Molecular Analysis of Sleep:Wake Cycles in Drosophila

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1 Molecular Analysis of Sleep:Wake Cycles in Drosophila A. SEHGAL,* W. JOINER,* A. CROCKER,* K. KOH,* S. SATHYANARAYANAN, Y. FANG,* M. WU,* J.A. WILLIAMS, AND X. ZHENG* *Howard Hughes Medical Institute, Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104; Molecular Oncology, Merck Research Laboratories, Boston, Massachusetts 02115; CABM, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey Sleep is controlled by two major regulatory systems: a circadian system that drives it with a 24-hour periodicity and a homeostatic system that ensures that adequate amounts of sleep are obtained. We are using the fruit fly Drosophila melanogaster to understand both types of regulation. With respect to circadian control, we have identified molecular mechanisms that are critical for the generation of a clock. Our recent efforts have focused on the analysis of posttranslational mechanisms, specifically the action of different phosphatases that control the phosphorylation and thereby the stability and/or nuclear localization of circadian clock proteins period (PER) and timeless (TIM). Resetting the clock in response to light is also mediated through posttranslational events that target TIM for degradation by the proteasome pathway; a recently identified ubiquitin ligase, jet lag (JET), is required for this response. Our understanding of the homeostatic control of sleep is in its early stages. We have found that mushroom bodies, which are a site of synaptic plasticity in the fly brain, are important for the regulation of sleep. In addition, through analysis of genes expressed under different behavioral states, we have identified some that are up-regulated during sleep deprivation. Thus, the Drosophila model allows the use of cellular and molecular approaches that should ultimately lead to a better understanding of sleep biology. INTRODUCTION To adapt to the cyclic environment in which they live, organisms have evolved endogenous timekeeping mechanisms that synchronize their behavior and physiology with the environmental 24-hour cycle. Of the many behaviors that occur with a circadian (or ~24 hour) periodicity, the best known is perhaps the sleep/wake cycle. Sleep-like states are displayed in many different species, but the regulation and function of such a state are poorly understood. Although the internal circadian clock controls the 24-hour rhythmicity of sleep, it is not required for the manifestation of sleep. The latter is controlled by a homeostatic system that drives the need to sleep based on prior wakefulness. Simply put, the circadian system is largely responsible for controlling the timing of sleep, whereas the homeostatic system controls the amount of sleep. We are using a simple model system, the fruit fly, Drosophila melanogaster, to understand the molecular basis of both these systems. Molecular Basis of the Clock In all organisms examined, the basic clock mechanism consists of one or more feedback loops in which cycling clock proteins rhythmically control the expression of their own mrnas, thereby maintaining cycles of gene expression (Sehgal 2004). In Drosophila, the major cycling components of the principal loop are the period and timeless proteins (PER and TIM) (see Fig. 1) (Hardin 2005). The per and tim genes are actively transcribed by the transcriptional factors, Clock (CLK) and cycle (CYC), during the day, which leads to peak levels of the respective mrnas in the early evening. At that point, the PER and TIM proteins start CIRCADIAN RHYTHMS IN DROSOPHILA Over the years, Drosophila has proved to be an extremely powerful model for the study of circadian rhythms (Hardin 2005). Currently, the assay of choice for measuring rhythms in Drosophila quantitates locomotor activity over time using an automated beam-break system. Flies are diurnal, and so most activity occurs during the day, with extended periods of rest (sleep) at night. The robust, approximately 24-hour rhythm observed in these behavioral assays provides an effective measurement for studying the genetic basis of the clock underlying this pattern of activity. Figure 1. Model for the Drosophila circadian clock. The model depicts the classical transcription-translation feedback loop for per and tim and a posttranslational loop through which rhythmic phosphorylation of PER and TIM may be maintained. For the sake of simplicity, the transcription loop that regulates expression of Clk is not shown. Cold Spring Harbor Symposia on Quantitative Biology, Volume LXXII Cold Spring Harbor Laboratory Press

2 558 SEHGAL ET AL. to accumulate and, at about the middle of the night, translocate into the nucleus to inhibit the activity of CLK and CYC. TIM is degraded in the late night/early morning, and PER a few hours later, which relieves the repression on CLK/CYC and leads to the start of a new cycle (Hardin 2005). It is clear that in order to generate a 24-hour rhythm from such a loop, multiple regulatory steps have to be built in. One such regulatory mechanism is phosphorylation; both PER and TIM are hyperphosphorylated at specific times of day and some of the kinases responsible for this phosphorylation have been identified (Fig. 1). PER is phosphorylated by casein kinase 1ε (CK1ε) and by casein kinase 2 and TIM is phosphorylated by glycogen synthase kinase 3β (GSK3β) (Kloss et al. 1998; Martinek et al. 2001; Lin et al. 2002; Akten et al. 2003). The different phosphorylation events regulate critical aspects of the feedback loop such as stability and nuclear expression of PER/TIM, indicating the importance of posttranslational control in the clock. In fact, transcriptional rhythms of per and tim may be dispensable to some extent. A few years ago, we generated flies in which levels of per and tim mrnas were held constant and found that the two proteins continued to cycle and drive behavioral rhythms in many of these flies (Yang and Sehgal 2001). This does not imply that rhythmic transcription does not serve a purpose. It (1) probably strengthens the amplitude and promotes the penetrance of the rhythm (a smaller percentage of the flies with noncycling per and tim mrnas showed behavioral rhythms), (2) confers temporal precision on to the rhythm (although the average period of these flies was ~24 hours, there was some variability from fly to fly), and (3) may regulate the differential response of the clock to light at different times of day (flies expressing noncycling per and tim mrnas showed aberrant responses to pulses of light delivered at different times during the night) (Yang and Sehgal 2001). Given that it is possible, however, to generate at least a partially functional clock without rhythmic expression of per and tim mrnas, the question arises as to how this is achieved. The cycling of the proteins under these conditions is most likely driven by posttranslational events such as phosphorylation. However, expression of the regulatory kinases mentioned above is not known to cycle. This led us to investigate a role of phosphatases in controlling the rhythmic phosphorylation states of PER and TIM. We found that both proteins, but predominantly PER, are dephosphorylated by protein phosphatase 2A (PP2A) (Fig. 1) (Sathyanarayanan et al. 2004). This dephosphorylation increases the stability of PER and also promotes its nuclear localization. Overexpression of wild-type or dominant-negative versions of the PP2A catalytic subunit in wild-type flies results in loss of rhythms accompanied by either constitutively elevated or greatly reduced PER expression, respectively. In addition, circadian periodicity is altered by changes in the levels of specific regulatory subunits of PP2A. Thus, overexpression of the widerborst (wdb) subunit produces a long period, and loss or gain of function of the twins (tws) subunit also results in circadian phenotypes (Sathyanarayanan et al. 2004; and data not shown). Note that we previously reported that overexpression of tws shortens circadian period, but we have since found that only the overexpression of the PP2A catalytic subunit produces a short period. Overexpression of tws results in a long period as does overexpression of the other regulatory subunit, wdb. Importantly, mrna levels of both regulatory subunits are expressed rhythmically, and the tws mrna cycles with a high amplitude. This cycling is eliminated in cyc 0 mutants, indicating that cycling of the phosphatase subunits constitutes another loop in the central clock, and one which controls the cycling of PER and TIM at a posttranslational level. Note that because the cycling of tws occurs at the level of the mrna, this loop may also require rhythmic transcription, although not of per and tim. Yet another transcription-based loop, which controls the expression of Clk, was described previously (Glossop et al. 1999, 2003; Cyran et al. 2003). We have recently found that protein phosphatase 1 (PP1) is also important for clock function. PP1 dephosphorylates PER and TIM and increases their stability (Fig. 1) (Fang et al. 2007). In this case, the primary target appears to be TIM, which also affects PP1 activity on PER. Thus, in cultured cells, in the absence of TIM, PER is destabilized by mild inhibition of PP1, presumably due to its increased phosphorylation. The presence of TIM stabilizes PER and renders it more resistant to inhibition of PP1. The effect is specific for PP1 because TIM does not affect the dephosphorylation of PER by PP2A (Fang et al. 2007). The stabilizing effect of TIM most likely involves a change in the phosphorylation state of PER: It may down-regulate kinase activity on PER or up-regulate phosphatase activity. A similar situation occurs in flies where PER depends on TIM for stability, but the mechanisms involved are not known (Price et al. 1995). Entrainment of the Clock to Light Entrainment of the Drosophila clock to light is achieved by light-induced degradation of TIM (Hunter- Ensor et al. 1996; Myers et al. 1996; Zeng et al. 1996). This effect of light fits well with the model which postulates that levels of PER and TIM constitute time-of-day signals; it follows then that anything that changes the time of the clock would do so by changing the levels of these molecules. Light is transmitted to TIM by the circadian photoreceptor, cryptochrome (CRY) (Fig. 2), which is Figure 2. Schematic of the Drosophila light response. In response to light, the circadian photoreceptor, CRY, transmits a signal to TIM that results in the degradation of TIM through the ubiquitinproteasome pathway and the action of a specific E3 ligase, JET. CRY is itself also degraded by light, but with a slower time course than TIM. The visual system (not shown here) can also entrain the clock to light:dark cycles, but the response to pulses of light and to constant light appears to require this pathway.

3 SLEEP:WAKE CYCLES IN DROSOPHILA 559 Figure 3. JET is required for the TIM response to light. (A) Activity records of jet flies in constant light. Wild-type flies (see representative example on the left) lose circadian rhythms in constant darkness, but jet mutants (representative example on right) retain rhythms under such conditions. (B) A cell culture assay for the circadian light response. TIM was transfected into Drosophila cultured cells along with other genes as indicated. When CRY and JET are coexpressed, TIM is degraded in response to light. coexpressed with clock proteins in the central brain (Stanewsky et al. 1998; Emery et al. 2000). CRY is itself degraded in response to light, which may serve to terminate the light response (Lin et al. 2001). Both CRY and TIM are degraded by the proteasome (Fig. 2) (Naidoo et al. 1999; Lin et al. 2001), but until recently, other molecular components of this response were not known. We recently identified one such component through the analysis of a mutation found in one of our fly stocks (Koh et al. 2006a). This particular stock showed reduced sensitivity to light such that it was rhythmic in the presence of constant light (wild-type flies are arrhythmic under these conditions) (Fig. 3A), and it took longer to adjust to a change in the light:dark schedule. This latter phenotype, which is analogous to extended jet lag, led us to term this mutant jet lag (jet). In addition to behavioral deficits, jet mutants were also aberrant in the molecular response to light, i.e., degradation of TIM by light was reduced in these flies. Cloning of the affected gene revealed that it encodes an E3 ligase, a molecule that typically targets specific substrates to the proteasome. In fact, JET turned out to be the E3 ubiquitin ligase that mediates the response of TIM to light. Using JET, we were able to reconstitute the TIM response to light in cultured cells. We transfected Drosophila S2 cells with TIM along with CRY and/or JET and found that upon exposure to light, TIM was degraded only if both CRY and JET were present (Fig. 3B) (Koh et al. 2006a). Mutant forms of JET, corresponding to the proteins in the jet mutant flies, were deficient in the cell culture assays mentioned above. In the course of cloning jet, we found that multiple stocks in the laboratory carried a mutation in this gene. Mutations in jet may have been selected for because they facilitate adaptation to laboratory conditions, i.e., they allow flies to retain rhythms despite the irregular light conditions usually found in laboratories. has not been demonstrated for Drosophila, our recent findings suggest that the Drosophila clock may be sensitive to metabolic activity. More specifically, we have found that an increase in oxidative stress compromises clock function. These findings were made with flies mutant for the metabolic gene foxo (Zheng et al. 2007). The FOXO protein is best known for its regulation by the insulin signaling pathway; it is a transcription factor that is excluded from the nucleus in response to insulin action (Neufeld 2003). When in the nucleus, FOXO controls the transcription of many genes, including those that have antioxidant activity (Kops et al. 2002). Thus, foxo mutants have increased oxidative stress. We found that the cycling of clock genes in peripheral clocks (clocks in nonbrain tissues) is dampened in foxo mutants (Zheng et al. 2007). Rest:activity rhythms are normal, indicating that central clock function is intact (Fig. 4). However, in response to low concentrations of paraquat (which further increases oxidative stress), the amplitude of the molecular oscillation in the central clock cells is also reduced, and rest:activity rhythms are rapidly abolished (Fig. 4). In contrast, wild-type flies retain rhythms in the presence of paraquat for up to 3 weeks (Koh et al. 2006b). These effects of FOXO occur in the fat Other Extrinsic Factors That Affect the Clock In addition to light, the clock can entrain to many other environmental factors such as temperature and social cues (Levine et al. 2002; Kaushik et al. 2007). In addition, metabolic activity may entrain the clock. Thus, mammals can be entrained to a restricted feeding paradigm (Damiola et al. 2000; Stokkan et al. 2001). Although this Figure 4. foxo mutants lose behavioral rhythms in the presence of paraquat. Activity rhythms were monitored for wild-type flies and foxo mutants in the presence or absence of 1 mm paraquat. Although wild-type flies are rhythmic under both conditions, foxo mutants lose rhythms when paraquat is added.

4 560 SEHGAL ET AL. body, which is the fly equivalent of the liver and adipose tissue. Given that rest:activity rhythms are driven by the central clock in the brain, these data demonstrate that a peripheral metabolic tissue can affect clock function in the brain. Because aging is typically associated with an increase in oxidative stress, and we have found that circadian rhythms also break down with age (Koh et al. 2006b), we also examined the effects of age on foxo mutants. As one might expect, they show a premature breakdown of rhythms (Zheng et al. 2007). SLEEP IN DROSOPHILA The basic mechanisms, as well as most of the molecules, underlying circadian rhythms in Drosophila are conserved in mammals and have even been implicated in human circadian disorders (Toh et al. 2001; Xu et al. 2005). The success of the fly system in elucidating the mechanisms of circadian biology prompted us to ask whether the fly could also be a model for sleep. As noted above, the timing of sleep is controlled by the circadian system, but the need for sleep, which in turn determines the amount of sleep, is controlled by a homeostatic system. The nature of this homeostatic control is poorly understood, as is the function(s) served by sleep. There is the expectation, however, that an understanding of the molecular mechanisms underlying sleep and sleep homeostasis may reveal the function of sleep (Hendricks et al. 2000a), hence, the recent efforts to study sleep in organisms that lend themselves to genetic analysis. To develop a Drosophila model for sleep, we sought to determine if criteria that have been proposed for sleep over the years are met by the rest phase in flies. Because electrophysiology experiments are difficult to conduct in the fly, we focused our efforts on behavioral characteristics of sleep. As described earlier, our circadian measurements had indicated the presence of well-consolidated daily rest periods. Thus, we knew that fly rest was a reversible state of behavioral quiescence regulated by the circadian clock. We also found that, like sleep, fly rest consists of long periods of behavioral immobility during which the arousal threshold (i.e., the minimum stimulus needed to invoke a response) is increased. More importantly, fly rest is controlled by homeostatic mechanisms which ensure that adequate amounts of rest are obtained. Thus, if flies are deprived of rest at night, they will make up for it by resting in the morning, a time at which they are normally active. These studies, and similar ones conducted simultaneously by another group, led to the fly rest state being established as a model for sleep (Hendricks et al. 2000b; Shaw et al. 2000). Interestingly, fly sleep can be pharmacologically manipulated by the same neurochemicals that affect mammalian sleep (Hendricks et al. 2000b; Shaw et al. 2000). In addition, since the original model was developed, electrophysiological correlates of the fly sleep state have been described (Nitz et al. 2002). We recently discovered another aspect of fly rest that is shared with mammalian sleep they both get fragmented with age. As flies get older, they show increased daytime sleep, decreased nighttime sleep, and decreased duration of sleep bouts (Koh et al. 2006b). Thus, in general, they have trouble maintaining a consolidated sleep state. Similar problems are known to occur in elderly humans, but the mechanisms are not wellunderstood (Pandi-Perumal et al. 2002). Our fly studies suggest that although circadian regulation may be disrupted with age, this is probably not the sole cause of the sleep disturbances in old flies. Young tim 01 mutants have more fragmented sleep than their wild-type counterparts, but their sleep patterns also get worse with age (Koh et al. 2006b). Because tim 01 flies have no clock to begin with, their age-induced defects must occur elsewhere, most likely in the homeostatic system. The fly model for sleep allows us to use genetic approaches to ask fundamental questions about sleep. Perhaps the most important questions in basic sleep biology concern the cellular location of the sleep homeostat, and the molecules that comprise the homeostat. Work in our laboratory is directed toward addressing both of these questions. Mapping Sleep-regulating Loci in the Fly Brain Shortly after developing a fly model for sleep, we began to test candidate molecules for sleep-promoting or sleep-inhibiting effects. The first pathway we studied in this context was the camp/pka (protein kinase A)/CREB (camp-response element-binding protein) pathway because we were interested in a possible connection between sleep and the consolidation of memory, and this pathway is implicated in learning and memory in all organisms studied (Mayford and Kandel 1999). We found that mutants of this pathway did indeed have effects on sleep, such that up-regulation of this pathway resulted in reduced sleep, whereas down-regulation was associated with increased sleep (Hendricks et al. 2001). The most dramatic phenotype was observed in flies that expressed a constitutively active form of protein kinase A (PKA). These flies had greatly reduced sleep. To identify regions of the fly brain that are important for the regulation of sleep, we made use of the constitutively active PKA molecule (Joiner et al. 2006). We first expressed it under control of an inducible panneural promoter and verified that induction of PKA in adult flies is sufficient to reduce sleep. We then drove its expression with promoters that are expressed in different parts of the fly brain and assayed the effects on sleep. We found that the most dramatic sleep phenotypes were obtained when PKA was expressed in the mushroom bodies (MBs), which are a site of synaptic plasticity in the fly brain. The MBs are made up of fiber tracts that are organized into different lobes. Based on the region of the MB targeted, PKA expression either increased or decreased sleep (Fig. 5). We concluded that MBs contain two types of sleepregulating neurons: those that promote sleep when PKA is increased and those that inhibit sleep under such conditions (Joiner et al. 2006). To ascertain whether PKA acts in adult MB neurons to regulate sleep, we also expressed it under control of a drug-inducible MB promoter. This driver is expressed in the same pattern as the sleep-inhibiting promoter and, as expected, its induction in adult flies carrying the upstream

5 SLEEP:WAKE CYCLES IN DROSOPHILA 561 Figure 5. Mushroom bodies (MBs) are a sleep-regulating structure in the fly brain. (A) Location of the MBs in the fly brain. The image depicts GFP expression driven by a panneuronal driver (elav-gene Switch). The MBs are encircled in white. The structures immediately below them are the antennal lobes. The punctate staining on the left and right corresponds to the optic lobes. As noted in the text, expression of PKA* with this driver inhibits sleep in a manner similar to that shown in the left profile in B. (B) Expressionofaconstitutively active PKA molecule (PKA*) is some areas of the MBs inhibits sleep (see sleep profile on the left), and in other areas it promotes sleep (profile on right). (Blue trace) Flies expressing PKA*; (green trace) controls. activation sequence (UAS)-PKA transgenic resulted in reduced sleep. Analysis of the sleep architecture in flies expressing PKA under control of the inducible MB promoter revealed specific defects in the homeostatic mechanisms. Despite the reduction in sleep, the number of sleep bouts did not increase to compensate, suggesting a deficit in the expression of the homeostat (Joiner et al. 2006). However, the homeostatic accrual of a sleep-promoting signal was intact in these flies, based on the compensatory sleep observed when the drug was removed (Joiner et al. 2006). We also used the MB promoter to probe the mechanisms perturbed by PKA action. To determine if PKA affects neuronal excitability, we directly manipulated electrical activity using the inducible promoter. To decrease firing, we employed a hyperpolarizing potassium channel, and to increase firing, we used a depolarizing sodium channel. Inducible expression of the former led to increased sleep, whereas the latter resulted in decreased sleep, mimicking the PKA phenotype. Thus, PKA most likely increases neuronal output in the sleepinhibiting neurons of the MBs (Joiner et al. 2006). Two other lines of evidence supported a role for the MBs in regulating sleep. (1) Ablation of the MBs decreased sleep (Joiner et al. 2006). It should be noted, however, that the decrease in sleep was smaller than that seen with other manipulations of the MBs, supporting the idea that MBs contain neurons with opposing effects on sleep. (2) We found that the effects of a serotonin receptor on sleep are mediated in MBs (Yuan et al. 2006). We proposed a model for how MBs regulate sleep taking into account these opposing influences and the effect of MB ablation. Our model postulates that the activity of sleeppromoting and sleep-inhibiting neurons in the MBs is Figure 6. Model for control of sleep by the MBs. As noted in the text, we found that the MBs contain sleep-promoting and sleepinhibiting neurons. Activity of these is most likely integrated to produce the overt behavioral state. In the default state (in the absence MBs), there is increased wakefulness. We propose that activity of the sleep-inhibiting neurons further increases wakefulness. The sleep-promoting neurons most likely exert an inhibitory influence on the default state and promote sleep. (Adapted from Joiner et al ) integrated to produce the overt behavioral state (Fig. 6). The default mode of the hypothetical integrator promotes wakefulness; thus, ablation of the MBs results in reduced sleep. The sleep-inhibiting neurons increase the activity of the integrator to further promote the wake state. The sleep-promoting neurons inhibit the integrator to reduce wakefulness and promote sleep. Molecules That Regulate Sleep To identify molecules that regulate sleep, we are taking the following multiple approaches. 1. Testing candidate molecules. Candidates are picked either because they have been implicated in sleep by work in other model systems or because of their link with some hypothesized function for sleep. As noted

6 562 SEHGAL ET AL. above, the camp pathway was tested as part of this approach. 2. Identifying targets of pharmacological reagents that affect sleep. 3. Conducting random mutagenesis screens for mutations that affect sleep and then cloning the genes mutated. 4. Looking for genes expressed differentially as a function of behavioral state. It is believed that sleep-promoting factors build up when an organism has been awake for a long period of time. This belief is based on experiments done many years ago in which cerebrospinal fluid extracted from sleep-deprived goats was shown to promote sleep in rested (i.e., nonsleep-deprived) controls (Pappenheimer et al. 1967). The nature of the sleep-promoting molecules is not known, although attempts were made to purify such molecules (Pappenheimer et al. 1975). We are interested in identifying homeostatic factors that promote sleep and, to this end, looked for changes in gene expression during sleep deprivation and sleep rebound (compensatory sleep following deprivation) in flies (Williams et al. 2007). It is possible that regulation of the critical sleep-promoting molecules does not occur at the transcriptional level, but earlier studies indicated that gene expression profiles do change with altered sleep regulation, although it is not yet known if they are the cause or the effect of the altered sleep (Cirelli and Tononi 1999). To identify changes in gene expression associated with sleep deprivation or sleep rebound, we conducted microarray analysis of the entire Drosophila genome (Williams et al. 2007). This resulted in the identification of a number of RNAs that are up-regulated or down-regulated during these behavioral states. The class of molecules that stood out among these was that of the immune response genes. Drosophila have an innate immune system that includes many components shared with the mammalian immune/inflammatory system (Brennan and Anderson 2004). Of note is the protein NFκb that mediates many immune/inflammatory responses. We found that sleep deprivation of Drosophila results in the up-regulation of Relish, one of the Drosophila NF-κb genes, and of many other immune response genes, some of which function downstream from Relish. Interestingly, inflammatory markers such as cytokines and NF-κb are also up-regulated in sleep-deprived mammals and are thought to promote sleep (Majde and Krueger 2005). Using the genetics available in Drosophila, wenext asked whether NF-κb promotes sleep in flies (Williams et al. 2007). We found that sleep is not reduced in flies that lack Relish, but it is reduced in flies heterozygous for a mutation in Relish. The lack of a phenotype in the homozygote may reflect compensation of some sort, not unlike what occurs with other phenotypes associated with processes such as aging (Rogina et al. 2000). The sleep phenotype in Relish mutants was rescued by expressing a wild-type Relish transgene in the fat body (Drosophila equivalent of the liver and adipose tissue and a major site of immune signaling), suggesting that the activity of peripheral tissues can affect the sleep state. We also asked Figure 7. Effects of sleep deprivation on the immune assay. Flies were injected with bacteria, and 24 hours later, they were homogenized and plated onto culture plates containing LB medium and ampicillin. The number of colony-forming units was scored for flies sleep deprived for several hours (DEP) and handled controls (HC). Sleep-deprived flies formed fewer colonies, indicating increased resistance to infection. the question of whether sleep deprivation affects the immune response. To our surprise, we found that resistance to infection was increased in sleep-deprived flies (Fig. 7). Although this seems to be counterintuitive, it is consistent with the up-regulated expression of immune genes under these conditions. We suggest that the enhancement of immune/inflammatory responses during acute sleep deprivation evolved as a mechanism to allow organisms to cope with intermittent periods of forced sleeplessness. We would predict that immune responses would be compromised with chronic sleep deprivation, but this has not yet been experimentally tested. CONCLUSIONS Using a Drosophila model, we have gained considerable insight into the mechanisms that constitute the clock and synchronize it to light. The importance of the transcription-based feedback loop is attested to by its conservation across species, but it is becoming increasingly clear that tightly controlled posttranslational modification of clock proteins is perhaps even more critical for timekeeping by the clock. The acute response of the Drosophila clock to light also involves changes at the posttranslational level, although these are probably followed rapidly by transcriptional effects. We are now also

7 SLEEP:WAKE CYCLES IN DROSOPHILA 563 realizing that extrinsic factors other than light can have profound effects on the clock. With respect to the outputs controlled by the clock, although we still understand little about the mechanisms used by the clock to transmit timeof-day signals, studies have been initiated to directly investigate the multilevel regulation of one output, sleep. Since the first demonstration that Drosophila rest is a sleeplike state controlled by homeostatic mechanisms, in addition to the well-known circadian regulation, Drosophila has become popular as a model for sleep. Studies of homeostatic control are revealing important sleep-regulating sites in the fly brain and also identifying molecules that affect sleep. It is expected that future work will identify the cellular and molecular circuits that underlie circadian and homeostatic control of sleep and indicate the point at which these systems intersect. 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