Sleep In Drosophila Research Articles

Sleep- and wake-dependent changes in neuronal activity and reactivity demonstrated in fly neurons using in vivo calcium imaging

Sleep in Drosophila shares many features with mammalian sleep, but it remains unknown whether spontaneous and evoked activity of individual neurons change with the sleep/wake cycle in flies as they do in mammals. Here we used calcium imaging to assess how the Kenyon cells in the fly mushroom bodies change their activity and reactivity to stimuli during sleep, wake, and after short or long sleep deprivation. As before, sleep was defined as a period of immobility of >5 min associated with a reduced behavioral response to a stimulus. We found that calcium levels in Kenyon cells decline when flies fall asleep and increase when they wake up. Moreover, calcium transients in response to two different stimuli are larger in awake flies than in sleeping flies. The activity of Kenyon cells is also affected by sleep/wake history: in awake flies, more cells are spontaneously active and responding to stimuli if the last several hours (5-8 h) before imaging were spent awake rather than asleep. By contrast, long wake (≥29 h) reduces both baseline and evoked neural activity and decreases the ability of neurons to respond consistently to the same repeated stimulus. The latter finding may underlie some of the negative effects of sleep deprivation on cognitive performance and is consistent with the occurrence of local sleep during wake as described in behaving rats. Thus, calcium imaging uncovers new similarities between fly and mammalian sleep: fly neurons are more active and reactive in wake than in sleep, and their activity tracks sleep/wake history.

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.

Sleep, Performance, and Memory in Flies.

Drosophila has proven to be a powerful model to identify genes and circuits that impact sleep. While the majority of studies have primarily been interested in identifying manipulations that alter sleep time, a growing body of work has begun to focus on how changing sleep influences functional outcomes such as cognitive performance, structural plasticity, and metabolism to name a few. Evaluating sleep time provides an appropriate entry point into elucidating sleep function. However, it is not possible to fully understand how a manipulation has impacted sleep regulation without first establishing how it has affected the animals’ well-being. Synaptic plasticity and memory are important functional outcomes that can be used to asses an animal’s status. In this manuscript, we review recent advances in studies examining sleep, memory, and performance. We conclude that as Drosophila sleep researchers expand their analysis beyond sleep time, the opportunities to discover the function of sleep will be enhanced.

Effects of light interruption on sleep and viability of Drosophila melanogaster.

Light is a very important regulator of the daily sleep rhythm. Here, we investigate the influence of nocturnal light stimulation on Drosophila sleep. Results showed that total daytime sleep was reduced due to a decrease in daytime sleep episode duration caused by discontinuous light stimulation, but sleep was not strongly impacted at nighttime although the discontinuous light stimulation occurred during the scotophase. During a subsequent recovery period without light interruption, the sleep quality of nighttime sleep was improved and of daytime sleep reduced, indicating flies have a persistent response to nocturnal light stimulation. Further studies showed that the discontinuous light stimulation damped the daily rhythm of a circadian light-sensitive protein cryptochrome both at the mRNA and protein levels, which subsequently caused disappearance of circadian rhythm of the core oscillator timeless and decrease of TIMLESS protein at nighttime. These data indicate that the nocturnal light interruption plays an important role in sleep through core proteins CRYTOCHROME and TIMLESS, Moreover, interruption of sleep further impacted reproduction and viability.

SIFamide and SIFamide receptor defines a novel neuropeptide signaling to promote sleep in Drosophila.

SIFamide receptor (SIFR) is a Drosophila G protein-coupled receptor for the neuropeptide SIFamide (SIFa). Although the sequence and spatial expression of SIFa are evolutionarily conserved among insect species, the physiological function of SIFa/SIFR signaling remains elusive. Here, we provide genetic evidence that SIFa and SIFR promote sleep in Drosophila. Either genetic ablation of SIFa-expressing neurons in the pars intercerebralis (PI) or pan-neuronal depletion of SIFa expression shortened baseline sleep and reduced sleep-bout length, suggesting that it caused sleep fragmentation. Consistently, RNA interference-mediated knockdown of SIFR expression caused short sleep phenotypes as observed in SIFa-ablated or depleted flies. Using a panel of neuron-specific Gal4 drivers, we further mapped SIFR effects to subsets of PI neurons. Taken together, these results reveal a novel physiological role of the neuropeptide SIFa/SIFR pathway to regulate sleep through sleep-promoting neural circuits in the PI of adult fly brains.

SLEEPLESS Is a Bifunctional Regulator of Excitability and Cholinergic Synaptic Transmission

Although sleep is conserved throughout evolution, the molecular basis of its control is still largely a mystery. We previously showed that the quiver/sleepless (qvr/sss) gene encodes a membrane-tethered protein that is required for normal sleep in Drosophila. SLEEPLESS (SSS) protein functions, at least in part, by upregulating the levels and open probability of Shaker (Sh) potassium channels to suppress neuronal excitability and enable sleep. Consistent with this proposed mechanism, loss-of-function mutations in Sh phenocopy qvr/sss-null mutants. However, sleep is more genetically modifiable in Sh than in qvr/sss mutants, suggesting that SSS may regulate additional molecules to influence sleep. Here we show that SSS also antagonizes nicotinic acetylcholine receptors (nAChRs) to reduce synaptic transmission and promote sleep. Mimicking this antagonism with the nAChR inhibitor mecamylamine or by RNAi knockdown of specific nAChR subunits is sufficient to restore sleep to qvr/sss mutants. Regulation of nAChR activity by SSS occurs posttranscriptionally, since the levels of nAChR mRNAs are unchanged in qvr/sss mutants. Regulation of nAChR activity by SSS may in fact be direct, since SSS forms a stable complex with and antagonizes nAChR function in transfected cells. Intriguingly, lynx1, a mammalian homolog of SSS, can partially restore normal sleep to qvr/sss mutants, and lynx1 can form stable complexes with Shaker-type channels and nAChRs. Together, our data point to an evolutionarily conserved, bifunctional role for SSS and its homologs in controlling excitability and synaptic transmission in fundamental processes of the nervous system such as sleep.

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.

Neuronal Machinery of Sleep Homeostasis in Drosophila

SummarySleep is under homeostatic control, but the mechanisms that sense sleep need and correct sleep deficits remain unknown. Here, we report that sleep-promoting neurons with projections to the dorsal fan-shaped body (FB) form the output arm of Drosophila’s sleep homeostat. Homeostatic sleep control requires the Rho-GTPase-activating protein encoded by the crossveinless-c (cv-c) gene in order to transduce sleep pressure into increased electrical excitability of dorsal FB neurons. cv-c mutants exhibit decreased sleep time, diminished sleep rebound, and memory deficits comparable to those after sleep loss. Targeted ablation and rescue of Cv-c in sleep-control neurons of the dorsal FB impair and restore, respectively, normal sleep patterns. Sleep deprivation increases the excitability of dorsal FB neurons, but this homeostatic adjustment is disrupted in short-sleeping cv-c mutants. Sleep pressure thus shifts the input-output function of sleep-promoting neurons toward heightened activity by modulating ion channel function in a mechanism dependent on Cv-c.

ERK Phosphorylation Regulates Sleep and Plasticity in Drosophila

Given the relationship between sleep and plasticity, we examined the role of Extracellular signal-regulated kinase (ERK) in regulating baseline sleep, and modulating the response to waking experience. Both sleep deprivation and social enrichment increase ERK phosphorylation in wild-type flies. The effects of both sleep deprivation and social enrichment on structural plasticity in the LNvs can be recapitulated by expressing an active version of ERK (UAS-ERK SEM) pan-neuronally in the adult fly using GeneSwitch (Gsw) Gsw-elav-GAL4. Conversely, disrupting ERK reduces sleep and prevents both the behavioral and structural plasticity normally induced by social enrichment. Finally, using transgenic flies carrying a cAMP response Element (CRE)-luciferase reporter we show that activating ERK enhances CRE-Luc activity while disrupting ERK reduces it. These data suggest that ERK phosphorylation is an important mediator in transducing waking experience into sleep.

DrosophilaNeuroligin 4 Regulates Sleep through Modulating GABA Transmission

Sleep is an essential and evolutionarily conserved behavior that is closely related to synaptic function. However, whether neuroligins (Nlgs), which are cell adhesion molecules involved in synapse formation and synaptic transmission, are involved in sleep is not clear. Here, we show that Drosophila Nlg4 (DNlg4) is highly expressed in large ventral lateral clock neurons (l-LNvs) and that l-LNv-derived DNlg4 is essential for sleep regulation. GABA transmission is impaired in mutant l-LNv, and sleep defects in dnlg4 mutant flies can be rescued by genetic manipulation of GABA transmission. Furthermore, dnlg4 mutant flies exhibit a severe reduction in GABAA receptor RDL clustering, and DNlg4 associates with RDLs in vivo. These results demonstrate that DNlg4 regulates sleep through modulating GABA transmission in l-LNvs, which provides the first known link between a synaptic adhesion molecule and sleep in Drosophila.

Regulation of Sleep by Neuropeptide Y-Like System in Drosophila melanogaster

Sleep is important for maintenance of normal physiology in animals. In mammals, neuropeptide Y (NPY), a homolog of Drosophila neuropeptide F (NPF), is involved in sleep regulation, with different effects in human and rat. However, the function of NPF on sleep in Drosophila melanogaster has not yet been described. In this study, we investigated the effects of NPF and its receptor-neuropeptide F receptor (NPFR1) on Drosophila sleep. Male flies over-expressing NPF or NPFR1 exhibited increased sleep during the nighttime. Further analysis demonstrated that sleep episode duration during nighttime was greatly increased and sleep latency was significantly reduced, indicating that NPF and NPFR1 promote sleep quality, and their action on sleep is not because of an impact of the NPF signal system on development. Moreover, the homeostatic regulation of flies after sleep deprivation was disrupted by altered NPF signaling, since sleep deprivation decreased transcription of NPF in control flies, and there were less sleep loss during sleep deprivation and less sleep gain after sleep deprivation in flies overexpressing NPF and NPFR1 than in control flies, suggesting that NPF system auto-regulation plays an important role in sleep homeostasis. However, these effects did not occur in females, suggesting a sex-dependent regulatory function in sleep for NPF and NPFR1. NPF in D1 brain neurons showed male-specific expression, providing the cellular locus for male-specific regulation of sleep by NPF and NPFR1. This study brings a new understanding into sleep studies of a sexually dimorphic regulatory mode in female and male flies.

Genome-wide association study of sleep in Drosophila melanogaster.

BackgroundSleep is a highly conserved behavior, yet its duration and pattern vary extensively among species and between individuals within species. The genetic basis of natural variation in sleep remains unknown.ResultsWe used the Drosophila Genetic Reference Panel (DGRP) to perform a genome-wide association (GWA) study of sleep in D. melanogaster. We identified candidate single nucleotide polymorphisms (SNPs) associated with differences in the mean as well as the environmental sensitivity of sleep traits; these SNPs typically had sex-specific or sex-biased effects, and were generally located in non-coding regions. The majority of SNPs (80.3%) affecting sleep were at low frequency and had moderately large effects. Additive models incorporating multiple SNPs explained as much as 55% of the genetic variance for sleep in males and females. Many of these loci are known to interact physically and/or genetically, enabling us to place them in candidate genetic networks. We confirmed the role of seven novel loci on sleep using insertional mutagenesis and RNA interference.ConclusionsWe identified many SNPs in novel loci that are potentially associated with natural variation in sleep, as well as SNPs within genes previously known to affect Drosophila sleep. Several of the candidate genes have human homologues that were identified in studies of human sleep, suggesting that genes affecting variation in sleep are conserved across species. Our discovery of genetic variants that influence environmental sensitivity to sleep may have a wider application to all GWA studies, because individuals with highly plastic genotypes will not have consistent phenotypes.

Nicotinamide mononucleotide adenylyltransferase maintains active zone structure by stabilizing Bruchpilot

Active zones are specialized presynaptic structures critical for neurotransmission. We show that a neuronal maintenance factor, nicotinamide mononucleotide adenylyltransferase (NMNAT), is required for maintaining active zone structural integrity in Drosophila by interacting with the active zone protein, Bruchpilot (BRP), and shielding it from activity-induced ubiquitin-proteasome-mediated degradation. NMNAT localizes to the peri-active zone and interacts biochemically with BRP in an activity-dependent manner. Loss of NMNAT results in ubiquitination, mislocalization and aggregation of BRP, and subsequent active zone degeneration. We propose that, as a neuronal maintenance factor, NMNAT specifically maintains active zone structure by direct protein-protein interaction.

Dopamine: On the Threshold of Sleep

A new study examining the neural circuitry regulating sleep in Drosophila has identified a pair of dopamine neurons that signal to the fan-shaped body to suppress sleep. These neurons are separate from the dopamine neurons that regulate motivation, memory, and feeding, suggesting that independent populations of dopamine neurons regulate distinct behaviors.

Identification of a dopamine pathway that regulates sleep and arousal in Drosophila

Sleep is required to maintain physiological functions, including memory, and is regulated by monoamines across species. Enhancement of dopamine signals by a mutation in the dopamine transporter (DAT) decreases sleep, but the underlying dopamine circuit responsible for this remains unknown. We found that the D1 dopamine receptor (DA1) in the dorsal fan-shaped body (dFSB) mediates the arousal effect of dopamine in Drosophila. The short sleep phenotype of the DAT mutant was completely rescued by an additional mutation in the DA1 (also known as DopR) gene, but expression of wild-type DA1 in the dFSB restored the short sleep phenotype. We found anatomical and physiological connections between dopamine neurons and the dFSB neuron. Finally, we used mosaic analysis with a repressive marker and found that a single dopamine neuron projecting to the FSB activated arousal. These results suggest that a local dopamine pathway regulates sleep.

Correction: High-Resolution Positional Tracking for Long-Term Analysis of Drosophila Sleep and Locomotion Using the “Tracker” Program

Correction: High-Resolution Positional Tracking for Long-Term Analysis of Drosophila Sleep and Locomotion Using the “Tracker” Program

High-Resolution Positional Tracking for Long-Term Analysis of Drosophila Sleep and Locomotion Using the “Tracker” Program

Drosophila melanogaster has been used for decades in the study of circadian behavior, and more recently has become a popular model for the study of sleep. The classic method for monitoring fly activity involves counting the number of infrared beam crosses in individual small glass tubes. Incident recording methods such as this can measure gross locomotor activity, but they are unable to provide details about where the fly is located in space and do not detect small movements (i.e. anything less than half the enclosure size), which could lead to an overestimation of sleep and an inaccurate report of the behavior of the fly. This is especially problematic if the fly is awake, but is not moving distances that span the enclosure. Similarly, locomotor deficiencies could be incorrectly classified as sleep phenotypes. To address these issues, we have developed a locomotor tracking technique (the “Tracker” program) that records the exact location of a fly in real time. This allows for the detection of very small movements at any location within the tube. In addition to circadian locomotor activity, we are able to collect other information, such as distance, speed, food proximity, place preference, and multiple additional parameters that relate to sleep structure. Direct comparisons of incident recording and our motion tracking application using wild type and locomotor-deficient (CASK-β null) flies show that the increased temporal resolution in the data from the Tracker program can greatly affect the interpretation of the state of the fly. This is especially evident when a particular condition or genotype has strong effects on the behavior, and can provide a wealth of information previously unavailable to the investigator. The interaction of sleep with other behaviors can also be assessed directly in many cases with this method.

Role of Homer Proteins in the Maintenance of Sleep-Wake States

Sleep is an evolutionarily conserved process that is linked to diurnal cycles and normal daytime wakefulness. Healthy sleep and wakefulness are integral to a healthy lifestyle; this occurs when an organism is able to maintain long bouts of both sleep and wake. Homer proteins, which function as adaptors for group 1 metabotropic glutamate receptors, have been implicated in genetic studies of sleep in both Drosophila and mouse. Drosophila express a single Homer gene product that is upregulated during sleep. By contrast, vertebrates express Homer as both constitutive and immediate early gene (H1a) forms, and H1a is up-regulated during wakefulness. Genetic deletion of Homer in Drosophila results in fragmented sleep and in failure to sustain long bouts of sleep, even under increased sleep drive. However, deletion of Homer1a in mouse results in failure to sustain long bouts of wakefulness. Further evidence for the role of Homer1a in the maintenance of wake comes from the CREB alpha delta mutant mouse, which displays a reduced wake phenotype similar to the Homer1a knockout and fails to up-regulate Homer1a upon sleep loss. Homer1a is a gene whose expression is induced by CREB. Sustained behaviors of the sleep/wake cycle are created by molecular pathways that are distinct from those for arousal or short bouts, and implicate an evolutionarily-conserved role for Homer in sustaining these behaviors.

Sleeping Around the Clock

Two proteins that regulate cell division also regulate sleep in Drosophila .

Factors that Differentially Affect Daytime and Nighttime Sleep in Drosophila melanogaster

Rest in the fruit fly Drosophila melanogaster has key characteristics of mammalian sleep and is thus considered as a fly version of sleep. Drosophila sleep has been studied extensively, with the aim of gaining fundamental insights into the evolutionarily conserved functions of sleep as well as the mechanisms that regulate it. An interesting question that has not yet been addressed is whether fly sleep can be classified into distinct sleep types, each having particular biological roles – like rapid eye movement (REM) and non-REM sleep in birds and mammals. Typically, Drosophila sleep displays a bimodal pattern, consisting of distinct daytime and nighttime components. Notably, daytime and nighttime sleep differ with respect to several qualities, such as sleep-bout lengths and arousal thresholds. In this short review, we describe several genetic and environmental factors that differentially affect daytime and nighttime sleep, highlighting the observations suggesting the notion that these temporally distinct components of Drosophila sleep may have unique biological functions and be regulated by different homeostatic regulatory mechanisms.