Download Using Drosophila to Understand the Genetics of Circadian Rhythms

REVIEW
Why a Fly? Using Drosophila to Understand the Genetics of Circadian Rhythms
and Sleep
Joan C. Hendricks, VMD, PhD1; Amita Sehgal, PhD1,2
1Center
for Sleep and Respiratory Neurobiology, 2Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, PA
Abstract: Among simple model systems, Drosophila has specific advantages for neurobehavioral investigations. It has been particularly useful for
understanding the molecular basis of circadian rhythms. In addition, the
genetics of fruit-fly sleep are beginning to develop. This review summarizes the current state of understanding of circadian rhythms and sleep in
the fruit fly for the readers of Sleep. We note where information is available in mammals, for comparison with findings in fruit flies, to provide an
evolutionary perspective, and we focus on recent findings and new ques-
tions. We propose that sleep-specific neural activity may alter cellular
function and thus accomplish the restorative function or functions of sleep.
In conclusion, we sound some cautionary notes about some of the complexities of working with this “simple” organism.
Citation: Hendricks JC; Sehgal A. Why a fly? Using Drosophila to understand the genetics of circadian rhythms and sleep. SLEEP
2004;27(2):334-42.
INTRODUCTION
gene silencing by expressing RNAi to interfere with specific gene products are also being used with increasing success in adult flies.6-8 This is
helpful because using mutants to analyze adult phenotypes has a number
of logical limits, despite their proven utility. In using a mutant adult to
study behavior, one must avoid making the unwarranted assumption that
any interesting phenotype is due to the absence of the gene product at the
time of the assay, without regard to developmental roles for the gene
product. This disadvantage also applies to many transgenic systems if
the gene is expressed or misexpressed throughout development. Further,
genes whose products are needed for normal development may have a
different role in adulthood that cannot be discerned if development is
prevented or grossly altered by a mutation or transgene that alters lifelong gene expression. Thus, these new tools should be particularly helpful for dissecting the genetics of sleep, as for other studies of adult
behavior. The fact that the human, mouse, and fly genomes are publicly
available and that reagents for the laboratory models are steadily being
developed should allow us to leverage discoveries made in Drosophila
into application in mammals. It took more than 20 years to find the 2d
core clock gene, timeless, after the discovery of period. Subsequent
progress, however, was rapid. Since we now stand on the shoulders of
both molecular circadian biology and the genomics revolution, the sleep
field may well be able to make relatively rapid progress. As our understanding of molecular biology advances, one question that begins to
seem within our grasp is discovering how changes in the CNS activity
that accompany sleep lead to changes in cellular function. We hypothesize that such changes must underlie the restorative function or functions
of sleep.
We will conclude this review with some caveats. Some elementary
principles are easy to overlook in the excitement of identifying interesting phenotypes in genetically modified animals. Flies are relatively easy
to work with, but rigorous testing and critical evaluation of the information is vital to solid findings and valid interpretations. The effects of
nongenetic influences and complex genetic interactions on adult behavior can profoundly alter phenotype. The ready availability of mutants,
transgenics, and other reagents does not remove the burden on the investigator to verify that any phenotype of interest is actually due to the
change in the specific gene of interest. Fortunately, the field has a tradition of rapid reproducibility that allows verification and refinement of
published data.
IN WRITING THIS REVIEW, THE AUTHORS ASKED OURSELVES, “WHAT CAN WE PROVIDE TO THE READERS OF
SLEEP THAT IS NOT ALREADY AVAILABLE IN THE MUCHREVIEWED AREA OF USING DROSOPHILA TO STUDY CIRCADIAN RHYTHMS AND SLEEP?” First, most reviews focus largely on
the molecular basis of circadian rhythms (see for example1,2)), so that a
review tailored for an audience interested in sleep may be appropriate.
Second, despite the frequency of reviews, new information is emerging
at such a rapid rate that it is worthwhile to highlight the current state of
knowledge and the tantalizing questions to which answers now seem
within our grasp.
We will take the time at the end of this review to provide caveats
about the limits and complexities of working with Drosophila. It is also
worth mentioning its advantages at the outset. Any simple model has the
advantages of it simplicity per se: a quantitatively smaller central nervous system (CNS) and genome ought to make it relatively easy to
unravel interactions than in any mammalian model. Further, simple
models tend to be both small and rapidly reproducing, providing large
numbers of animals and inexpensive maintenance and, thus, both statistical power and ready duplication of results. In the last decade of the 20th
century, organisms as distant from humans as yeast, worms, and flies
were extensively used to work out the basics of cell function. When
viewed in the company of worms and yeast, Drosophila has the additional advantage of its complex behavior. Flies have the ability to learn,
court, run, fly, and sleep, and they have advanced and well-studied sensory systems. A further benefit is the accrual of technical advances from
a century-long focus on fruit flies as a model for genetics. Not only was
the Drosophila melanogaster genome the first of a laboratory animal to
be sequenced,3 but the open culture of the world of fly research has been
instrumental in providing the best-annotated genome, providing quality
control and bolstering confidence that predicted genes have a basis in
reality. Further, the ability to manipulate the genome is increasingly
sophisticated. In addition to mutants, spatially and temporally inducible
transgenic systems have recently been reported.4,5 Genetic methods for
Disclosure Statement
No significant financial interest/other relationship to disclose.
DROSOPHILA AS A MODEL FOR CIRCADIAN RHYTHMS
Submitted for publication December 2003
Accepted for publication January 2004
Address correspondence to: Joan C. Hendricks, Center for Sleep, 991 Maloney
Building, 3600 Spruce St, Philadelphia, PA 19104, Ph: (215) 615-3156;
Fax: (215) 662-7749; E-mail: [email protected]
SLEEP, Vol. 27, No. 2, 2004
While fruit-fly research has contributed tremendously to virtually all
aspects of biology, its importance to the circadian field is particularly
noteworthy. Firstly, as opposed to the traditional use of Drosophila to
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Why a Fly?—Hendricks and Sehgal
study development, circadian biology exploited this organism to determine the molecular underpinnings of behavior. Secondly, because mammalian homologs of the first Drosophila clock gene, period, were not
found for many years, a substantial body of Drosophila work accumulated before any of this could be applied to mammals. As a result,
Drosophila circadian research managed to stay ahead of, and influence,
the work done in mammals for many years. At this point, even while
rapid progress is being made in the mammalian circadian arena,
Drosophila biologists continue to make important strides in our understanding of circadian rhythms. As elaborated below, every aspect of the
Drosophila circadian system shares mechanistic features and molecules
with its mammalian counterpart.
In addition to the PER-TIM loop, there is a second loop that involves
the other cycling component of the clock, namely Clk. In flies and mammals, CLK and BMAL1 activate transcription by binding to a specific
promoter element called an E box.2 Among the genes activated thus by
CLK/BMAL1 are 2, vrille (vri) and Pdp1, that, in turn, regulate Clk
expression.18,19 The expression of Clk is activated by PDP1 and
repressed by VRI. PER-TIM are positive regulators of Clk expression,
most likely because they inhibit the activation of the CLK repressor, VRI
(this indicates that VRI dominates over PDP1 in the control of Clk
expression).19 Cyclic expression of Clk is maintained by this loop. In
mammals, as in flies, only 1 component of the transcriptional heterodimer cycles, but in this case it is BMAL1.20 BMAL1, like
Drosophila CLK, functions in a second feedback loop in which it negatively regulates its own expression by activating the expression of its
repressor, rev-erb α.21 mPER2 positively regulates BMAL1 expression,
again probably by inhibiting the activation of its repressor.
In both flies and mammals, it appears that the second loop, composed
of the transcriptional activator, is not essential for overt rhythms. This is
not to say that the proteins in this loop are not required, merely that their
cycling may not be critical. Thus, a knockout of rev-erbα, which eliminates cycling of BMAL1 mRNA (the protein cycles with very low
amplitude even in wild-type mice) and increases levels of the protein,
produces only subtle behavioral phenotypes.21 The stability, and perhaps
the precision, of the period are affected, indicating that this loop may be
a fine-tuning mechanism that serves to maintain stability and accuracy.
Likewise in flies, overexpression of Clk, such that its cycling is blunted,
does not result in a decrement of rhythms, although similar manipulations of per and tim result in deficits in the free-running rhythm.22,23 It
appears that Clk determines amplitude, but not periodicity or phase, of
the rhythm.24 This would be consistent with the lack of a significant
dosage effect.
Clock Mechanisms and Molecules are Conserved From Flies to Mammals
Fruit-fly research identified the first clock genes, showed how they
cycle and function in a feedback loop, addressed the regulation and the
importance of transcriptional and posttranscriptional control, demonstrated the effects of specific kinases on clock proteins, and identified
secondary loops that interact with the primary loops.1,2 The well-known
loop in which the cycling period (PER) and tim (TIM) proteins negatively regulate the synthesis of their own transcription by inhibiting the
activity of transcriptional activators, Clock (CLK) and BMAL1, now has
to be supplemented with many other additional components (Figure 1).
PER is phosphorylated by casein kinase 1ε, product of the double-time
(dbt) gene, and by casein kinase 2, the alpha and beta components of
which are encoded by the Timekeeper and Andante genes respectively.912 These phosphorylation events affect the stability and the timing of
nuclear localization of PER. Likewise, in mammals, stability and nuclear expression of mPER1 and mPER2 are affected by casein kinase 1ε
and perhaps casein kinase 1δ.13 Drosophila TIM is phosphorylated by
glycogen synthase kinase (GSK)3β, which also serves to regulate the
timed nuclear expression of PER-TIM.14 Although the exact mechanism
that controls the timing of nuclear expression is not known, it involves
active nuclear export in both systems. Interestingly, the targets of the
nuclear export machinery are TIM in Drosophila and PER in mammals.15,16 Of the components noted here, the one whose role in mammals
was debatable was TIM. As a result, there was no concerted effort to
identify a role for GSK3β. However, a recent study indicates that mTIM
may, in fact, function as a clock component.17
Redundant Pathways Entrain the Clock to Light
The visual photoreceptors are not essential for entrainment of circadian rhythms. This was first noted in flies many years ago when eyeless
flies were tested for behavioral rhythms.25 These results were confirmed
when a molecular assay for circadian entrainment became available. The
TIM protein is degraded in response to light, and it mediates resetting of
the molecular clock and of the ensuing behavioral rhythm.26,27 Flies lacking eyes or some of the components of the visual system are able to
degrade TIM and reset their clocks.26,27 However, deficits in entrainment
were noted in flies mutant for the trp (transient receptor potential) proteins, which are channels required for visual transduction, suggesting
that the visual system might play a role when present.27
Work done in the late 1990s led to the identification of a dedicated circadian photoreceptor, a flavin-binding protein called cryptochrome
(CRY).28 CRY functions within the clock cells and interacts directly with
TIM in response to light.29,30 This is followed by an unidentified
sequence of events that leads to the degradation of both TIM and CRY.31
Flies lacking CRY show severe deficits in their response to pulses of
light—TIM is not degraded and the behavioral rhythm fails to reset.31,32
However, cry mutant flies can entrain their circadian rhythms to
light:dark cycles.32 Circadian entrainment is completely eliminated
when the cry mutation is coupled with the glass (gl) mutation, which is
required for the development of all the visual structures in the fly—the
compound eyes, the ocelli, and an extraretinal photosensory structure
called the Hofbauer-Buchner eyelet.33 In the absence of gl, all opsinmediated transduction ceases. This, together with the elimination of
CRY-mediated photoreception, results in a circadian-blind fly. However,
the mechanisms by which the visual system resets the clock are not
known (Figure 2).
This redundant nature of circadian photoreception is also seen in the
mammalian system. Although the eyes are clearly required for entrainment of the central clock within the suprachiasmatic nucleus (SCN), the
cells that mediate visual transduction (rods and cones) can be eliminated.34 An opsin expressed in retinal ganglion cells, melanopsin, appears
Figure 1—The molecular clock mechanism in Drosophila. The loop involving per and tim
is shown in solid lines, the interlocked Clock (Clk) loop is indicated by dashed lines. DOUBLETIME (DBT) affects TIMELESS (TIM)-dependent PERIOD (PER) stability. CASEIN
KINASE 2 (CK2) and GSK3β affect the timing of nuclear entry of PER/TIM and, therefore,
are shown to act on the heterodimer (since nuclear expression of PER affects TIM and vice
versa) although they may actually phosphorylate the monomeric proteins. VRILLE (VRI)
and PDP1 repress and activate CLK, respectively.
SLEEP, Vol. 27, No. 2, 2004
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Why a Fly?—Hendricks and Sehgal
nents that function in the circadian system are not known, peptides such
as these typically act through signaling pathways such as Ras/MAPK. In
addition, the Ras/MAPK pathway is implicated in signaling in the SCN
in both circadian input and output.51 Finally, in identifying parallels
between flies and mammals with respect to circadian output, it is intriguing that circadian rhythm and sleep deficits have been noted in patients
with Fragile X syndrome.52
to serve as a photoreceptor dedicated to nonvisual functions.35,36
However, as in the fly, the entrainment pathway is redundant, with both
visual components as well as melanopsin contributing to the signal from
the eye to the SCN.37,38 The redundancy of the system is most likely an
adaptive mechanism that facilitates synchrony with the environment.
Output from the Central Clock is Mediated, at Least in Part, by Secreted
Peptides
Central and Peripheral Clocks Function as Part of a System
The major output of the central clock cells in Drosophila is an 18
amino-acid peptide termed pigment-dispersing factor (PDF). The accumulation of PDF cycles at specific nerve terminals, which may be
indicative of cyclic release, and flies lacking PDF can not sustain
rhythms in constant darkness.39,40 Both rest-activity and eclosion (hatching of adult flies from pupae) rhythms are affected, indicating that PDF
is required for more than 1 output.39-41 The function of PDF appears to
include synchronizing oscillations among different brain clock cells
under free-running (constant-darkness) conditions.42 Although the
mechanisms that affect PDF release are not known, they appear to not
involve fast synaptic transmission.43 However, the integrity of the PDF
projections appears to be important, as excessive branching of these, as
caused by the loss of the Drosophila Fragile X (dfmr1) gene, results in
arrhythmic behavior.44 Also, overexpression of PDF results in significant
arrhythmia, but only when the extraneous expression is in cells that project to the relevant region (ie, the region where it normally cycles).45
Thus, the molecule is capable of limited diffusion.
The PDF receptor has not yet been identified. However, some of the
components that function downstream of PDF in a circadian output pathway are known. The Drosophila homolog of the Neurofibromatosis 1
(NF1) gene is required for rest-activity rhythms in a pathway that
includes components of the Ras/MAPK pathway.46 MAPK activity
cycles in the region of the brain where PDF accumulation cycles. Thus,
this region (the dorsal region) is an important part of the output circuit.
The function of the SCN also involves secreted peptides.
Interestingly, a knockout of the VPAC2 peptide receptor, which binds 2
peptides implicated in photic entrainment, results in the loss of synchronous clock-gene oscillations in the SCN, similar to the effects seen
in PDF-null flies.47 In addition, the rest-activity output rhythm only
requires a diffusible molecule as opposed to an elaborate synaptic network. Transplantation experiments have shown that a transplanted SCN
could restore rhythms in an SCN-lesioned animal even when it was
encapsulated in a membrane.48 Since then, there has been considerable
effort to identify these secreted molecules that drive rest-activity
rhythms. As of this writing, 2 are known—transforming growth factor
alpha (TGF-α) and prokineticin.49,50 Although the downstream compo-
This is an increasingly popular area of circadian biology. Although
many different aspects of physiology were known to occur with a circadian rhythm, it was the discovery of clock gene expression in various
body tissues that lead to the concept of “peripheral clocks” that control
local tissue-specific functions. Since the outputs of the different tissues
vary greatly, the genes that cycle in these tissues also vary considerably.53,54
As for most other advances, the presence of clocks in different tissues
was also first identified in the fly. Use of a luciferase reporter showed
that per expression cycles in isolated head and body tissues.55
Experiments to determine whether these peripheral clocks are
autonomous or under control of the central clock in the brain have produced mixed results. The clocks in the Malphigian tubules (the fly kidney) and the antenna (which houses an olfactory clock) are independent
of the central clock in the brain.56,57 They are equipped with their own
photoreceptors and are able to drive local molecular or physiologic
rhythms even in the absence of the brain clock. In this respect, they are
different from peripheral clocks in mammals, which appear to require
the SCN, at least for synchrony if not for clock function per se.58,59
A more recent study of Drosophila eclosion indicates that this autonomy is not true of all Drosophila clocks. As noted above, eclosion is the
hatching of an adult fly from its pupa, and it is gated to the early daylight hours. Although it occurs only once in a fly’s lifetime, it can be
measured as a rhythm in a population of flies. It turns out that eclosion
is controlled by a clock in the prothoracic gland (PG).41 This PG clock
is not autonomous; it requires input, including PDF, from the central
clock in the brain. Again, it is not clear whether this input is required for
basic clock function or for synchrony. Since the measurement of a PG
rhythm requires the coordination of multiple cells and animals, an
arrhythmic phenotype does not allow one to distinguish between a complete lack of rhythm versus the loss of synchrony among cells or animals. Regardless, it is now clear that, in Drosophila, the degree of autonomy of peripheral clocks varies (Figure 2). Mammalian circadian
research has now moved on to the effects of clock genes on the cell cycle
and on tumor growth.60,61 Although many of these types of experiments,
particularly the latter, are difficult with the fly,
they are not impossible. There have been recent
advances in the use of the fly for tumor biology.62 Thus, effects of circadian mutants on these
processes may be studied. Finally, the fly may
also prove to be a useful system for assaying
the connection between feeding or metabolism
and the clock, another subject that is currently
of interest in the mammalian field. We already
know of 1 starvation-induced gene in
Drosophila, takeout, that is regulated by the circadian clock.63 Other links will most likely be
found.
DROSOPHILA AS A MODEL FOR SLEEP
Figure 2—Organization of the circadian system in Drosophila. Central clock cells in the fly brain contain a photoreceptor,
cryptochrome. In addition, the visual system can also entrain the clock although the mechanisms are not known. The output
from the central clock includes PIGMENT DISPERSING FACTOR (PDF) which acts through the Ras/MAPK pathway and
other unknown components and cells (indicated by the ?) to drive rest:activity rhythms. PDF also acts, either directly or indirectly, on a peripheral clock in the prothoracic gland (PG) to control eclosion rhythms. The PG may express a photoreceptor
also, but requires PDF to sustain eclosion rhythms in constant darkness. Other peripheral clocks in the fly are photoreceptive
and autonomous.
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336
Sleep-like behavior in Drosophila was first
reported in 2000. A brief summary of the published findings using this model is presented in
Table 1. Tobler and colleagues had long noted
that sleep-like behavior is manifested across the
animal kingdom, regardless of our inability to
Why a Fly?—Hendricks and Sehgal
but the components of the cAMP-PKA signaling pathway were also
implicated.67 A screen using baseline rest as an assay has independently
confirmed that PKA is important in regulating fly sleep.83 The fly studies of CREB were useful in guiding mammalian studies using CREBmutant mice that revealed a conserved involvement of CREB in sleep
regulation.84 Since CREB is a transcription factor that is part of a complex network of cell signaling, pinpointing the specificity of its involvement in states of sleep and wakefulness is the next challenge.
Spurred by an the interest in the genetic mechanisms underlying the 2
regulatory systems (circadian and homeostatic), 2 laboratories investigated how rest was altered in null clock mutants.68,69 The clock-related
transcription factor cycle (homolog of mammalian BMAL1) was shown
in these 2 independent studies to be implicated in rest regulation. 68,69
Interestingly, other clock genes were found to have minimal or no effects
on rest either at baseline or after deprivation, 68,69 and abolishing the central clock cells (lateral neurons, the fly homolog of the SCN) did not
have a major effect.68 Thus, a noncircadian role of cycle was implicated
in sleep regulation. In principle, this finding provides the basis for future
studies examining the interaction of the circadian and homeostatic components of rest regulation in detail. One interesting prospect is to use the
relatively simple fly CNS to understand the anatomic interconnections in
detail. However, although much is known of the neuroanatomy of the fly
clock, fundamental progress in understanding the neuroanatomy of the
homeostatic regulatory system will be required before substantial studies can be conducted. There are additional novel implications of these
studies. Since all of the null clock mutants lack a functional clock, but
only the cycle mutants exhibit profound alterations in rest duration and
homeostasis, a noncircadian role of cycle has been implicated in sleep
regulation. Focusing on the abnormally prolonged rebound in female
cycle mutants, Shaw used multiple genetic tools to implicate changes in
deprivation-related induction of the heat-shock factor hsp83.69 The
mechanism linking cycle to hsp83 was not elucidated but should be readily discernible if it is direct. Further, Shaw showed that prolonged sleep
deprivation was lethal to both wild-type flies and to cycle mutants, providing a link to the fact that prolonged sleep deprivation is lethal in
rats.85
The role of clock genes in sleep regulation in mammals is still under
investigation. In mice, reports of PERIOD86 and CLOCK87 mutants have
been published and are generally consistent with the fly studies. As in
flies, PERIOD mutants have no phenotype68,69 and CLOCK mutants
have a subtle decrease of baseline sleep.68 A change in rapid eye movement (REM) sleep homeostasis was noted in CLOCK mutant mice,
which of course would not be discernible in flies, since no substates have
been identified in fly sleep. Since CLOCK is the dimer partner of
CYCLE, one might expect that the transcriptional activation produced
by this dimer to be implicated in the changes in sleep, as well as the firmly established role in circadian rhythms. It may be, alternatively, that
these PAS-containing proteins pair up with different partners to affect
sleep duration and homeostasis. The BMAL1-mutant phenotype in
mammals seems unlikely to resemble the null cycle-mutant phenotype in
flies, as these mice have profoundly decreased locomotor activity.88 If
BMAL1 affects sleep in the opposite direction as CLOCK, this would
argue of course that these 2 proteins do not act as partners in exerting
their effect on mammalian sleep. There is a precedent for this, as the
clock genes in peripheral clocks also seem to make use of alternative
partners in both mammals89,90 and flies.57 It will be interesting to follow
future studies of the role of clock genes in noncircadian parameters of
sleep and other behaviors as these investigations continue in flies and
mammals. The sense had been that the endogenous clock uses dedicated
genes that have no other functions. This idea is being modified, as it is
obvious that these proteins can have multiple functions in addition to
their role as central clock genes. As 2 interesting examples, the period
gene has now been shown to have a role in tumor suppression in mammals,60 and microarray data in Clock-mutant flies revealed altered (mostly increased) levels of expression of many genes that do not have circadian cycles.91
document a sleeplike electroencephalogram signature.75 Further, large
insects such as honeybees and cockroaches had been shown to have
behavioral features of sleep.76-79 Probably because of their small size,
fruit flies were not studied earlier, although the presumption that they
were sleeping during their prolonged period of circadian inactivity was
implicit in some early descriptions of their behavior.80 In 2000, two laboratories independently published data showing that fruit flies exhibit
behavioral features of a sleep-like state.64,65 While the methods differed
in detail, the findings were remarkably consistent: wild-type flies of both
sexes exhibit prolonged nocturnal periods of immobility, in both conditions of alternating light-dark and constant darkness. During this period
of consolidated rest lasting several hours, the arousal threshold to a variety of stimuli is elevated. If consolidated rest is prevented by mechanical stimulation for as little as 3 hours, a compensatory rebound follows,
providing evidence of homeostatic regulation. Even in these initial
descriptions, evidence was also provided that genes could modify the
rest state: genes that altered amine metabolism65 and the circadian
clock64 were reported to alter the homeostatic response to deprivation.
Another early finding was that the circadian system and homeostatic
systems interact in regulate sleep duration in flies,64 as they do in mammals.81 In a triumph of timing, the completed Drosophila genome was
also released in 2000,3 and the theoretical utility of Drosophila as a
genetic organism was reinforced by the undeniable fact that many genes
of medical importance were conserved, including genes involved in
many human CNS diseases.3,82 While many questions remain to be
answered regarding the nature of the fly’s sleep state (are there substates
within the apparently unitary fly sleep state? What duration is minimally required to accomplish the restorative function? What anatomic substrates underlie sleep?), the existence of a behavioral state similar to
sleep is sufficiently well received that we will refer to the state interchangeably as “sleep” or “rest” for the remainder of this review.
Pharmacology of Fly Sleep
The initial reports both showed a wake-promoting effect of caffeine64,65 and also showed a sleep-promoting effect of an α1-receptor
agonist64 or an antihistamine.65 Similarly, we have found that modafinil,
a novel wake-promoting agent that acts through unknown mechanisms,
produces sleeplessness in flies similar to the sleeplessness it produces in
mammals.66 All of these responses resemble those in mammals and are
suggestive that the mechanisms of action are conserved. This presumption has yet to be verified.
Beginning to Understand the Genetics of Fly Sleep
It is now hard to believe that there was initially some skepticism that
sleep need would prove to be a genetically regulated behavior.
Subsequent to the initial reports, a candidate-gene approach showed that
changing the expression or activity of specific genes and their products
could alter baseline rest and homeostatic rebound. Interestingly, while it
might seem obvious that baseline sleep and compensatory rebound
would be linked, this is not always the case.65,67,68,73 The array of
reagents available in Drosophila allowed these investigations to move
beyond implicating individual genes. Not only was the transcription factor CREB shown to be important for recovery from sleep deprivation,
Table 1—What have we learned about sleep in flies?
1. Wake-promoting drugs (caffeine and modafinil) are effective in flies64-66
2. Changes in the function of a single gene can alter sleep duration and homeostasis65,67-69
3. Gender affects sleep regulation68-71
4. Many genes are induced by sleep deprivation64,69; the pattern mimics cellular stress65
5. Few genes are induced by sleep64,65
6. Prolonged mechanical sleep deprivation is lethal69
7. Baseline and rebound regulation can be dissociated67,68,72,73
8. Energy stores in the central nervous system are repleted during sleep74
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Why a Fly?—Hendricks and Sehgal
The possible involvement of the heat-shock proteins noted by Shaw
and coauthors in rest regulation69 also has interesting implications. Heatshock proteins act as chaperones to optimize proper protein folding,
which is especially important during heat stress. Gene-expression studies after sleep deprivation in flies65 and mammals92,93 (and personal communications, Miroslaw Mackiewicz, PhD, Center for Sleep and
Respiratory Neurobiology, University of Pennsylvania, 2003) have consistently suggested that prolonged waking, at least when produced by
external stimulation, leads to changes in gene expression that resemble
the response to cellular stressors such as heat or reactive oxygen. CREB,
of course, is also involved in stress responses,94,95 leading to the question
of whether waking inevitably involves at least a mild form of cellular
stress. If so, then prolonged waking would presumably exacerbate this
stress. Do the results of admittedly unnatural experimental sleep-deprivation paradigms have relevance for such human conditions as natural
short sleepers, shift workers, or insomniacs? Or is unrelenting, unavoidable, and involuntary laboratory-based sleep deprivation a special case
that is outside the realm of ordinary human experience? Parallel studies
on human volunteers will likely be required to resolve this question.
Regardless, these findings reinforce the commonsense assessment that a
prudent approach to reducing one’s sleep is warranted. Cumulative cellular stress, especially oxidative stress, is generally implicated in aging
and neurodegenerative disease.96-101 If sleep deprivation acts synergistically with chronologic age, more-rapid deterioration in CNS function
would be expected in individuals who also do not get sufficient sleep—
whatever “sufficient” means for an individual. Interestingly, every study
of sleep-related gene expression has shown that while many genes are
upregulated during sleep deprivation, few are induced by sleep.64,92,102,103
Clearly, we are just beginning to identify genes involved in sleep regulation, but the fact that 2 transcription factors have been implicated is
hopeful for the future. Combining microarray techniques with phenotyping of genetically modified mice and flies will help us to verify and
extend these initial findings. In mice, strain differences have been
shown,104,105 and at least 1 interesting gene locus has been identified by
QTL mapping.104 Studies of the heritability of human sleep need are also
underway at many institutions including the University of Pennsylvania.
mented in humans,109,110 sex influences on sleep regulation has been surprisingly little studied. There is a striking difference in the rebound
response of male and female cycle mutant flies, with females showing
increased and males showing decreased rebound responses.68,69 Wildtype males have a more marked daytime nap and less consolidated nighttime sleep.70,71 Interestingly, male flies have been consistently and
almost exclusively used to study circadian rhythms, and both sexes are
routinely combined in molecular studies, so that important differences
may have been missed in most published studies of circadian rhythms
and other behaviors; this is an issue discussed in more detail elsewhere.111 The anatomic substrate for the differences between wild-type
males and females has been pursued by artificially expressing a feminizing gene in specific regions of the CNS in male flies.70 We also have
preliminary evidence that the response of Drosophila to pharmacologic
agents can be sex-dimorphic (P. Schotland, Ph.D., University of
Pennsylvania, unpublished observations, 2003). While interesting per se,
sex dimorphism in sleep regulation is also a specific instance of the general cautionary rule that behavior, including sleep, is highly susceptible
to background genetic modulation. That is, not surprisingly, many genes
can alter sleep, and sex, with its global influence on cellular function,
may also have global influences on the genetics of sleep regulation.
Neural Activity During Fly Sleep and Waking
For any neuroscientist accustomed to working in mammalian systems,
the fly’s CNS is bewildering, not only because it bears little resemblance
to the mammalian CNS, but also because it has been little investigated,
especially in adults. The application of spatially and temporally specific
gene-expression systems is increasingly refining our understanding of
the functional anatomy of the fly CNS, since cellular function can be
altered in specific regions or at specific times.43,112,113 Some of the most
striking examples have been in the area of learning and memory.114,115 A
more familiar approach for the mammalian neuroscientist is direct
recordings, understandably a daunting task give the tiny size of the fruit
fly. However, some interesting progress has been made in recording
from the adult fly’s CNS.116-118 Imaging of calcium flux in the living
adult fly has also been reported,119,120 and a culture system that allows
both visualization of calcium flux and measurement of synaptic activity121,122 offers great potential. However, the restraint and dissection necessary to perform both of these latter methods do not permit observation
of any behavioral signs of sleep or arousal. Even the electrophysiologic
studies in the intact fruit fly require tethering the fly. Although in larger
organisms (eg, honeybees76) such a preparation can be maintained for
days and permits the expression of circadian rhythmicity in locomotor
rest and activity,77 fruit flies only sleep for approximately 20% of the
night in this situation. With sleep defined as periods of complete quiescence and reduced responsiveness, bouts are usually a little longer than
5 minutes, with a maximum of about 15 minutes in duration. Thus, the
ideal tool for monitoring state-related brain activity in the same fashion
as with a chronically implanted electroencephalogram is still not available in fruit flies. The great utility of the tethered preparation to date has
been to establish that changes in oscillatory frequency accompany perception of relevant environmental stimuli in the waking fly.117,118 To
date, no mass neural signature has been detected in this preparation that
is correlated with the fly’s sleep state.118 It does not appear that synchronous slowing of the frequency of neural activity heralds or accompanies the periods of immobility displayed by the tethered fly118 (and
personal communications, B. van Swinderen, Ph.D., 2003). It is always
difficult and unsatisfying to make definitive conclusions based on a lack
of information; one could easily imagine that technical problems, a lack
of prolonged consolidated sleep, or the sheer lack of sufficient time for
adaptation (flies are highly sensitive to environmental changes as
innocuous as a change in food) might prevent successful recording of a
sleep-specific waveform using the tethered fly preparation. Nonetheless,
we cannot at present document that changes analogous to mammalian
electroencephalographic sleep signatures exist in the fly. Interestingly, it
does appear that prior to entering a period of immobility, the fly’s neu-
CNS Energy Use in Fly Sleep and Waking
Sleep would intuitively seem likely to allow or perhaps actively promote repletion of energy stores that are depleted during waking. Can we
document that waking CNS activity depletes, and sleep repletes, neural
energy stores? While this theory has been recognized as a very attractive
concept ever since it was introduced in 1995,106 it has been difficult to
answer the question conclusively in mammals.107,108 There are serious
methodologic considerations for the measurement of glycogen in the relatively large brain of mammals that do not apply to flies. Metabolism in
the mammalian brain continues after death unless death is produced by
very rapid heating of the brain, which is difficult to achieve. However,
near-instantaneous heating of the entire brain is trivial in flies, due to the
small size and low thermal inertia of the tissue. The glycogen level of the
fly brain has been shown to vary inversely with sleep in undisturbed
flies, and repletion of depleted glycogen after sleep deprivation has also
been shown to occur during rebound sleep.74 Mere stimulation was not
responsible for the depletion of glycogen during sleep deprivation, as the
same stimuli applied during the normal active period did not alter brain
glycogen. This opens the possibility that studies of the oxidative stress
accompanying wakefulness can be studied in detail in flies. Presumably,
an interacting cascade of compensatory, protective, and modulating
influences are involved. Since oxidative stress and aging have been usefully studied in flies (see, for example101), this is likely to be an interesting avenue to pursue.
Sex Interactions with Genetic Sleep Regulation
Another area that may also be easier to manipulate in flies is sex interactions with genotype. While male-female differences have been docuSLEEP, Vol. 27, No. 2, 2004
338
Why a Fly?—Hendricks and Sehgal
ral activity is uncoupled from environmental stimuli (B. van Swinderen,
Ph.D., personal communications, 2003), presumably a neural correlate
of the lack of behavioral responsiveness. Further, while overall power is
maintained during brief (5- to 20-second) pauses in activity that occur
during the waking state, overall power across the frequency spectrum
declines when the fly is in the more prolonged (5- to 15-minute) quiescent, nonresponsive state defined as sleep. Thus, current information
indicates that, even if there are not discernible synchronous high-amplitude discharges that characterize fly sleep, there is nonetheless a global
change in brain electrical activity during the sleep state.
Posttranscriptional modifications may, of course, be at least as important
as transcriptional changes. Further, the issue of compartmentalization
and movement may be almost as important as the gene-product profiles.
For example, the normal circadian changes in subcellular distribution of
the clock protein TIMELESS do not occur when the electrical activity in
Drosophila clock neurons is silenced.113 The ability to visualize specific
reporter-tagged molecules in the Drosophila brain might be combined
with the newly developed techniques to culture brains to answer to the
question of state-related changes in subcellular localization. For example, brains from “sleepless” mutants or modafinil-fed brains might
exhibit consistent changes in the pattern of distribution of specific
molecules that contrasts with the pattern in normal animals. How would
changes in electrical activity become changes in cell function? Given
that it seems inevitable that intracellular messengers must link sleeprelated changes in activity to gene expression and posttranscriptional
changes (Figure 3B), is there any current information to guide future
investigations into such a mechanism? Given current information, we
favor the possibility that the link is made through changes in Ca++
(Figure 3C). Changes in Ca++ levels have been noted to be promoted by
sleeping waveforms.124 Clearly, Ca++ can influence gene expression acting through a number of transcription factors,133-135 with the frequency
of Ca++ oscillations being an important signal to specify the pattern of
gene transcription.136 Ca++ oscillations have been noted in the
Drosophila CNS (both larval and adult),119 and intracellular Ca++ is
activity dependent in cultured Drosophila CNS.121,122 Whatever the signaling mechanism, we hypothesize that the endogenous signaling is optimized and the signal-to-noise ratio optimized by the sleeping state of the
brain, such that specific (repair and plasticity-related) genes are transcribed best during sleep. In this scenario, longer consolidated sleep
bouts would best permit the completion of these programs of gene transcription and related cellular-repair functions. The activity of the repair
genes might persist during waking and thus contribute to the improvement of waking function that follows a prolonged restorative bout of
sleep.
Speculation: Can Flies Help Us to Answer the Question Why Animals Need
Sleep Instead of Mere Rest?
It is easy to understand the need for inactivity for somatic and neural
recovery from exhaustion, but the need for sleep—a homeostatically
regulated period of unresponsiveness—is harder to understand. Several
putative biologic functions for sleep have, of course, been advanced.123126 We presume that the function of sleep must be fostered by sleep-specific changes in brain activity, whether sleep-specific waveforms, as in
mammals, or a decrease of global activity, as shown in flies (Figure 3A).
A hallmark of mammalian sleep is high-amplitude, slow, synchronous
discharges during sleep. In contrast to waking, where the waveforms are
in the faster alpha, beta, and gamma ranges, spindles, K complexes, and
delta waves characterizes non-REM, and pontogeniculooccipital spikes
occur just prior to and during REM sleep. In addition, a slower (0.1Hz)
waveform has been noted that underlies the occurrence of spindles.124,127132 As noted above, flies have not been shown to have a specific neural
waveform during their sleep. However, responsiveness to environmental
stimuli is greatly decreased during the sleep state by either neural or
behavioral measures. Together with the fact that glycogen is repleted
during rest74 and the overall power of neural activity at all frequencies
declines when flies are sleeping (B. van Swinderen, Ph.D., personal
communications, 2003), it is reasonable to conclude that fly sleep is a
time of greatly reduced neural activity. Since cell function during the
sleep state is altered by the sleep-related changes in activity, it seems
logical to assume that changes in cellular function are fundamental to the
function of sleep (Figure 3B). The conversion of state-related changes in
activity to changes in gene expression must rely on intracellular messenger systems. Sleep-related gene expression is under investigation in
several labs, and the proteome during sleep will surely soon be attacked.
CONCLUSIONS, NUANCES, AND NUISANCES
We conclude with some caveats. We hope that fruit flies will increasingly be used to probe the genetics and cell biology of sleep. However,
some basic cautions may help the investigator who is new to the use of
Drosophila. Despite the fact that fruit flies are renowned for the ease of
their cultivation, reproduction, and maintenance, they are actually quite sensitive and
finicky about culture conditions. Behavior is
readily influenced by environmental influences
such as light, temperature, humidity, food,
social crowding, and reproductive status. More
subtle effects of stimuli such as noise, mechanical disruptions, odors, and electrical fields
have not been well documented but may play a
role in the notorious variability of assays of
complex behaviors. One special instance that is
highly relevant to the study of sleep is the possibility that insects have a stress response analogous to the mammalian adrenocortical
response: stress is a serious issue for all studies
of sleep deprivation, and there simply is very
little information about this in insects. Using a
variety of methods to produce sleep deprivation
will be necessary to better understand this
issue, as all studies to date have used mechanical stimuli.
Figure 3—Are sleep-specific changes in neural activity related to restorative sleep functions? A. Sleep-specific brain activity
changes are well documented, and many putative restorative functions have been proposed, can mechanisms to link them be
Another fundamental issue is that we still
identified? B. We posit that changes in electrical activity must lead to changes in cell function (note that we are not limiting
have
no definitive temporal criterion for fly
this to neurons; glial function could also be involved). C. Sleep-specific activity changes are proposed to be converted into
sleep. The initial descriptions used a 30-minute
restorative cellular functions through changes in intracellular signaling. We suggest here that Ca++ levels (or, probably more
importantly, frequency and perhaps amplitude of Ca++ oscillations) provide information that changes both gene transcription
duration as the definition of rest64,65; however,
and post-transcriptional modifications. For further details see text.
subsequent studies have also used 5-minute
SLEEP, Vol. 27, No. 2, 2004
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Why a Fly?—Hendricks and Sehgal
measures.66,69 In normal flies, most rest in a 24-hour period is accomplished in bouts lasting > 30 minutes,64 but this bout length can be significantly shortened by gene mutations68 and drugs.66 We need to know
whether there is a minimal duration of immobility that meets the definition of sleep. That is, what is the shortest bout length that is accompanied by behavioral unresponsiveness and provides a restorative function? We have noted that modafinil decreases consolidated (30 minutes)
but not brief (5 minutes) sleep over a 24-hour day in flies.66 Since withdrawal of modafinil after several days results in a slight but statistically
significant rebound of sleep, we concluded that some but not all of the
restorative function of sleep can be accomplished during these shorter
bouts.66
Finally, while breeding flies is rather straightforward, and many
mutants and transgenics are readily available either through public
sources or from colleagues, interpreting the data must be critical and rigorous. Flies cannot be stored as frozen organisms but, rather, must be
maintained as breeding colonies. Thus, genetic abnormalities can be lost
or acquired. Any mutant or transgenic should be verified to have the
abnormality of interest, and any role for genetic background should be
ruled out before attributing a particular phenotype to a particular gene.
For example, many lines of flies have a mutation that lengthens the
endogenous circadian period (J. Hendricks, personal observations), and
both baseline rest and amplitude of the circadian rhythm are quite variable among individual wild-type flies (J. Hendricks, personal observations). There are well-established methods for mapping a phenotype to a
particular gene locus: rescuing the phenotype of a null mutant by transgenically restoring the mutant gene is one such method. We have also
noted above that sex can interact profoundly with genotype. The commonsense warning to the novice fly researcher is simply to realize that
many additional steps must follow the initial observation that a genetically altered line of flies has an abnormal phenotype. We have noted, for
example, that whereas the baseline rest duration mapped to the cycle
locus, the effect of cycle on homeostatic rebound and on lifespan in
cycle-null flies is modified by genetic background in addition to the
cycle mutation.68 This sensitivity to background does not change the fact
that longevity, response to stress, and gene expression are altered in
cycle mutants, but gene interactions must be considered before attributing these aspects of the phenotype simply to a single gene.
In conclusion, flies helped to identify molecular mechanisms underlying the endogenous clock that are largely conserved in mammals. What
we know so far encourages us to think that fly rest is a very useful
homolog to mammalian sleep: in both mammals and flies, sleep loss
resembles cellular stress and can be lethal; flies respond like mammals
to several pharmacologic agents, and the CREB transcription pathway
seems to be involved in promoting wakefulness in both flies and mammals. We do not yet have thoroughly elucidated pathways for sleep regulation or function, but we clearly have begun to take the first steps.
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