAL4/UAS (upstream activating sequence) system6 to express acatalytic subunit of PKA in various regions of the fly brain. Wefirst expressed PKA under the control of the RU486-inducible pan-neuronal driver elavGeneSwitch7. Restricting the expression of PKAto adult neurons decreased daily sleep, supporting earlier studieswith mutants such as dunce5 that increase PKA levels, and showingthat PKAdirectly regulates sleep rather than a developmental processthat might affect sleep (Fig. 1a). We next expressed PKA under thecontrol of different GAL4 drivers and examined the changes in totaldaily sleep in the different driver/transgene combinations relative todriver/background and background/transgene controls (Fig. 1b).When both controls were taken into account, the expression ofPKA by only two drivers led to changes in sleep that exceeded 2 s.d.These were 201Y, which increased sleep by 75 ^ 3% and 93 ^ 4%respectively, and c309, which decreased sleep by 46 ^ 11% and43 ^ 14% per day compared with the two sets of controls. Changesin sleep caused by all other GAL4 drivers remained within 1 s.d. of themean.We next examined whether activity levels during wake periods

were affected by the 201Yand c309 drivers. Many GAL4 driver/UAS–PKA lines were hypoactive, but line 201Y had normal waking activity(Fig. 1c). Similarly, activity normalized to waking time in c309 wasnot significantly higher in PKA-driven animals than in either control(Fig. 1c), indicating that c309 was not hyperactive. We conclude thatthe sleep phenotypes of animals expressing PKA under control of the201Y and c309 drivers are not associated with abnormal wakingactivity. Interestingly, both these drivers are known to be expressedin the mushroom bodies (MBs)8, a brain region implicated inassociative learning.Given the strong, yet opposite, effects that 201Y and c309 had on

sleep, we further characterized their expression patterns in the flybrain by crossing them into animals bearing a UAS transgene for

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Figure 1 | Expression of PKA in various regions of the fly brain affects sleepdifferentially. a, The pan-neuronal driver elavGeneSwitch7 was used toexpress UAS-mc*, which contains a constitutively active subunit of PKAdownstream of an upstream activating sequence (UAS)30. Sleep, defined as5min of immobility, was assayed in flies in which expression of PKA wasinduced with 500mMRU486 (dashed line) and in uninduced controls (solidline). b, Progeny were collected from crosses between 21 GAL4 driversand y w;UAS-mc*. For controls, each GAL4 line was crossed to y w flies(the background for the UAS-transgenic line), and y w;UAS-mc* was crossedto w1118 (the background for the GAL4 drivers). Daily sleep was averagedover 4 days. Sleep from control animals was subtracted from theexperimental group, then divided by the amount of control sleep to calculatenet percentage changes. Grey bars, sleep inw1118/y w flies carrying UAS-mc*and a GAL4 transgene relative to sleep in w1118/y w flies carrying therespective GAL4 transgene alone; white bars, sleep in w1118/y w fliescarrying UAS-mc* and a GAL4 transgene relative to sleep in w1118/y w fliescarrying only UAS-mc*. c, For each of the crosses in b, average dailyactivity (beam crossings) was divided by average total wake time.Differences in waking activity between GAL4/UAS-mc* and control flieswere calculated as in b. For each group, n $ 25. Where errors are shown,they are s.e.m.

1Center for Sleep and Respiratory Neurobiology and 2Howard Hughes Medical Institute, University of Pennsylvania Medical School, Philadelphia, Pennsylvania 19104, USA.3Laboratory of Molecular Biology, National Institute of Mental Health, Bethesda, Maryland 20892, USA.

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green fluorescent protein (GFP). We found that 201Y is expressedlargely in the g lobes and the core region of the a/b lobes of the MBs,whereas c309 is expressed in the a/b and g lobes but not in the coreregion of the a/b lobes (Fig. 2a, b, and Supplementary Fig. S1). Thisdifferential expression pattern within the MBs indicates that PKAmight affect the regulation of sleep by the MBs in both a positive anda negative fashion by using anatomically distinct classes of neurons.Consistent with this notion of heterogeneous cell types within theMBs9–11, some MB drivers, such as 30Y and 238Y, promoted sleepduring the day but inhibited sleep during the night, leading to onlymarginal overall changes in daily sleep (Fig. 1, and SupplementaryFig. S2). This effect was not observed with any driver that wasexpressed exclusively outside the MBs (Fig. 2c and SupplementaryTable 1). A small increase in daytime sleep was also frequentlyproduced by the pan-neuronal elavGeneSwitch driver, whichdecreased overall sleep (data not shown, and Fig. 1a). The expressionpatterns of 238Yand 30Yoverlap those of 201Yand c309, supportingthe idea that 238Y and 30Y are expressed in both sleep-promotingand sleep-inhibiting areas (see Supplementary Table S1 and Sup-plementary Fig. S1 for expression patterns of drivers used in thisstudy).To test the hypothesis that PKA expression in MBs regulates adult

sleep, we expressed the PKA transgene under the control of anRU486-activatable MB GAL4 driver, P{MB-Switch}12. We confirmedselective expression of this driver in the MBs by coupling it to a GFPreporter, and found inducible expression in the MBs (Fig. 2d, e, andSupplementary Fig. S1). Sleep was significantly reduced in responseto RU486 in MB-Switch/PKA animals (Fig. 3a, b) but was unaffectedby the drug in control animals harbouring either the driver or thetransgene alone (Fig. 3c). Thus, PKA overexpressed preferentially inspecific neurons of adult MBs is sufficient to reduce sleep.Next we compared sleep structure in the hyposomnolent animals

with that of controls. In both MB-Switch/PKA animals and c309/PKAanimals, loss of sleep was caused by a shortened sleep bout durationwithout a concomitant increase in the sleep bout number (Fig. 3d, e,and Supplementary Fig. S3a, b). The underlying cause of reducedsleep in both sets of animals therefore seems to be impaired sleepneed, because the alternative—normal sleep need, but an inability to

maintain the sleep state—would be expected to produce an increasein sleep bout number. In contrast, in 201Y sleep bout durationremained unchanged (Supplementary Fig. S3c, d).We then asked whether the reduction of sleep in MB-Switch/PKA

Figure 2 | Localization of GAL4-dependent brain expression. A nuclear-targeted GFP (GFPn) was expressed under the control of different GAL4drivers. a, c309/GFPn; upper arrows refer to a lobes and lower arrows to blobes of MBs. b, 201Y/GFPn; upper, middle and lower arrows point to a, gand b lobes, respectively, withinMBs. c, 104Y/GFPn; the fan-shaped body ofthe central complex and cells rimming the optic lobes and anterior brain areilluminated. d, P{MB-Switch}/GFPn with RU486 in the food; labelling is asin b. e, P{MB-Switch}/GFPn without RU486 in the food.

Figure 3 | Inducible expression of PKA in the MBs leads to decreased sleepbout duration and accumulation of sleep debt. a, Sleep was monitored inw1118/y w;UAS-mc*/þ;P{MB-Switch}/þ females in the presence (dashedline) or absence (solid line) of 500mM RU486 (n $ 16 for each group). Atthe end of day 3, animals were transferred to tubes lacking RU486 (arrow).Grey bars represent 12-h dark periods. b, Sleep was measured for females inLD (n ¼ 61 or 62) and in constant darkness (DD, n ¼ 28–30), and for malesin LD (n ¼ 45 or 46) and inDD (n ¼ 15 or 16). Black bars represent animalsfed RU486; white bars are controls. Plotted values represent averages overthree days. c, w1118/y w;;P{MB-Switch}/þ or w1118/y w;UAS-mc*/þ femalecontrols were placed in LD for 3 days with (black bars) or without (whitebars) RU486 in their food. d, Sleep bouts in LD were binned according toduration for P{MB-Switch}/UAS-mc* females with (black bars) or without(white bars) RU486 (n ¼ 43–45 for each group). e, Sleep bout number in LDis similar for P{MB-Switch}/UAS-mc* females treated with RU486 (blackbars) to that in genotypically identical uninduced animals (white bars).n ¼ 43–45 for each group. f, Flies were monitored for four days in thepresence or absence of 500mMRU486 and then transferred to tubes with nodrug at diurnal time 0 (ZT0; when the light was turned on). Rebound sleepduring the following 12 hours was determined by subtracting sleep in the ‘nodrug to no drug’ group from sleep in ‘drug to no drug’ animals. n . 64 forw1118/y w;UAS-mc*/þ;P{MB-Switch}/þ experimental animals; n . 30 forw1118/y w;;P{MB-Switch}/þ and w1118/y w;UAS-mc*/þ controls. g, elav-GeneSwitch/UAS-mc* females were switched at ZT0 from drug to drug(black bar), from no drug to no drug (white bar) or from drug to no drug(that is rebound conditions, grey bar). After 2 h, PKA activity was measuredin heads from each group. Significance was determined with a one-wayanalysis of variance. Asterisk, P , 0.05; two asterisks, p , 0.01; n.s.,p . 0.05 by unpaired t-test. Where errors are shown, they are s.e.m.

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animals was due to an impaired accrual of a sleep-inducing signal. Ifthis were so, then a hallmark of sleep, homeostatic rebound—sleepthat exceeds baseline to compensate for lost sleep—should not occuron relief of induced PKA expression. However, when RU486 waswithdrawn after about three days of sleep deprivation, an averagerebound of 156 ^ 38min was observed (Fig. 3a, f). This is a robustrebound, comparable to that producedwhen genetically identical butuninduced flies were submitted to a standard 12 h of mechanicaldeprivation (137 ^ 26min; Supplementary Fig. S4). Behaviouralrebound was also observed in animals expressing elavGeneSwitch-driven PKA, after withdrawal of RU486 (data not shown), and wasaccompanied by a decrease in PKA activity in fly heads (Fig. 3g).Rebound after withdrawal of RU486 indicates that PKA might notprevent the accrual of sleep-promoting signals but might suppresshomeostatic output.To determine whether PKA affects sleep by regulating synaptic

output in MB neurons, we inducibly expressed either of two Kþ

channels, Kir2.1 (ref. 13) or EKO14, under the control of theMB-Switch driver. Such transgenic expression should suppressaction-potential firing by hyperpolarizing neurons and decreasingmembrane resistance, thus leading to reduced synaptic transmission.We found that induction of either Kir2.1 or EKO caused a significantincrease in sleep (Fig. 4a, b, and Supplementary Fig. S5). Because theopposite was observed with PKA expression in the same neurons,

it indicates that PKA might decrease sleep by increasing eitherexcitability or synaptic transmission. To address this issue further,we inducibly expressed a sodium channel (NaChBac), whichdepolarizes neurons and increases excitability15. When expressedunder the control of the MB-Switch driver, the sodium channelcaused a decrease in sleep (Fig. 4c, d), similar to that produced byPKA, confirming that PKA increases the output of these neurons.The MB-Switch driver is expressed in a subpopulation of MB

neurons similar to those labelled by c309 (Supplementary Fig. S1),and both drivers had sleep-inhibiting effects. As noted above, thispattern of expression differed from that of other drivers (Supplemen-tary Fig. S1), which had sleep-promoting or bidirectional effects onsleep (Fig. 1b, and Supplementary Fig. S2), thus leading us to proposethat the MBs contain sleep-inhibiting and sleep-promoting neurons.To determine the overall effect of MBs on sleep, we ablated themwithhydroxyurea and examined sleep and activity in adult flies. Consist-ent with previous reports16,17 was our observation of an overallincrease in activity (Supplementary Fig. S6a). However, normal-ization of this activity to waking time indicates that the phenotypederives less from hyperactivity than from a reduction in sleep(Supplementary Fig. S6b, c). Even so, the reduction in sleep wasmuch less than that seen with other manipulations of the MBs (seeabove) or in short-sleep mutants such as minisleep18. This supportsour conclusion that MBs exert both positive and negative influenceson sleep that are integrated to produce the overt behavioural state.Our model (Supplementary Fig. S7) takes into account our results;notably the integrator downstream of the MBs promotes activity inthe default state. Thus, when MBs are ablated the overall effect isincreased wakefulness.Opposing effects of the c309 and 201Ydrivers are also observed in a

different behavioural model. They parallel MB-dependent changes inbrain activity during the sleep/wake cycle that are associated withsalience, a behavioural trait that may correspond to arousal. Con-sistent with our data was the observation that reducing synaptictransmission using the c309 driver inhibited salience, whereas the201Y driver in the same type of experiment yielded no change. Wewould predict increased arousal with 201Y, but in those experimentsthe animals were already awake4.Because MBs receive and transduce considerable sensory, particu-

larly olfactory, input to the fly brain, we speculate that they promotearousal or sleep by allowing or inhibiting the throughput of sensoryinformation. In addition, given the major function that MBs have inregulating plasticity in the fly brain, it is likely that this is linked totheir role in sleep. In mammals, sleep deprivation suppresses theperformance of learned tasks, and sleep permits memory consolida-tion19. Sleep and sleep deprivation also differentially affect corticalsynaptic plasticity20. InDrosophila, MBs participate in the consolida-tion or retrieval of memories involving olfactory cues21–24, courtshipconditioning8,25 and context-dependent visual cues26,27 by mecha-nisms that include cAMP signalling. Distinct anatomical regions ofthe MBs have been shown to be important for at least some forms ofmemory23,28, as we have now also shown for sleep. Thus, memory andsleep may involve similar molecular pathways (cAMP signalling) andanatomical regulatory loci (MBs).

METHODSSleep assays. Female flies 3–7 days old were placed in 65mm £ 5mm glass tubescontaining 5% sucrose/2% agarose with or without 500mM RU486. Flies wereacclimated for about 36 h at 25 8C in 12 h light/12 h dark (LD) conditions.Locomotor activity was collected in LD with DAMS monitors (Trikinetics) asdescribed previously1,5. Sleep was measured as bouts of 5min of inactivity, asdescribed previously29, using a moving window over 30-s intervals. In someexperiments RU486 was removed at the time points indicated in figure legends.Localization of GAL4 expression. Many of the GAL4 lines from Fig. 1 werecrossed to a fly line with a transgene encodingGFP fused to a nuclear localizationsignal (GFPn). Brains were dissected and fixed for 20–30min in 4% parafor-maldehyde in PBS before photography of serial z-sections of whole mounts withthe use of confocal microscopy. In some cases, images were deconvolved to allow

Figure 4 |Decreasing and increasing excitability in a subset of MB neuronshave opposite effects on sleep. a, In P{MB-Switch}/UAS-Kir2.1 animals inwhich Kþ channel expression in the MBs was induced by RU486 (dashedline), sleep exceeded that of control animals of identical genotype (solidline). b, RU486 significantly increased sleep in MBSwitch/Kir2.1 females(black bars); white bars are uninduced controls. From left to right: forw1118/y w;;MBSwitch/Kir2.1, n ¼ 34–47; for w1118/y w;;MBSwitch/þ,n ¼ 30 or 31; for w1118/y w;;UAS-Kir2.1/þ, n ¼ 11–26. c, In P{MB-Switch)/UAS-NaChBac flies in which Naþ channel expression in the MBs wasinduced by RU486 (dashed line), sleep was significantly suppressed incomparisonwith control animals of identical genotype (solid line). d, RU486significantly suppressed sleep in MBswitch/UAS-NaChBac females. Fromleft to right: for w1118/y w;UAS-NaChBac/þ;MBSwitch/þ, n ¼ 46 or 47; forw1118/y w;;MBSwitch/þ, n ¼ 31; for w1118/y w;UAS-NaChBac/þ, n ¼ 59 or60. Two asterisks, P , 0.01 by unpaired t-test. Where errors are shown, theyare s.e.m.

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virtual transverse sections to be taken through MB peduncles.PKA assays. After 1 week in LD, elav-GeneSwitch/UAS-mc* females wereswitched at ZT0 from drug to drug, from no drug to no drug or from drug tono drug. After 2 h, heads were isolated and homogenized on ice in bufferconsisting of (in mM): 10 HEPES pH7.5, 100 KCl, 1 EDTA, 5 dithiothreitol, 5phenylmethylsulphonyl fluoride, 10% glycerol, 0.1% Triton X-100 and onetablet of Complete (Promega). Debris was pelleted for 5min at 4 8C in amicrocentrifuge and discarded. A protein assay (Dc kit; Bio-Rad) was performedon supernatant from each sample, and homogenization buffer was added asnecessary to make each sample equal in concentration. PKA activity wasmeasured as ATP-dependent quenching of fluorescent kemptide in accordancewith the manufacturer’s instructions (IQ assay; Pierce) and normalized to valuesdetermined for animals maintained on RU486 throughout the experiment.Hydroxyurea-dependent ablation ofMBs.Mated Canton-S flies were placed onstandard grape juice/agar plates to induce egg-laying over a 1-h period. On thenext day, first-instar larvae were collected within 1 h of hatching and placed in50% yeast paste in the presence or absence of 50mgml21 hydroxyurea for 4 hbefore being transferred to standard food vials, as described previously21. Aftereclosion, adult male flies were aged for one week and then placed in glass tubes tomeasure sleep/activity patterns over a three-day period in LD.

Received 25 January; accepted 12 April 2006.

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

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

3. Nitz, D. A., van Swinderen, B., Tononi, G. & Greenspan, R. J.Electrophysiological correlates of rest and activity in Drosophila melanogaster.Curr. Biol. 12, 1934–-1940 (2002).

4. van Swinderen, B. & Greenspan, R. J. Salience modulates 20–-30 Hz brainactivity in Drosophila. Nature Neurosci. 6, 579–-586 (2003).

5. Hendricks, J. C. et al. A non-circadian role for cAMP signaling and CREBactivity in Drosophila rest homeostasis. Nature Neurosci. 4, 1108–-1115 (2001).

6. Brand, A. H. & Perrimon, N. Targeted expression as a means of altering cellfates and generating dominant phenotypes. Development 118, 401–-415 (1993).

7. Osterwalder, T., Yoon, K. S., White, B. H. & Keshishian, H. A conditionaltissue-specific transgene expression system using inducible GAL4. Proc. NatlAcad. Sci. USA 98, 12596–-12601 (2001).

8. Joiner, M. A. & Griffith, L. C. Mapping of the anatomical circuit of CaMkinase-dependent courtship conditioning in Drosophila. Learn. Mem. 6, 177–-192(1999).

9. Armstrong, J. D., de Belle, J. S., Wang, Z. & Kaiser, K. Metamorphosis of themushroom bodies; large-scale rearrangements of the neural substrates forassociative learning and memory in Drosophila. Learn. Mem. 5, 102–-114 (1998).

10. Zhu, S., Chiang, A. S. & Lee, T. Development of the Drosophila mushroombodies: elaboration, remodeling and spatial organization of dendrites in thecalyx. Development 130, 2603–-2610 (2003).

11. Crittenden, J. R., Skoulakis, E. M., Han, K. A., Kalderon, D. & Davis, R. L.Tripartite mushroom body architecture revealed by antigenic markers. Learn.Mem. 5, 38–-51 (1998).

12. Mao, Z., Roman, G., Zong, L. & Davis, R. L. Pharmacogenetic rescue in time andspace of the rutabaga memory impairment using Gene-Switch. Proc. Natl Acad.Sci. USA 101, 198–-203 (2004).

13. Baines, R. A., Uhler, J. P., Thompson, A., Sweeney, S. T. & Bate, M. Alteredelectrical properties in Drosophila neurons developing without synaptictransmission. J. Neurosci. 21, 1523–-1531 (2001).

14. White, B. H. et al. Targeted attenuation of electrical activity in Drosophila usinga genetically modified Kþ channel. Neuron 31, 699–-711 (2001).

15. Luan, H. et al. Functional dissection of a neuronal network required forcuticle tanning and wing expansion in Drosophila. J. Neurosci. 26, 573–-584(2006).

16. Martin, J. R., Ernst, R. & Heisenberg, M. Mushroom bodies suppress locomotoractivity in Drosophila melanogaster. Learn. Mem. 5, 179–-191 (1998).

17. Helfrich-Forster, C., Wulf, J. & de Belle, J. S. Mushroom body influence onlocomotor activity and circadian rhythms in Drosophila melanogaster.J. Neurogenet. 16, 73–-109 (2002).

18. Cirelli, C. et al. Reduced sleep in Drosophila Shaker mutants. Nature 434,1087–-1092 (2005).

19. Walker, M. P. & Stickgold, R. Sleep-dependent learning and memoryconsolidation. Neuron 44, 121–-133 (2004).

20. Frank, M. G., Issa, N. P. & Stryker, M. P. Sleep enhances plasticity in thedeveloping visual cortex. Neuron 30, 275–-287 (2001).

21. de Belle, J. S. & Heisenberg, M. Associative odor learning in Drosophila abolishedby chemical ablation of mushroom bodies. Science 263, 692–-695 (1994).

22. Dubnau, J., Grady, L., Kitamoto, T. & Tully, T. Disruption of neurotransmissionin Drosophila mushroom body blocks retrieval but not acquisition of memory.Nature 411, 476–-480 (2001).

23. McGuire, S. E., Le, P. T. & Davis, R. L. The role of Drosophila mushroom bodysignaling in olfactory memory. Science 293, 1330–-1333 (2001).

24. Zars, T., Fischer, M., Schulz, R. & Heisenberg, M. Localization of a short-termmemory in Drosophila. Science 288, 672–-675 (2000).

25. McBride, S. M. et al. Mushroom body ablation impairs short-term memory andlong-term memory of courtship conditioning in Drosophila melanogaster. Neuron24, 967–-977 (1999).

26. Liu, L., Wolf, R., Ernst, R. & Heisenberg, M. Context generalization in Drosophilavisual learning requires the mushroom bodies. Nature 400, 753–-756 (1999).

27. Tang, S. & Guo, A. Choice behavior of Drosophila facing contradictory visualcues. Science 294, 1543–-1547 (2001).

28. Pascual, A. & Preat, T. Localization of long-term memory within the Drosophilamushroom body. Science 94, 1115–-1117 (2001).

29. Huber, R. et al. Sleep homeostasis in Drosophila melanogaster. Sleep 27,628–-639 (2004).

30. Li, W., Ohlmeyer, J. T., Lane, M. E. & Kalderon, D. Function of protein kinase Ain hedgehog signal transduction and Drosophila imaginal disc development. Cell80, 553–-562 (1995).

Supplementary Information is linked to the online version of the paper atwww.nature.com/nature.

Acknowledgements We thank G. Roman and R. Davis for P{MB-Switch}, andL. Griffith, T. Kitamoto, R. Greenspan, J. D. Armstrong, K. Kaiser, R. Allada andC. Hellfrich-Forster for other GAL4 lines; J. Hendricks for advice on studying flysleep; E. Friedman, S. Harbison, K. Ho, K. Koh, S. Sathyanarayanan, M. Wu,Q. Yuan and X. Zheng for critical comments on the manuscript; and K. Liu forassistance with animal maintenance. We also thank J. Pitman and R. Allada forcommunicating results before publication. W.J.J. and A.C. were supported bytraining grants to the Center for Sleep and Respiratory Neurobiology at theUniversity of Pennsylvania, and W.J.J. was also supported by a PickwickFellowship from the National Sleep Foundation. Partial support was provided bya programme project grant from the NIA. B.H.W. was supported by theIntramural Research Program of the NIH, NIMH.

Author Contributions W.J.J. conducted the behavioural experiments, wrote thesleep analysis software, analysed the sleep data, prepared fly brains and headsfor confocal microscopy and PKA assays, and co-wrote the manuscript. A.C.assisted with the behavioural experiments and sleep analysis, prepared flybrains for imaging, performed all confocal microscopy, and provided feedbackon the manuscript. B.H.W. provided NaChBac transgenic animals and feedbackon the manuscript. A.S. conceived the project, provided guidance and criticalinterpretation during the project, co-wrote the manuscript and providedfinancial support.

Author Information Reprints and permissions information is available atnpg.nature.com/reprintsandpermissions. The authors declare no competingfinancial interests. Correspondence and requests for materials should beaddressed to A.S. (amita@mail.med.upenn.edu).

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