Waking Up the Sleep Field: An Overview on the Implications of Genetics and Bioinformatics of Sleep.

Sleep genetics is an intriguing, as yet less understood, understudied, emerging area of biological and medical discipline. A generalist may not be aware of the current status of the field given the variety of journals that have published studies on the genetics of sleep and the circadian clock over the years. For researchers venturing into this fascinating area, this review thus includes fundamental features of circadian rhythm and genetic variables impacting sleep-wake cycles. Sleep/wake pathway medication exposure and susceptibility are influenced by genetic variations, and the responsiveness of sleep-related medicines is influenced by several functional polymorphisms. This review highlights the features of the circadian timing system and then a genetic perspective on wakefulness and sleep, as well as the relationship between sleep genetics and sleep disorders. Neurotransmission genes, as well as circadian and sleep/wake receptors, exhibit functional variability. Experiments on animals and humans have shown that these genetic variants impact clock systems, signaling pathways, nature, amount, duration, type, intensity, quality, and quantity of sleep. In this regard, the overview covers research on sleep genetics, the genomic properties of several popular model species used in sleep studies, homologs of mammalian genes, sleep disorders, and related genes. In addition, the study includes a brief discussion of sleep, narcolepsy, and restless legs syndrome from the viewpoint of a model organism. It is suggested that the understanding of genetic clues on sleep function and sleep disorders may, in future, result in an evidence-based, personalized treatment of sleep disorders.

Altered EEG power spectrum, but not sleep-wake architecture, in HCN1 knockout mice.

Sleep is a complex biological state characterized by large populations of neurons firing in a rhythmic or synchronized manner. HCN channels play a critical role in generating and sustaining synchronized neuronal firing and are involved in the actions of anaesthetics. However, the role of these channels in sleep-wakefulness per se has yet to be studied. We conducted polysomnographic recordings of Hcn1 constitutive knockout (Hcn1 KO) and wild-type (WT) mice in order to investigate the potential role of HCN1 channels in sleep/wake regulation. EEG and EMG data were analysed using the Somnivore™ machine learning algorithm. Time spent in each vigilance state, bout number and duration, and EEG power spectral activity were compared between genotypes. There were no significant differences in the time spent in wake, rapid eye movement (REM) or non-REM (NREM) sleep between Hcn1 KO and WT mice. Wake bout duration during the inactive phase was significantly shorter in Hcn1 KO mice whilst no other bout parameters were affected by genotype. Hcn1 KO mice showed a reduction in overall EEG power which was particularly prominent in the theta (5-9 Hz) and alpha (9-15 Hz) frequency bands and most evident during NREM sleep. Together these data suggest that HCN1 channels do not play a major role in sleep architecture or modulation of vigilance states. However, loss of these channels significantly alters underlying neuronal activity within these states which may have functional consequences.

Kinase signalling in excitatory neurons regulates sleep quantity and depth.

Progress has been made in the elucidation of sleep and wakefulness regulation at the neurocircuit level1,2. However, the intracellular signalling pathways that regulate sleep and the neuron groups in which these intracellular mechanisms work remain largely unknown. Here, using a forward genetics approach in mice, we identify histone deacetylase 4 (HDAC4) as a sleep-regulating molecule. Haploinsufficiency of Hdac4, a substrate of salt-inducible kinase 3 (SIK3)3, increased sleep. By contrast, mice that lacked SIK3 or its upstream kinase LKB1 in neurons or with a Hdac4S245A mutation that confers resistance to phosphorylation by SIK3 showed decreased sleep. These findings indicate that LKB1-SIK3-HDAC4 constitute a signalling cascade that regulates sleep and wakefulness. We also performed targeted manipulation of SIK3 and HDAC4 in specific neurons and brain regions. This showed that SIK3 signalling in excitatory neurons located in the cerebral cortex and the hypothalamus positively regulates EEG delta power during non-rapid eye movement sleep (NREMS) and NREMS amount, respectively. A subset of transcripts biased towards synaptic functions was commonly regulated in cortical glutamatergic neurons through the expression of a gain-of-function allele of Sik3 and through sleep deprivation. These findings suggest that NREMS quantity and depth are regulated by distinct groups of excitatory neurons through common intracellular signals. This study provides a basis for linking intracellular events and circuit-level mechanisms that control NREMS.

Orexin 2 receptor-selective agonist danavorexton improves narcolepsy phenotype in a mouse model and in human patients.

Narcolepsy type 1 (NT1) is a sleep disorder caused by a loss of orexinergic neurons. Narcolepsy type 2 (NT2) is heterogeneous; affected individuals typically have normal orexin levels. Following evaluation in mice, the effects of the orexin 2 receptor (OX2R)-selective agonist danavorexton were evaluated in single- and multiple-rising-dose studies in healthy adults, and in individuals with NT1 and NT2. In orexin/ataxin-3 narcolepsy mice, danavorexton reduced sleep/wakefulness fragmentation and cataplexy-like episodes during the active phase. In humans, danavorexton administered intravenously was well tolerated and was associated with marked improvements in sleep latency in both NT1 and NT2. In individuals with NT1, danavorexton dose-dependently increased sleep latency in the Maintenance of Wakefulness Test, up to the ceiling effect of 40 min, in both the single- and multiple-rising-dose studies. These findings indicate that OX2Rs remain functional despite long-term orexin loss in NT1. OX2R-selective agonists are a promising treatment for both NT1 and NT2.

The evolutionarily conserved miRNA-137 targets the neuropeptide hypocretin/orexin and modulates the wake to sleep ratio.

Hypocretin (Hcrt), also known as orexin, neuropeptide signaling stabilizes sleep and wakefulness in all vertebrates. A lack of Hcrt causes the sleep disorder narcolepsy, and increased Hcrt signaling has been speculated to cause insomnia, but while the signaling pathways of Hcrt are relatively well-described, the intracellular mechanisms that regulate its expression remain unclear. Here, we tested the role of microRNAs (miRNAs) in regulating Hcrt expression. We found that miR-137, miR-637, and miR-654-5p target the human HCRT gene. miR-137 is evolutionarily conserved and also targets mouse Hcrt as does miR-665. Inhibition of miR-137 specifically in Hcrt neurons resulted in Hcrt upregulation, longer episodes of wakefulness, and significantly longer wake bouts in the first 4 h of the active phase. IL-13 stimulation upregulated endogenous miR-137, while Hcrt mRNA decreased both in vitro and in vivo. Furthermore, knockdown of miR-137 in zebrafish substantially increased wakefulness. Finally, we show that in humans, the MIR137 locus is genetically associated with sleep duration. In conclusion, these results show that an evolutionarily conserved miR-137:Hcrt interaction is involved in sleep­wake regulation.

The sleep-wake distribution contributes to the peripheral rhythms in PERIOD-2.

In the mouse, Period-2 (Per2) expression in tissues peripheral to the suprachiasmatic nuclei (SCN) increases during sleep deprivation and at times of the day when animals are predominantly awake spontaneously, suggesting that the circadian sleep-wake distribution directly contributes to the daily rhythms in Per2. We found support for this hypothesis by recording sleep-wake state alongside PER2 bioluminescence in freely behaving mice, demonstrating that PER2 bioluminescence increases during spontaneous waking and decreases during sleep. The temporary reinstatement of PER2-bioluminescence rhythmicity in behaviorally arrhythmic SCN-lesioned mice submitted to daily recurring sleep deprivations substantiates our hypothesis. Mathematical modeling revealed that PER2 dynamics can be described by a damped harmonic oscillator driven by two forces: a sleep-wake-dependent force and an SCN-independent circadian force. Our work underscores the notion that in peripheral tissues the clock gene circuitry integrates sleep-wake information and could thereby contribute to behavioral adaptability to respond to homeostatic requirements.

Circadian rhythms are daily cycles in behavior and physiology which repeat approximately every 24 hours. The master regulator of these rhythms is located in a small part of the brain called the supra-chiasmatic nucleus. This brain structure regulates the timing of sleep and wakefulness and is also thought to control the daily rhythms of cells throughout the body on a molecular level. It does this by synchronizing the activity of a set of genes called clock genes. Under normal conditions, the levels of proteins coded for by clock genes change throughout the day following a rhythm that matches sleep-wake patterns. However, keeping animals and humans awake at their preferred sleeping times affects the protein levels of clock genes in many tissues of the body. This suggests that, in addition to the supra-chiasmatic nucleus, sleep-wake cycles may also influence clock-gene rhythms throughout the body. To test this theory, Hoekstra, Jan et al. measured the levels of PERIOD-2, a protein coded for by the clock gene Period-2, while tracking sleep-wake states in mice. They did this by imaging a bioluminescent version of the PERIOD-2 protein in the brain and the kidneys, at the same time as they recorded the brain activity, movement and muscle response of animals. Results showed that PERIOD-2 increased on waking and decreased when mice fell asleep. Additionally, in mice lacking a circadian rhythm in sleep-wake behavior ­ whose changes in PERIOD-2 levels with respect to time were greatly reduced ­ imposing a regular sleep-wake cycle restored normal PERIOD-2 rhythmicity. Next, Hoekstra, Jan et al. developed a mathematical model to understand how sleep-wake cycles together with circadian rhythms affect clock-gene activity in the brain and kidneys. Computer simulations suggested that sleep-wake cycles and circadian factors act as forces of comparable strength driving clock-gene dynamics. Both need to act in concert to keep clock-genes rhythmic. The model also predicted the large and immediate effects of sleep deprivation on PERIOD-2 levels, giving further credence to the idea that waking accelerated clock-gene rhythms while sleeping slowed them down. Modelling also suggested that having regular clock-gene rhythms protects against sleep disturbances. In summary, this work shows how sleep patterns contribute to the daily rhythms in clock genes in the brain and body. The findings support the idea that well-timed sleep-wake schedules could help people to adjust to new time zones. It might also be useful to inform other strategies to reduce the health impacts of shift work.

Chemogenetic modulation of histaminergic neurons in the tuberomamillary nucleus alters territorial aggression and wakefulness.

Designer receptor activated by designer drugs (DREADDs) techniques are widely used to modulate the activities of specific neuronal populations during behavioural tasks. However, DREADDs-induced modulation of histaminergic neurons in the tuberomamillary nucleus (HATMN neurons) has produced inconsistent effects on the sleep-wake cycle, possibly due to the use of Hdc-Cre mice driving Cre recombinase and DREADDs activity outside the targeted region. Moreover, previous DREADDs studies have not examined locomotor activity and aggressive behaviours, which are also regulated by brain histamine levels. In the present study, we investigated the effects of HATMN activation and inhibition on the locomotor activity, aggressive behaviours and sleep-wake cycle of Hdc-Cre mice with minimal non-target expression of Cre-recombinase. Chemoactivation of HATMN moderately enhanced locomotor activity in a novel open field. Activation of HATMN neurons significantly enhanced aggressive behaviour in the resident-intruder test. Wakefulness was increased and non-rapid eye movement (NREM) sleep decreased for an hour by HATMN chemoactivation. Conversely HATMN chemoinhibition decreased wakefulness and increased NREM sleep for 6 h. These changes in wakefulness induced by HATMN modulation were related to the maintenance of vigilance state. These results indicate the influences of HATMN neurons on exploratory activity, territorial aggression, and wake maintenance.

Genomic heterogeneity affects the response to Daylight Saving Time.

Circadian clocks control the timing of many physiological events in the 24-h day. When individuals undergo an abrupt external shift (e.g., change in work schedule or travel across multiple time zones), circadian clocks become misaligned with the new time and may take several days to adjust. Chronic circadian misalignment, e.g., as a result of shift work, has been shown to lead to several physical and mental health problems. Despite the serious health implications of circadian misalignment, relatively little is known about how genetic variation affects an individual's ability to entrain to abrupt external changes. Accordingly, we used the one-hour advance from the onset of daylight saving time (DST) as a natural experiment to comprehensively study how individual heterogeneity affects the shift of sleep/wake cycles in response to an abrupt external time change. We found that individuals genetically predisposed to a morning tendency adjusted to the advance in a few days, while genetically predisposed evening-inclined individuals had not shifted. Observing differential effects by genetic disposition after a one-hour advance underscores the importance of heterogeneity in adaptation to external schedule shifts. These genetic differences may affect how individuals adjust to jet lag or shift work as well.

AANAT1 functions in astrocytes to regulate sleep homeostasis.

How the brain controls the need and acquisition of recovery sleep after prolonged wakefulness is an important issue in sleep research. The monoamines serotonin and dopamine are key regulators of sleep in mammals and in Drosophila. We found that the enzyme arylalkylamine N-acetyltransferase 1 (AANAT1) is expressed by Drosophila astrocytes and specific subsets of neurons in the adult brain. AANAT1 acetylates monoamines and inactivates them, and we found that AANAT1 limited the accumulation of serotonin and dopamine in the brain upon sleep deprivation (SD). Loss of AANAT1 from astrocytes, but not from neurons, caused flies to increase their daytime recovery sleep following overnight SD. Together, these findings demonstrate a crucial role for AANAT1 and astrocytes in the regulation of monoamine bioavailability and homeostatic sleep.

Sleep is essential for our physical and mental health. A lack of sleep can affect our energy and concentration levels and is often linked to chronic illnesses and mood disorders. Sleep is controlled by an internal clock in our brain that operates on a 24-hour cycle, telling our bodies when we are tired and ready for bed, or fresh and alert to start a new day. In addition, the brain tracks the need for sleep and drives the recovery of sleep after periods of prolonged wakefulness ­ a process known as sleep-wake homeostasis. Chemical messengers in the brain such as dopamine and serotonin also play an important part in regulating our sleep drive. While dopamine keeps us awake, serotonin can both prevent us from and help us falling asleep, depending on the part of the brain in which it is released. Most research has focused on the role of different brain circuits on sleep, but it has been shown that a certain type of brain cell, known as astrocyte, may also be important for sleep regulation. So far, it has been unclear if astrocytes could be involved in regulating the need for recovery sleep after a sleep-deprived night ­ also known as rebound sleep. Now, Davla, Artiushin et al. used sleep-deprived fruit flies to investigate this further. The flies were kept awake over 12 hours (from 6pm to 6am), using intermittent physical agitation. The researchers found that astrocytes in the brains of fruit flies express a molecule called AANAT1, which peaked at the beginning of the night, declined as the night went on and recovered by morning. In sleep deprived flies, it inactivated the chemical messengers and so lowered the amount of dopamine and serotonin in the brain. However, in mutant flies that lacked AANAT1, both dopamine and serotonin levels increased in the brain after sleep deprivation. When AANAT1 was selectively removed from astrocytes only, sleep-deprived flies needed more rebound sleep during the day to make up for lost sleep at night. This shows that both astrocytes and AANAT1 play a crucial role in sleep homeostasis. Molecules belonging to the AANAT family exist in both flies and humans, and these results could have important implications for the science of sleep. The study of Davla, Artiushin et al. paves the way for understanding the mechanisms of sleep homeostasis that are similar in both organisms, and may in the future, help to identify sleep drugs that target astrocytes and the molecules they express.

Forward genetics identifies a novel sleep mutant with sleep state inertia and REM sleep deficits.

Switches between global sleep and wakefulness states are believed to be dictated by top-down influences arising from subcortical nuclei. Using forward genetics and in vivo electrophysiology, we identified a recessive mouse mutant line characterized by a substantially reduced propensity to transition between wake and sleep states with an especially pronounced deficit in initiating rapid eye movement (REM) sleep episodes. The causative mutation, an Ile102Asn substitution in the synaptic vesicular protein, VAMP2, was associated with morphological synaptic changes and specific behavioral deficits, while in vitro electrophysiological investigations with fluorescence imaging revealed a markedly diminished probability of vesicular release in mutants. Our data show that global shifts in the synaptic efficiency across brain-wide networks leads to an altered probability of vigilance state transitions, possibly as a result of an altered excitability balance within local circuits controlling sleep-wake architecture.

Loss of Arc attenuates the behavioral and molecular responses for sleep homeostasis in mice.

The activity-regulated cytoskeleton-associated protein (Arc) gene is a neural immediate early gene that is involved in synaptic downscaling and is robustly induced by prolonged wakefulness in rodent brains. Converging evidence has led to the hypothesis that wakefulness potentiates, and sleep reduces, synaptic strengthening. This suggests a potential role for Arc in these and other sleep-related processes. However, the role of Arc in sleep remains unknown. Here, we demonstrated that Arc is important for the induction of multiple behavioral and molecular responses associated with sleep homeostasis. Arc knockout (KO) mice displayed increased time spent in rapid eye movement (REM) sleep under baseline conditions and marked attenuation of sleep rebound to both 4 h of total sleep deprivation (SD) and selective REM deprivation. At the molecular level, the following homeostatic sleep responses to 4-h SD were all blunted in Arc KO mice: increase of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluA1 and its phosphorylation in synaptoneurosomes; induction of a subset of SD-response genes; and suppression of the GluA1 messenger RNA in the cortex. In wild-type brains, SD increased Arc protein expression in multiple subcellular locations, including the nucleus, cytoplasm, and synapse, which is reversed in part by recovery sleep. Arc is critical for these behavioral and multiple molecular responses to SD, thus providing a multifunctional role for Arc in the maintenance of sleep homeostasis, which may be attributed by the sleep/wake-associated changes in subcellular location of Arc.

Loss of the conserved PKA sites of SIK1 and SIK2 increases sleep need.

Although sleep is one of the most conserved behaviors, the intracellular mechanism regulating sleep/wakefulness remains unknown. We recently identified a protein kinase, SIK3, as a sleep-regulating molecule. Mice that lack a well-conserved protein kinase A (PKA) phosphorylation site, S551, showed longer non-rapid eye movement (NREM) sleep and increased NREMS delta density. S551 of SIK3 is conserved in other members of the SIK family, such as SIK1 (S577) and SIK2 (S587). Here, we examined whether the PKA phosphorylation sites of SIK1 and SIK2 are involved in sleep regulation by generating Sik1S577A and Sik2S587A mice. The homozygous Sik1S577A mice showed a shorter wake time, longer NREMS time, and higher NREMS delta density than the wild-type mice. The heterozygous and homozygous Sik2S587A mice showed increased NREMS delta density. Both the Sik1S577A and Sik2S587A mice exhibited proper homeostatic regulation of sleep need after sleep deprivation. Despite abundant expression of Sik1 in the suprachiasmatic nucleus, the Sik1S577A mice showed normal circadian behavior. Although Sik2 is highly expressed in brown adipose tissue, the male and female Sik2S587A mice that were fed either a chow or high-fat diet showed similar weight gain as the wild-type littermates. These results suggest that PKA-SIK signaling is involved in the regulation of sleep need.

Evolutionarily Conserved Regulation of Sleep by the Protein Translational Regulator PERK.

Sleep is a cross-species phenomenon whose evolutionary and biological function remain poorly understood. Clinical and animal studies suggest that sleep disturbance is significantly associated with disruptions in protein homeostasis-or proteostasis-in the brain, but the mechanism of this link has not been explored. In the cell, the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) pathway modulates proteostasis by transiently inhibiting protein synthesis in response to proteostatic stress. In this study, we examined the role of the PERK pathway in sleep regulation and provide the first evidence that PERK signaling is required to regulate normal sleep in both vertebrates and invertebrates. We show that pharmacological inhibition of PERK reduces sleep in both Drosophila and zebrafish, indicating an evolutionarily conserved requirement for PERK in sleep. Genetic knockdown of PERK activity also reduces sleep in Drosophila, whereas PERK overexpression induces sleep. Finally, we demonstrate that changes in PERK signaling directly impact wake-promoting neuropeptide expression, revealing a mechanism through which proteostatic pathways can affect sleep and wake behavior. Taken together, these results demonstrate that protein synthesis pathways like PERK could represent a general mechanism of sleep and wake regulation and provide greater insight into the relationship between sleep and proteostasis.

Sleep-wake-driven and circadian contributions to daily rhythms in gene expression and chromatin accessibility in the murine cortex.

The timing and duration of sleep results from the interaction between a homeostatic sleep-wake-driven process and a periodic circadian process, and involves changes in gene regulation and expression. Unraveling the contributions of both processes and their interaction to transcriptional and epigenomic regulatory dynamics requires sampling over time under conditions of unperturbed and perturbed sleep. We profiled mRNA expression and chromatin accessibility in the cerebral cortex of mice over a 3-d period, including a 6-h sleep deprivation (SD) on day 2. We used mathematical modeling to integrate time series of mRNA expression data with sleep-wake history, which established that a large proportion of rhythmic genes are governed by the homeostatic process with varying degrees of interaction with the circadian process, sometimes working in opposition. Remarkably, SD caused long-term effects on gene-expression dynamics, outlasting phenotypic recovery, most strikingly illustrated by a damped oscillation of most core clock genes, including Arntl/Bmal1, suggesting that enforced wakefulness directly impacts the molecular clock machinery. Chromatin accessibility proved highly plastic and dynamically affected by SD. Dynamics in distal regions, rather than promoters, correlated with mRNA expression, implying that changes in expression result from constitutively accessible promoters under the influence of enhancers or repressors. Serum response factor (SRF) was predicted as a transcriptional regulator driving immediate response, suggesting that SRF activity mirrors the build-up and release of sleep pressure. Our results demonstrate that a single, short SD has long-term aftereffects at the genomic regulatory level and highlights the importance of the sleep-wake distribution to diurnal rhythmicity and circadian processes.

TAK-925, an orexin 2 receptor-selective agonist, shows robust wake-promoting effects in mice.

Orexin-producing neurons in the lateral hypothalamus are a critical regulator of sleep/wake states, and their loss is associated with narcolepsy type 1 (NT1). Orexin peptides act on two G protein-coupled receptors: orexin 1 receptor (OX1R) and orexin 2 receptor (OX2R). OX2R knockout (KO) mice, but not OX1R KO mice, showed clear narcolepsy-like phenotypes, including fragmented sleep-wake cycles. Moreover, OX2R-selective antagonists have been shown to induce sleepiness in mice, and activation of OX2R has been reported to increase wakefulness. In this study, we characterized in vitro and in vivo profiles of a novel, highly selective OX2R agonist, TAK-925 [methyl (2R,3S)-3-[(methylsulfonyl)amino]-2-{[(cis-4-phenylcyclohexyl)oxy]methyl}piperidine-1-carboxylate]. TAK-925 activated human recombinant OX2R with 50% effective concentration value of 5.5 nM, and showed >5,000-fold selectivity over OX1R in calcium mobilization assays. TAK-925 induced OX2R-downstream signals similar to those displayed by orexin peptides in Chinese hamster ovary cells stably expressing human OX2R. In an electrophysiological study, TAK-925 activated physiological OX2R on histaminergic neurons in the mouse tuberomammillary nucleus (TMN). Subcutaneous (SC) administration of TAK-925 also modulated neuronal activity in various brain regions, including TMN, as measured by an immunohistochemical analysis using an anti-c-fos antibody. TAK-925 (SC) increased wakefulness in wild-type mice, but not in OX2R KO mice, during their sleep phase, demonstrating that a highly selective OX2R agonist can increase wakefulness in mice via OX2R activation. TAK-925 may have therapeutic potential to reduce hypersomnia in multiple disorders including NT1.

The Neuropeptide Galanin Is Required for Homeostatic Rebound Sleep following Increased Neuronal Activity.

Sleep pressure increases during wake and dissipates during sleep, but the molecules and neurons that measure homeostatic sleep pressure remain poorly understood. We present a pharmacological assay in larval zebrafish that generates short-term increases in wakefulness followed by sustained rebound sleep after washout. The intensity of global neuronal activity during drug-induced wakefulness predicted the amount of subsequent rebound sleep. Whole-brain mapping with the neuronal activity marker phosphorylated extracellular signal-regulated kinase (pERK) identified preoptic Galanin (Galn)-expressing neurons as selectively active during rebound sleep, and the relative induction of galn transcripts was predictive of total rebound sleep time. Galn is required for sleep homeostasis, as galn mutants almost completely lacked rebound sleep following both pharmacologically induced neuronal activity and physical sleep deprivation. These results suggest that Galn plays a key role in responding to sleep pressure signals derived from neuronal activity and functions as an output arm of the vertebrate sleep homeostat.

A Rare Mutation of ß1-Adrenergic Receptor Affects Sleep/Wake Behaviors.

Sleep is crucial for our survival, and many diseases are linked to long-term poor sleep quality. Before we can use sleep to enhance our health and performance and alleviate diseases associated with poor sleep, a greater understanding of sleep regulation is necessary. We have identified a mutation in the ß1-adrenergic receptor gene in humans who require fewer hours of sleep than most. In vitro, this mutation leads to decreased protein stability and dampened signaling in response to agonist treatment. In vivo, the mice carrying the same mutation demonstrated short sleep behavior. We found that this receptor is highly expressed in the dorsal pons and that these ADRB1+ neurons are active during rapid eye movement (REM) sleep and wakefulness. Activating these neurons can lead to wakefulness, and the activity of these neurons is affected by the mutation. These results highlight the important role of ß1-adrenergic receptors in sleep/wake regulation.

Methodology and theoretical basis of forward genetic screening for sleep/wakefulness in mice.

The regulatory network of genes and molecules in sleep/wakefulness remains to be elucidated. Here we describe the methodology and workflow of the dominant screening of randomly mutagenized mice and discuss theoretical basis of forward genetics research for sleep in mice. Our high-throughput screening employs electroencephalogram (EEG) and electromyogram (EMG) to stage vigilance states into a wake, rapid eye movement sleep (REMS) and non-REM sleep (NREMS). Based on their near-identical sleep/wake behavior, C57BL/6J (B6J) and C57BL/6N (B6N) are chosen as mutagenized and counter strains, respectively. The total time spent in the wake and NREMS, as well as the REMS episode duration, shows sufficient reproducibility with small coefficients of variance, indicating that these parameters are most suitable for quantitative phenotype-driven screening. Coarse linkage analysis of the quantitative trait, combined with whole-exome sequencing, can identify the gene mutation associated with sleep abnormality. Our simulations calculate the achievable LOD score as a function of the phenotype strength and the numbers of mice examined. A pedigree showing a mild decrease in total wake time resulting from a heterozygous point mutation in the Cacna1a gene is described as an example.

The Serotonergic Raphe Promote Sleep in Zebrafish and Mice.

The role of serotonin (5-HT) in sleep is controversial: early studies suggested a sleep-promoting role, but eventually the paradigm shifted toward a wake-promoting function for the serotonergic raphe. Here, we provide evidence from zebrafish and mice that the raphe are critical for the initiation and maintenance of sleep. In zebrafish, genetic ablation of 5-HT production by the raphe reduces sleep, sleep depth, and the homeostatic response to sleep deprivation. Pharmacological inhibition or ablation of the raphe reduces sleep, while optogenetic stimulation increases sleep. Similarly, in mice, ablation of the raphe increases wakefulness and impairs the homeostatic response to sleep deprivation, whereas tonic optogenetic stimulation at a rate similar to baseline activity induces sleep. Interestingly, burst optogenetic stimulation induces wakefulness in accordance with previously described burst activity of the raphe during arousing stimuli. These results indicate that the serotonergic system promotes sleep in both diurnal zebrafish and nocturnal rodents. VIDEO ABSTRACT.

Loss of DmGluRA exacerbates age-related sleep disruption and reduces lifespan.

Declines in sleep amount and quality-characterized by excessive daytime sleepiness and an inability to sleep at night-are common features of aging. Sleep dysfunction is also associated with age-related ailments and diseases, suggesting that sleep is functionally relevant to the aging process. Metabotropic glutamate receptors (mGluRs)-which are critical regulators of neurotransmission and synaptic plasticity-have been implicated in both age-related disease and sleep regulation. Therefore, in this study, we examined the sleep and aging effect of complete genetic loss of mGluR signaling in Drosophila melanogaster. Genetic knockdown of the sole Drosophila mGluR-known as DmGluRA-reduced daytime wakefulness and nighttime sleep, recapitulating age-related sleep changes that occur across species. Furthermore, loss of DmGluRA significantly reduced lifespan and exacerbated age-related sleep loss in older flies. Thus, we identify DmGluRA as a novel regulator of sleep whose loss results in an age-relevant sleep phenotype that is associated with shortened lifespan. This is the first evidence that mGluR signaling regulates sleep/wake in a manner that is relevant to the aging process.