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Sleep and the fruit fly
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Opinion
TRENDS in Neurosciences Vol.24 No.3 March 2001
Sleep and the fruit fly Ralph J. Greenspan, Giulio Tononi, Chiara Cirelli and Paul J. Shaw
deprivation results in death in ~17 days, about as long as it takes to die from food deprivation. Remarkably, the cause of death remains unknown in spite of much effort to identify it. Thus, discovering the functions of sleep constitutes a challenge for biological research in general and for neuroscience in particular. Flies – a possible solution
The function of sleep remains a long-standing mystery in neurobiology. The presence of a sleep-like state has recently been demonstrated in the fruit fly, Drosophila melanogaster, meeting the essential behavioral criteria for sleep and also showing pharmacological and molecular correlates of mammalian sleep. This development opens up the possibility of applying genetic analysis to the identification of key molecular components of sleep. A mutant of monoamine metabolism has already been found to affect the homeostatic regulation of sleeplike behavior in the fly. The record of Drosophila in laying the foundations for subsequent studies in mammals argues in favor of the force of this new approach.
Fruit flies and neurobiologists have had a dysfunctional relationship for many years. Since its debut in the field during the late 1960s, Drosophila has been occasionally embraced, but often rejected, as a fitting subject of research. Yet it persists in coming back for more. The homology of its genes to those of mammals is now well demonstrated1, but the similarity of its behaviors and higher brain functions is still viewed skeptically by many. The recent observation of sleep-like behavior in Drosophila signals a new approach to a previously intractable problem of higher brain function and augurs a new era of fly relevance to human neurobiology. Sleep – the problem
Ralph J. Greenspan* Giulio Tononi Chiara Cirelli Paul J. Shaw The Neurosciences Institute, 10640 John Jay Hopkins Dr., San Diego, CA 92121, USA. *e-mail: greenspan@ nsi.edu
Sleep is an essential biological process whose function remains unknown in spite of decades of vigorous research. Although many interesting hypotheses have been proposed, none have been comprehensive enough to gain general acceptance2. Nonetheless, sleep is believed to be important for several reasons. It is ubiquitous, having been identified in all species of mammals, birds, and reptiles that have been properly investigated3. Furthermore, sleep has persevered even though it is a potentially costly behavior. During sleep, an animal cannot forage for food, take care of its young, procreate or avoid the dangers of predation, indicating that in comparison to these essential behaviors, sleep must serve an important function. Moreover, the evolution of remarkable adaptations, including sleep restricted to one brain hemisphere at a time in some marine mammals and birds, supports the observation that it might not be feasible to survive without sleep, even in potentially life-threatening situations. The importance of sleep is also suggested by prolonged sleep deprivation experiments in both humans and rats. After just one day of sleep deprivation in humans, the maintenance of waking becomes increasingly difficult and the pressure to sleep becomes overwhelming. In rats, chronic total sleep
The utility of the fruit fly to study sleep depends on whether it actually sleeps and, if so, on whether sleep is a phenomenon that will be amenable to genetic analysis. The universal way to define sleep is by behavioral criteria3,4. These include prolonged periods of quiescence; a reduced responsiveness to external stimuli; rapid reversibility, which distinguishes sleep, for example, from hibernation or coma; and homeostatic regulation – the increased need for sleep that follows sleep deprivation. Another characteristic of sleep is its independent regulation that is not merely tied to the circadian clock. This refers to the fact that sleep is clearly a circadian rhythm, but has more complex regulation. Recently, an additional characteristic has been provided as a result of the systematic screening of gene expression, where it has been shown that the expression of certain genes is higher in waking than in sleep in many brain regions5.
‘During sleep, an animal cannot forage for food, take care of its young, procreate or avoid the dangers of predation, …sleep must serve an indicating… important function.’ Using these criteria, we and others have evaluated rest in Drosophila melanogaster to determine if it could be used as an effective model system for studying sleep. Careful observations of fly behavior using visual, infrared, and ultrasound activity monitoring systems6,7 have shown that Drosophila exhibit consolidated periods of both activity and more importantly rest (Fig. 1). During periods of quiescence that last five minutes or longer, flies are unresponsive to mild external stimuli but can be quickly aroused with stronger stimulation. Furthermore, when the flies are individually deprived of a night’s rest by gently tapping on their containers, they exhibit a large compensatory increase in rest the next day (Fig. 1). This homeostatic rest ‘rebound’ appears to be correlated with the loss of rest, not with the deprivation stimuli or with the increase in motor activity per se. Another striking similarity between mammalian sleep and fly rest is that the young need more of it than the old, and it is reduced and fragmented in older flies. Moreover, adenosine
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Fig. 1. (a) Representative activity record of a single fly maintained on a 12:12 hr light (horizontal white bar): dark (horizontal black bar) cycle. Activity was monitored on day one with the infrared system (black) and then the same fly was evaluated again on day two with the highresolution ultrasound system (gray). Both systems were in good agreement. Activity predominates during the light phase, whereas consolidated periods of rest predominate during the dark phase. (b) Rest was monitored using the infrared system. The amount of rest in mins (mean ± SEM) is shown for each hour of a 24 hr day. During baseline flies rest little during the light phase, obtaining ~90% of their daily rest during the dark period (white circles). After 12 hr of rest deprivation, by gentle handling, during the dark period (black squares), flies show a large increase in rest compared with baseline.
TRENDS in Neurosciences Vol.24 No.3 March 2001
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antagonists such as caffeine increase waking6,7, whereas antihistamines increase rest and reduce its latency7. These pharmacological similarities are somewhat surprising and warrant further investigation in order to determine the extent of conservation. In addition to the behavioral, ontogenetic and pharmacological studies described above, we also identified molecular correlates of behavioral state in Drosophila, as has been previously reported for mammals5. Approximately 10 000 RNA species were screened in extracts of fly heads from resting, awake, or rest-deprived flies. In agreement with previous results in the rat, the majority of transcripts were not modulated by the rest-activity cycle. However, a minority of genes were differentially expressed during rest and waking, irrespective of circadian factors. Most significantly, we found that several genes that were upregulated during waking versus rest in the fly correspond to genes that are upregulated during waking versus sleep in the rat (Table 1). Altogether, behavioral, ontogenetic, pharmacological, and molecular criteria indicate that rest in Drosophila shares many of the critical features of mammalian sleep.
Table 1. Gene expression correlates of waking versus rest/sleep Gene
Gene expression in waking versus rest/sleepb
Gene expression in deprivation versus rest/sleepb
Fly
Rat
Fly
Rat
BiP (Hsc70-3)
+
+
++
++
Cytochrome oxidase C
+
++
++
+
Arylalkylamine N-acetyltransferase (Dat)a
+
+
+
Arylsulfotransferasea
+
Fatty acid synthase
++
+
Cytochrome P450 (Cyp4e2)
++
+
aInvolved b+
in the catabolism of monoamines. and ++ indicate relative magnitude of change from waking to rest/sleep.
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One of the genes exhibiting state-dependent expression is an enzyme involved in the catabolism of serotonin, dopamine, and octopamine, Arylalkyamine N-acetyl transferase (aaNAT1), also called Dopamine acetyltransferase (Dat) (Refs 8,9). In comparison with the rest state, its expression is ~70% higher during waking and ~50% higher after rest deprivation. By contrast, Dat mRNA is not regulated in a circadian manner7,10. The state-dependent modulation of an enzyme involved in the catabolism of monoamines has precedent in mammalian sleep studies. In mammals, monoaminergic activity is high during waking, declines dramatically during non-rapid eye movement (NREM) sleep, and is virtually absent during rapid eye movement (REM) sleep11,12. This has led to the suggestion that sleep might serve to counteract the effects of continued monoaminergic discharge during waking. According to this hypothesis, an impaired catabolism of monoamines should result in an increased need for sleep13,14. Indeed, several hours of sleep deprivation, which are associated with a homeostatic response, result in an increase in the mRNA for arylsulfotransferase (AST), an enzyme implicated in the catabolism of monoamines in the rat15. Moreover, AST levels increase further following prolonged sleep deprivation lasting several days. Flies homozygous for a mutation at the Dopamine acetyltransferase locus (Datlo) (Refs 8,10) display a rest rebound that is greater than rest-deprived controls (Fig. 2). By contrast, their baseline amounts of activity and rest are normal. These homozygotes show an ~90% reduction in one of two Dat mRNA isoforms that results in an ~70% reduction in enzyme activity10. By constructing flies with even less Dat activity, in which the mutant allele is placed heterozygous with a chromosomal deletion that completely removes the locus (Datlo/Df), we observed an even larger and longer lasting homeostatic response (Fig. 2). The proportionality of sleep rebound to amount of Dat activity in the fly argues, at a minimum, for a modulatory role of monoamines in the homeostatic response. Fly mutants have also made it possible to test the independence of sleep mechanisms from the circadian clock7. In mammals the independence of these two systems has been demonstrated by evaluating sleep following lesions of the suprachiasmatic nucleus (SCN), the site of the principal circadian pacemaker. Following such lesions, sleep no longer shows a circadian organization4. When SCN-lesioned animals are deprived of sleep they exhibit an increase in sleep duration and intensity, indicating that homeostatic mechanisms are not dependent upon an intact circadian system. In the fly, it is not necessary to ablate the structure responsible for the generation of the circadian rhythm because the contribution of the clock can be evaluated in mutant but anatomically intact animals. Arrhythmic mutants of the period locus16, which regulates circadian rhythms, have the
TRENDS in Neurosciences Vol.24 No.3 March 2001
(a) Rest (mins)
Fig. 2. (a) The amount of rest in mins is shown for each hour of a 24 hr day for flies homozygous for a mutation at the Dopamine acetyltransferase locus (Dat lo ) and for flies carrying the Dat lo mutation heterozygous with a chromosomal deletion that completely removes the locus (Dat lo/Df). During baseline Dat lo and Dat lo/Df flies obtained similar amounts of rest (pooled data open circles). Following 12 hr of rest deprivation with the automated system, Dat lo flies display a rest rebound that is greater compared with baseline (black squares) and restdeprived controls (data not shown). In Dat lo/Df flies, an even larger and longer lasting homeostatic response was observed (gray triangles). (b) Under constant darkness, arrhythmic per 01 flies had the same amount of rest compared with light–dark conditions (open circles). Following 6 hrs of rest deprivation by gentle tapping (gray bar) there is a significant increase in rest during the first 6 hrs of recovery (black squares) compared with baseline.
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same amount of rest under constant darkness compared with light–dark conditions, but rest is evenly distributed across the 24 hr day. Most importantly, twelve hours of rest deprivation results in a significant increase in rest during recovery compared with baseline (Fig. 2), thus demonstrating that the homeostatic response is intact in the absence of a circadian rhythm. The genetic approach
The demonstration of a sleep-like state in Drosophila opens the way for a genetic assault on the problem of the function of sleep. The suitability of a genetic approach can only be proven by doing it successfully, but the question merits discussion nonetheless. If sleep is primarily a change in physiological state, it is not obvious that such a change will have genetically separable components. Similar doubts preceded two earlier cases in which Drosophila genetics were applied to behaviors of relevance to humans, namely circadian rhythms and learning. In both cases, the expectation was that mutations in many genes unrelated to the primary mechanisms would produce spurious behavioral defects, or that such mutants would inevitably be lethal and thus unanalyzable (discussed in Ref. 17). Neither scenario was borne out. Instead, the mutants opened the way to fundamental insights into the mechanisms of these complex phenomena16,18. With specific reference to sleep, there are several human genetic disorders whose effects are relatively restricted to sleep19, and there are strain variations in mice for the amounts of the various stages of sleep20. Moreover, the demonstration of state-dependent changes in gene expression in the brain associated with sleep, waking and sleep deprivation5,15,21–23 argues in favor of a genetic component. Similarly, the identification of hypocretin/orexin and its receptor as the genes http://tins.trends.com
responsible for murine and canine models of hereditary narcolepsy24,25, while not necessarily central to the function of sleep, demonstrate the feasibility of obtaining sleep mutants. In the past, genetic assaults on difficult problems have proven most valuable in providing the first opening into questions that were otherwise inaccessible (‘Genetics are the shock-troops of biology’26). In the face of skepticism about the feasibility of such an approach17, Konopka and Benzer isolated the first circadian rhythm mutants and identified the period gene27, thus setting the stage for cracking open the mechanism of circadian rhythms. The delineation of the clock as a transcriptional feedback loop with time delays in translation and transport back into the nucleus was accomplished through a multi-disciplinary approach, which depended heavily on the continued isolation of mutants to identify key genes16,28. The fly has subsequently served as both the paradigm and the source of DNA sequences for genetic and molecular studies of mammalian circadian rhythms29. In the realm of learning and memory, there were questions as to whether the fly was even capable of learning, let alone whether one could obtain informative mutants (discussed in Ref. 17). The demonstration of associative conditioning30,31, with its attendant phenomena of temporal memory phases32 and contextual learning33, established the fly as a bona fide learner, if not a genius. The isolation of dunce17 represented the first in a sporadic series of mutations affecting various components of acquisition and retention, some of which alter the cAMP signal transduction pathway, others of which affect development of the pertinent brain structures18. These studies have served as a paradigm for mammalian studies of memory consolidation34. The key to the success of this approach is the ability to isolate mutants without bias as to the genes involved, their level of expression, or the extent of their pleiotropic involvement in other processes. These features distinguish it from the ‘knockout’ approach in mice. As it turned out, none of the pertinent genes were known beforehand for rhythms and few for learning. Doubts about the possibility of obtaining mutants that were restricted enough in their phenotype to be informative were subsequently dispelled by two important observations. First, these behaviors appear relatively rarely in mutagenesis screens, indicating that not just any mutation will impair the behavior35. Second, tests of a wide range of existing mutants producing a variety of metabolic, developmental and physiological defects showed normal learning, indicating robustness and resistance to trivial mutant effects36. This is not the same as saying that there are dedicated genes for rhythms or for learning and memory – virtually all of
Opinion
TRENDS in Neurosciences Vol.24 No.3 March 2001
these genes have been shown to affect other additional processes. Many result in lethality if completely knocked out but produce restricted, informative phenotypes if less severely mutated37,38. The obvious question that arises from the foregoing is what will sleep mutants look like? We already have a clue from the Dat mutant that the homeostatic regulation of sleep might be selectively effected, leading to an altered rebound after deprivation in the absence of other changes. Beyond this, we can only speculate, but it might be instructive to consider some of the relevant features of mutants affecting rhythms and learning. The time-keeping function of the circadian clock is a cell-autonomous mechanism. This might not apply to all manifestations of rhythmicity in the nervous system, some of which might require communication between cells, but it accurately describes the basic clock mechanism itself. Thus, mutations in clock components are less complicated by obligate cell interactions than if the fundamental time-keeping mechanism were multicellular. Neuronal plasticity, the underlying mechanism that is altered in learning mutants, is also cell-autonomous in the sense that alterations in the signaling characteristics of circuits stimulated by training occur autonomously at References 1 Rubin, G.M. et al. (2000) Comparative genomics of the eukaryotes. Science 287, 2204–2215 2 Rechtschaffen, A. (1998) Current perspectives on the function of sleep. Perspect. Biol. Med. 41, 359–390 3 Campbell, S.S. and Tobler, I. (1984) Animal sleep: a review of sleep duration across phylogeny. Neurosci. Biobehav. Rev. 8, 269–300 4 Tobler, I. (2000) Phylogeny of sleep regulation. In Principles and Practice of Sleep Medicine (3rd edn) (Kryger, M.H. et al. eds), Saunders 5 Cirelli, C. and Tononi, G. (1999) Differences in brain gene expression between sleep and waking as revealed by mRNA differential display and cDNA microarray technology. J. Sleep Res. 8 (Suppl. 1), 44–52 6 Hendricks, J.C. et al. (2000) Rest in Drosophila is a sleep-like state. Neuron 25, 129–138 7 Shaw, P.J. et al. (2000) Correlates of sleep and waking in Drosophila melanogaster. Science 287, 183–1837 8 Maranda, B. and Hodgetts, R. (1977) A characterization of dopamine acetyltransferase in Drosophila melanogaster. Insect Biochem. 7, 33–43 9 Hintermann, E. et al. (1996) Cloning of an arylalkylamine N-acetyltransferase (aaNAT1) from Drosophila melanogaster expressed in the nervous system and the gut. Proc. Natl. Acad. Sci. U. S. A. 93, 12315–12320 10 Brodbeck, D. et al. (1998) Molecular and biochemical characterization of the aaNAT1 (Dat) locus in Drosophila melanogaster, differential expression of two gene products. DNA Cell Biol. 17, 621–633 11 McGinty, D.J. and Harper, R.M. (1976) Dorsal raphe neurons, depression of firing during sleep in cats. Brain Res. 101, 569–575 12 Aston-Jones, G. and Bloom, F.E. (1981) Activity of norepinephrine-containing locus coeruleus http://tins.trends.com
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16 17 18
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25
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individual synapses. The mutant screens might actually have selected for mutations that are relatively restricted to effects on the rate-limiting synapses involved in learning. These would be the cells experiencing the greatest plasticity and thus exerting the largest influence on phenotype. Mutations in genes that affect these cells selectively could be tolerated and still produce a relatively specific phenotype. It is possible that sleep is not a cell autonomous process but instead is a coordinated change in the state of the whole brain. If true, this will certainly have correlates at the individual cellular level, but unlike the clock and plasticity, it might require obligate interactions between cells to be initiated or maintained. Will this hinder the prospects for isolating sleep mutants? No more so than it has for development, which also depends on obligate cell interactions and for which myriad mutants with specific phenotypes have been isolated. In conclusion, we believe that the possibilities for making new inroads into the long-standing mystery of why we sleep have now been improved with the enlisting of the fruit fly. Once again flies are proving that they are more like us than one might think.
neurons in behaving rats anticipates fluctuations in the sleep-waking cycle. J. Neurosci. 1, 876–886 Siegel, J.M. and Rogawski, M.A. (1988) A function for REM sleep: regulation of noradrenergic receptor sensitivity. Brain Res. 472, 213–233 Jouvet, M. (1999) Sleep and serotonin, an unfinished story. Neuropsychopharmacology 21 (Suppl. 2), 24S–27S Cirelli, C. and Tononi, G. (1998) Changes in gene expression after long-term sleep deprivation in rats. Soc. Neurosci. Abstr. 24, 1430 Hall, J.C. (1998) Genetics of biological rhythms in Drosophila. Adv. Genet. 38, 135–184 Greenspan, R.J. (1990) The emergence of neurogenetics. Semin. Neurosci. 2, 145–157 Dubnau, J. and Tully, T. (1998) Gene discovery in Drosophila, new insights for learning and memory. Ann. Rev. Neurosci. 21, 407–444 Parkes, J.D. (1999) Genetic factors in human sleep disorders with special reference to Norrie disease, Prader-Willi syndrome and Moebius syndrome. J. Sleep Res. 8 (Suppl.), 14–22 Franken, P. et al. (1999) Genetic determinants of sleep regulation in inbred mice. Sleep 22, 155–169 Rhyner, T.A. et al. (1990) Molecular cloning of forebrain mRNAs which are modulated by sleep deprivation. Eur. J. Neurosci. 2, 1063–1073 Cirelli, C. et al. (1993) Fos-like immunoreactivity in the rat brain in spontaneous wakefulness and sleep. Arch. Ital. Biol. 131, 327–330 Pompeiano, M. et al. (1994) Immediate-early genes in spontaneous wakefulness and sleep, expression of c-fos and NGFI-A mRNA and protein. J. Sleep Res. 3, 80–96 Chemelli, R.M. et al. (1999) Narcolepsy in orexin knockout mice, molecular genetics of sleep regulation. Cell 8, 437–451 Lin, L. et al. (1999) The sleep disorder canine narcolepsy is caused by a mutation in the hypocretin (orexin) receptor 2 gene. Cell 98, 365–376
26 Edgar, R.S. and Epstein, R.H. (1965) The genetics of a bacterial virus. Sci. Am. 212, 70–76 27 Konopka, R.J. and Benzer, S. (1971) Clock mutants of Drosophila melanogaster. Proc. Natl. Acad. Sci. U. S. A. 68, 2112–2116 28 Young, M.W. (1998) The molecular control of circadian behavioral rhythms and their entrainment in Drosophila. Annu. Rev. Biochem. 67, 135–152 29 Wilsbacher, L.D. and Takahashi, J.S. (1998) Circadian rhythms molecular basis of the clock. Curr. Opin. Genet. Dev. 8, 595–602 30 Quinn, W.G. et al. (1974) Conditioned behavior in Drosophila melanogaster. Proc. Natl. Acad. Sci. U. S. A. 71, 708–712 31 Tully, T. and Quinn, W.G. (1985) Classical conditioning and retention in normal and mutant Drosophila melanogaster. J. Comp. Physiol. 157, 263–277 32 Tully, T. et al. (1994) Genetic dissection of consolidated memory in Drosophila. Cell 79, 35–47 33 Liu, L. et al. (1999) Context generalization in Drosophila visual learning requires the mushroom bodies. Nature 400, 753–756 34 Mayford, M. and Kandel, E.R. (1999) Genetic approaches to memory storage. Trends Genet. 15, 463–470 35 Hall, J.C. (1990) Genetics of circadian rhythms. Annu. Rev. Genet. 24, 659–697 36 Dudai, Y. (1977) Properties of learning and memory in Drosophila melanogaster. J. Comp. Physiol. 114, 69–90 37 Hall, J.C. (1994) Pleiotropy of behavioral genes. In Flexibility and Constraint in Behavioral Systems (Greenspan, R.J. and Kyriacou. C.P. eds), Dahlem Konferenzen Publications. 38 Greenspan, R.J. (1997) A kinder, gentler genetic analysis of behavior: Dissection gives way to modulation. Curr. Opin. Neurobiol. 7, 805–811
Opinion
TRENDS in Neurosciences Vol.24 No.3 March 2001
Sleep and the fruit fly Ralph J. Greenspan, Giulio Tononi, Chiara Cirelli and Paul J. Shaw
deprivation results in death in ~17 days, about as long as it takes to die from food deprivation. Remarkably, the cause of death remains unknown in spite of much effort to identify it. Thus, discovering the functions of sleep constitutes a challenge for biological research in general and for neuroscience in particular. Flies – a possible solution
The function of sleep remains a long-standing mystery in neurobiology. The presence of a sleep-like state has recently been demonstrated in the fruit fly, Drosophila melanogaster, meeting the essential behavioral criteria for sleep and also showing pharmacological and molecular correlates of mammalian sleep. This development opens up the possibility of applying genetic analysis to the identification of key molecular components of sleep. A mutant of monoamine metabolism has already been found to affect the homeostatic regulation of sleeplike behavior in the fly. The record of Drosophila in laying the foundations for subsequent studies in mammals argues in favor of the force of this new approach.
Fruit flies and neurobiologists have had a dysfunctional relationship for many years. Since its debut in the field during the late 1960s, Drosophila has been occasionally embraced, but often rejected, as a fitting subject of research. Yet it persists in coming back for more. The homology of its genes to those of mammals is now well demonstrated1, but the similarity of its behaviors and higher brain functions is still viewed skeptically by many. The recent observation of sleep-like behavior in Drosophila signals a new approach to a previously intractable problem of higher brain function and augurs a new era of fly relevance to human neurobiology. Sleep – the problem
Ralph J. Greenspan* Giulio Tononi Chiara Cirelli Paul J. Shaw The Neurosciences Institute, 10640 John Jay Hopkins Dr., San Diego, CA 92121, USA. *e-mail: greenspan@ nsi.edu
Sleep is an essential biological process whose function remains unknown in spite of decades of vigorous research. Although many interesting hypotheses have been proposed, none have been comprehensive enough to gain general acceptance2. Nonetheless, sleep is believed to be important for several reasons. It is ubiquitous, having been identified in all species of mammals, birds, and reptiles that have been properly investigated3. Furthermore, sleep has persevered even though it is a potentially costly behavior. During sleep, an animal cannot forage for food, take care of its young, procreate or avoid the dangers of predation, indicating that in comparison to these essential behaviors, sleep must serve an important function. Moreover, the evolution of remarkable adaptations, including sleep restricted to one brain hemisphere at a time in some marine mammals and birds, supports the observation that it might not be feasible to survive without sleep, even in potentially life-threatening situations. The importance of sleep is also suggested by prolonged sleep deprivation experiments in both humans and rats. After just one day of sleep deprivation in humans, the maintenance of waking becomes increasingly difficult and the pressure to sleep becomes overwhelming. In rats, chronic total sleep
The utility of the fruit fly to study sleep depends on whether it actually sleeps and, if so, on whether sleep is a phenomenon that will be amenable to genetic analysis. The universal way to define sleep is by behavioral criteria3,4. These include prolonged periods of quiescence; a reduced responsiveness to external stimuli; rapid reversibility, which distinguishes sleep, for example, from hibernation or coma; and homeostatic regulation – the increased need for sleep that follows sleep deprivation. Another characteristic of sleep is its independent regulation that is not merely tied to the circadian clock. This refers to the fact that sleep is clearly a circadian rhythm, but has more complex regulation. Recently, an additional characteristic has been provided as a result of the systematic screening of gene expression, where it has been shown that the expression of certain genes is higher in waking than in sleep in many brain regions5.
‘During sleep, an animal cannot forage for food, take care of its young, procreate or avoid the dangers of predation, …sleep must serve an indicating… important function.’ Using these criteria, we and others have evaluated rest in Drosophila melanogaster to determine if it could be used as an effective model system for studying sleep. Careful observations of fly behavior using visual, infrared, and ultrasound activity monitoring systems6,7 have shown that Drosophila exhibit consolidated periods of both activity and more importantly rest (Fig. 1). During periods of quiescence that last five minutes or longer, flies are unresponsive to mild external stimuli but can be quickly aroused with stronger stimulation. Furthermore, when the flies are individually deprived of a night’s rest by gently tapping on their containers, they exhibit a large compensatory increase in rest the next day (Fig. 1). This homeostatic rest ‘rebound’ appears to be correlated with the loss of rest, not with the deprivation stimuli or with the increase in motor activity per se. Another striking similarity between mammalian sleep and fly rest is that the young need more of it than the old, and it is reduced and fragmented in older flies. Moreover, adenosine
http://tins.trends.com 0166-2236/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0166-2236(00)01719-7
Opinion
20 10 0 00.00 04.00 08.00 12.00 16.00 20.00 Time (hrs)
3000 2000 1000 0
Ultrasound
Infrared
(a)
(b) Rest (min)
Fig. 1. (a) Representative activity record of a single fly maintained on a 12:12 hr light (horizontal white bar): dark (horizontal black bar) cycle. Activity was monitored on day one with the infrared system (black) and then the same fly was evaluated again on day two with the highresolution ultrasound system (gray). Both systems were in good agreement. Activity predominates during the light phase, whereas consolidated periods of rest predominate during the dark phase. (b) Rest was monitored using the infrared system. The amount of rest in mins (mean ± SEM) is shown for each hour of a 24 hr day. During baseline flies rest little during the light phase, obtaining ~90% of their daily rest during the dark period (white circles). After 12 hr of rest deprivation, by gentle handling, during the dark period (black squares), flies show a large increase in rest compared with baseline.
TRENDS in Neurosciences Vol.24 No.3 March 2001
60 40 20 0 00.00 04.00 08.00 12.00 16.00 20.00 24.00 Time (hrs) TRENDS in Neurosciences
antagonists such as caffeine increase waking6,7, whereas antihistamines increase rest and reduce its latency7. These pharmacological similarities are somewhat surprising and warrant further investigation in order to determine the extent of conservation. In addition to the behavioral, ontogenetic and pharmacological studies described above, we also identified molecular correlates of behavioral state in Drosophila, as has been previously reported for mammals5. Approximately 10 000 RNA species were screened in extracts of fly heads from resting, awake, or rest-deprived flies. In agreement with previous results in the rat, the majority of transcripts were not modulated by the rest-activity cycle. However, a minority of genes were differentially expressed during rest and waking, irrespective of circadian factors. Most significantly, we found that several genes that were upregulated during waking versus rest in the fly correspond to genes that are upregulated during waking versus sleep in the rat (Table 1). Altogether, behavioral, ontogenetic, pharmacological, and molecular criteria indicate that rest in Drosophila shares many of the critical features of mammalian sleep.
Table 1. Gene expression correlates of waking versus rest/sleep Gene
Gene expression in waking versus rest/sleepb
Gene expression in deprivation versus rest/sleepb
Fly
Rat
Fly
Rat
BiP (Hsc70-3)
+
+
++
++
Cytochrome oxidase C
+
++
++
+
Arylalkylamine N-acetyltransferase (Dat)a
+
+
+
Arylsulfotransferasea
+
Fatty acid synthase
++
+
Cytochrome P450 (Cyp4e2)
++
+
aInvolved b+
in the catabolism of monoamines. and ++ indicate relative magnitude of change from waking to rest/sleep.
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One of the genes exhibiting state-dependent expression is an enzyme involved in the catabolism of serotonin, dopamine, and octopamine, Arylalkyamine N-acetyl transferase (aaNAT1), also called Dopamine acetyltransferase (Dat) (Refs 8,9). In comparison with the rest state, its expression is ~70% higher during waking and ~50% higher after rest deprivation. By contrast, Dat mRNA is not regulated in a circadian manner7,10. The state-dependent modulation of an enzyme involved in the catabolism of monoamines has precedent in mammalian sleep studies. In mammals, monoaminergic activity is high during waking, declines dramatically during non-rapid eye movement (NREM) sleep, and is virtually absent during rapid eye movement (REM) sleep11,12. This has led to the suggestion that sleep might serve to counteract the effects of continued monoaminergic discharge during waking. According to this hypothesis, an impaired catabolism of monoamines should result in an increased need for sleep13,14. Indeed, several hours of sleep deprivation, which are associated with a homeostatic response, result in an increase in the mRNA for arylsulfotransferase (AST), an enzyme implicated in the catabolism of monoamines in the rat15. Moreover, AST levels increase further following prolonged sleep deprivation lasting several days. Flies homozygous for a mutation at the Dopamine acetyltransferase locus (Datlo) (Refs 8,10) display a rest rebound that is greater than rest-deprived controls (Fig. 2). By contrast, their baseline amounts of activity and rest are normal. These homozygotes show an ~90% reduction in one of two Dat mRNA isoforms that results in an ~70% reduction in enzyme activity10. By constructing flies with even less Dat activity, in which the mutant allele is placed heterozygous with a chromosomal deletion that completely removes the locus (Datlo/Df), we observed an even larger and longer lasting homeostatic response (Fig. 2). The proportionality of sleep rebound to amount of Dat activity in the fly argues, at a minimum, for a modulatory role of monoamines in the homeostatic response. Fly mutants have also made it possible to test the independence of sleep mechanisms from the circadian clock7. In mammals the independence of these two systems has been demonstrated by evaluating sleep following lesions of the suprachiasmatic nucleus (SCN), the site of the principal circadian pacemaker. Following such lesions, sleep no longer shows a circadian organization4. When SCN-lesioned animals are deprived of sleep they exhibit an increase in sleep duration and intensity, indicating that homeostatic mechanisms are not dependent upon an intact circadian system. In the fly, it is not necessary to ablate the structure responsible for the generation of the circadian rhythm because the contribution of the clock can be evaluated in mutant but anatomically intact animals. Arrhythmic mutants of the period locus16, which regulates circadian rhythms, have the
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Fig. 2. (a) The amount of rest in mins is shown for each hour of a 24 hr day for flies homozygous for a mutation at the Dopamine acetyltransferase locus (Dat lo ) and for flies carrying the Dat lo mutation heterozygous with a chromosomal deletion that completely removes the locus (Dat lo/Df). During baseline Dat lo and Dat lo/Df flies obtained similar amounts of rest (pooled data open circles). Following 12 hr of rest deprivation with the automated system, Dat lo flies display a rest rebound that is greater compared with baseline (black squares) and restdeprived controls (data not shown). In Dat lo/Df flies, an even larger and longer lasting homeostatic response was observed (gray triangles). (b) Under constant darkness, arrhythmic per 01 flies had the same amount of rest compared with light–dark conditions (open circles). Following 6 hrs of rest deprivation by gentle tapping (gray bar) there is a significant increase in rest during the first 6 hrs of recovery (black squares) compared with baseline.
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same amount of rest under constant darkness compared with light–dark conditions, but rest is evenly distributed across the 24 hr day. Most importantly, twelve hours of rest deprivation results in a significant increase in rest during recovery compared with baseline (Fig. 2), thus demonstrating that the homeostatic response is intact in the absence of a circadian rhythm. The genetic approach
The demonstration of a sleep-like state in Drosophila opens the way for a genetic assault on the problem of the function of sleep. The suitability of a genetic approach can only be proven by doing it successfully, but the question merits discussion nonetheless. If sleep is primarily a change in physiological state, it is not obvious that such a change will have genetically separable components. Similar doubts preceded two earlier cases in which Drosophila genetics were applied to behaviors of relevance to humans, namely circadian rhythms and learning. In both cases, the expectation was that mutations in many genes unrelated to the primary mechanisms would produce spurious behavioral defects, or that such mutants would inevitably be lethal and thus unanalyzable (discussed in Ref. 17). Neither scenario was borne out. Instead, the mutants opened the way to fundamental insights into the mechanisms of these complex phenomena16,18. With specific reference to sleep, there are several human genetic disorders whose effects are relatively restricted to sleep19, and there are strain variations in mice for the amounts of the various stages of sleep20. Moreover, the demonstration of state-dependent changes in gene expression in the brain associated with sleep, waking and sleep deprivation5,15,21–23 argues in favor of a genetic component. Similarly, the identification of hypocretin/orexin and its receptor as the genes http://tins.trends.com
responsible for murine and canine models of hereditary narcolepsy24,25, while not necessarily central to the function of sleep, demonstrate the feasibility of obtaining sleep mutants. In the past, genetic assaults on difficult problems have proven most valuable in providing the first opening into questions that were otherwise inaccessible (‘Genetics are the shock-troops of biology’26). In the face of skepticism about the feasibility of such an approach17, Konopka and Benzer isolated the first circadian rhythm mutants and identified the period gene27, thus setting the stage for cracking open the mechanism of circadian rhythms. The delineation of the clock as a transcriptional feedback loop with time delays in translation and transport back into the nucleus was accomplished through a multi-disciplinary approach, which depended heavily on the continued isolation of mutants to identify key genes16,28. The fly has subsequently served as both the paradigm and the source of DNA sequences for genetic and molecular studies of mammalian circadian rhythms29. In the realm of learning and memory, there were questions as to whether the fly was even capable of learning, let alone whether one could obtain informative mutants (discussed in Ref. 17). The demonstration of associative conditioning30,31, with its attendant phenomena of temporal memory phases32 and contextual learning33, established the fly as a bona fide learner, if not a genius. The isolation of dunce17 represented the first in a sporadic series of mutations affecting various components of acquisition and retention, some of which alter the cAMP signal transduction pathway, others of which affect development of the pertinent brain structures18. These studies have served as a paradigm for mammalian studies of memory consolidation34. The key to the success of this approach is the ability to isolate mutants without bias as to the genes involved, their level of expression, or the extent of their pleiotropic involvement in other processes. These features distinguish it from the ‘knockout’ approach in mice. As it turned out, none of the pertinent genes were known beforehand for rhythms and few for learning. Doubts about the possibility of obtaining mutants that were restricted enough in their phenotype to be informative were subsequently dispelled by two important observations. First, these behaviors appear relatively rarely in mutagenesis screens, indicating that not just any mutation will impair the behavior35. Second, tests of a wide range of existing mutants producing a variety of metabolic, developmental and physiological defects showed normal learning, indicating robustness and resistance to trivial mutant effects36. This is not the same as saying that there are dedicated genes for rhythms or for learning and memory – virtually all of
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these genes have been shown to affect other additional processes. Many result in lethality if completely knocked out but produce restricted, informative phenotypes if less severely mutated37,38. The obvious question that arises from the foregoing is what will sleep mutants look like? We already have a clue from the Dat mutant that the homeostatic regulation of sleep might be selectively effected, leading to an altered rebound after deprivation in the absence of other changes. Beyond this, we can only speculate, but it might be instructive to consider some of the relevant features of mutants affecting rhythms and learning. The time-keeping function of the circadian clock is a cell-autonomous mechanism. This might not apply to all manifestations of rhythmicity in the nervous system, some of which might require communication between cells, but it accurately describes the basic clock mechanism itself. Thus, mutations in clock components are less complicated by obligate cell interactions than if the fundamental time-keeping mechanism were multicellular. Neuronal plasticity, the underlying mechanism that is altered in learning mutants, is also cell-autonomous in the sense that alterations in the signaling characteristics of circuits stimulated by training occur autonomously at References 1 Rubin, G.M. et al. (2000) Comparative genomics of the eukaryotes. Science 287, 2204–2215 2 Rechtschaffen, A. (1998) Current perspectives on the function of sleep. Perspect. Biol. Med. 41, 359–390 3 Campbell, S.S. and Tobler, I. (1984) Animal sleep: a review of sleep duration across phylogeny. Neurosci. Biobehav. Rev. 8, 269–300 4 Tobler, I. (2000) Phylogeny of sleep regulation. In Principles and Practice of Sleep Medicine (3rd edn) (Kryger, M.H. et al. eds), Saunders 5 Cirelli, C. and Tononi, G. (1999) Differences in brain gene expression between sleep and waking as revealed by mRNA differential display and cDNA microarray technology. J. Sleep Res. 8 (Suppl. 1), 44–52 6 Hendricks, J.C. et al. (2000) Rest in Drosophila is a sleep-like state. Neuron 25, 129–138 7 Shaw, P.J. et al. (2000) Correlates of sleep and waking in Drosophila melanogaster. Science 287, 183–1837 8 Maranda, B. and Hodgetts, R. (1977) A characterization of dopamine acetyltransferase in Drosophila melanogaster. Insect Biochem. 7, 33–43 9 Hintermann, E. et al. (1996) Cloning of an arylalkylamine N-acetyltransferase (aaNAT1) from Drosophila melanogaster expressed in the nervous system and the gut. Proc. Natl. Acad. Sci. U. S. A. 93, 12315–12320 10 Brodbeck, D. et al. (1998) Molecular and biochemical characterization of the aaNAT1 (Dat) locus in Drosophila melanogaster, differential expression of two gene products. DNA Cell Biol. 17, 621–633 11 McGinty, D.J. and Harper, R.M. (1976) Dorsal raphe neurons, depression of firing during sleep in cats. Brain Res. 101, 569–575 12 Aston-Jones, G. and Bloom, F.E. (1981) Activity of norepinephrine-containing locus coeruleus http://tins.trends.com
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individual synapses. The mutant screens might actually have selected for mutations that are relatively restricted to effects on the rate-limiting synapses involved in learning. These would be the cells experiencing the greatest plasticity and thus exerting the largest influence on phenotype. Mutations in genes that affect these cells selectively could be tolerated and still produce a relatively specific phenotype. It is possible that sleep is not a cell autonomous process but instead is a coordinated change in the state of the whole brain. If true, this will certainly have correlates at the individual cellular level, but unlike the clock and plasticity, it might require obligate interactions between cells to be initiated or maintained. Will this hinder the prospects for isolating sleep mutants? No more so than it has for development, which also depends on obligate cell interactions and for which myriad mutants with specific phenotypes have been isolated. In conclusion, we believe that the possibilities for making new inroads into the long-standing mystery of why we sleep have now been improved with the enlisting of the fruit fly. Once again flies are proving that they are more like us than one might think.
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26 Edgar, R.S. and Epstein, R.H. (1965) The genetics of a bacterial virus. Sci. Am. 212, 70–76 27 Konopka, R.J. and Benzer, S. (1971) Clock mutants of Drosophila melanogaster. Proc. Natl. Acad. Sci. U. S. A. 68, 2112–2116 28 Young, M.W. (1998) The molecular control of circadian behavioral rhythms and their entrainment in Drosophila. Annu. Rev. Biochem. 67, 135–152 29 Wilsbacher, L.D. and Takahashi, J.S. (1998) Circadian rhythms molecular basis of the clock. Curr. Opin. Genet. Dev. 8, 595–602 30 Quinn, W.G. et al. (1974) Conditioned behavior in Drosophila melanogaster. Proc. Natl. Acad. Sci. U. S. A. 71, 708–712 31 Tully, T. and Quinn, W.G. (1985) Classical conditioning and retention in normal and mutant Drosophila melanogaster. J. Comp. Physiol. 157, 263–277 32 Tully, T. et al. (1994) Genetic dissection of consolidated memory in Drosophila. Cell 79, 35–47 33 Liu, L. et al. (1999) Context generalization in Drosophila visual learning requires the mushroom bodies. Nature 400, 753–756 34 Mayford, M. and Kandel, E.R. (1999) Genetic approaches to memory storage. Trends Genet. 15, 463–470 35 Hall, J.C. (1990) Genetics of circadian rhythms. Annu. Rev. Genet. 24, 659–697 36 Dudai, Y. (1977) Properties of learning and memory in Drosophila melanogaster. J. Comp. Physiol. 114, 69–90 37 Hall, J.C. (1994) Pleiotropy of behavioral genes. In Flexibility and Constraint in Behavioral Systems (Greenspan, R.J. and Kyriacou. C.P. eds), Dahlem Konferenzen Publications. 38 Greenspan, R.J. (1997) A kinder, gentler genetic analysis of behavior: Dissection gives way to modulation. Curr. Opin. Neurobiol. 7, 805–811