Julie A. Williams

Rest in Drosophila Is a Sleep-like State

To facilitate the genetic study of sleep, we documented that rest behavior in Drosophila melanogaster is a sleep-like state. The animals choose a preferred location, become immobile for periods of up to 157 min at a particular time in the circadian day, and are relatively unresponsive to sensory stimuli. Rest is affected by both homeostatic and circadian influences: when rest is prevented, the flies increasingly tend to rest despite stimulation and then exhibit a rest rebound. Drugs acting on a mammalian adenosine receptor alter rest as they do sleep, suggesting conserved neural mechanisms. Finally, normal homeostatic regulation depends on the timeless but not the period central clock gene. Understanding the molecular features of Drosophila rest should shed new light on the mechanisms and function of sleep.

A non-circadian role for cAMP signaling and CREB activity in Drosophila rest homeostasis

In the fruit fly, Drosophila melanogaster, rest shares features with mammalian sleep, including prolonged immobility, decreased sensory responsiveness and a homeostatic rebound after deprivation. To understand the molecular regulation of sleep-like rest, we investigated the involvement of a candidate gene, cAMP response-element binding protein (CREB). The duration of rest was inversely related to cAMP signaling and CREB activity. Acutely blocking CREB activity in transgenic flies did not affect the clock, but increased rest rebound. CREB mutants also had a prolonged and increased homeostatic rebound. In wild types, in vivo CREB activity increased after rest deprivation and remained elevated for a 72-hour recovery period. These data indicate that cAMP signaling has a non-circadian role in waking and rest homeostasis in Drosophila.

FMRFamide signaling promotes stress-induced sleep in Drosophila

Enhanced sleep in response to cellular stress is a conserved adaptive behavior across multiple species, but the mechanism of this process is poorly understood. Drosophila melanogaster increases sleep following exposure to septic or aseptic injury, and Caenorhabditis elegans displays sleep-like quiescence following exposure to high temperatures that stress cells. We show here that, similar to C. elegans, Drosophila responds to heat stress with an increase in sleep. In contrast to Drosophila infection-induced sleep, heat-induced sleep is not sensitive to the time-of-day of the heat pulse. Moreover, the sleep response to heat stress does not require Relish, the NFκB transcription factor that is necessary for infection-induced sleep, indicating that sleep is induced by multiple mechanisms from different stress modalities. We identify a sleep-regulating role for a signaling pathway involving FMRFamide neuropeptides and their receptor FR. Animals mutant for either FMRFamide or for the FMRFamide receptor (FR) have a reduced recovery sleep in response to heat stress. FR mutants, in addition, show reduced sleep responses following infection with Serratia marcescens, and succumb to infection at a faster rate than wild-type controls. Together, these findings support the hypothesis that FMRFamide and its receptor promote an adaptive increase in sleep following stress. Because an FMRFamide-like neuropeptide plays a similar role in C. elegans, we propose that FRMFamide neuropeptide signaling is an ancient regulator of recovery sleep which occurs in response to cellular stress.

Acute Sleep Deprivation Enhances Post-Infection Sleep and Promotes Survival during Bacterial Infection in Drosophila

STUDY OBJECTIVES Sleep is known to increase as an acute response to infection. However, the function of this behavioral response in host defense is not well understood. To address this problem, we evaluated the effect of acute sleep deprivation on post-infection sleep and immune function in Drosophila. SETTING Laboratory. PARTICIPANTS Drosophila melanogaster. METHODS AND RESULTS Flies were subjected to sleep deprivation before (early DEP) or after (late DEP) bacterial infection. Relative to a non-deprived control, flies subjected to early DEP had enhanced sleep after infection as well as increased bacterial clearance and survival outcome. Flies subjected to late DEP experienced enhanced sleep following the deprivation period, and showed a modest improvement in survival outcome. Continuous DEP (early and late DEP) throughout infection also enhanced sleep later during infection and improved survival. However, improved survival in flies subjected to late or continuous DEP did not occur until after flies had experienced sleep. During infection, both early and late DEP enhanced NFκB transcriptional activity as measured by a luciferase reporter (κB-luc) in living flies. Early DEP also increased NFκB activity prior to infection. Flies that were deficient in expression of either the Relish or Dif NFκB transcription factors showed normal responses to early DEP. However, the effect of early DEP on post-infection sleep and survival was abolished in double mutants, which indicates that Relish and Dif have redundant roles in this process. CONCLUSIONS Acute sleep deprivation elevated NFκB-dependent activity, increased post-infection sleep, and improved survival during bacterial infection.

Increased sleep promotes survival during a bacterial infection in Drosophila.

STUDY OBJECTIVES The relationship between sleep and immune function is not well understood at a functional or molecular level. We therefore used a genetic approach in Drosophila to manipulate sleep and evaluated effects on the ability of flies to fight bacterial infection. SETTING Laboratory. PARTICIPANTS Drosophila melanogaster. METHODS AND RESULTS We used a genetic approach to transiently alter neuronal excitability in the mushroom body, a region in the central brain that is known to regulate sleep. Flies with increased sleep for up to two days prior to a bacterial infection showed increased resistance to the infection and improved survival. These flies also had increased expression levels of a subset of anti-microbial peptide mRNA prior to infection, as well as increased NFκB activity during infection as indicated by in vivo luciferase reporter activity. In contrast, flies that experienced reduced sleep for up to two days prior to infection had no effect on survival or on NFκB activity during infection. However, flies with reduced sleep showed an altered defense mechanism, such that resistance to infection was increased, but at the expense of reduced tolerance. This effect was dependent on environmental condition. CONCLUSIONS Increasing sleep enhanced activity of an NFκB transcription factor, increased resistance to infection, and strongly promoted survival. Together, these findings support the hypothesis that sleep is beneficial to the host by maintaining a robust immune system.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals (1) and has also recently been described in Drosophila melanogaster(2). It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system(3,4). However, experimental evidence that supports this hypothesis is limited(4), and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NFκB, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system(5). Real-time monitoring of NFκB activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Sleep Homeostasis and General Anesthesia: Are Fruit Flies Well Rested after Emergence from Propofol?

Background:Shared neurophysiologic features between sleep and anesthetic-induced hypnosis indicate a potential overlap in neuronal circuitry underlying both states. Previous studies in rodents indicate that preexisting sleep debt discharges under propofol anesthesia. The authors explored the hypothesis that propofol anesthesia also dispels sleep pressure in the fruit fly. To the authors’ knowledge, this constitutes the first time propofol has been tested in the genetically tractable model, Drosophila melanogaster. Methods:Daily sleep was measured in Drosophila by using a standard locomotor activity assay. Propofol was administered by transferring flies onto food containing various doses of propofol or equivalent concentrations of vehicle. High-performance liquid chromatography was used to measure the tissue concentrations of ingested propofol. To determine whether propofol anesthesia substitutes for natural sleep, the flies were subjected to 10-h sleep deprivation (SD), followed by 6-h propofol exposure, and monitored for subsequent sleep. Results:Oral propofol treatment causes anesthesia in flies as indicated by a dose-dependent reduction in locomotor activity (n = 11 to 41 flies from each group) and increased arousal threshold (n = 79 to 137). Recovery sleep in flies fed propofol after SD was delayed until after flies had emerged from anesthesia (n = 30 to 48). SD was also associated with a significant increase in mortality in propofol-fed flies (n = 44 to 46). Conclusions:Together, these data indicate that fruit flies are effectively anesthetized by ingestion of propofol and suggest that homologous molecular and neuronal targets of propofol are conserved in Drosophila. However, behavioral measurements indicate that propofol anesthesia does not satisfy the homeostatic need for sleep and may compromise the restorative properties of sleep.

Wolbachia affects sleep behavior in Drosophila melanogaster

Wolbachia are endosymbiotic bacteria present in a wide range of insects. Although their dramatic effects on host reproductive biology have been well studied, the effects of Wolbachia on sleep behavior of insect hosts are not well documented. In this study, we report that Wolbachia infection caused an increase of total sleep time in both male and female Drosophila melanogaster. The increase in sleep was associated with an increase in the number of nighttime sleep bouts or episodes, but not in sleep bout duration. Correspondingly, Wolbachia infection also reduced the arousal threshold of their fly hosts. However, neither circadian rhythm nor sleep rebound following deprivation was influenced by Wolbachia infection. Transcriptional analysis of the dopamine biosynthesis pathway revealed that two essential genes, Pale and Ddc, were significantly upregulated in Wolbachia-infected flies. Together, these results indicate that Wolbachia mediates the expression of dopamine related genes, and decreases the sleep quality of their insect hosts. Our findings help better understand the host-endosymbiont interactions and in particular the Wolbachias impact on behaviors, and thus on ecology and evolution in insect hosts.

A Conserved Circadian Function for the Neurofibromatosis 1 Gene

SUMMARY Loss of the Neurofibromatosis 1 (Nf1) protein, neurofibromin, in Drosophila disrupts circadian rhythms of locomotor activity without impairing central clock function, suggesting effects downstream of the clock. However, the relevant cellular mechanisms are not known. Leveraging the discovery of output circuits for locomotor rhythms, we dissected cellular actions of neurofibromin in recently identified substrates. Herein, we show that neurofibromin affects the levels and cycling of calcium in multiple circadian peptidergic neurons. A prominent site of action is the pars intercerebralis (PI), the fly equivalent of the hypothalamus, with cell-autonomous effects of Nf1 in PI cells that secrete DH44. Nf1 interacts genetically with peptide signaling to affect circadian behavior. We extended these studies to mammals to demonstrate that mouse astrocytes exhibit a 24-hr rhythm of calcium levels, which is also attenuated by lack of neurofibromin. These findings establish a conserved role for neurofibromin in intracellular signaling rhythms within the nervous system.