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Sleep is widely recognized as essential, but without a clear singular function. Inadequate sleep impairs cognition, metabolism, immune function, and many other processes. Work in genetic model systems has greatly expanded our understanding of basic sleep neurobiology as well as introduced new concepts for why we sleep. Among these is an idea with its roots in human work nearly 50 years old: sleep in early life is crucial for normal brain maturation. Nearly all known species that sleep do so more while immature, and this increased sleep coincides with a period of exuberant synaptogenesis and massive neural circuit remodeling. Adequate sleep also appears critical for normal neurodevelopmental progression. This article describes recent findings regarding molecular and circuit mechanisms of sleep, with a focus on development and the insights garnered from models amenable to detailed genetic analyses.

Significance Reduced sleep duration is a hallmark of modern-day society and is increasingly associated with medical conditions, such as diabetes, obesity, metabolic syndrome, and cardiovascular disease. Here we present data from a rat model and human clinical study of chronic sleep restriction, both revealing that two metabolites in blood, oxalic acid and diacylglycerol 36:3, are quantitatively depleted under sleep-restricted conditions and restored after recovery sleep. Our findings also reveal a significant overall shift in lipid metabolism, with higher levels of phospholipids in both species and evidence of a systemic oxidative environment. This work provides a potential link between the known pathologies of reduced sleep duration and metabolic dysfunction. Sleep is an essential biological process that is thought to have a critical role in metabolic regulation. In humans, reduced sleep duration has been associated with risk for metabolic disorders, including weight gain, diabetes, obesity, and cardiovascular disease. However, our understanding of the molecular mechanisms underlying effects of sleep loss is only in its nascent stages. In this study we used rat and human models to simulate modern-day conditions of restricted sleep and addressed cross-species consequences via comprehensive metabolite profiling. Serum from sleep-restricted rats was analyzed using polar and nonpolar methods in two independent datasets ( n = 10 per study, 3,380 measured features, 407 identified). A total of 38 features were changed across independent experiments, with the majority classified as lipids (18 from 28 identified). In a parallel human study, 92 metabolites were identified as potentially significant, with the majority also classified as lipids (32 of 37 identified). Intriguingly, two metabolites, oxalic acid and diacylglycerol 36:3, were robustly and quantitatively reduced in both species following sleep restriction, and recovered to near baseline levels after sleep restriction ( P < 0.05, false-discovery rate < 0.2). Elevated phospholipids were also noted after sleep restriction in both species, as well as metabolites associated with an oxidizing environment. In addition, polar metabolites reflective of neurotransmitters, vitamin B3, and gut metabolism were elevated in sleep-restricted humans. These results are consistent with induction of peroxisome proliferator-activated receptors and disruptions of the circadian clock. The findings provide a potential link between known pathologies of reduced sleep duration and metabolic dysfunction, and potential biomarkers for sleep loss.

Idiopathic hypersomnia (IH) is a poorly understood sleep disorder characterized by excessive daytime sleepiness despite normal nighttime sleep. Combining human genomics with behavioral and mechanistic studies in fish and flies, we uncover a role for beat-Ia/CADM2 , synaptic adhesion molecules of the immunoglobulin superfamily, in excessive sleepiness. Neuronal knockdown of Drosophila beat-Ia results in sleepy flies and loss of the vertebrate ortholog of beat-Ia , CADM2 , results in sleepy fish. We delineate a developmental function for beat-Ia in synaptic elaboration of neuropeptide F (NPF) neurites projecting to the suboesophageal zone (SEZ) of the fly brain. Brain connectome and experimental evidence demonstrate these NPF outputs synapse onto a subpopulation of SEZ GABAergic neurons to stabilize arousal. NPF is the Drosophila homolog of vertebrate neuropeptide Y (NPY), and an NPY receptor agonist restores sleep to normal levels in zebrafish lacking CADM2 . These findings point towards NPY modulation as a treatment target for human hypersomnia. Cross-species studies show that loss of the synaptic adhesion molecules beat-Ia/CADM2 causes excessive sleepiness by disrupting development of wake-promoting NPF/NPY brain circuits. Targeting NPY signaling may offer treatment for human hypersomnia.

Sleep and feeding patterns lack strong daily rhythms during early life. As diurnal animals mature, feeding is consolidated to the day and sleep to the night. In Drosophila , circadian sleep patterns are initiated with formation of a circuit connecting the central clock to arousal output neurons; emergence of circadian sleep also enables long-term memory (LTM). However, the cues that trigger the development of this clock-arousal circuit are unknown. Here, we identify a role for nutritional status in driving sleep-wake rhythm development in Drosophila larvae. We find that in the 2nd instar larval period (L2), sleep and feeding are spread across the day; these behaviors become organized into daily patterns by the 3rd instar larval stage (L3). Forcing mature (L3) animals to adopt immature (L2) feeding strategies disrupts sleep-wake rhythms and the ability to exhibit LTM. In addition, the development of the clock (DN1a)-arousal (Dh44) circuit itself is influenced by the larval nutritional environment. Finally, we demonstrate that larval arousal Dh44 neurons act through glucose metabolic genes to drive onset of daily sleep-wake rhythms. Together, our data suggest that changes to energetic demands in developing organisms trigger the formation of sleep-circadian circuits and behaviors. Like most young animals, babies must obtain enough nutrients and energy to grow, yet they also need to rest for their brains to mature properly. As many exhausted new parents know first-hand, balancing these conflicting needs results in frequent, rapid switches between eating and sleeping. Eventually, new-borns’ internal biological clock system, which is aligned with the 24-hour light cycle, becomes fully operational. Exactly how this then translates into allowing them to stay alert during the day and be sleepy at night is still unclear. Like humans, the larvae of fruit flies first sleep haphazardly before developing a circadian pattern whereby they sleep at night and eat during the day. This shift occurs when a group of nerve cells called DN1a, whose job is to ‘keep time’, connects with Dh44, a subset of neurons which, when active, promote wakefulness. The trigger for these changes, however, has remained elusive. In response, Poe et al. hypothesized that feeding behaviour and nutrient availability coordinated the emergence of sleep rhythms in fruit flies. Forcing fruit fly larvae to keep feeding in an ‘immature’ pattern – by either genetic manipulations or reducing the sugar content of their food – not only prevented them from developing ‘mature’ sleeping rhythms but also resulted in memory problems. These experiments also showed that the DN1a-Dh44 connection depends on nutrient availability, as it did not form in larvae raised on the low-sugar food. Further genetic experiments showed that the Dh44 cells themselves act like nutrient sensors during the emergence of sleeping patterns. These results shed new light on the factors triggering sleep rhythm development. Poe et al. hope that the understanding gained can be extended to humans and eventually help manage nervous system disorders and health problems associated with disrupted sleep during early life.

Sleep is nearly universal among animals, yet remains poorly understood. Recent work has leveraged simple model organisms, such as Caenorhabditis elegans and Drosophila melanogaster larvae, to investigate the genetic and neural bases of sleep. However, manual methods of recording sleep behavior in these systems are labor intensive and low in throughput. To address these limitations, we developed methods for quantitative imaging of individual animals cultivated in custom microfabricated multiwell substrates, and used them to elucidate molecular mechanisms underlying sleep. Here, we describe the steps necessary to design, produce, and image these plates, as well as analyze the resulting behavioral data. We also describe approaches for experimentally manipulating sleep. Following these procedures, after ~2 h of experimental preparation, we are able to simultaneously image 24 C. elegans from the second larval stage to adult stages or 20 Drosophila larvae during the second instar life stage at a spatial resolution of 10 or 27 µm, respectively. Although this system has been optimized to measure activity and quiescence in Caenorhabditis larvae and adults and in Drosophila larvae, it can also be used to assess other behaviors over short or long periods. Moreover, with minor modifications, it can be adapted for the behavioral monitoring of a wide range of small animals. Individual animals are cultivated in custom microfabricated multiwell substrates. This protocol describes how to design, produce, and image the plates, as well as analyze the resulting behavioral data.

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. We know from personal experience that sleepless nights can change the way we behave, sometimes making us more irritable and less adept at social interactions. However, it can be difficult to establish cause and effect: does a lack of sleep lead to altered behavior, or vice versa? The fruit fly, Drosophila melanogaster, is a popular model organism for studying questions like this because its neural circuitry is relatively well understood. To explore the effects of lack of sleep on social behaviors, and in particular on aggression, Kayser et al. disrupted the sleep of male fruit flies using various techniques, such as shaking them during the night, and then observed how they behaved. The experiments revealed that sleep-deprived flies were less aggressive than flies with undisturbed sleep. Furthermore, sleep-deprived male flies were less successful at mating with female flies when they were in direct competition with a rested male fly. Normal behavior was restored when the sleep-deprived flies were allowed to recover lost sleep for as little as six hours before the next aggression assay. To investigate how sleep loss leads to a decrease in aggressive behavior, Kayser et al. used different drugs to treat the sleep-deprived flies. A drug activating the equivalent of the noradrenergic system in flies helped them to recover normal fighting behaviors despite a lack of sleep. In mammals, noradrenaline is a chemical that affects heart rate, sleep-wake patterns, aggression and a number of other phenomena. Although aggressive behavior is often perceived as negative in humans, it can be important for survival. Human brains and behaviors are obviously more complex than those of Drosophila. However, learning more about the neuronal circuits that control sleep and social behavior in fruit flies may lead to an improved understanding of these phenomena in humans and, in the longer term, the development of drugs that can influence or modulate aggressive behaviors.

Sleep during development is involved in refining brain circuitry, but a role for sleep in the earliest periods of nervous system elaboration, when neurons are first being born, has not been explored. Here we identify a sleep state in Drosophila larvae that coincides with a major wave of neurogenesis. Mechanisms controlling larval sleep are partially distinct from adult sleep: octopamine, the Drosophila analog of mammalian norepinephrine, is the major arousal neuromodulator in larvae, but dopamine is not required. Using real-time behavioral monitoring in a closed-loop sleep deprivation system, we find that sleep loss in larvae impairs cell division of neural progenitors. This work establishes a system uniquely suited for studying sleep during nascent periods, and demonstrates that sleep in early life regulates neural stem cell proliferation. Nearly all animals sleep more while they are still developing, suggesting that sleep is important in early life. Previous studies have shown that sleep may be required for building connections in the brain. However, it has been difficult to study the effects of sleep in earlier stages of brain development, when stem cells divide to create brain cells in a process known as “neurogenesis”. This is partly because, in mammals, most neurogenesis occurs in the womb. Scientists have successfully studied sleep using the common fruit fly. But these studies have so far focused on adult flies, in which neurogenesis is mostly complete. Fly larvae, on the other hand, are widely used to study brain development and neurogenesis. Compared to mammals in the womb, fruit fly larvae are very easy to access and manipulate. However, unlike adult flies, no one had previously looked to see if larvae even display a behaviour that would fit the definition of sleep. To see if fruit fly larvae do sleep, Szuperak et al. created the “LarvaLodge”, an apparatus in which individual larvae can be housed while having their activity monitored over time. In these lodges, a bright light was used to test how hard it is to arouse inactive fruit fly larvae, and further experiments asked what happens when larvae are prevented from resting. Then, to look at neurogenesis in the larvae, Szuperak et al. used a stain that labels dividing stem cells within the nervous system. Those cells could then be seen and counted when a larva was dissected and examined under a microscope. The results from the LarvaLodge showed that fruit fly larvae do indeed sleep: they have extended periods of rest during which they react less to outside disturbances and adopt a particular posture (they retract their heads towards their bodies). Also when larvae were deprived of sleep, by shining a light or shaking, they compensated by sleeping more afterwards. Importantly, depriving the larvae of sleep also led to lower levels of neurogenesis. These findings establish the fruit fly larva as a new and useful system for studying the role of sleep in early development, and may help shed light on the role sleep plays in disorders affecting brain development.

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.

During sleep, animals do not eat, reproduce or forage. Sleeping animals are vulnerable to predation. Yet, the persistence of sleep despite evolutionary pressures, and the deleterious effects of sleep deprivation, indicate that sleep serves a function or functions that cannot easily be bypassed. Recent research demonstrates sleep to be phylogenetically far more pervasive than previously appreciated; it is possible that the very first animals slept. Here, we give an overview of sleep across various species, with the aim of determining its original purpose. Sleep exists in animals without cephalized nervous systems and can be influenced by non-neuronal signals, including those associated with metabolic rhythms. Together, these observations support the notion that sleep serves metabolic functions in neural and non-neural tissues.

The development of central nervous system synapses requires precise coordination between presynaptic and postsynaptic components. The EphB family controls postsynaptic development by interacting with glutamate receptors and regulating dendritic filopodia motility, but how EphBs induce the formation of presynaptic specializations is less well understood. Here, we show that knockdown of presynaptic ephrin-B1, ephrin-B2, or syntenin-1, but not ephrin-B3, prevents EphB-dependent presynaptic development. Ephrin-B1, ephrin-B2, and syntenin-1 are clustered together with presynaptic markers, suggesting that these molecules function jointly in presynaptic development. Knockdown of ephrin-B1 or ephrin-B2 reduces the number of synaptic specializations and the colocalization of syntenin-1 with synaptic markers. Simultaneous knockdown of ephrin-B1 and ephrin-B2 suggests that they function independently in the formation of synaptic contacts, but act together to recruit syntenin-1 to presynaptic terminals. Taken together, these results demonstrate that ephrin-B1 and ephrin-B2 function with EphB to mediate presynaptic development via syntenin-1.