Sleep In Drosophila Research Articles

High calorie diet augments age-associats sleep impairment in Drosophila

The fruit fly, Drosophila melanogaster is an established model used for aging and longevity studies and more recently for sleep studies. Mammals and Drosophila share various physiological, pathological, pharmacological and genetic similarities in these processes. In particular, sleep is essential for survival in both species and both have age-associated sleep quality alterations. Here we report that a high calorie diet, which accelerates the aging process and reduces lifespan across species, also accelerates age-associated sleep changes in Drosophila. These changes are more evident in the dopamine transporter mutant, fumin, that displays a short sleep phenotype due to enhanced dopaminergic signaling. With normal food, fumin mutants sleep for only one third of the time that the control flies do, but still show equivalent longevity. However, when on a mildly high calorie diet, their sleep length shows a marked decrease and they have a reduced longevity. These data indicate that the age-associated change in sleep in Drosophila is a physiologically regulated aging process that is tightly linked to calorie intake and that the dopamine level plays an important role. In addition, this provides another evidence that sleep is essential for the longevity of Drosophila.

Pan-neuronal knockdown of the c-Jun N-terminal Kinase (JNK) results in a reduction in sleep and longevity in Drosophila

Sleep is a unique behavioral state that is conserved between species, and sleep regulation is closely associated to metabolism and aging. The fruit fly, Drosophila melanogaster has been used to study the molecular mechanism underlying these physiological processes. Here we show that the c-Jun N-terminal Kinase (JNK) gene, known as basket (bsk) in Drosophila, functions in neurons to regulate both sleep and longevity in Drosophila. Pan-neuronal knockdown of JNK mRNA expression by RNA interference resulted in a decrease in both sleep and longevity. A heterozygous knockout of JNK showed similar effects, indicating the molecular specificity. The JNK knockdown showed a normal arousal threshold and sleep rebound, suggesting that the basic sleep mechanism was not affected. JNK is known to be involved in the insulin pathway, which regulates metabolism and longevity. A JNK knockdown in insulin-producing neurons in the pars intercerebralis had slight effects on sleep. However, knocking down JNK in the mushroom body had a significant effect on sleep. These data suggest a unique sleep regulating pathway for JNK.

Insomniac and Cullin-3 Regulate Sleep and Wakefulness in Drosophila

In a forward genetic screen in Drosophila, we have isolated insomniac, a mutant that severely reduces the duration and consolidation of sleep. Anatomicallyrestricted genetic manipulations indicate that insomniac functions within neurons to regulate sleep. insomniac expression does not oscillate in a circadian manner, and conversely, the circadian clock is intact in insomniac mutants, suggesting that insomniac regulates sleep by pathways distinct from the circadian clock. The protein encoded by insomniac is a member of the BTB/POZ superfamily, which includes many proteins that function as adaptors for the Cullin-3 (Cul3) ubiquitin ligase complex. We show that Insomniac can physically associate with Cul3, and that reduction of Cul3 activity in neurons recapitulates the insomniac phenotype. The extensive evolutionary conservation of insomniac and Cul3 suggests that protein degradation pathways may have a general role in governing the sleep and wakefulness of animals.

A Conserved Long Noncoding RNA Affects Sleep Behavior inDrosophila

Metazoan genomes encode an abundant collection of mRNA-like, long noncoding (lnc)RNAs. Although lncRNAs greatly expand the transcriptional repertoire, we have a limited understanding of how these RNAs contribute to developmental regulation. Here, we investigate the function of the Drosophila lncRNA called yellow-achaete intergenic RNA (yar). Comparative sequence analyses show that the yar gene is conserved in Drosophila species representing 40–60 million years of evolution, with one of the conserved sequence motifs encompassing the yar promoter. Further, the timing of yar expression in Drosophila virilis parallels that in D. melanogaster, suggesting that transcriptional regulation of yar is conserved. The function of yar was defined by generating null alleles. Flies lacking yar RNAs are viable and show no overt morphological defects, consistent with maintained transcriptional regulation of the adjacent yellow (y) and achaete (ac) genes. The location of yar within a neural gene cluster led to the investigation of effects of yar in behavioral assays. These studies demonstrated that loss of yar alters sleep regulation in the context of a normal circadian rhythm. Nighttime sleep was reduced and fragmented, with yar mutants displaying diminished sleep rebound following sleep deprivation. Importantly, these defects were rescued by a yar transgene. These data provide the first example of a lncRNA gene involved in Drosophila sleep regulation. We find that yar is a cytoplasmic lncRNA, suggesting that yar may regulate sleep by affecting stabilization or translational regulation of mRNAs. Such functions of lncRNAs may extend to vertebrates, as lncRNAs are abundant in neural tissues.

Pan-Neuronal Knockdown of Calcineurin Reduces Sleep in the Fruit Fly,Drosophila melanogaster

Sleep is a unique physiological state, which is behaviorally defined, and is broadly conserved across species from mammals to invertebrates such as insects. Because of the experimental accessibility provided by various novel animal models including the fruit fly, Drosophila melanogaster, there have been significant advances in the understanding of sleep. Although the physiological functions of sleep have not been fully elucidated, accumulating evidence indicates that sleep is necessary to maintain the plasticity of neuronal circuits and, hence, is essential in learning and memory. Calcineurin (Cn) is a heterodimeric phosphatase composed of CnA and CnB subunits and known to function in memory consolidation in the mammalian brain, but its neurological functions in the fruit fly are largely unknown. Here, we show that Cn is an important regulator of sleep in Drosophila. A pan-neuronal RNA interference-mediated knockdown of Cn expression resulted in sleep loss, whereas misexpression of the constitutively active form of a CnA protein led to increased sleep. Furthermore, CnA knockdown also impaired the retention of aversive olfactory memory. These results indicate a role for Cn and calcium-dependent signal transduction in sleep and memory regulation and may bring insight into the relationship between them.

DrosophilaQVR/SSS Modulates the Activation and C-Type Inactivation Kinetics of Shaker K+Channels

The quiver/sleepless (qvr/sss) gene encodes a small, glycosylphosphatidylinositol-anchored protein that plays a critical role in the regulation of sleep in Drosophila. Loss-of-function mutations in qvr/sss severely suppress sleep and effect multiple changes in in situ Shaker K(+) currents, including decreased magnitude, slower time-to-peak, and cumulative inactivation. Recently, we demonstrated that SLEEPLESS (SSS) protein modulates Shaker channel activity, possibly through a direct interaction at the plasma membrane. We show here that SSS accelerates the activation of heterologously expressed Shaker channels with no effect on deactivation or fast N-type inactivation. Furthermore, this SSS-induced acceleration is sensitive to the pharmacological disruption of lipid rafts and sufficiently accounts for the slower time-to-peak of in situ Shaker currents seen in qvr/sss mutants. We also find that SSS decreases the rate of C-type inactivation of heterologously expressed Shaker channels, providing a potential mechanism for the cumulative inactivation phenotype induced by qvr/sss loss-of-function mutations. Kinetic modeling based on the in vitro results suggests that the SSS-dependent regulation of channel kinetics accounts for nearly 40% of the decrease in Shaker current magnitude in flies lacking SSS. Sleep duration in qvr/sss-null mutants is restored to normal by a qvr/sss transgene that fully rescues the Shaker kinetic phenotypes but only partially rescues the decrease in current magnitude. Together, these results suggest that the role of SSS in the regulation of sleep in Drosophila correlates more strongly with the effects of SSS on Shaker kinetics than current magnitude.

Notch Signaling Modulates Sleep Homeostasis and Learning after Sleep Deprivation in Drosophila

The role of the transmembrane receptor Notch in the adult brain is poorly understood. Here, we provide evidence that bunched, a negative regulator of Notch, is involved in sleep homeostasis. Genetic evidence indicates that interfering with bunched activity in the mushroom bodies (MBs) abolishes sleep homeostasis. Combining bunched and Delta loss-of-function mutations rescues normal homeostasis, suggesting that Notch signaling may be involved in regulating sensitivity to sleep loss. Preventing the downregulation of Delta by overexpressing a wild-type transgene in MBs reduces sleep homeostasis and, importantly, prevents learning impairments induced by sleep deprivation. Similar resistance to sleep loss is observed with Notch(spl-1) gain-of-function mutants. Immunohistochemistry reveals that the Notch receptor is expressed in glia, whereas Delta is localized in neurons. Importantly, the expression in glia of the intracellular domain of Notch, a dominant activated form of the receptor, is sufficient to prevent learning deficits after sleep deprivation. Together, these results identify a novel neuron-glia signaling pathway dependent on Notch and regulated by bunched. These data highlight the emerging role of neuron-glia interactions in regulating both sleep and learning impairments associated with sleep loss.

Go Signaling in Mushroom Bodies Regulates Sleep in Drosophila

Sleep is a fundamental physiological process and its biological mechanisms are poorly understood. In Drosophila melanogaster, heterotrimeric Go protein is abundantly expressed in the brain. However, its post-developmental function has not been extensively explored. Locomotor activity was measured using the Drosophila Activity Monitoring System under a 12:12 LD cycle. Sleep was defined as periods of 5 min with no recorded activity. Pan-neuronal elevation of Go signaling induced quiescence accompanied by an increased arousal threshold in flies. By screening region-specific GAL4 lines, we mapped the sleep-regulatory function of Go signaling to mushroom bodies (MBs), a central brain region which modulates memory, decision making, and sleep in Drosophila. Up-regulation of Go activity in these neurons consolidated sleep while inhibition of endogenous Go via expression of Go RNAi or pertussis toxin reduced and fragmented sleep, indicating that the Drosophila sleep requirement is affected by levels of Go activity in the MBs. Genetic interaction results showed that Go signaling serves as a neuronal transmission inhibitor in a cAMP-independent pathway. Go signaling is a novel signaling pathway in MBs that regulates sleep in Drosophila.

Dietary Modulation of Drosophila Sleep-Wake Behaviour

BackgroundA complex relationship exists between diet and sleep but despite its impact on human health, this relationship remains uncharacterized and poorly understood. Drosophila melanogaster is an important model for the study of metabolism and behaviour, however the effect of diet upon Drosophila sleep remains largely unaddressed.Methodology/Principal FindingsUsing automated behavioural monitoring, a capillary feeding assay and pharmacological treatments, we examined the effect of dietary yeast and sucrose upon Drosophila sleep-wake behaviour for three consecutive days. We found that dietary yeast deconsolidated the sleep-wake behaviour of flies by promoting arousal from sleep in males and shortening periods of locomotor activity in females. We also demonstrate that arousal from nocturnal sleep exhibits a significant ultradian rhythmicity with a periodicity of 85 minutes. Increasing the dietary sucrose concentration from 5% to 35% had no effect on total sucrose ingestion per day nor any affect on arousal, however it did lengthen the time that males and females remained active. Higher dietary sucrose led to reduced total sleep by male but not female flies. Locomotor activity was reduced by feeding flies Metformin, a drug that inhibits oxidative phosphorylation, however Metformin did not affect any aspects of sleep.ConclusionsWe conclude that arousal from sleep is under ultradian control and regulated in a sex-dependent manner by dietary yeast and that dietary sucrose regulates the length of time that flies sustain periods of wakefulness. These findings highlight Drosophila as an important model with which to understand how diet impacts upon sleep and wakefulness in mammals and humans.

Persistence of Morning Anticipation Behavior and High Amplitude Morning Startle Response Following Functional Loss of Small Ventral Lateral Neurons in Drosophila

Light-activated large ventral lateral clock neurons (large LNv) modulate behavioral arousal and sleep in Drosophila while their counterparts, the small LNv (s-LNv) are important for circadian behavior. Recently, it has been proposed that the pattern of day-night locomotor behavioral activity is mediated by two anatomically distinct oscillators composed of a morning oscillator in the small LNv and an evening oscillator in the lateral dorsal neurons and an undefined number of dorsal pacemaker neurons. This contrasts with a circuit described by network models which are not as anatomically constrained. By selectively ablating the small LNv while sparing the large LNv, we tested the relative importance of the small and large LNv for regulating morning behavior of animals living in standard light/dark cycles. Behavioral anticipation of the onset of morning and the high amplitude morning startle response which coincides with light onset are preserved in small LNv functionally-ablated animals. However, the amplitude of the morning behavioral peak is severely attenuated in these animals during the transition from regular light/dark cycles to constant darkness, providing further support that small LNv are necessary for circadian behavior. The large LNv, in combination with the network of other circadian neurons, in the absence of functional small LNv are sufficient for the morning anticipation and the high amplitude light-activated morning startle response.

Modulation of the Frequency Response of Shaker Potassium Channels by the Quiver Peptide Suggesting a Novel Extracellular Interaction Mechanism

:Recent studies have indicated that the Shaker potassium channel regulates sleep in Drosophila. The Drosophila quiver (qvr) gene encodes a novel potassium channel subunit that modulates the Shaker potassium channel. The Qvr peptide contains a signal sequence for extracellular localization and may regulate a unique feature of the Shaker IA current that confers special neuronal excitability patterns. Thus, studies of the Shaker channel properties in the qvr mutant background should provide an opportunity to uncover a new form of physiologic modulation of potassium channels. We have begun to investigate the impact of qvr protein on the Shaker channel properties and its implications in synaptic function in vivo. We studied synaptic transmission at the larval neuromuscular junction and characterized the transient potassium current IA in larval muscles. We identified two different functional states of IA in qvr larval muscles, as reflected by two distinct components, IAF and IAS, differing in their kinetics of recovery from inactivation and sensitivity to a K+ channel blocker. Correspondingly, qvr mutant larvae exhibit multiple synaptic discharges following individual nerve stimuli during repetitive activity.

Sleep, aging, and lifespan in Drosophila

BackgroundEpidemiological studies in humans suggest that a decrease in daily sleep duration is associated with reduced lifespan, but this issue remains controversial. Other studies in humans also show that both sleep quantity and sleep quality decrease with age. Drosophila melanogaster is a useful model to study aging and sleep, and inheriting mutations affecting the potassium current Shaker results in flies that sleep less and have a shorter lifespan. However, whether the link between short sleep and reduced longevity exists also in wild-type flies is unknown. Similarly, it is unknown whether such a link depends on sleep amount per se, rather than on other factors such as waking activity. Also, sleep quality has been shown to decrease in old flies, but it remains unclear whether aging-related sleep fragmentation is a generalized phenomenon.ResultsWe compared 3 short sleeping mutant lines (Hk1, HkY and Hk2) carrying a mutation in Hyperkinetic, which codes for the beta subunit of the Shaker channel, to wild-type siblings throughout their entire lifespan (all flies kept at 20°C). Hk1 and HkY mutants were short sleeping relative to wild-type controls from day 3 after eclosure, and Hk2 flies became short sleepers about two weeks later. All 3 Hk mutant lines had reduced lifespan relative to wild-type flies. Total sleep time showed a trend to increase in all lines with age, but the effect was most pronounced in Hk1 and HkY flies. In both mutant and wild-type lines sleep quality did not decay with age, but the strong preference for sleep at night declined starting in "middle age". Using Cox regression analysis we found that in Hk1 and HkY mutants and their control lines there was a negative relationship between total sleep amount during the first 2 and 4 weeks of age and hazard (individual risk of death), while no association was found in Hk2 flies and their wild-type controls. Hk1 and HkY mutants and their control lines also showed an association between total daily wake activity over the first 2 and 4 weeks of age and hazard. However, when both sleep duration and wake activity were used in the same regression, the effects of activity were much reduced, while most of the sleep effects remained significant. Finally, Hk1 flies and wild-type siblings were also tested at 25°C, and results were similar to those at 20°C. Namely, Hk1 mutants were short sleeping, hyperactive, and short lived relative to controls, and sleep quality in both groups did not decrease with age.ConclusionsDifferent Hk mutations affect the sleep phenotype, and do so in an age-dependent manner. In 4 of the 6 lines tested sleep associates significantly with lifespan variation even after any effect of activity is removed, but activity does not associate significantly with lifespan after the effects of sleep are removed. Thus, in addition to environmental factors and genetic background, sleep may also affect longevity. Sleep quality does not necessarily decay as flies age, suggesting that aging-related sleep fragmentation may also depend on many factors, including genetic background and rearing conditions.

Calcineurin regulates sleep and memory in Drosophila

Calcineurin regulates sleep and memory in Drosophila

SLEEPLESS, a Ly-6/neurotoxin family member, regulates the levels, localization and activity of Shaker

Sleep is a whole–organism phenomenon accompanied by global changes in neural activity. We previously identified SLEEPLESS (SSS) as a novel glycosylphosphatidyl–inositol–anchored protein required for sleep in Drosophila. Here, we demonstrate a critical role for SSS in regulating the sleep–modulating potassium channel, Shaker. SSS and Shaker exhibit similar expression patterns in the brain and specifically affect each other’s expression levels. sss mutants exhibit altered Shaker localization, reduced Shaker current density, and slower Shaker current kinetics. Transgenic expression of sss in sss mutants rescues defects in Shaker expression and activity cell–autonomously and also suggests that SSS functions in wake–promoting, cholinergic neurons. Importantly, in heterologous cells, SSS accelerates kinetics of Shaker currents and can be co–immunoprecipitated with Shaker, suggesting that SSS interacts with Shaker and modulates its activity. SSS is predicted to belong to the Ly–6/neurotoxin superfamily, suggesting a novel mechanism for regulation of neuronal excitability by endogenous toxin–like molecules.

Co-regulated transcriptional networks contribute to natural genetic variation in Drosophila sleep

Sleep disorders are common in humans, and sleep loss increases the risk of obesity and diabetes1. Studies in Drosophila2, 3 have revealed molecular pathways4–7 and neural tissues8–10 regulating sleep; however, genes that maintain genetic variation for sleep in natural populations are unknown. Here, we characterized sleep in 40 wild-derived Drosophila lines and observed abundant genetic variation in sleep architecture. We associated sleep with genome-wide variation in gene expression11 to identify candidate genes. We independently confirmed that molecular polymorphisms in Catecholamines up are associated with variation in sleep; and that P-element mutations in four candidate genes affect sleep and gene expression. Transcripts associated with sleep grouped into biologically plausible genetically correlated transcriptional modules. We confirmed co-regulated gene expression using P-element mutants. Genes associated with sleep duration are evolutionarily conserved. Quantitative genetic analysis of natural phenotypic variation is an efficient method for revealing candidate genes and pathways.

The GABAA Receptor RDL Acts in Peptidergic PDF Neurons to Promote Sleep in Drosophila

Sleep is regulated by a circadian clock that times sleep and wake to specific times of day and a homeostat that drives sleep as a function of prior wakefulness. To analyze the role of the circadian clock, we have used the fruit fly Drosophila. Flies display the core behavioral features of sleep, including relative immobility, elevated arousal thresholds, and homeostatic regulation. We assessed sleep-wake modulation by a core set of circadian pacemaker neurons that express the neuropeptide PDF. We find that disruption of PDF function increases sleep during the late night in light:dark and the first subjective day of constant darkness. Flies deploy genetic and neurotransmitter pathways to regulate sleep that are similar to those of their mammalian counterparts, including GABA. We find that RNA interference-mediated knockdown of the GABA(A) receptor gene, Resistant to dieldrin (Rdl), in PDF neurons reduces sleep, consistent with a role for GABA in inhibiting PDF neuron function. Patch-clamp electrophysiology reveals GABA-activated picrotoxin-sensitive chloride currents on PDF+ neurons. In addition, RDL is detectable most strongly on the large subset of PDF+ pacemaker neurons. These results suggest that GABAergic inhibition of arousal-promoting PDF neurons is an important mode of sleep-wake regulation in vivo.

This Month in Anesthesiology

The authors examined the epidemiology of perioperative AIS in three common surgeries in adult patients: hemicolectomy, total hip replacement, and lobectomy/segmental lung resection. Data were extracted from a national database in the United States for the years 2000 to 2004. Patients with perioperative AIS were identified as were comorbid conditions that may be risk factors for perioperative AIS. Those who developed perioperative AIS were as follows: 0.7% of hemicolectomy patients, 0.2% of total hip replacement patients, and 0.6% of lobectomy/segmental lung resection patients. For patients older than 65, AIS rose to 1.0% for hemicolectomy, 0.3% for hip replacement, and 0.8% for pulmonary resection. Perioperative AIS is an important source of morbidity and mortality associated with noncardiac, nonvascular surgery particularly in elderly patients. See the accompanying Editorial View on page 209 Anesthetic requirements of the fruit fly mutant minisleeper and other Drosophila mutants were determined based on the response of the flies at different concentrations of isoflurane and sevoflurane. The average amount of daily sleep in wild-type Drosophila was 965 minutes but only 584 minutes in minisleeper mutant flies. The EC50values in wild-type Drosophila were 0.706 for isoflurane and 1.298 for sevoflurane; however, EC50values in the minisleeper mutant flies for isoflurane and sevoflurane were 1.306 and 2.013, respectively. A single-gene mutation in Drosophila that causes an extreme reduction in daily sleep is responsible for a significant increase in the requirement of volatile anesthetics. This study has implications for sleep and mechanisms of anesthesia and sedation.Hyperglycemia in perioperative patients has been identified as a risk factor for morbidity and mortality. Intensive insulin therapy (IIT) has been shown to reduce morbidity and mortality among the critically ill, decrease infection rates and improve survival after cardiac surgery, and improve outcomes in acute neurological injury and acute myocardial infarction. However, severe hypoglycemia and adverse events associated with IIT are a concern. The authors summarize the mechanisms and rationale of hyperglycemia and IIT, and review the evidence behind the use of IIT in the perioperative period. While avoidance of hyperglycemia is clearly beneficial, the appropriate glucose target and specific subpopulations that might benefit from IIT have yet to be identified. See the accompanying Editorial View on page 204 The authors hypothesized that the use of factor XIII would delay the decrease in clot firmness in high-risk patients with high fibrin monomer levels. This concept was tested in a prospective, randomized, double-blind, placebo-controlled trial in elective cancer surgery. Patients were randomized to receive factor XIII (30 U/kg) or placebo and standard therapy. During the interim analysis, clot firmness was evaluated. Patients receiving placebo lost 38% firmness whereas those receiving factor XIII did not. This proof of concept study confirmed that patients at high risk for intraoperative blood loss showed reduced loss of clot firmness when factor XIII was administered. Further clinical trials are needed to assess this treatment on blood loss and use of blood products. See the accompanying Editorial View on page 212 An updated report by the American Society of Anesthesiologists Task Force on Neuraxial Opioids.In multivariate models, body mass index is a weak but significant predictor of difficult and failed intubation.Tighter glucose control may improve renal function in potential organ donors. See the accompanying Editorial View on page 207 Early transfusion is an independent predictor of adult respiratory distress syndrome in adult trauma. See the accompanying Editorial View on page 216

Increased Volatile Anesthetic Requirement in Short-sleeping Drosophila Mutants

Anesthesia and sleep share physiologic and behavioral similarities. The anesthetic requirement of the recently identified Drosophila mutant minisleeper and other Drosophila mutants was investigated. Sleep and wakefulness were determined by measuring activity of individual wild-type and mutant flies. Based on the response of the flies at different concentrations of the volatile anesthetics isoflurane and sevoflurane, concentration-response curves were generated and EC50 values were calculated. The average amount of daily sleep in wild-type Drosophila (n = 64) was 965 +/- 15 min, and 1,022 +/- 29 in Na[har](P > 0.05; n = 32) (mean +/- SEM, all P compared to wild-type and other shaker alleles). Sh flies slept 584 +/- 13 min (n = 64, P < 0.01), Sh flies 412 +/- 22 min (n = 32, P < 0.01), and Sh flies 782 +/- 25 min (n = 32, P < 0.01). The EC50 values for isoflurane were 0.706 (95% CI 0.649 to 0.764, n = 661) and for sevoflurane 1.298 (1.180 to 1.416, n = 522) in wild-type Drosophila; 1.599 (1.527 to 1.671, n = 308) and 2.329 (2.177 to 2.482, n = 282) in Sh, 1.306 (1.212 to 1.400, n = 393) and 2.013 (1.868 to 2.158, n = 550) in Sh, 0.957 (0.860 to 1.054, n = 297) and 1.619 (1.508 to 1.731, n = 386) in Sh, and 0.6154 (0.581 to 0.649, n = 360; P < 0.05) and 0.9339 (0.823 to 1.041, n = 274) in Na[har], respectively (all P < 0.01). A single-gene mutation in Drosophila that causes an extreme reduction in daily sleep is responsible for a significant increase in the requirement of volatile anesthetics. This suggests that a single gene mutation affects both sleep behavior and anesthesia and sedation.

Widespread Changes in Synaptic Markers as a Function of Sleep and Wakefulness in Drosophila

Widespread Changes in Synaptic Markers as a Function of Sleep and Wakefulness in Drosophila

A Video Method to Study Drosophila Sleep

To use video to determine the accuracy of the infrared beam-splitting method for measuring sleep in Drosophila and to determine the effect of time of day, sex, genotype, and age on sleep measurements. A digital image analysis method based on frame subtraction principle was developed to distinguish a quiescent from a moving fly. Data obtained using this method were compared with data obtained using the Drosophila Activity Monitoring System (DAMS). The location of the fly was identified based on its centroid location in the subtracted images. The error associated with the identification of total sleep using DAMS ranged from 7% to 95% and depended on genotype, sex, age, and time of day. The degree of the total sleep error was dependent on genotype during the daytime (P < 0.001) and was dependent on age during both the daytime and the nighttime (P < 0.001 for both). The DAMS method overestimated sleep bout duration during both the day and night, and the degree of these errors was genotype dependent (P < 0.001). Brief movements that occur during sleep bouts can be accurately identified using video. Both video and DAMS detected a homeostatic response to sleep deprivation. Video digital analysis is more accurate than DAMS in fly sleep measurements. In particular, conclusions drawn from DAMS measurements regarding daytime sleep and sleep architecture should be made with caution. Video analysis also permits the assessment of fly position and brief movements during sleep.