The impact of Nrf2 knockout on the neuroprotective effects of dexmedetomidine in a mice model of cognitive impairment.

The nuclear factor erythroid 2-related factor 2 (Nrf2) signalling pathway represents a crucial intrinsic protective system against oxidative stress and inflammation and plays a significant role in various neurological disorders. However, the effect of Nrf2 signalling on the regulation of cognitive impairment remains unknown. Dexmedetomidine (DEX) has neuroprotective effects and can ameliorate lipopolysaccharide (LPS)-induced cognitive dysfunction. Our objective was to observe whether Nrf2 knockout influences the efficacy of DEX in improving cognitive impairment and to attempt to understand its underlying mechanisms. An LPS-induced cognitive dysfunction model in wild-type and Nrf2 knockout mice (Institute of Cancer Research background; male; 8-12 weeks) was used to observe the impact of DEX on cognitive dysfunction. LPS was intraperitoneally injected, followed by novel object recognition and morris water maze experiments 24 h later. Hippocampal tissues were collected for histopathological and molecular analyses. Our research findings suggest that DEX enhances the expression of NQO1, HO-1, PSD95, and SYP proteins in hippocampal tissue, inhibits microglial proliferation, reduces pro-inflammatory cytokines IL-1ß and TNF-ɑ, increases anti-inflammatory cytokine IL-10, and improves dendritic spine density, thereby alleviating cognitive dysfunction induced by LPS. However, the knockout of the Nrf2 gene negated the aforementioned effects of DEX. In conclusion, DEX alleviates cognitive deficits induced by LPS through mechanisms of anti-oxidative stress and anti-inflammation, as well as by increasing synaptic protein expression and dendritic spine density. However, the knockout of the Nrf2 gene reversed the effects of DEX. The Nrf2 signaling pathway plays a crucial role in the mitigation of LPS-induced cognitive impairment by DEX.

A neuron-glia lipid metabolic cycle couples daily sleep to mitochondrial homeostasis.

Sleep is thought to be restorative to brain energy homeostasis, but it is not clear how this is achieved. We show here that Drosophila glia exhibit a daily cycle of glial mitochondrial oxidation and lipid accumulation that is dependent on prior wake and requires the Drosophila APOE orthologs NLaz and GLaz, which mediate neuron-glia lipid transfer. In turn, a full night of sleep is required for glial lipid clearance, mitochondrial oxidative recovery and maximal neuronal mitophagy. Knockdown of neuronal NLaz causes oxidative stress to accumulate in neurons, and the neuronal mitochondrial integrity protein, Drp1, is required for daily glial lipid accumulation. These data suggest that neurons avoid accumulation of oxidative mitochondrial damage during wake by using mitophagy and passing damage to glia in the form of lipids. We propose that a mitochondrial lipid metabolic cycle between neurons and glia reflects a fundamental function of sleep relevant for brain energy homeostasis.

Sleepiness, not total sleep amount, increases seizure risk.

Sleep loss has been associated with increased seizure risk since antiquity. Despite this observation standing the test of time, how poor sleep drives susceptibility to seizures remains unclear. To identify underlying mechanisms, we restricted sleep in Drosophila epilepsy models and developed a method to identify spontaneous seizures using quantitative video tracking. Here we find that sleep loss exacerbates seizures but only when flies experience increased sleep need, or sleepiness , and not necessarily with reduced sleep quantity. This is supported by the paradoxical finding that acute activation of sleep-promoting circuits worsens seizures, because it increases sleep need without changing sleep amount. Sleep-promoting circuits become hyperactive after sleep loss and are associated with increased whole-brain activity. During sleep restriction, optogenetic inhibition of sleep-promoting circuits to reduce sleepiness protects against seizures. Downregulation of the 5HT1A serotonin receptor in sleep-promoting cells mediates the effect of sleep need on seizures, and we identify an FDA-approved 5HT1A agonist to mitigate seizures. Our findings demonstrate that while homeostatic sleep is needed to recoup lost sleep, it comes at the cost of increasing seizure susceptibility. We provide an unexpected perspective on interactions between sleep and seizures, and surprisingly implicate sleep- promoting circuits as a therapeutic target for seizure control.

Ecdysone acts through cortex glia to regulate sleep in Drosophila.

Steroid hormones are attractive candidates for transmitting long-range signals to affect behavior. These lipid-soluble molecules derived from dietary cholesterol easily penetrate the brain and act through nuclear hormone receptors (NHRs) that function as transcription factors. To determine the extent to which NHRs affect sleep:wake cycles, we knocked down each of the 18 highly conserved NHRs found in Drosophila adults and report that the ecdysone receptor (EcR) and its direct downstream NHR Eip75B (E75) act in glia to regulate the rhythm and amount of sleep. Given that ecdysone synthesis genes have little to no expression in the fly brain, ecdysone appears to act as a long-distance signal and our data suggest that it enters the brain more at night. Anti-EcR staining localizes to the cortex glia in the brain and functional screening of glial subtypes revealed that EcR functions in adult cortex glia to affect sleep. Cortex glia are implicated in lipid metabolism, which appears to be relevant for actions of ecdysone as ecdysone treatment mobilizes lipid droplets (LDs), and knockdown of glial EcR results in more LDs. In addition, sleep-promoting effects of exogenous ecdysone are diminished in lsd-2 mutant flies, which are lean and deficient in lipid accumulation. We propose that ecdysone is a systemic secreted factor that modulates sleep by stimulating lipid metabolism in cortex glia.

A circadian clock regulates efflux by the blood-brain barrier in mice and human cells.

The blood-brain barrier (BBB) is critical for neural function. We report here circadian regulation of the BBB in mammals. Efflux of xenobiotics by the BBB oscillates in mice, with highest levels during the active phase and lowest during the resting phase. This oscillation is abrogated in circadian clock mutants. To elucidate mechanisms of circadian regulation, we profiled the transcriptome of brain endothelial cells; interestingly, we detected limited circadian regulation of transcription, with no evident oscillations in efflux transporters. We recapitulated the cycling of xenobiotic efflux using a human microvascular endothelial cell line to find that the molecular clock drives cycling of intracellular magnesium through transcriptional regulation of TRPM7, which appears to contribute to the rhythm in efflux. Our findings suggest that considering circadian regulation may be important when therapeutically targeting efflux transporter substrates to the CNS.

Successful intraoperative management in patients with abdominal compartment syndrome induced by giant liposarcomas: Two case reports.

RATIONALE: Giant intra-abdominal liposarcomas weighing over 20 kg often increase the intra-abdominal pressure (IAP), which has severe effects on the cardiovascular and respiratory systems. Abdominal compartment syndrome is defined typically as the combination of a raised IAP of 20 mm Hg or higher and new onset of organ dysfunction or failure. The anesthetic management and perioperative management are very challenging. PATIENTS CONCERNS: We presented 2 patients with rare giant growing liposarcoma of the abdomen, weighing 21 kg and over 35 kg, respectively. Circulatory management was particularly difficult in the first case, while respiratory management and massive blood loss was very challenging in the second one. DIAGNOSIS: With a computed tomography scan and peritoneal-to-abdominal height ratio measurement, preoperatively the risk of developing intra-abdominal hypertension and abdominal compartment syndrome was recognized early in each patient. The inferior vena cava and right atrium of the first patient was compressed and malformed due to the uplifted diaphragm, while there was severe decreased lung volume and increased airway resistance, because of rare giant retroperitoneal liposarcomas in the second case. Histologic examination revealed dedifferentiated liposarcoma in both cases. INTERVENTIONS: Both of the patients underwent resection surgery with multiple monitoring; transesophageal echocardiography monitoring in the first case and pressure-controlled ventilation volume guaranteed mechanical ventilation mode in both cases. OUTCOMES: Intraoperatively and postoperatively no cardiopulmonary complications in both patients. The first patient was discharged without any complications on postoperative day 10, and the second patient underwent another surgery because of anastomotic leakage resulting from bowel resection. LESSONS: Multiple monitorings, in particular transesophageal echocardiography should be considered in patients with increased IAP due to a giant mass, while an appropriate lung protection ventilation strategy is crucial in these patients.

Misregulation of Drosophila Myc Disrupts Circadian Behavior and Metabolism.

Drosophila Myc (dMyc) is highly conserved and functions as a transcription factor similar to mammalian Myc. We previously found that oncogenic Myc disrupts the molecular clock in cancer cells. Here, we demonstrate that misregulation of dMyc expression affects Drosophila circadian behavior. dMyc overexpression results in a high percentage of arrhythmic flies, concomitant with increases in the expression of clock genes cyc, tim, cry, and cwo. Conversely, flies with hypomorphic mutations in dMyc exhibit considerable arrhythmia, which can be rescued by loss of dMnt, a suppressor of dMyc activity. Metabolic profiling of fly heads revealed that loss of dMyc and its overexpression alter steady-state metabolite levels and have opposing effects on histidine, the histamine precursor, which is rescued in dMyc mutants by ablation of dMnt and could contribute to effects of dMyc on locomotor behavior. Our results demonstrate a role of dMyc in modulating Drosophila circadian clock, behavior, and metabolism.

A Circadian Clock in the Blood-Brain Barrier Regulates Xenobiotic Efflux.

Endogenous circadian rhythms are thought to modulate responses to external factors, but mechanisms that confer time-of-day differences in organismal responses to environmental insults/therapeutic treatments are poorly understood. Using a xenobiotic, we find that permeability of the Drosophila "blood"-brain barrier (BBB) is higher at night. The permeability rhythm is driven by circadian regulation of efflux and depends on a molecular clock in the perineurial glia of the BBB, although efflux transporters are restricted to subperineurial glia (SPG). We show that transmission of circadian signals across the layers requires cyclically expressed gap junctions. Specifically, during nighttime, gap junctions reduce intracellular magnesium ([Mg2+]i), a positive regulator of efflux, in SPG. Consistent with lower nighttime efflux, nighttime administration of the anti-epileptic phenytoin is more effective at treating a Drosophila seizure model. These findings identify a novel mechanism of circadian regulation and have therapeutic implications for drugs targeted to the central nervous system.

Genetic Dissociation of Daily Sleep and Sleep Following Thermogenetic Sleep Deprivation in Drosophila.

STUDY OBJECTIVES: Sleep rebound-the increase in sleep that follows sleep deprivation-is a hallmark of homeostatic sleep regulation that is conserved across the animal kingdom. However, both the mechanisms that underlie sleep rebound and its relationship to habitual daily sleep remain unclear. To address this, we developed an efficient thermogenetic method of inducing sleep deprivation in Drosophila that produces a substantial rebound, and applied the newly developed method to assess sleep rebound in a screen of 1,741 mutated lines. We used data generated by this screen to identify lines with reduced sleep rebound following thermogenetic sleep deprivation, and to probe the relationship between habitual sleep amount and sleep following thermogenetic sleep deprivation in Drosophila. METHODS: To develop a thermogenetic method of sleep deprivation suitable for screening, we thermogenetically stimulated different populations of wake-promoting neurons labeled by Gal4 drivers. Sleep rebound following thermogenetically-induced wakefulness varies across the different sets of wake-promoting neurons that were stimulated, from very little to quite substantial. Thermogenetic activation of neurons marked by the c584-Gal4 driver produces both strong sleep loss and a substantial rebound that is more consistent within genotypes than rebound following mechanical or caffeine-induced sleep deprivation. We therefore used this driver to induce sleep deprivation in a screen of 1,741 mutagenized lines generated by the Drosophila Gene Disruption Project. Flies were subjected to 9 h of sleep deprivation during the dark period and released from sleep deprivation 3 h before lights-on. Recovery was measured over the 15 h following sleep deprivation. Following identification of lines with reduced sleep rebound, we characterized baseline sleep and sleep depth before and after sleep deprivation for these hits. RESULTS: We identified two lines that consistently exhibit a blunted increase in the duration and depth of sleep after thermogenetic sleep deprivation. Neither of the two genotypes has reduced total baseline sleep. Statistical analysis across all screened lines shows that genotype is a strong predictor of recovery sleep, independent from effects of genotype on baseline sleep. CONCLUSIONS: Our data show that rebound sleep following thermogenetic sleep deprivation can be genetically separated from sleep at baseline. This suggests that genetically controlled mechanisms of sleep regulation not manifest under undisturbed conditions contribute to sleep rebound following thermogenetic sleep deprivation.

Independent Effects of γ-Aminobutyric Acid Transaminase (GABAT) on Metabolic and Sleep Homeostasis.

Breakdown of the major sleep-promoting neurotransmitter, γ-aminobutyric acid (GABA), in the GABA shunt generates catabolites that may enter the tricarboxylic acid cycle, but it is unknown whether catabolic by-products of the GABA shunt actually support metabolic homeostasis. In Drosophila, the loss of the specific enzyme that degrades GABA, GABA transaminase (GABAT), increases sleep, and we show here that it also affects metabolism such that flies lacking GABAT fail to survive on carbohydrate media. Expression of GABAT in neurons or glia rescues this phenotype, indicating a general metabolic function for this enzyme in the brain. As GABA degradation produces two catabolic products, glutamate and succinic semialdehyde, we sought to determine which was responsible for the metabolic phenotype. Through genetic and pharmacological experiments, we determined that glutamate, rather than succinic semialdehyde, accounts for the metabolic phenotype of gabat mutants. This is supported by biochemical measurements of catabolites in wild-type and mutant animals. Using in vitro labeling assays, we found that inhibition of GABAT affects energetic pathways. Interestingly, we also observed that gaba mutants display a general disruption in bioenergetics as measured by altered levels of tricarboxylic acid cycle intermediates, NAD(+)/NADH, and ATP levels. Finally, we report that the effects of GABAT on sleep do not depend upon glutamate, indicating that GABAT regulates metabolic and sleep homeostasis through independent mechanisms. These data indicate a role of the GABA shunt in the development of metabolic risk and suggest that neurological disorders caused by altered glutamate or GABA may be associated with metabolic disruption.

Sleep deprivation suppresses aggression in Drosophila.

Sleep disturbances negatively impact numerous functions and have been linked to aggression and violence. However, a clear effect of sleep deprivation on aggressive behaviors remains unclear. We find that acute sleep deprivation profoundly suppresses aggressive behaviors in the fruit fly, while other social behaviors are unaffected. This suppression is recovered following post-deprivation sleep rebound, and occurs regardless of the approach to achieve sleep loss. Genetic and pharmacologic approaches suggest octopamine signaling transmits changes in aggression upon sleep deprivation, and reduced aggression places sleep-deprived flies at a competitive disadvantage for obtaining a reproductive partner. These findings demonstrate an interaction between two phylogenetically conserved behaviors, and suggest that previous sleep experiences strongly modulate aggression with consequences for reproductive fitness.

A critical period of sleep for development of courtship circuitry and behavior in Drosophila.

Most animals sleep more early in life than in adulthood, but the function of early sleep is not known. Using Drosophila, we found that increased sleep in young flies was associated with an elevated arousal threshold and resistance to sleep deprivation. Excess sleep results from decreased inhibition of a sleep-promoting region by a specific dopaminergic circuit. Experimental hyperactivation of this circuit in young flies results in sleep loss and lasting deficits in adult courtship behaviors. These deficits are accompanied by impaired development of a single olfactory glomerulus, VA1v, which normally displays extensive sleep-dependent growth after eclosion. Our results demonstrate that sleep promotes normal brain development that gives rise to an adult behavior critical for species propagation and suggest that rapidly growing regions of the brain are most susceptible to sleep perturbations early in life.

Identification of Redeye, a new sleep-regulating protein whose expression is modulated by sleep amount.

In this study, we report a new protein involved in the homeostatic regulation of sleep in Drosophila. We conducted a forward genetic screen of chemically mutagenized flies to identify short-sleeping mutants and found one, redeye (rye) that shows a severe reduction of sleep length. Cloning of rye reveals that it encodes a nicotinic acetylcholine receptor α subunit required for Drosophila sleep. Levels of RYE oscillate in light-dark cycles and peak at times of daily sleep. Cycling of RYE is independent of a functional circadian clock, but rather depends upon the sleep homeostat, as protein levels are up-regulated in short-sleeping mutants and also in wild type animals following sleep deprivation. We propose that the homeostatic drive to sleep increases levels of RYE, which responds to this drive by promoting sleep. DOI: http://dx.doi.org/10.7554/eLife.01473.001.

[Treatment strategy for the floating shoulder injury].

OBJECTIVE: To explore the clinical features and operative treatment of floating shoulder injuries. METHODS: The clinical data of 25 patients with floating shoulder injuries that had been admitted to the hospital from July 2000 to May 2011 were retrospectively analyzed. There were 18 males and 7 females,wirh an average age of (36.2 +/- 2.3) years ranging from 17 to 56 years. The scapular neck fractures associated with clavicle fractures were in 21 cases and acromioclavicular joint dislocation in 4 cases. All cases were accompanied by associated injuries. Among of them, 7 cases were conservative treatment, 7 cases were fixed clavicle only, 11 cases were clavicle and scapular. All datum were rated according to Herscovici, Constant and Murley, Rowe rate system. RESULTS: All patients were followed up for 4.6 years (range 11 months to 10 years). All fractures were healed except for 1 clavicle was delay healed. There were not infections and fixation fracture,2 of conservative treatment were dropping shoulder, 2 of 3 brachial plexus injuries were recovered 3 months later, 1 was 6 months. Suprascapular nerve injury was recoved 1 year later. Herscovici evaluation: 18 excellent, 5 good, 1 fair, 1 poor;Constant and Murley shoulder score: (83.2 +/- 5.7), pain score (12.1 + 2.5), activities of daily living score (17.2 +/- 3.2), range of motion score (32.1 +/- 3.5), strength score (18.5 +/- 2.1); Rowe evaluation: 18 excellent, 5 fair, 2 poor. CONCLUSION: Floating shoulder injuries is high energy injury, destroy superior shoulder suspensory complex stability ,appropriate treatment should be choosen according to fractures displacement and smash level, patients' professional and anticipation

Evoked bursting in injured Aß dorsal root ganglion neurons: a mechanism underlying tactile allodynia.

Chronic compression of rat dorsal root ganglion (CCD) produced tactile allodynia accompanied with hyperexcitability of the myelinated Aß dorsal root ganglion (DRG) neurons. The Aß DRG neuron hyperexcitability exhibits as bursting discharges in response to peripherally evoked action potentials (evoked bursting [EB]). The incidence of EB was significantly increased after chronic compression of DRG (CCD) (43.3%) vs control (13.3%). EB was maintained by oscillation of the membrane potential, and its duration was increased when the membrane potential was depolarized. EB was found to coexist in some neurons with spontaneous bursting (SB), but EB always occurred at a more negative membrane potential than SB. Afterdischarges of the wide dynamic range neurons of the dorsal horn in the spinal cord in response to electrical stimulation of Aß afferent nerve fibers were suppressed by blocking EB of the DRG neurons. CCD neurons with EB exhibited increased current density of persistent sodium current (I(Nap)) and hyperpolarization-activated cation current (I(h)) and decreased α-dendrotoxin (α-DTX) sensitive current (I(DTX)). The increased I(h) activated by afterhyperpolarization of peripheral afferent action potential was necessary for EB generation and a balance between I(DTX) and I(Nap) might be necessary for EB maintenance. This study may suggest a role of EB of myelinated DRG neurons in development of allodynia after nerve injury and a potential pharmaceutical therapy in treating neuropathic allodynia.

Old flies have a robust central oscillator but weaker behavioral rhythms that can be improved by genetic and environmental manipulations.

Sleep-wake cycles break down with age, but the causes of this degeneration are not clear. Using a Drosophila model, we addressed the contribution of circadian mechanisms to this age-induced deterioration. We found that in old flies, free-running circadian rhythms (behavioral rhythms assayed in constant darkness) have a longer period and an unstable phase before they eventually degenerate. Surprisingly, rhythms are weaker in light-dark cycles and the circadian-regulated morning peak of activity is diminished under these conditions. On a molecular level, aging results in reduced amplitude of circadian clock gene expression in peripheral tissues. However, oscillations of the clock protein PERIOD (PER) are robust and synchronized among different clock neurons, even in very old, arrhythmic flies. To improve rhythms in old flies, we manipulated environmental conditions, which can have direct effects on behavior, and also tested a role for molecules that act downstream of the clock. Coupling temperature cycles with a light-dark schedule or reducing expression of protein kinase A (PKA) improved behavioral rhythms and consolidated sleep. Our data demonstrate that a robust molecular timekeeping mechanism persists in the central pacemaker of aged flies, and reducing PKA can strengthen behavioral rhythms.

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 glycosylphosphatidyl inositol-anchored protein required for sleep in Drosophila. Here we found that SSS is critical for regulating the sleep-modulating potassium channel Shaker. SSS and Shaker shared similar expression patterns in the brain and specifically affected each other's expression levels. sleepless (sss) loss-of-function mutants exhibited altered Shaker localization, reduced Shaker current density and slower Shaker current kinetics. Transgenic expression of sss in sss mutants rescued defects in Shaker expression and activity cell-autonomously and suggested that SSS functions in wake-promoting, cholinergic neurons. In heterologous cells, SSS accelerated the kinetics of Shaker currents and was co-immunoprecipitated with Shaker, suggesting that SSS modulates Shaker activity via a direct interaction. SSS is predicted to belong to the Ly-6/neurotoxin superfamily, suggesting a mechanism for regulation of neuronal excitability by endogenous toxin-like molecules.

The effects of caffeine on sleep in Drosophila require PKA activity, but not the adenosine receptor.

Caffeine is one of the most widely consumed stimulants in the world and has been proposed to promote wakefulness by antagonizing function of the adenosine A2A receptor. Here, we show that chronic administration of caffeine reduces and fragments sleep in Drosophila and also lengthens circadian period. To identify the mechanisms underlying these effects of caffeine, we first generated mutants of the only known adenosine receptor in flies (dAdoR), which by sequence is most similar to the mammalian A2A receptor. Mutants lacking dAdoR have normal amounts of baseline sleep, as well as normal homeostatic responses to sleep deprivation. Surprisingly, these mutants respond normally to caffeine. On the other hand, the effects of caffeine on sleep and circadian rhythms are mimicked by a potent phosphodiesterase inhibitor, IBMX (3-isobutyl-1-methylxanthine). Using in vivo fluorescence resonance energy transfer imaging, we find that caffeine induces widespread increase in cAMP levels throughout the brain. Finally, the effects of caffeine on sleep are blocked in flies that have reduced neuronal PKA activity. We suggest that chronic administration of caffeine promotes wakefulness in Drosophila, at least in part, by inhibiting cAMP phosphodiesterase activity.

Identification of SLEEPLESS, a sleep-promoting factor.

Sleep is an essential process conserved from flies to humans. The importance of sleep is underscored by its tight homeostatic control. Through a forward genetic screen, we identified a gene, sleepless, required for sleep in Drosophila. The sleepless gene encodes a brain-enriched, glycosylphosphatidylinositol-anchored protein. Loss of SLEEPLESS protein caused an extreme (>80%) reduction in sleep; a moderate reduction in SLEEPLESS had minimal effects on baseline sleep but markedly reduced the amount of recovery sleep after sleep deprivation. Genetic and molecular analyses revealed that quiver, a mutation that impairs Shaker-dependent potassium current, is an allele of sleepless. Consistent with this finding, Shaker protein levels were reduced in sleepless mutants. We propose that SLEEPLESS is a signaling molecule that connects sleep drive to lowered membrane excitability.

A genetic screen for sleep and circadian mutants reveals mechanisms underlying regulation of sleep in Drosophila.

STUDY OBJECTIVES: In order to characterize the genetic mechanisms underlying sleep, we have carried out a large-scale screen in Drosophila to identify short-sleeping mutants. The objectives of this study were as follows: (1) characterize the phenotypes of the shortest-sleeping mutants; (2) examine whether changes in arousal threshold or sleep homeostasis underlie short-sleeping phenotypes; (3) clone a gene affected in one of the shortest sleepers; and (4) investigate whether circadian mutants can be identified using light:dark (L:D) locomotor data obtained for studying sleep behavior. DESIGN: Locomotor activity was measured using the Drosophila Activity Monitoring System in a 12:12 L:D cycle. SETTING: Drosophila research laboratory. PARTICIPANTS: Adult flies from the 2nd chromosome Zuker collection, which contain mutations in most of the nonessential genes on the Drosophila 2nd chromosome. MEASUREMENTS AND RESULTS: Our analysis of sleep characteristics suggests that daily activity (but not waking activity) correlates with daily sleep time and that defects in sleep maintenance are more common than defects in sleep initiation. Our shortest sleepers have intact or increased sleep rebound following sleep deprivation but show reduced thresholds for arousal. Molecular analysis of one of the short-sleeping lines indicates that it is a novel allele of a dopamine transporter (DAT). Finally, we describe a novel approach for identifying circadian mutants using L:D data. CONCLUSIONS: Our data suggest that most short-sleeping mutant phenotypes in Drosophila are characterized by an inability to stay asleep, most likely because of a reduced arousal threshold.