Exploring phylogeny to find the function of sleep.

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.

A Drosophila model of sleep restriction therapy for insomnia.

Insomnia is the most common sleep disorder among adults, especially affecting individuals of advanced age or with neurodegenerative disease. Insomnia is also a common comorbidity across psychiatric disorders. Cognitive behavioral therapy for insomnia (CBT-I) is the first-line treatment for insomnia; a key component of this intervention is restriction of sleep opportunity, which optimizes matching of sleep ability and opportunity, leading to enhanced sleep drive. Despite the well-documented efficacy of CBT-I, little is known regarding how CBT-I works at a cellular and molecular level to improve sleep, due in large part to an absence of experimentally-tractable animals models of this intervention. Here, guided by human behavioral sleep therapies, we developed a Drosophila model for sleep restriction therapy (SRT) of insomnia. We demonstrate that restriction of sleep opportunity through manipulation of environmental cues improves sleep efficiency in multiple short-sleeping Drosophila mutants. The response to sleep opportunity restriction requires ongoing environmental inputs, but is independent of the molecular circadian clock. We apply this sleep opportunity restriction paradigm to aging and Alzheimer's disease fly models, and find that sleep impairments in these models are reversible with sleep restriction, with associated improvement in reproductive fitness and extended lifespan. This work establishes a model to investigate the neurobiological basis of CBT-I, and provides a platform that can be exploited toward novel treatment targets for insomnia.

Starvation resistance is associated with developmentally specified changes in sleep, feeding and metabolic rate.

Food shortage represents a primary challenge to survival, and animals have adapted diverse developmental, physiological and behavioral strategies to survive when food becomes unavailable. Starvation resistance is strongly influenced by ecological and evolutionary history, yet the genetic basis for the evolution of starvation resistance remains poorly understood. The fruit fly Drosophila melanogaster provides a powerful model for leveraging experimental evolution to investigate traits associated with starvation resistance. While control populations only live a few days without food, selection for starvation resistance results in populations that can survive weeks. We have previously shown that selection for starvation resistance results in increased sleep and reduced feeding in adult flies. Here, we investigate the ontogeny of starvation resistance-associated behavioral and metabolic phenotypes in these experimentally selected flies. We found that selection for starvation resistance resulted in delayed development and a reduction in metabolic rate in larvae that persisted into adulthood, suggesting that these traits may allow for the accumulation of energy stores and an increase in body size within these selected populations. In addition, we found that larval sleep was largely unaffected by starvation selection and that feeding increased during the late larval stages, suggesting that experimental evolution for starvation resistance produces developmentally specified changes in behavioral regulation. Together, these findings reveal a critical role for development in the evolution of starvation resistance and indicate that selection can selectively influence behavior during defined developmental time points.

Oxalic acid and diacylglycerol 36:3 are cross-species markers of sleep debt.

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.

Energetic demands regulate sleep-wake rhythm circuit development.

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.

TDP-43 impairs sleep in Drosophila through Ataxin-2-dependent metabolic disturbance.

Neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia are associated with substantial sleep disruption, which may accelerate cognitive decline and brain degeneration. Here, we define a role for trans-activation response element (TAR) DNA binding protein 43 (TDP-43), a protein associated with human neurodegenerative disease, in regulating sleep using Drosophila. Expression of TDP-43 severely disrupts sleep, and the sleep deficit is rescued by Atx2 knockdown. Brain RNA sequencing revealed that Atx2 RNA interference regulates transcripts enriched for small-molecule metabolic signaling in TDP-43 brains. Focusing on these Atx2-regulated genes, we identified suppressors of the TDP-43 sleep phenotype enriched for metabolism pathways. Knockdown of Atx2 or treatment with rapamycin attenuated the sleep phenotype and mitigated the disruption of small-molecule glycogen metabolism caused by TDP-43. Our findings provide a connection between toxicity of TDP-43 and sleep disturbances and highlight key aspects of metabolism that interplay with TDP-43 toxicity upon Atx2 rescue.

Shared and unique 3D genomic features of substance use disorders across multiple cell types.

Recent genome-wide association studies (GWAS) have revealed shared genetic components among alcohol, opioid, tobacco and cannabis use disorders. However, the extent of the underlying shared causal variants and effector genes, along with their cellular context, remain unclear. We leveraged our existing 3D genomic datasets comprising high-resolution promoter-focused Capture-C/Hi-C, ATAC-seq and RNA-seq across >50 diverse human cell types to focus on genomic regions that coincide with GWAS loci. Using stratified LD regression, we determined the proportion of genomewide SNP heritability attributable to the features assayed across our cell types by integrating recent GWAS summary statistics for the relevant traits: alcohol use disorder (AUD), tobacco use disorder (TUD), opioid use disorder (OUD) and cannabis use disorder (CanUD). Statistically significant enrichments (P<0.05) were observed in 14 specific cell types, with heritability reaching 9.2-fold for iPSC-derived cortical neurons and neural progenitors, confirming that they are crucial cell types for further functional exploration. Additionally, several pancreatic cell types, notably pancreatic beta cells, showed enrichment for TUD, with heritability enrichments up to 4.8-fold, suggesting genomic overlap with metabolic processes. Further investigation revealed significant positive genetic correlations between T2D with both TUD and CanUD (FDR<0.05) and a significant negative genetic correlation with AUD. Interestingly, after partitioning the heritability for each cell type's cis-regulatory elements, the correlation between T2D and TUD for pancreatic beta cells was greater (r=0.2) than the global genetic correlation value. Our study provides new genomic insights into substance use disorders and implicates cell types where functional follow-up studies could reveal causal variant-gene mechanisms underpinning these disorders.

Energetic Demands Regulate Sleep-Wake Rhythm Circuit Development.

Sleep and feeding patterns lack a clear daily rhythm during early life. As diurnal animals mature, feeding is consolidated to the day and sleep to the night. Circadian sleep patterns begin 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 (L2) period, sleep and feeding are spread across the day; these behaviors become organized into daily patterns by 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 triggers the formation of sleep-circadian circuits and behaviors.

Developmental emergence of sleep rhythms enables long-term memory in Drosophila.

In adulthood, sleep-wake rhythms are one of the most prominent behaviors under circadian control. However, during early life, sleep is spread across the 24-hour day. The mechanism through which sleep rhythms emerge, and consequent advantage conferred to a juvenile animal, is unknown. In the second-instar Drosophila larvae (L2), like in human infants, sleep is not under circadian control. We identify the precise developmental time point when the clock begins to regulate sleep in Drosophila, leading to emergence of sleep rhythms in early third-instars (L3). At this stage, a cellular connection forms between DN1a clock neurons and arousal-promoting Dh44 neurons, bringing arousal under clock control to drive emergence of circadian sleep. Last, we demonstrate that L3 but not L2 larvae exhibit long-term memory (LTM) of aversive cues and that this LTM depends upon deep sleep generated once sleep rhythms begin. We propose that the developmental emergence of circadian sleep enables more complex cognitive processes, including the onset of enduring memories.

Stem cell-specific ecdysone signaling regulates the development and function of a Drosophila sleep homeostat.

Complex behaviors arise from neural circuits that are assembled from diverse cell types. Sleep is a conserved and essential behavior, yet little is known regarding how the nervous system generates neuron types of the sleep-wake circuit. Here, we focus on the specification of Drosophila sleep-promoting neurons-long-field tangential input neurons that project to the dorsal layers of the fan-shaped body neuropil in the central complex (CX). We use lineage analysis and genetic birth dating to identify two bilateral Type II neural stem cells that generate these dorsal fan-shaped body (dFB) neurons. We show that adult dFB neurons express Ecdysone-induced protein E93, and loss of Ecdysone signaling or E93 in Type II NSCs results in the misspecification of the adult dFB neurons. Finally, we show that E93 knockdown in Type II NSCs affects adult sleep behavior. Our results provide insight into how extrinsic hormonal signaling acts on NSCs to generate neuronal diversity required for adult sleep behavior. These findings suggest that some adult sleep disorders might derive from defects in stem cell-specific temporal neurodevelopmental programs.

Neurofibromin 1 regulates early developmental sleep in Drosophila.

Sleep disturbances are common in neurodevelopmental disorders, but knowledge of molecular factors that govern sleep in young animals is lacking. Evidence across species, including Drosophila, suggests that juvenile sleep has distinct functions and regulatory mechanisms in comparison to sleep in maturity. In flies, manipulation of most known adult sleep regulatory genes is not associated with sleep phenotypes during early developmental (larval) stages. Here, we examine the role of the neurodevelopmental disorder-associated gene Neurofibromin 1 (Nf1) in sleep during numerous developmental periods. Mutations in Neurofibromin 1 (Nf1) are associated with sleep and circadian disorders in humans and adult flies. We find in flies that Nf1 acts to regulate sleep across the lifespan, beginning during larval stages. Nf1 is required in neurons for this function, as is signaling via the Alk pathway. These findings identify Nf1 as one of a small number of genes positioned to regulate sleep across developmental periods.

EphB controls NMDA receptor function and synaptic targeting in a subunit-specific manner.

Dynamic regulation of the localization and function of NMDA receptors (NMDARs) is critical for synaptic development and function. The composition and localization of NMDAR subunits at synapses are tightly regulated and can influence the ability of individual synapses to undergo long-lasting changes in response to stimuli. Here, we examine mechanisms by which EphB2, a receptor tyrosine kinase that binds and phosphorylates NMDARs, controls NMDAR subunit localization and function at synapses. We find that, in mature neurons, EphB2 expression levels regulate the amount of NMDARs at synapses, and EphB activation decreases Ca(2+)-dependent desensitization of NR2B-containing NMDARs. EphBs are required for enhanced localization of NR2B-containing NMDARs at synapses of mature neurons; triple EphB knock-out mice lacking EphB1-3 exhibit homeostatic upregulation of NMDAR surface expression and loss of proper targeting to synaptic sites. These findings demonstrate that, in the mature nervous system, EphBs are key regulators of the synaptic localization of NMDARs.

Ephrin-B1 and ephrin-B2 mediate EphB-dependent presynaptic development via syntenin-1.

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.

Getting into rhythm: developmental emergence of circadian clocks and behaviors.

Circadian clocks keep time to coordinate diverse behaviors and physiological functions. While molecular circadian rhythms are evident during early development, most behavioral rhythms, such as sleep-wake, do not emerge until far later. Here, we examine the development of circadian clocks, outputs, and behaviors across phylogeny, with a particular focus on Drosophila. We explore potential mechanisms for how central clocks and circadian output loci establish communication, and discuss why from an evolutionary perspective sleep-wake and other behavioral rhythms emerge long after central clocks begin keeping time.

Intrinsic maturation of sleep output neurons regulates sleep ontogeny in Drosophila.

The maturation of sleep behavior across a lifespan (sleep ontogeny) is an evolutionarily conserved phenomenon. Mammalian studies have shown that in addition to increased sleep duration, early life sleep exhibits stark differences compared with mature sleep with regard to sleep states. How the intrinsic maturation of sleep output circuits contributes to sleep ontogeny is poorly understood. The fruit fly Drosophila melanogaster exhibits multifaceted changes to sleep from juvenile to mature adulthood. Here, we use a non-invasive probabilistic approach to investigate the changes in sleep architecture in juvenile and mature flies. Increased sleep in juvenile flies is driven primarily by a decreased probability of transitioning to wake and characterized by more time in deeper sleep states. Functional manipulations of sleep-promoting neurons in the dorsal fan-shaped body (dFB) suggest that these neurons differentially regulate sleep in juvenile and mature flies. Transcriptomic analysis of dFB neurons at different ages and a subsequent RNAi screen implicate the genes involved in dFB sleep circuit maturation. These results reveal that the dynamic transcriptional states of sleep output neurons contribute to the changes in sleep across the lifespan.

Synaptic dysfunction connects autism spectrum disorder and sleep disturbances: A perspective from studies in model organisms.

Sleep disturbances (SD) accompany many neurodevelopmental disorders, suggesting SD is a transdiagnostic process that can account for behavioral deficits and influence underlying neuropathogenesis. Autism Spectrum Disorder (ASD) comprises a complex set of neurodevelopmental conditions characterized by challenges in social interaction, communication, and restricted, repetitive behaviors. Diagnosis of ASD is based primarily on behavioral criteria, and there are no drugs that target core symptoms. Among the co-occurring conditions associated with ASD, SD are one of the most prevalent. SD often arises before the onset of other ASD symptoms. Sleep interventions improve not only sleep but also daytime behaviors in children with ASD. Here, we examine sleep phenotypes in multiple model systems relevant to ASD, e.g., mice, zebrafish, fruit flies and worms. Given the functions of sleep in promoting brain connectivity, neural plasticity, emotional regulation and social behavior, all of which are of critical importance in ASD pathogenesis, we propose that synaptic dysfunction is a major mechanism that connects ASD and SD. Common molecular targets in this interplay that are involved in synaptic function might be a novel avenue for therapy of individuals with ASD experiencing SD. Such therapy would be expected to improve not only sleep but also other ASD symptoms.

The emerging link between autoimmune disorders and neuropsychiatric disease.

Abnormal autoimmune activity has been implicated in a number of neuropsychiatric disorders. In this review, the authors discuss a newly recognized class of synaptic autoimmune encephalitides as well as behavioral and cognitive manifestations of systemic autoimmune diseases.

Randomized Noninferiority Trial of Telehealth Delivery of Cognitive Behavioral Treatment of Insomnia Compared to In-Person Care.

Objective: Insomnia is prevalent and is associated with a range of negative sequelae. Cognitive behavioral treatment for insomnia (CBT-I) is the recommended intervention, but availability is limited. Telehealth provides increased access, but its efficacy is not certain. The objective of this study was to compare the efficacy of CBT-I delivered by telehealth to in-person treatment and to a waitlist control.Methods: Individuals with DSM-5 insomnia disorder (n = 60) were randomized to telehealth CBT-I, in-person CBT-I, or 8-week waitlist control. CBT-I was delivered over 6-8 weekly sessions by video telehealth or in-person in an outpatient clinic. Follow-up assessments were at 2 weeks and 3 months posttreatment. The Insomnia Severity Index (ISI) was the primary outcome. Change in ISI score was compared between the CBT-I group in an intent-to-treat, noninferiority analysis using an a priori margin of -3.0 points. All analyses were conducted using mixed-effects models. Data collection occurred from November 2017-July 2020.Results: The mean (SD) change in ISI score from baseline to 3-month follow-up was -7.8 (6.1) points for in-person CBT-I, -7.5 (6.9) points for telehealth, and -1.6 (2.1) for waitlist, and the difference between the CBT-I groups was not statistically significant (t28 = -0.98, P = .33). The lower confidence limit of this between-group difference in the mean ISI changes was greater than the a priori margin of -3.0 points, indicating that telehealth treatment was not inferior to in-person treatment. There were significant improvements on most secondary outcome measures but no group differences.Conclusions: Telehealth CBT-I may produce clinically significant improvements in insomnia severity that are noninferior to in-person treatment. CBT-I is also associated with significant gains across a range of domains of functioning. Telehealth is a promising option for increasing access to treatment without loss of clinical gains.Trial Registration: ClinicalTrials.gov identifier: NCT03328585.

Treatment of Insomnia with Zaleplon in HIV+ Significantly Improves Sleep and Depression.

More than 50% of individuals who are HIV positive report insomnia, which can reduce HIV treatment adherence, impair quality of life, and contribute to metabolic dysfunction. Major depressive disorder is also highly comorbid in this population, leading to further impairment. There is evidence that treating insomnia may improve not only sleep, but depression and metabolic function, as well. The present study aimed to examine the effects of pharmacotherapeutic treatment of insomnia on sleep, depression, and metabolic functioning in individuals with HIV. 20 individuals with asymptomatic seropositive HIV and comorbid insomnia and depression were administered zaleplon for 6 weeks. Insomnia severity was assessed using the Insomnia Severity Index and Epworth Sleepiness Scale, and depression severity was assessed using the Quick Inventory of Depression, both prior to treatment and 6 weeks post treatment. Metabolomic changes were assessed using a comprehensive platform measuring ~2000 lipid features and polar metabolites. Linear mixed effects models demonstrated that 6 weeks of treatment with zaleplon significantly improved symptoms of both insomnia and depression. Metabolomic analyses also demonstrated that changes in insomnia severity were associated with significant changes in key branched chain amino acid metabolites. Our results show that improvement in insomnia is associated with reduced depressive symptoms and beneficial metabolomic changes. Additionally, changes in key branched chain amino acid metabolites following treatment may serve as useful biomarkers of treatment response.

The chromatin remodeler ISWI acts during Drosophila development to regulate adult sleep.

Sleep disruptions are among the most commonly reported symptoms across neurodevelopmental disorders (NDDs), but mechanisms linking brain development to normal sleep are largely unknown. From a Drosophila screen of human NDD-associated risk genes, we identified the chromatin remodeler Imitation SWItch/SNF (ISWI) to be required for adult fly sleep. Loss of ISWI also results in disrupted circadian rhythms, memory, and social behavior, but ISWI acts in different cells and during distinct developmental times to affect each of these adult behaviors. Specifically, ISWI expression in type I neuroblasts is required for both adult sleep and formation of a learning-associated brain region. Expression in flies of the human ISWI homologs SMARCA1 and SMARCA5 differentially rescues adult phenotypes, while de novo SMARCA5 patient variants fail to rescue sleep. We propose that sleep deficits are a primary phenotype of early developmental origin in NDDs and point toward chromatin remodeling machinery as critical for sleep circuit formation.