HIGHLIGHTED TOPICS
Genetic Models in Applied Physiology
Invited Review: Sleeping flies don't lie: the use of Drosophila melanogaster to study sleep and circadian rhythms

Center for Sleep and Respiratory Neurobiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104

	ABSTRACT

TOP ABSTRACT INTRODUCTION CIRCADIAN RHYTHMS SLEEP FUTURE DIRECTIONS CONCLUDING REMARKS REFERENCES

During the past century, flies thoroughly proved their value as an animal model for the study of the genetics of development and basic cell processes. During the past three decades, they have also been extensively used to study the genetics of behavior. For both circadian rhythms and for sleep, flies are helping us to understand the genetic mechanisms that underlie these complex behaviors. Since 1971, discoveries in the fly have led the way to a number of significant discoveries, establishing a mechanistic framework that is now known to be conserved in the mammalian clock. The highlights of this history are described. For sleep, the use of the fly as a model is relatively new, that is, only within the past 2 yr. Nonetheless, studies have already established that two transcription factors alter rest and rest homeostasis. The implications of these advances for the future of sleep research are summarized.

PAS domain; clock; genes; homeostasis; stress; sleeplike rest

	INTRODUCTION

TOP ABSTRACT INTRODUCTION CIRCADIAN RHYTHMS SLEEP FUTURE DIRECTIONS CONCLUDING REMARKS REFERENCES

SINCE EARLY IN THE 20th century, fruit flies have been extraordinarily useful for the study of genetics of development and cell signaling. By the end of the century, not only was the contribution of Drosophila melanogaster to science amply documented in the scientific literature but also a series of books for the general public celebrated the fruit fly and its devotees (17, 71, 156). Why have flies been so successful in the laboratory? Besides economic reasons and the efficiency of studying a small, rapidly reproducing animal for genetics, simpler animals also have scientific advantages. The reduced gene number means that there is less redundancy and less compensation. The relevance of studying genes in model organisms has been further highlighted by the evidence that the genomes of species of biomedical interest, from yeast to humans, are widely conserved; among these, the fly's genome is the closest to that of mammals (116). Furthermore, even where flies do not suffer from mammalian diseases, disease cognate genes can be identified (21, 109).

The leadership of Seymour Benzer (13, 14, 156) was instrumental in establishing the view that behavior was just another set of phenotypes amenable to genetic analysis. The fly has specific advantages for the study of neurobehavioral genetics: despite their relatively simple central nervous system (CNS), they still have a repertoire of behaviors that are complex enough to be readily recognized as analogous to interesting human behaviors [even "embarrassingly" so (14)]. Flies run, explore, respond to their environment, court, groom, and learn. Many of these behaviors have yielded to genetic dissection (5, 19, 87, 88, 149, 164), producing substantial insights into the strategies that the nervous system uses to perform complex functions.

With this background, one can easily see why flies were chosen to study the molecular basis of circadian rhythms and, more recently, sleep. This review will first focus on circadian rhythms, where the contribution of flies has illuminated the path to remarkable progress in understanding the mammalian clock, including the first mechanistic explanation of a human sleep disorder at the molecular level. Next, the new discovery that rest in flies is a sleeplike state and the emerging evidence that flies may also help us understand this in mammals are presented.

	CIRCADIAN RHYTHMS

TOP ABSTRACT INTRODUCTION CIRCADIAN RHYTHMS SLEEP FUTURE DIRECTIONS CONCLUDING REMARKS REFERENCES

Part I: The Fly Reveals Conservation of the Central Clock

The beginning: a gene that controls circadian rhythms. Flies have readily observed locomotor activity that has long been known to have a circadian rhythm (99, 101, 115). A second overt rhythm is the emergence of adults from their pupal cases (eclosion) in the early morning hours (100). Automated devices were introduced to monitor each of these rhythms almost from the beginning of their discovery (115, 168), and further improvements (49, 73) increased the efficiency of monitors so that large-scale ("high-throughput" in today's jargon) studies were relatively easy. A commercially available computerized version of both monitors with software is now available to download the data for computer analysis (Trikinetics, Waltham, MA), providing a uniform methodology that certainly has facilitated conducting and reproducing large-scale studies. In 1971, the seminal publication that established circadian rhythms as a genetic field (73) used the fact that a mutation of a single gene (period, abbreviated per) changed both of these independent rhythms, showing that the gene must be involved in the central clock rather than in any effector system or "output" of the clock. Konopka and Benzer (73) screened a population of 2,000 mutagenized flies and identified not only a null allele that abolished locomotor and eclosion rhythms but also mutations in the same gene that shortened or lengthened the period (length of the endogenous day), solidifying the evidence that this single gene is fundamental in determining 24-h timing. The principle of a "monogenic" basis for such a complex behavior was thus established and became the basis for the entire field of Drosophila behavioral neurogenetics (14). More narrowly, this study demonstrated that a gene controlling circadian rhythms could be neatly removed without other obvious effects on the animal. The gene was neither pleiotropic (involved in multiple pathways or cellular functions) nor redundant (readily replaced by other genes). The proof of the principle that circadian rhythmicity per se could be abolished by mutating a single "private" gene was the fly's first major contribution (Table 1) to the field.

View this table: [in this window] [in a new window]   Table 1.   Timeline of fly contributions to the clock

During the 23 years that period was the only known central clock gene in any animal, it was extensively studied. After the gene was cloned in 1984 (107), the distribution of its protein and mRNA were found to be widespread and largely nuclear (83) and found to exhibit circadian oscillations in the head (50, 134). The functional localization of the clock was established in period mosaics (35). The expression of PER in a small number of neurons at the edge of the central brain, termed "lateral neurons," was sufficient to drive rest-activity rhythms (35, 40, 52). PER was found to be temporally phosphorylated and degraded (32).

The discovery of the eponymous "PAS" domain (named because of its initial description in PERIOD, ARNT, and SINGLEMINDED proteins) (60) turned out to have profound implications beyond the circadian field. The PAS domain was originally shown to mediate protein-protein interactions, permitting homo- and heterodimerization (60). Now that more than 200 PAS-containing proteins have been discovered (23), it is clear that PAS is an ancient motif present in archaea, bacteria, and plants as well as in animals (9, 23, 102, 142, 143). PAS-containing proteins are receptors, signal transducers, and transcription factors that provide information about the environment to the organism and mediate adaptive responses. Crews and Fan (23) suggested that the general function of PAS-containing proteins is to "monitor the energy state of cells." For example, mutations of the PAS domain of the bacterial Aer gene have shown that Aer is a "transducer for redox taxis." That is, it provides information that guides bacteria to move to the optimal redox potential in a fluid medium (111, 142, 143). The canonical PAS-containing proteins in eukaryotes mediate information about light, temperature (PERIOD), oxygen (hypoxia-inducible factors such as SINGLEMINDED), and xenobiotics (ARNT/AHR). They dimerize, enter the nucleus, and change gene transcription (23). PAS proteins have also been postulated to regulate homeostasis in mammalian development as well as in adult adaptive responses (23).

A clock mechanism revealed: discovery of the second central clock gene. In 1994, the second bona fide central clock gene in flies, timeless (tim), was finally identified in a mutagenesis screen. Similar to that for PERIOD, locomotion and eclosion were both arrhythmic in flies lacking functional TIMELESS (124), and both the mRNA and the protein cycled (125). When PERIOD heterodimerized with TIMELESS, in part through the PAS domain (42, 118), PERIOD was protected from degradation (104). The two proteins accumulated in the cytoplasm over a 6-h period and could then enter the nucleus and inhibit the transcription of both per and tim (154). Mathematical models showed that two interacting genes could account for the lag between RNA and protein peaks necessary for robust, stable, high-amplitude 24-h oscillations (81, 98). At this point, two more puzzles of the clock were provided with likely solutions: 1) negative feedback is accomplished by a heterodimer of two clock proteins and 2) the lag between per transcription and PER accumulation and nuclear entry is explained by the required interaction between these two proteins (Table 1 and Fig. 1).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Schematic of mechanisms in Drosophila (bold typeface) and mammalian variants (normal typeface). 1: The dCLOCK:CYCLE [homologs of mammalian CLOCK, BMAL-1 (for brain, muscle, ARNT-like-1)] dimer binds to the E-box motif and activates gene expression of period (per) and timeless (tim) (mammalian pers and crys). 2: doubletime phosphorylates monomeric PER, which is then degraded. 3: PER:TIM (or PER:CRY) dimers are protected from phosphorylation so that nuclear entry can occur. 4: Once within the nucleus, PER (or CRY in mammals) interferes with the dCLOCK:CYCLE (homologs of mammalian CLOCK:BMAL) activation of per, tim (or per, cry in mammals) gene expression. See text for further description. m, murine; d, Drosophila; PER, per, PERIOD protein and mRNA; TIM, tim, TIMELESS protein and mRNA; DBT, DOUBLETIME kinase (casein kinase I); CRY, cry, CRYPTOCHROME protein and mRNA.

A second, bonus function for TIM gave us insight into the link between light signals and behavioral timing. Even a brief light pulse at the right time of day (or more accurately the night, as light pulses are ineffective during daylight hours) changes the timing of circadian behavior rhythms for the synchronization in expecting dawn and/or dusk (see Ref. 38 for review). TIM levels in the animal (but not in vitro) were found to be rapidly decreased by a light pulse (61, 80, 92, 165). This degradation, accomplished by proteasomal mechanisms (93), was tightly related to behavioral resetting (140, 160). This provided another key insight: animals use relevant environmental variables to change the level of a core clock component, in this case, of the TIMELESS protein, which changes the timing of gene expression and thus resets the timing of rhythmic behavior to synchronize it to the environment (Table 1).

Mammals catch up. In the mid-1990s, despite the excitement generated by the increasingly complete understanding of clock mechanisms in Drosophila, there was some uneasiness about the lack of mammalian homologs. In the absence of evidence of molecular mechanisms, some doubted that the elegantly described fruit fly mechanisms were relevant to mammals. Other strategies were being discovered in other models. For example, another insect, the silk moth, was found to have per and tim and to use them in the clock (122), but the mechanism was completely different, relying on cyclical posttranslational modulation rather than cyclical transcription. Work in Neurospora (see Ref. 31 for review) revealed that the fungal clock shared with Drosophila autoregulatory feedback and proteins with PAS domains (24), but the proteins were not per or tim homologs. When the mouse Clock gene, the first mammalian clock gene discovered (153), was cloned in 1997 (69), it was not a per or tim homolog, although it contained a PAS domain as well as a basic helix-loop-helix (bHLH) DNA-binding motif.

Until 1997, mammalian homologs for per or tim and a fly homolog for Clock were all among the missing. These gaps were filled in what one review titled a "clockwork explosion" (112). The entire framework of both the murine and the Drosophila were laid out in numerous important publications (1, 4, 26, 43, 58, 59, 70, 72, 78, 104, 117, 129, 131, 144, 170; see also Refs. 31, 112, 114, 123 for reviews). Three per homologs were identified and found to cycle in mammals (139, 144). Among these, mPer2 is the closest molecular and functional homolog to Drosophila per (167); it can even rescue robust rhythms in per-null flies (130).

The roles of the multiple per genes are not completely clear. Two of the mammalian per genes are induced by light (although the dynamics and degree vary) (3, 10, 137, 170). mPer1 and mPer2 appear to interact to provide the "morning" and "evening" clocks (137). Alternative splice forms of Drosophila PER have been shown to perform a parallel function, allowing the animal to alter the timing of its activity to changing light and temperature conditions (86, 132). This most likely provides a mechanism for adapting to seasonal changes of both of these inputs (16). This is a specific example of a general phenomenon: a role played by multiple mammalian genes is served in flies by one gene with multiple splice isoforms.

A tim homolog was also discovered in mammals. However, despite some early evidence that it was a component of the clock (72, 120, 145), the preponderance of evidence now supports only a developmental role (46). Subsequently, a second tim gene in the fly was identified and found to be a closer homolog to mammalian tim (12). Insects may have expanded the more primitive developmental role to provide a light-responsive partner for PER (113). As described below, another molecule, cryptochrome, which plays a role in both the fly and the mammal clock, appears to have taken on the role of PER's partner in mammals (Table 1, Fig. 1).

The positive arm of the feedback loop. Once the role of the PER/TIM dimer in negative feedback was established, the theoretical need for a positive mechanism to activate transcription was manifest. Although they did not lead the way, flies have participated in "closing the loop" (26). Parallel and complementary studies in flies, mammals, and in vitro systems established that the molecules and mechanisms for this arm of molecular cycling have remarkable conservation (Table 1, Fig. 1). CLOCK [or dCLOCK, the Drosophila homolog (4)] has a dimer partner that was first identified in mammalian brain and muscle and was named BMAL-1 (for brain, muscle, ARNT-like-1) (1, 59, 62) and subsequently identified as a CLOCK-interacting protein (43). The Drosophila homolog is CYCLE (117). These proteins have both a PAS and a bHLH domain. In both flies and mammals, they dimerize and bind to "E-box" DNA sequences to provide activation of the downstream genes (per and tim in flies; per's and cry's in mammals). The activity of this dimer is cyclically repressed by PER when it enters the nucleus with its dimer partner.

Some interesting differences in details have also emerged. In flies, both the protein and the mRNA of CLOCK cycle but neither the mRNA nor the transcript of the paradoxically named cycle oscillates (see Ref. 15 for review). In mammals, BMAL-1 mRNA cycles (1, 141) and its protein appears to be degraded by light (141). Murine CLOCK has minimal (103) or no (69) cycling. Such findings have led to the question, Can we define a single feature (e.g., cycling of a transcript, protein, or dimer) that is sufficient to provide an endogenous timing mechanism? We do not yet have an answer, but, clearly, proteins can cycle in the absence of transcriptional cycling, and not all proteins need to cycle (161).

Part II: Variations on a Theme: The Story of Cryptochrome

The importance of light for resetting the clock to synchronize rhythms to the sun's rising and setting prompted investigations into the anatomic and molecular input pathways. In the fly, all visual input is dispensable: electrical activity responsive to light in the eyes and other light-sensing tissues was shown to be unnecessary for photic resetting, including TIM degradation (140, 160). The threshold for resetting the Drosophila circadian clock is lowest in the range of blue-green light. Presumably not coincidentally, blue-green light predominates during twilight (38, 140, 169). This led to the proposal that a blue-light-responsive molecule described in plants and flies (20), cryptochrome, might be involved in circadian light responsiveness (160). With the identification of a fly cryptochrome mutant, "CRY-BABY" (cry^b) (136), it was shown that these animals indeed have altered light-sensing abilities in that they did not reset their internal clock normally when exposed to brief flashes of light. However, they could be entrained by light so that they were synchronized to the normal oscillations of light and dark. Also, they exhibited normal locomotor rhythms in constant light and temperature conditions. Overexpressing the gene produced similarly limited defects (33). Flies are circadian blind only when the cry^b mutation is combined with a mutation that abolishes all known photosensitive structures (53). This demonstrates that the light-sensing structures and molecular mechanisms must normally interact to synchronize behavior in flies. The same interaction is suggested in mammals (169).

In mammals, the discovery of two cryptochrome homologs (CRY1 and CRY2) (77) led to the expectation of a similar photosensor role, but a double mutant lacking both proteins was found to be arrhythmic (150). Furthermore, CRY cycles in abundance in the mouse suprachiasmatic nucleus (SCN), the locus of the mammalian central clock. Finally, CRY and PER appear to interact in a fashion reminiscent of the TIM-PER interactions in flies (79). Thus, although mammals may not use TIM in the clock, its functional role is retained by swapping TIM for CRY to provide a dimer partner for PER (Table 1, Fig. 1).

Whether cryptochromes also play a role in mammalian photic resetting has not been elucidated to date, as resetting cannot be detected in arrhythmic animals (126). CRY could play both a photosensing and a central clock role (113). An opsin (perhaps melanopsin) could also be a good mammalian circadian photosensor candidate (169).

Part III: From Flies to Clinical Sleep Disorders

Exciting discoveries have continued into the new millennium with the discovery of a clinical role for the kinase that phosphorylates PER. The kinase was first described in Drosophila when a mutation caused a short endogenous day length of only 18 h. The gene causing this short-period phenotype was thus named doubletime (104). Researchers (104) also described a long-period mutation in the same gene with an endogenous day nearly 27 h long and a low-viability severe hypomorph. The gene turned out to be a casein kinase I, a highly conserved gene already known to have a role in development (70). The dynamics of phosphorylation determine the accumulation of PER and thus the period length of the circadian rhythm.

Conservation of this function was established when the gene responsible for the first mammalian clock mutant ever described, the short-period tau hamster (106), was cloned and found to be a mutation of the homologous casein kinase gene (84). The excitement generated by this finding was followed by an even more astonishing one: a defect in PER phosphorylation is implicated in an inherited human sleep disorder. In 1999, a family was identified with several members who could not stay awake past the early evening and who also awoke during early morning. Physiological tests of body temperature and melatonin levels confirmed that their endogenous clocks were set ahead by 3-4 h compared with most people. For sufferers of this lifelong condition, the irresistible urge to sleep was advanced ahead of the normal; therefore, the disorder was termed familial advanced sleep-phase syndrome (65). In constant light conditions, one elderly family member showed a short circadian period, indicating that her internal clock was running more than 1 h faster than normal (65). The mutation was identified by linkage analysis and found to be in the predicted casein-kinase binding region of the hPer2 protein (148), most likely leading to deficient phosphorylation and altered turnover (148). It is becoming clear that this phenotype and similar genetic variants in circadian period are not rare in humans. Additional families have been identified both by the original workers and in other clinics (Ref. 108 and Louis Ptacek, personal communications). "Delayed-phase sleep syndrome," in which family members fall asleep and awaken extremely late, have also been discovered (Phyllis Zee, personal communications). Further delineation of the molecular mechanisms will clearly lead to an improved understanding of how circadian clock genes interact with environmental and social conditions in humans.

	SLEEP

TOP ABSTRACT INTRODUCTION CIRCADIAN RHYTHMS SLEEP FUTURE DIRECTIONS CONCLUDING REMARKS REFERENCES

Part I: Getting Out of the Clock: Output Mechanisms

Our understanding of the molecular basis of the central timekeeping mechanism has far outstripped our understanding of how the cycling of clock molecules in a relatively small group of brain cells is translated into overt physiological and behavioral rhythms of the whole animal. The molecular connections between the metaphorical clock "gears" and its "hands" remain only sketchy. In no case is the complete pathway from the clock, through intermediaries, to a behavioral or physiological output completely understood. Two of the best described Drosophila output molecules are the neuropeptide pigment-dispersing factor or PDF (52, 110) and the lipocalin takeout (121, 135). Neither of these has a mammalian homolog, judged by sequence conservation, but functional conservation may occur. PDF was identified quite early as being present in all Drosophila clock neurons, with a very limited distribution beyond these neurons (52). Interestingly, some of the best-established outputs from the mammalian clock, the SCN, are neuropeptides (20a, 64, 74). The second well-described output molecule, takeout, links the clock to nutrient intake. The takeout transcript cycles with a 24-h rhythm in the male head, and it is induced by starvation and becomes widely distributed in the gastrointestinal tract. Although takeout mutants are perfectly rhythmic, they fail to show the normal locomotor patterns of starving flies and succumb to starvation more quickly (121). Thus a normal role in connecting the clock to feeding or metabolism is suggested. The lipocalin family is a poorly conserved group of humoral signaling molecules that includes retinol binding protein, among others (36, 37, 119, 158). A role for the retinoic acid receptor in mediating clock information in the murine cardiovascular system has been reported (90), suggesting that additional members of this family are involved in circadian rhythms and that a general role for lipocalins is conserved. Indeed, a large number of members of this family were described in a genome-wide search for cycling or clock-regulated genes in flies (89).

A number of other output molecules have been identified in the fly. The RNA binding protein lark is involved in eclosion but not locomotion (95, 166). A mutation of nuclear factor-1 (NF-1) (157), the Drosophila homolog of the neurofibromatosis gene, leads to arrhythmia of locomotion but not eclosion. NF-1 seems to act through the mitogen-activated protein kinase signaling pathway (157). A number of studies implicate the cAMP signaling cascade in the clock (11, 82, 85); as described below, Hendricks et al. (57) have shown a noncircadian role for the same cascade in rest and rest homeostasis.

Advances in the ability to screen the entire genome for genes cycling with a 24-h rhythm have led to two recent publications that used microarrays for gene expression profiling (22, 89). As in earlier studies with older techniques to identify cycling genes (151, 152), the link to the clock might be very indirect. That is, clock-regulated physiological or behavioral outputs could secondarily lead to changes in gene expression. Indeed, the evidence is that only a small minority of cycling genes is directly clock controlled (22, 89). The cycling genes, exclusive of those already well established to be part of the clock, did not have a statistically increased number of binding sites for the Clock/cycle dimer when investigated in silico (that is, with the use of bioinformatics) (22). Similarly, a subset of cycling genes was investigated for evidence of direct regulation by the dimer in vitro (89), with largely negative results. Nonetheless, an enormous number of new candidates for clock regulation have been generated, and understanding whether and how these are part of the biological clock will be a challenge for the future.

Such comprehensive studies of the genome provide evidence that levels of complexity will be added to the model of clock function as we move from a reductionist approach to assembling the entire organism. The independence of the central clock from influence by the outputs it regulates has traditionally been emphasized. In the intact animal in a natural setting, we may find that the central clock can be modulated by peripheral oscillations. Behaviors that normally occur with a 24-h cycle but are not themselves dependent on the central clock, for example, feeding and sleep, normally occur at times gated by the clock. However, these behaviors are obviously in themselves qualitatively normal in the absence of a clock, whether due to surgical ablation of the SCN or molecular mutation of central clock genes. Similarly, the clock itself clearly continues to function even if its outputs are altered. That is, starvation or prolonged wakefulness, for example, do not abolish the central circadian rhythm. Nonetheless, interesting interactions and modulating influences may exist in the normal animal in its natural environment. Although not as important as light, both feeding and sleep deprivation have been shown experimentally to provide timing information to the clock (25, 91, 138). Perhaps these and other behaviors, including social and reproductive activities, normally feed back positively on the clock, reinforcing the normal timing and increasing the amplitude of the cycles. This premise is being extensively pursued, in part because of the implication that behavior that drives peripheral clocks out of phase with the central molecular clock could underlie some of the misery of the modern maladies of jet lag and shift work (25, 103, 138).

Part II: Getting to Sleep

Do flies sleep? One of the most intriguing and well-conserved behavioral outputs of the clock is the basic rest-activity cycle or, in mammals, the sleep-wake cycle, whose fundamental molecular basis remains largely a mystery. Sleep is a complex state required for normal waking function and perhaps even for life itself (30, 34). One fundamental feature that has been extensively described is the interaction between a daily occurrence of sleep (circadian regulation), which is independent of the prior history of sleep accumulation, and the need for sleep (homeostatic regulation), which is profoundly influenced by the prior sleep history (29). That is, the initiation, duration, and intensity of sleep are influenced both by the circadian time of day and by the animal's sleep need. To be considered sleeplike, an inactive state should have the following features (18, 56): 1) consolidated circadian periods of immobility, 2) a species-specific posture and/or resting place, 3) an increased arousal threshold (although the state can be reversed by intense stimulation), and 4) a homeostatic regulatory mechanism. We found that, according to these criteria, rest in Drosophila is a sleeplike state (54). Rest in Drosophila shares with sleep the intriguing features of prolonged immobility, lack of sensory responses, and homeostatic rebound in response to rest deprivation, in addition to the well-known circadian regulatory influences on activity. These flies also show conservation of behavioral changes in response to caffeine and to an adenosine agonist that produces sleep in mammals. We concluded that rest in these simple animals may be considered a primordial form of the sleep state. A second independent study reported nearly identical behavioral and pharmacological findings and also showed that genes upregulated in response to rest deprivation were conserved between flies and mammals (127).

Perhaps the most surprising thing about using fruit flies to study sleep is that it took so long. In 1971, Seymour Benzer (14) noted that flies appeared "as if asleep on their feet" during the night. Irene Tobler and others had earlier noted sleeplike features of rest in invertebrates, including insects (18, 66, 67, 146, 147).

Part II: Using Flies to Demystify Sleep: Two State-Related Molecular Pathways So Far

The cAMP cascade and rest in flies. To understand the molecular regulation of sleeplike rest, we investigated the role of a candidate gene, cAMP-response element binding protein (CREB). A number of features of this signaling pathway drew our attention. First, the cAMP-protein kinase A-CREB signaling pathway has been shown to have both molecular and functional conservation from invertebrates to mammals (2, 133). Second, CREB mediates neural adaptation to various inputs (39, 48), including the consolidation of long-term memory in Drosophila and mammals (19, 133, 162, 163). One idea with a long history and considerable experimental support is that sleep's restorative function involves adaptive neural plasticity (see Ref. 56 for review). Because sleep is thought to promote normal CNS maturation and may aid in long-term memory consolidation and recovery from neural injury (56), we were curious about whether there might also be some functional relationship between sleep and CREB activity. Finally, CREB activity has a circadian activity pattern in Drosophila (11), suggesting to us an interaction with the rest-activity cycle. We exploited the powerful genetics and the ready availability of reagents to modify gene expression in Drosophila to establish a functional relationship between the cAMP signaling pathway and rest (11). We found that decreasing or increasing cAMP and CREB activity decreased or increased waking, respectively. Rest rebound was increased when CREB activity was blocked or defective (11). Thus changes in cAMP signaling and CREB activity were inversely related to changes in the need for rest, suggesting perhaps that normal rest fosters normal CREB activity. To discover whether changing rest altered CREB activity, we monitored CREB-dependent gene expression in vivo (11). We found that CREB activity was increased immediately after rest deprivation and also during the subsequent 3-day recovery period when a rest rebound occurs (11). This is consistent with the possibility that CREB activity increases during the rest rebound, helping the animal to recover its normal wakefulness after the rebound. These results support a functional role for CREB both in maintaining waking and in a restorative function of the sleeplike rest state of Drosophila during recovery from rest deprivation. It is not yet know whether this restorative function does, indeed, include adaptive neural plasticity, such as that involved in long-term memory consolidation. Our colleagues have established a similar role for CREB in mice (Graves L, Hellman K, Veasey S, Blendy J, Pack A, and Abel E, unpublished observation).

A role for the clock transcription factor cycle/BMAL-1. In studying the clock mutants to further understand the relationship between the central clock and rest, Paul Shaw and co-workers (128) found that cycle-null mutants had a particularly prominent phenotype. In contrast to other clock mutants, female cycle-null mutants exhibited an abnormally exaggerated rebound after rest deprivation and also succumbed much more rapidly to a lethal effect of rest deprivation (128). Shaw et al. searched for a molecular basis for the increased vulnerability to sleep loss and found that cycle-null mutants had a relative decrease in heat shock protein induction by rest deprivation. To take this observation beyond the correlative level, they induced heat shock proteins in cycle nulls and found that they could "rescue" the exaggerated rebound. They also established that specific heat shock mutants had a phenotype similar to that of the cycle nulls.

These workers conclude that this may provide the first hint about the functional targets of sleep and its molecular mechanisms. We believe that there are additional lessons to be gleaned from the cycle mutant (55). The function of CYCLE is clearly clock independent, as mutations of its partner CLOCK do not produce the same phenotype (55, 128). More broadly, the rest regulation role of cycle must be independent of clock function per se, since rest is not always altered by the absence of a molecular clock. That is, although one clock mutant (tim) has a quantitative deficit in rest rebound, other arrhythmic mutants have relatively normal rest homeostasis (54, 55, 128). Other studies in both flies and mice have shown that noncircadian behaviors can be differentially altered in different clock mutants (6, 94). This may be explained in part by the fact that different clock genes modulate different downstream targets, as revealed by microarray studies (22, 89). Perhaps even more relevant is the fact that the loss of a functional dClock gene led to steady-state changes in a number of genes that do not have cyclic transcription (89). Clock genes are present in a number of tissues beyond the central clock (68), and in some cases their expression is cytoplasmic (83), pointing to additional nonclock functions for these genes.

A second issue highlighted by the study of cycle mutants is that of gender dimorphism. The cycle-null phenotype in males and females is strikingly different (55, 128). Partly because male flies are generally used for locomotor studies, and molecular studies are conducted in mixed populations, such phenomena may have been overlooked in the past. Gender differences in circadian cycling have only recently received attention (51). Interestingly, takeout, in addition to being regulated by both the clock and starvation (135), is regulated in a sex-specific fashion by the fruitless gene (27, 28). Our laboratory has not found a role for takeout in rest regulation (Hendricks, unpublished observations), but the integrated action of multiple influences (in this case, gender, feeding state, and circadian time) provides an example of the sort of molecular mechanism that could underlie gender-related differences in both rest and circadian functions. The large number of clock-regulated takeout family members may merit further investigation. Somewhat surprisingly, gender dimorphism in human sleep homeostasis is also a newly emerging area. Women have a different time course of delta and non-rapid eye movement sleep during recovery sleep after deprivation (7, 8). Because flies have an exquisitely well-understood genetic basis of cellular gender, with tools that can be used to change the genetic gender of individual cells or of groups of cells or tissues, this will be a fascinating area to study in this organism. This sort of partial neural feminization has already shown that central motor programming of locomotion is gender dimorphic in the CNS (41, 87, 88). Perhaps, similar approaches would be useful to identify, first, which neurons are responsible for gender differences in rest and rest homeostasis and then how these differences are mediated on a molecular level.

In its brief history, the new "fly sleep" field has already taken advantage of the power of sophisticated fly genetics. The studies moved quickly beyond the correlative level by using genetically modified flies to establish that state-related changes in gene expression (128) or transcriptional activity (57) had functional significance.

	FUTURE DIRECTIONS

TOP ABSTRACT INTRODUCTION CIRCADIAN RHYTHMS SLEEP FUTURE DIRECTIONS CONCLUDING REMARKS REFERENCES

Flies proved to be extraordinarily useful to establish the strategies by which genes establish self-sustaining 24-h oscillations of transcription and protein accumulation, but there are still limits to our understanding (15). Do the flies have more to teach us? There are some interesting questions presently being investigated: How are the humoral and electrical signals linked among neurons in the anatomical clock (96, 155)? How does the central clock interact with the numerous peripheral clocks (63, 75, 76, 159)? In addition to the feedback loops described above, there are clearly additional feedback loops and there are additional cycling elements. How do these multiple molecular clocks interact (15, 45, 103)? Finally, do some clock genes have an even more fundamental relationship to the life cycle? There is recent evidence that the mammalian tim homolog has a role in early development rather than the clock and newer evidence for a role of the clock in fly development (105, 166). At the opposite end of life, a surprising recent result is that the tau mutation in the hamster not only leads to a short endogenous circadian day but also increases longevity (97).

What might we expect with regard to the molecular biology of sleep? Genome-wide approaches to characterizing genes related to Drosophila rest and rest homeostasis, including whole-genome expression profiling and systematic mutagenesis, are already underway. From initial results, it seems certain that suites of genes that are up- or downregulated during sleep loss and genes that are up- or downregulated during the recovery from sleep loss will be identified. To go beyond the correlative level, the use of mutants and overexpression transgenics that are readily available or easily generated in flies will of course be useful. It is worth noting that sleep-related genes and molecular pathways identified to date have multiple functions; i.e., they are pleiotropic. This means that studying mutants in all species could be problematic because crucial steps in development may be disrupted, interfering with detection of an adult behavioral phenotype. Unlike the Drosophila clock genes, sleep-related genes may be redundant as well as pleiotropic. Judging by the success to date in identifying roles of CREB and cycle, even if this is the case for some genes, it will not prevent at least some significant pathways from being discovered. It is notable that the CREB-deficient mouse phenotype is much more subtle than the fly CREB mutant phenotype (Graves L, Hellman K, Veasey S, Blendy J, Pack A, and Abel E, unpublished observation), presumably precisely because of residual CREB function and compensation by homologs such as cAMP response element modulator (CREM) (J. Blendy, personal communications). In general, the molecular basis of sleep may differ from the molecular basis of circadian timing in the same fashion as the anatomic substrates differ. That is, although a specific discrete brain region (the SCN or the lateral neurons) is necessary and sufficient for circadian timing of behavioral and physiological rhythms, no specific brain region has ever been identified as the "sleep center." Rather, every area that has a role in sleep also has additional functions, and destroying a single discrete region does not abolish all aspects of sleep. By analogy, perhaps a distributed network of sleep-related cell signaling pathways and cellular functions should be anticipated.

	CONCLUDING REMARKS

TOP ABSTRACT INTRODUCTION CIRCADIAN RHYTHMS SLEEP FUTURE DIRECTIONS CONCLUDING REMARKS REFERENCES

General interest in understanding the biological clock is most often linked to the human health implications of sleep deprivation and sleep disorders. Clock studies that originated with discoveries in Drosophila are beginning to explain clinical disorders of sleep timing. As mentioned, a number of families with inherited abnormalities of sleep timing and duration have already been identified (65, 108), and, for some of these, the responsible alleles in the central clock genes have been defined (148). It may be that a range of sleep propensities in the general population is related to allelic variation among the human population. A number of lines of evidence are also beginning to link the coordination between the central SCN clock and the peripheral clocks in organs such as liver and lung (103, 138) to such modern problems as jet lag and sleep disorders related to shift work. In these disorders, the timing information provided by the environment and by voluntary shifts in behaviors such as feeding and sleep are temporally out of phase with the central clock, leading to desynchrony among the body's clocks. How this molecular desynchrony leads to subjective and objective abnormalities is still not known. Another aspect with clinical relevance is the molecular biology underlying the circadian occurrence of some disorders. For example, the physiology underlying the timing of cardiovascular accidents may be related to a molecular clock in the vascular endothelium that interacts with humoral factors (90). Studies of Drosophila rest have not yet verified a link to any clinical sleep disorder, but those of us studying Drosophila are keenly interested in obtaining such data. As we improve our understanding of the molecular basis of sleep and sleep homeostasis, we hope to assemble information about the function of sleep that can explain abnormalities of the quality, as well as the timing, of sleep.

To conclude with a balanced outlook, we note that, despite their many advantages, flies will always have some shortcomings. The most absolute is that the specific details of clinically important rhythms, such as those of the cardiovascular system, cannot be meaningfully studied in flies. To date, flies have not been particularly useful to study physiology, including hormonal cycling. This is to some degree a matter of focus, since Drosophila has traditionally been used by geneticists. However, as with some other issues such as detailed neuroanatomy, the size of the fly makes such studies technically challenging. Similar issues are important to some degree in limiting the knowledge of peripheral clocks and even of multiple oscillators within the CNS. Nonetheless, using clever approaches (44, 96), principles of interactions still can be investigated. The essential importance of using the fly (or any other species evolutionarily distant from the species of the investigator) to study complex behaviors or functions that are of interest in mammals is twofold. 1) The simplicity of the fly can provide a map that is useful to guide studies in mammals. 2) The evolutionary distance between mammals and flies provides a comparative perspective. If we find that functions or pathways are conserved, this reinforces the conclusion that these are particularly efficient and advantageous. Where they differ, we gain new ideas about alternative solutions to the same survival challenge.

	ACKNOWLEDGEMENTS

The author is grateful to Drs. Amita Sehgal and Cindy Otto for generous donation of time and helpful comments, and to Dr. Allan Pack for continuing support in all scientific endeavors.

	FOOTNOTES

This work was supported in part by National Institutes of Health Grants SCOR HL-60287 and P01 AG-17628.

Address for reprint requests and other correspondence: J. C. Hendricks, 991 Maloney Bldg., Center for Sleep and Respiratory Neurobiology, Univ. of Pennsylvania, 3600 Spruce St., Philadelphia, PA 19104 (E-mail: jch{at}mail.med.upenn.edu).

10.1152/japplphysiol.00904.2002

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