Clock and Cycle Limit Starvation-Induced Sleep Loss in Drosophila

Alex C. Keene,1,* Erik R. Duboue´,1 Daniel M. McDonald,1 changes in sleep during 24 hr of food deprivation (Figure 1A). Monica Dus,2 Greg S.B. Suh,2,4 Scott Waddell,3,4 We found that similar to mammals, both male (Figures 1B and Justin Blau1,4 and 1C) and female (Figure 1D) flies dramatically suppressed 1Biology Department, New York University, New York, NY their sleep during starvation compared to control flies fed ad 10003, USA libitum. Determining the percentage change in sleep within 2Skirball Institute for Biomolecular Medicine, New York individual animals revealed that starved flies robustly sup- University School of Medicine, New York, NY 10016, USA pressed sleep compared to baseline control levels 3Department of Neurobiology, University of Massachusetts (Figure 1E). Flies housed with sucrose did not suppress their Medical School, Worcester, MA 01605, USA sleep, indicating that caloric intake with no amino acids is sufficient to support normal levels of sleep. Flies housed with the nonmetabolizable sweetener sucralose suppressed Summary sleep (Figures S1A and S1B available online), confirming that the effects of starvation on sleep are due to a caloric deficit Neural systems controlling the vital functions of sleep and rather than sensory systems detecting the absence of food feeding in mammals are tightly interconnected: sleep depri- in an environment. Sleep suppression during starvation is vation promotes feeding, whereas starvation suppresses a generalizable phenomenon in Drosophilidae; we observed sleep. Here we show that starvation in Drosophila potently it in multiple D. melanogaster laboratory lines and Drosophila suppresses sleep, suggesting that these two homeostati- species when tested with the same assay (Figures S1C and cally regulated behaviors are also integrated in flies. The S1D). sleep-suppressing effect of starvation is independent of Because starvation has been reported to induce hyperac- the mushroom bodies, a previously identified sleep locus tivity [10], we examined whether increased activity during star- in the fly brain, and therefore is regulated by distinct neural vation could account for the observed lack of sleep. We found circuitry. The circadian clock genes Clock (Clk) and cycle that food deprivation both suppresses sleep and induces (cyc) are critical for proper sleep suppression during starva- hyperactivity (Figures S1E and S1F). We also asked whether tion. However, the sleep suppression is independent of light flies compensated for lost sleep during food deprivation with cues and of circadian rhythms as shown by the fact that a homeostatic rebound by increasing their sleep on recovery. starved period mutants sleep like wild-type flies. By selec- Analysis in the DAMS and visual inspection revealed that male tively targeting subpopulations of Clk-expressing neurons, and female flies rebound in the 4 hr after food deprivation (data we localize the observed sleep phenotype to the dorsally not shown). located circadian neurons. These findings show that Clk We speculated that changes in sleep and activity result from and cyc act during starvation to modulate the conflict of the initiation of foraging behavior. A polymorphism in the whether flies sleep or search for food. foraging (for) cGMP-dependent protein kinase (PKG) gene is one determinant of foraging strategy in both larvae and adult rover Results and Discussion Drosophila [11, 12]. We found that for flies enhanced sleep suppression compared to the forsitter strain (Figure S1G), sup- Studies in mammals suggest that the homeostatic regulation porting the notion that flies suppress sleep during starvation in of feeding and sleep involves functionally interconnected order to search for food. mechanisms [1]. This overlap is evident at the clinical, physio- Sleep profiles revealed that male flies sleep relatively nor- logical, cellular, and molecular levels. Longitudinal studies in mally during the initial 10–12 hr of starvation (Figure 1B). To humans reveal a strong correlation between sleep patterns test whether the lag in sleep suppression is due to light cues and body mass index, leptin, and ghrelin levels [2]. In or to the metabolic effects of starvation, we shifted the start mammals, long-term sleep deprivation stimulates appetite time of the 24 hr food deprivation period to ZT12 (end of [3], whereas food deprivation suppresses sleep [4]. Further- lights-on) (Figure 1F). No significant differences in sleep during more, the hypothalamic neuropeptides orexin/hypocretin the initial 12 hr of starvation were apparent between the ZT0 and neuropeptide Y promote both food intake and wakeful- and ZT12 groups and fed control flies. However, sleep was ness [5, 6]. significantly suppressed over hours 12–24 of starvation in Fruit flies exhibit all the behavioral hallmarks of sleep both the ZT0 and ZT12 groups. These results indicate that including prolonged periods of behavioral quiescence, sleep suppression is independent of light cues and more prob- a species-specific postural change, increased arousal ably results from the time of starvation. threshold, and rebound after sleep deprivation [7, 8]. To deter- We repeated our experiments with the recently developed mine whether flies suppress sleep during starvation, we video tracking software pySolo [13]. We assayed male flies in 00 00 00 measured their locomotor activity in the Drosophila activity the same tubes used in DAMS and in 1 3 1 square 3 0.5 monitor system (DAMS) and analyzed sleep as previously deep arenas containing either agar or food. Male flies also described [9]. By using a 3-day protocol, we monitored robustly suppressed their sleep upon starvation in these experiments (Figure 1G). Therefore, the sleep-suppressing effects of starvation can be measured via independent anal- *Correspondence: [email protected] ysis, are generalizable to a second environment, and are not 4These authors contributed equally to this work an artifact of the activity monitoring system. Current Biology Vol 20 No 13 1210

Male Figure 1. Food Deprivation Suppresses Sleep A B 100 Food (A) Paradigm for measuring the effects of starva- Sucrose 80 Agar tion on sleep. Experiments took place over a 3-day period that began 1 day after the loading 60 of ﬂies into standard DAMS tubes. Baseline

% sleep 40 (Day 1): all groups were kept on food. Experiment (Day 2): flies were transferred to fresh food 20 (control, yellow), 150 mM sucrose (red), or agar 0 (food deprivation, blue). Recovery (Day 3): All 0 122436486072Time (hr) LD groups were transferred to fresh food. F/S (B) Sleep profile of male flies reveals that starva- D C Female Light tion (agar group, blue line) suppresses sleep 100 Male Light 80 Dark compared to food- (black line) and sucrose- Dark 80 (red line) fed flies during the experimental day 60 (gray bar). Days on food (1 and 3) are depicted 60 by yellow bars. White/black bars indicate lights ** 40 ** on/lights off. % Sleep % Sleep 40 (C and D) Quantification of day (white bars) and 20 20 night (black bars) sleep over 24 hr reveals ** decreased sleep during the night in male (C) 0 0 and female (D) flies in the agar group compared Day 123 123 123 Day 123 123 123 to food-fed (male, n R 49, for all groups, Food Sucrose Agar Food Sucrose Agar R G p < 0.001; female, n 42, for all groups, E 20 p < 0.001) and sucrose-fed (male, p < 0.001; Food female, p < 0.001) controls. Male flies fed agar Sucrose 60 0 Agar did not differ in daytime sleep compared to baseline (p > 0.146) while the female agar group sup- -20 pressed sleep during the day (p < 0.001). Sucrose % Change % -40 40 is sufficient to support normal sleep in daytime ** ** ** and nighttime (male, p > 0.35; female, p > 0.084). ** -60 (E) Percentage change from baseline sleep Male Female ** % sleep ** reveals that male and female flies on agar 20 suppress sleep compared to the food and ** sucrose groups (p < 0.001, all groups). F 80 Light (F) Starvation starting at ZT0 (n R 26) suppresses Dark sleep during the following night (ZT12-24; 60 ** 0 ** Food Agar Food Agar Sucrose Sucralose p < 0.0001), while flies starved at ZT12 suppress 40 Square Arena DAMS cuvettes sleep during the following day (ZT0-12;

% Sleep p < 0.001). In both cases, no statistical difference 20 is observed for hours 0–12 of starvation (p > 0.171, p > 0.089). 0 Food Suc Agar Food Suc Agar (G) Male flies were tracked in square arenas ZT0 ZT12 (n R 31; left) or tubes (n R 25; right). Daytime and nighttime sleep was significantly suppressed in the agar groups (square arena, p < 0.01; tube, p < 0.01). Sucralose-fed flies in tubes suppressed both daytime and nighttime sleep compared to food- and sucrose-fed controls (p < 0.001). Daytime sleep (ZT0-12) did not differ between the agar and sucralose groups (p > 0.63). Asterisk denotes significant difference (p < 0.01, ANOVA) from control groups. Data are mean 6 SEM. See also Figure S1.

We next asked which neural populations mediate this silencing method and expressed a dominant-negative tem- behavior. The mushroom bodies are involved in regulation of perature-sensitive UAS-ShibireTS1 (ShiTS1) in the mushroom sleep and locomotor activity [9, 14, 15]. They are also a site bodies [19]. Expressing ShiTS1 in mushroom body neurons for the integration of multimodal sensory information and for reversibly blocks their neurotransmission above the restric- the gating of behavioral responses involving olfactory memory tive temperature of 29C[9]. Because flies did not tolerate and complex visually guided behavior [16–18], and thus are 24 hr of starvation at 30C, we developed a more restricted a candidate for a neural locus regulating sleep suppression starvation protocol to allow us to silence neurons during star- during starvation. We chemically ablated the mushroom vation. After 18 hr of baseline activity measurement, flies bodies with hydroxyurea (HU) treatment (Figure 2A) to test were transferred to agar tubes for food deprivation (ZT18) their role in promoting wakefulness upon starvation. Consis- and maintained at 22C through ZT24/0 (Figure 2E). At tent with previous findings [9, 14, 15], HU-treated (+HU) male ZT24/0 the temperature was increased to 30C and sleep and female flies are more active and sleep less than untreated was recorded through ZT12. To determine the effects of control flies (2HU) (Figures 2B and 2C). However, both +HU neural silencing on sleep, daytime sleep (ZT0-12) of male flies and –HU flies slept less when housed with agar than with on agar was compared to controls of the same genotype kept food (Figures 2C and 2D), demonstrating that flies with or on food and to the baseline recording at the permissive without mushroom bodies suppress sleep equally in response temperature (22C). Starved flies expressing UAS-ShiTS1 to starvation. These data suggest that although the mushroom under control of the mushroom body GAL4 drivers OK107, bodies promote sleep, they are not involved in starvation- c747, and H24 all suppressed sleep compared to fed flies induced sleep suppression. of the same genotype (Figure 2F), confirming that the mush- To further test for a role of the mushroom bodies in room bodies are dispensable for suppression of sleep during starvation-induced suppression of sleep, we used a neural starvation. Analysis of the Sleep-Feeding Conflict in Drosophila 1211

Figure 2. Mushroom Bodies Are Dispensable for A B 5000 Food Sleep-Feeding Interactions ** 4000 Agar ** (A) Hydroxyurea ablation of the mushroom 3000 bodies. Anti-FASII staining labels the mushroom ** bodies and central complex of a representative 2000 ** brain from an untreated –HU ﬂy (left). Although

Total activity/24hr Total 1000 the central complex is intact, the mushroom 50 m bodies are not detectable in brains from +HU flies 0 -HU +HU -HU +HU (right). (B) Total 24 hr activity reveals that both 2HU and Male Female C D +HU flies are more active on agar than on food 80 (n R 33 for all groups; male, 2HU, p < 0.001, Male Female Food 0 +HU, p < 0.004; female, 2HU, p < 0.002, +HU, -HU Agar p < 0.001). 2HU flies are also less active than 60 +HU -20 +HU flies (male, p < 0.001; female, p < 0.001). ** (C) Percentage of sleep per 24 hr reveals that 40 -40 ** both 2HU and +HU flies suppress sleep on agar % sleep ** -60 compared to flies on food (male, 2HU, 20 % Change -80 p < 0.001, +HU, p < 0.001; female, 2HU, ** p < 0.001, +HU, p < 0.003). 0 -100 (D) Suppression of sleep calculated as mean -HU +HU -HU +HU percentage change/fly reveals that in both male Male Female and female flies’ sleep suppression during starvation is not significantly affected by HU treat- E F 100 ment (male, p > 0.84; female, p > 0.54). 22 C-Food (E) Schematic of the heat and feeding protocol for 30 C-Food 80 TS1 30 C-Agar silencing the mushroom body with Shi . Flies in 60 the agar group are starved beginning at ZT18, 6 hr prior to temperature shift.

% sleep 40 (F) Flies with silenced mushroom body neurons ** ** suppress sleep on agar at 30C compared to ﬂies 20 ** on food at 30C. Data depict the percentage of ** time spent sleeping from ZT0 to ZT12 (n R 16 0 TS1 WT OK107 c747 H24 for all groups; Shi /+ = p < 0.006; H24 = p < 0.001; OK107 = p < 0.01; c747 = p < 0.01). UAS-ShiTS1 Asterisk denotes signiﬁcant difference (p < 0.01, ANOVA) from control groups. Data are mean 6 SEM.

In an effort to examine the genetic components modulating starvation that is independent of their effect on circadian regu- sleep during starvation, we identified the circadian rhythm- lation. defective cyc0, ClkJrk, and Clkar mutant fly strains that ex- Clk and cyc have previously been implicated in promoting hibited enhanced suppression of sleep when starved (Figures sleep and flies mutant for these genes are short sleepers 3A and 3B). Importantly, whereas the period01 (per01), Pigment [23]. To test whether enhanced suppression of sleep during dispersing factor (Pdf01), ClkJrk, Clkar, and cyc0 mutant fly starvation is a general property of short sleeping flies, we as- strains are all arrhythmic in constant darkness, per and Pdf sayed sleep suppression in the short sleeping mutants fumin mutant flies suppressed their sleep similar to wild-type flies, (fmn) and shakerminisleep (Shmns). We were unable to test sleep- suggesting that the sleep phenotypes observed in Clk and less mutant flies because of lethality during the 24 hr starva- cyc mutants can be differentiated from defects in circadian tion protocol. Whereas Clk and cyc mutants enhance sleep behavior (Figures S2A and S2B). We were unable to test time- suppression during starvation, both fmn and Shmns sup- less01 mutants because of high levels of lethality during the pressed sleep similar to wild-type controls (Figure 3C), sug- 24 hr starvation protocol. To confirm that the enhanced sleep gesting that the enhanced sleep suppression observed in Clk suppression observed in Clk and cyc mutants is not due to and cyc mutants is not due to their short-sleep phenotype. hyperactivity, we analyzed waking activity in ClkJrk, Clkar, We next sought to localize Clk and cyc function in starvation- and cyc0 mutants and found that all mutants tested have induced sleep suppression. For this, we used a previously waking activity that is comparable or less than wild-type flies described dominant-negative Clk transgene (ClkDN) lacking in both fed and starved states (Table S1). In agreement with its basic DNA-binding domain but retaining its protein interac- previous reports of circadian phenotypes, we found the sleep tion domains [24]. Disrupting Clk function in peripheral tissues, phenotype of the ClkJrk allele to be dominant while Clkar and sensory neurons, and the glucagon-like AKH neurons had no cyc0 phenotypes were recessive [20, 21](Figures 3A and 3B). effect on response to starvation (Figures S2C and S2D), sug- Because Clk and cyc encode transcription factors that hetero- gesting that Clk function in promoting sleep during starvation dimerize to activate the circadian genes per and tim [22], is not in peripheral sensory neurons. we tested Clkar/+,cyc0/+ transheterozygous flies for sleep Within the fly brain, Clk is expressed in circadian oscillator suppression. We found that Clkar/+,cyc0/+ flies suppressed cells, some of which are the small and large ventrolateral sleep to a greater degree than did Clkar/+ and cyc0/+, suggest- neurons (LNvs) that express the neuropeptide Pigment ing that these genes functionally interact to promote sleep dispersing factor (Pdf). Clk is also expressed in dorsally during starvation (Figures 3A and 3B). These data are consis- located circadian neurons and peripheral cells that include tent with a novel role for Clk and cyc in promoting sleep during populations of dorsal neurons (DNs) and dorsally located Current Biology Vol 20 No 13 1212

ABFigure 3. Mutations in Clk and cyc Enhance 80 0 Sleep Suppression during Starvation Food (A) Male flies with mutations in Clk and cyc were Agar -20 60 tested for sleep in tubes containing food or ** agar. In all groups tested, flies slept less on ** -40 agar than on food (n R 41; p < 0.001 for all 40 groups). -60 % sleep ** % change (B) Calculating percentage change in sleep ar Jrk 0 ** reveals that Clk , Clk , and cyc flies signifi- 20 -80 cantly enhanced sleep suppression during star- ** ** ** ** ** vation compared to wild-type controls ** -100 0 r ar k 0 a (p < 0.001 for all groups). Sleep suppression in ar jrk 0 ar /+ /+ Jr 0 /+ 0 /+ 0 /+ /+ /+ 0 /+ 0 /+ 0 /+ WT ar Jrk WT ar Jrk Clk cyc Jrk Jrk Clk Clk cyc Clk /Clk /Clk Clk Clk cyc Jrk flies heterozygous for Clk (Clk /+) does not Clk Clk cyc Jrk /+, cyc ar /+,cyc /+, cyc ar /+,cyc Jrk Clk Jrk Jrk Clk differ from Clk homozygous flies (p > 0.38) Clk k Clk Clk Cl and is greater than wild-type controls (p < 0.001). Clkar/+,cyc0/+ heterozygous flies C C D Pdf-GAL4 0 enhance sleep suppression compared to wild- type and Clkar/+ and cyc0/+ heterozygous flies -10 (p < 0.01 for all groups). (C) The short-sleep mutants fmn and Shmns -20 suppress sleep comparably to wild-type flies (p > 0.75). -30 cry-GAL4

% change (D) Whole-brain confocal images of Pdf-GAL4 and cry-Gal4 driving UAS-mCD8:GFP reveals -40 the expression pattern of each driver. -50 (E) Male flies expressing UAS-ClkDN with tim- WT fmn Shmns GAL4 and cry-GAL4 show enhanced sleep suppression when starved. All groups tested significantly suppress sleep on agar compared EF0 to food (n R 28; p < 0.001 for both groups). 80 Food (F) Analyzing data in (E) as percentage of sleep Agar -20 suppression between flies on food or agar 60 reveals that tim-GAL4;Pdf-GAL80;UAS-ClkDN -40 ** ** flies suppress sleep significantly more than ** ** 40 ** wild-type flies and those harboring UAS-ClkDN

% sleep * -60

% change or GAL4 transgenes alone (p < 0.001 for all

20 ** ** groups). Asterisks denote signiﬁcant difference **** ** -80 ** (p < 0.01, ANOVA) from control groups. ** Data are mean 6 SEM. See also Figure S2 and 0 -100 WT ClkDN tim Pdf tim Pdf tim; cry tim; Table S1. Pdf-GAL80 cry-GAL80 WT ClkDN tim Pdf tim Pdf tim; cry tim; Pdf-GAL80 cry-GAL80 UAS-ClkDN UAS-ClkDN

LNs (LNds) [25]. Specifically disrupting Clk function in the LNvs in all tim cells except those expressing cry (tim-GAL4,cry- abolishes circadian rhythm [24], suggesting that the LNvs are GAL80) [27] does not enhance sleep suppression. Taken critical for pacemaker function. We first ectopically expressed together, these results suggest that a population of dorsally a dominant-negative Clk (ClkDN) isoform in either all circadian located Clk-expressing neurons promote sleep during starva- neurons or selectively in subpopulations of Clk-expressing tion. Therefore, we have identified a novel mechanism through cells [24]. Disrupting Clk function in all circadian neurons which circadian and feeding systems modulate sleep. with tim-GAL4 (tim-GAL4; UAS-ClkDN) enhanced sleep sup- To support this anatomical localization of Clk function, we pression during starvation compared to flies harboring either selectively silenced or activated different populations of Clk- tim-GAL4 or UAS-ClkDN single transgenes alone (Figures 3E expressing neurons in adulthood. Expressing UAS-ShiTS1 in and 3F). all clock cells with tim-GAL4 or Clk-GAL4 enhanced sleep To further refine the population of tim-expressing neurons suppression, whereas selectively silencing LNvs via Pdf- regulating sleep during starvation, we used GAL4 and GAL80 GAL4 did not affect sleep during starvation (Figures 4A–4C), in combinations to express ClkDN in subpopulations of Clk fortifying the conclusion that a population of Clk-expressing neurons. Flies with disrupted Clk function in all tim-expressing Pdf-negative neurons promote sleep during starvation. cells except PDF neurons (tim-GAL4, Pdf-GAL80;UAS-ClkDN) We therefore reasoned that activation of these neurons showed enhanced sleep suppression, suggesting that the would cause starved flies to sleep as if they were fed. We primary pacemaker neurons do not modulate sleep during ectopically expressed the high heat-sensitive cation channel food deprivation. Fortifying this conclusion, we found that dTrpA1 to activate Clk-expressing neurons with regional and expressing ClkDN only in LNvs (Pdf-Gal4; UAS-ClkDN) did temporal specificity [28]. Activation of all Clk neurons caused not affect suppression of sleep (Figures 3E and 3F). The cry- lethality in both fed and starved flies at 28C so we therefore GAL4 driver labels the large and small LNvs, the DN1 subpop- used the more restricted cry-GAL4 driver. Activation of cry- ulation of DNs, and the LNds ([26] and Figure 3D). cry-GA- GAL4-expressing neurons during starvation abolished sleep L4;UAS-ClkDN flies enhanced sleep suppression, suggesting suppression (Figures 4D–4F), confirming a role for these that either a single PDF-negative small-LNvs and/or the neurons in promoting sleep during starvation. Activating LNds and/or the DN1s, which are all cry+, Pdf2, promote sleep LNvs alone (Pdf-GAL4;UAS-dTrpA1) during starvation did during starvation. Supporting this notion, expression of ClkDN not affect sleep suppression compared to wild-type controls. Analysis of the Sleep-Feeding Conflict in Drosophila 1213

Food-30 C AB C 40 Agar-30 C 80 Food-22 C 20 Food-30 C Food 60 Agar-30 C 0 Agar ** 40 -20 % change % sleep ** 20 ** -40

** ** 0 -60 WT Pdf-GAL4 tim-GAL4 Clk-GAL4 WT Pdf-GAL4 tim-GAL4 Clk-GAL4 LD ZT 0 12 24/0 UAS-ShiTS1 UAS-ShiTS1

DE F 80 Food-28 C Food-22 C 40 Agar-28 C Food Food-28 C 60 ** 20 Agar-28 C Agar ** 0 40 **

% sleep -20 % change 20 -40 **

LD 0 -60 ** ZT 0 12 24/0 WT Pdf-GAL4 cry-GAL4 WT Pdf-GAL4 cry-GAL4 UAS-dTrpA1 UAS-dTrpA1

Figure 4. Clk/cyc Expressing Neurons Acutely Regulate Sleep during Starvation (A) Schematic of the restricted temperature shift and feeding protocol for ShiTS1 manipulations. Flies on agar were food deprived for 12 hr during the temperature shift (n R 22; ZT0-12). (B) Blocking transmission from tim or Clk-GAL4 neurons suppressed sleep in starved flies. All flies harbor UAS-ShiTS1. Control (UAS-ShiTS1/+) and Pdf-GA- L4;UAS-ShiTS1 flies do not suppress sleep at 30C in the restricted starvation protocol (p > 0.11; p > 0.79), whereas tim-GAL4;UAS-ShiTS1 and Clk-GA- L4;UAS-ShiTS1 flies sleep significantly less on agar than on food (p < 0.001; p < 0.001). (C) Analyzing data as percentage change from fed flies at 22C reveals that starved UAS-ShiTS1/+ and Pdf-GAL4;UAS-ShiTS1 did not differ from fed flies (p > 0.62) whereas tim-GAL4;UAS-ShiTS1 and Clk-GAL4;UAS-ShiTS1 suppress sleep when starved (p > 0.001). (D) Schematic of the temperature shift and feeding protocol for dTrpA1 manipulations. Flies on agar were food deprived for 18 hr beginning at ZT18, 6 hr prior to the temperature shift (n R 23; ZT0-12). (E) Acute excitation of cry-GAL4-expressing neurons blocks the effects of starvation on sleep. All flies harbor UAS-dTrpA1. Control flies (UAS-dTrpA1/+) and Pdf-GAL4;UAS -dTrpA1 flies suppress sleep at 28C (p < 0.003; p < 0.007), whereas cry-GAL4;UAS-dTrpA1 flies do not suppress sleep during starvation (p > 0.71). (F) Analyzing data as percentage change from fed flies at 22C reveals that starved UAS-TrpA1/+ and Pdf-GAL4;UAS-TrpA1 suppress sleep compared to fed controls at 30C (p < 0.001), whereas starved cry-GAL4;UAS-TrpA1 do not differ from fed controls (p > 0.56). Asterisks denote significant difference (p < 0.01, ANOVA) from control groups. Data are mean 6 SEM.

Therefore, neurotransmission from cells other than the central these genes and the role of the PI may advance our under- pacemaker neurons act acutely during adulthood to promote standing of genes selectively modulating sleep during food wakefulness during starvation. deprivation. In addition to modulating circadian rhythms, core circadian genes and neurons have been implicated in numerous behav- Experimental Procedures iors including cocaine sensitivity, feeding, courtship, and memory [29–33]. We find the DN1s or LNds that express circa- Food Deprivation Experiments dian genes promote sleep during starvation. These neurons For food deprivation experiments, 2- to 4-day-old males or mated females are functionally distinct from the LNvs that control behavioral (unless otherwise stated) were loaded into tubes containing standard brown rhythms during constant darkness. These findings are consis- for acclimation. After 1 day of acclimation in DAMS tubes with standard fly food, baseline sleep was measured for 24 hr. Flies were then transferred at tent with the existence of a neural mechanism mediating a ZT0 (start of lights on, Day 2) to a tube containing either standard fly food (ad behavioral conflict that determines whether a fly sleeps or libitum control) or agar supplemented with 150 mM sucrose, 1 mM sucra- seeks food. lose, or 1% agar, alone for 24 hr. Flies were then transferred back to food- We find that for kinase activity is positively correlated with containing vials, and activity was recorded for an additional 24 hr recovery sleep suppression [34], suggesting that for may counteract period (Day 3). Tubes were maintained in a 25C incubator with 12:12 LD Clk and cyc function. In addition to for, feeding-related genes cycles. All data presented result from at least two independent experiments. such as neuropeptide F (NPF) and metabolism-related genes including Drosophila insulin-like peptides (DILPs), take out, Sleep Analysis and Drosophila p70/S6 Kinase have been implicated in control For all experiments except those with pySolo, sleep was analyzed with the Excel-based ‘‘Sleep Counting Macro’’ [9] generously supplied by R. Allada of feeding [35–37]. DILPs are expressed in the pars intercere- (Northwestern University). For video monitoring experiments, we used py- bralis (PI), a brain region previously implicated in sleep regula- Solo analysis suite generously supplied by G. Gilestro and C. Chirelli tion and starvation response [38, 39]. Future studies examining (University of Wisconsin, Madison, WI). The within-fly percentage change Current Biology Vol 20 No 13 1214

in sleep was calculated as ((% sleep starved 2 % sleep baseline)/% sleep 13. Gilestro, G.F., and Cirelli, C. (2009). pySolo: A complete suite for sleep baseline) 3 100. analysis in Drosophila. Bioinformatics 25, 1466–1467. 14. Joiner, W.J., Crocker, A., White, B.H., and Sehgal, A. (2006). Sleep in ShibireTS1 and dTrpA1 Experiments Drosophila is regulated by adult mushroom bodies. Nature 441, Flies were prepared as described for standard food deprivation experi- 757–760. ments, except that flies were kept at 22C for acclimation and for 24 hr of 15. Martin, J.R., Ernst, R., and Heisenberg, M. (1998). Mushroom bodies baseline recordings. For UAS-ShiTS1 experiments, flies were then trans- suppress locomotor activity in Drosophila melanogaster. Learn. Mem. ferred at ZT0 to tubes containing either food (fed group) or 1% agar (control 5, 179–191. group) and sleep was recorded for 12 hr with the incubator temperature at 16. Krashes, M.J., DasGupta, S., Vreede, A., White, B., Armstrong, J.D., and 30C. For dTrpA1 experiments, flies were transferred to agar at ZT18 and Waddell, S. (2009). A neural circuit mechanism integrating motivational maintained at 22C. The following day, the temperature was increased to state with memory expression in Drosophila. Cell 139, 416–427. 28 C at ZT0 and the experiment proceeded through ZT12. Only the light 17. Liu, L., Wolf, R., Ernst, R., and Heisenberg, M. (1999). Context general- phase (ZT0-12) was analyzed in both protocols. ization in Drosophila visual learning requires the mushroom bodies. Nature 400, 753–756. Supplemental Information 18. Zhang, K., Guo, J.Z., Peng, Y., Xi, W., and Guo, A. (2007). Dopamine- mushroom body circuit regulates saliency-based decision-making in Supplemental Information includes Supplemental Experimental Proce- Drosophila. Science 316, 1901–1904. dures, two figures, and one table and can be found with this article online 19. Kitamoto, T. (2001). Conditional modification of behavior in Drosophila at doi:10.1016/j.cub.2010.05.029. by targeted expression of a temperature-sensitive shibire allele in defined neurons. J. Neurobiol. 47, 81–92. Acknowledgments 20. Allada, R., Kadener, S., Nandakumar, N., and Rosbash, M. (2003). A recessive mutant of Drosophila Clock reveals a role in circadian This work was funded by an NIGM NRSA 5F32GM086207 to A.C.K.; NIH rhythm amplitude. EMBO J. 22, 3367–3375. grant 5R01GM063911 to J.B.; NIH grant 5R01MH081982 to S.W.; Alfred P. 21. Rutila, J.E., Suri, V., Le, M., So, W.V., Rosbash, M., and Hall, J.C. (1998). Sloan, Whitehall foundation, and Whitehead President Award to G.S.B.S.; CYCLE is a second bHLH-PAS clock protein essential for circadian and Hilda and Preston Davis Foundation Postdoctoral Fellowship to M.D. rhythmicity and transcription of Drosophila period and timeless. Cell The authors are grateful to Giorgio Gilestro (U. Wisconsin) and Jena Pitman 93, 805–814. 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