Crucial role of TFAP2B in the nervous system for regulating NREM sleep.

The AP-2 transcription factors are crucial for regulating sleep in both vertebrate and invertebrate animals. In mice, loss of function of the transcription factor AP-2ß (TFAP2B) reduces non-rapid eye movement (NREM) sleep. When and where TFAP2B functions, however, is unclear. Here, we used the Cre-loxP system to generate mice in which Tfap2b was specifically deleted in the nervous system during development and mice in which neuronal Tfap2b was specifically deleted postnatally. Both types of mice exhibited reduced NREM sleep, but the nervous system-specific deletion of Tfap2b resulted in more severe sleep phenotypes accompanied by defective light entrainment of the circadian clock and stereotypic jumping behavior. These findings indicate that TFAP2B in postnatal neurons functions at least partly in sleep regulation and imply that TFAP2B also functions either at earlier stages or in additional cell types within the nervous system.

ER proteostasis regulators cell-non-autonomously control sleep.

Sleep is regulated by peripheral tissues under fatigue. The molecular pathways in peripheral cells that trigger systemic sleep-related signals, however, are unclear. Here, a forward genetic screen in C. elegans identifies 3 genes that strongly affect sleep amount: sel-1, sel-11, and mars-1. sel-1 and sel-11 encode endoplasmic reticulum (ER)-associated degradation components, whereas mars-1 encodes methionyl-tRNA synthetase. We find that these machineries function in non-neuronal tissues and that the ER unfolded protein response components inositol-requiring enzyme 1 (IRE1)/XBP1 and protein kinase R-like ER kinase (PERK)/eukaryotic initiation factor-2α (eIF2α)/activating transcription factor-4 (ATF4) participate in non-neuronal sleep regulation, partly by reducing global translation. Neuronal epidermal growth factor receptor (EGFR) signaling is also required. Mouse studies suggest that this mechanism is conserved in mammals. Considering that prolonged wakefulness increases ER proteostasis stress in peripheral tissues, our results suggest that peripheral ER proteostasis factors control sleep homeostasis. Moreover, based on our results, peripheral tissues likely cope with ER stress not only by the well-established cell-autonomous mechanisms but also by promoting the individual's sleep.

Impaired wakefulness and rapid eye movement sleep in dopamine-deficient mice.

Despite the established roles of the dopaminergic system in promoting arousal, the effects of loss of dopamine on the patterns of sleep and wakefulness remain elusive. Here, we examined the sleep architecture of dopamine-deficient (DD) mice, which were previously developed by global knockout of tyrosine hydroxylase and its specific rescue in noradrenergic and adrenergic neurons. We found that DD mice have reduced time spent in wakefulness. Unexpectedly, DD mice also exhibited a marked reduction in the time spent in rapid eye movement (REM) sleep. The electroencephalogram power spectrum of all vigilance states in DD mice were also affected. These results support the current understanding of the critical roles of the dopaminergic system in maintaining wakefulness and also implicate its previously unknown effects on REM sleep.

Sleep Architecture in Mice Is Shaped by the Transcription Factor AP-2ß.

The molecular mechanism regulating sleep largely remains to be elucidated. In humans, families that carry mutations in TFAP2B, which encodes the transcription factor AP-2ß, self-reported sleep abnormalities such as short-sleep and parasomnia. Notably, AP-2 transcription factors play essential roles in sleep regulation in the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster Thus, AP-2 transcription factors might have a conserved role in sleep regulation across the animal phyla. However, direct evidence supporting the involvement of TFAP2B in mammalian sleep was lacking. In this study, by using the CRISPR/Cas9 technology, we generated two Tfap2b mutant mouse strains, Tfap2bK144 and Tfap2bK145 , each harboring a single-nucleotide mutation within the introns of Tfap2b mimicking the mutations in two human kindreds that self-reported sleep abnormalities. The effects of these mutations were compared with those of a Tfap2b knockout allele (Tfap2b-). The protein expression level of TFAP2B in the embryonic brain was reduced to about half in Tfap2b+/- mice and was further reduced in Tfap2b-/- mice. By contrast, the protein expression level was normal in Tfap2bK145/+ mice but was reduced in Tfap2bK145/K145 mice to a similar extent as Tfap2b-/- mice. Tfap2bK144/+ and Tfap2bK144/K144 showed normal protein expression levels. Tfap2b+/- female mice showed increased wakefulness time and decreased nonrapid eye movement sleep (NREMS) time. By contrast, Tfap2bK145/+ female mice showed an apparently normal amount of sleep but instead exhibited fragmented NREMS, whereas Tfap2bK144/+ male mice showed reduced NREMS time specifically in the dark phase. Finally, in the adult brain, Tfap2b-LacZ expression was detected in the superior colliculus, locus coeruleus, cerebellum, and the nucleus of solitary tract. These findings provide direct evidence that TFAP2B influences NREMS amounts in mice and also show that different mutations in Tfap2b can lead to diverse effects on sleep architecture.

Progressive Changes in Sleep and Its Relations to Amyloid-ß Distribution and Learning in Single App Knock-In Mice.

Alzheimer's disease (AD) patients often suffer from sleep disturbances. Alterations in sleep, especially rapid eye movement sleep (REMS), can precede the onset of dementia. To accurately characterize the sleep impairments accompanying AD and their underlying mechanisms using animal models, it is crucial to use models in which brain areas are affected in a manner similar to that observed in the actual patients. Here, we focused on AppNL-G-F mice, in which expression levels and patterns of mutated amyloid precursor protein (APP) follow the endogenous patterns. We characterized the sleep architecture of male AppNL-G-F homozygous and heterozygous mice at two ages (six and 12 months). At six months, homozygous mice exhibited reduced REMS, which was further reduced at 12 months together with a slight reduction in non-REMS (NREMS). By contrast, heterozygous mice exhibited an overall normal sleep architecture. Homozygous mice also exhibited decreased electroencephalogram γ to δ power ratio during REMS from six months, resembling the electroencephalogram slowing phenomenon observed in preclinical or early stages of AD. In addition, homozygous mice showed learning and memory impairments in the trace fear conditioning (FC) at both ages, and task performance strongly correlated with REMS amount at 12 months. Finally, histologic analyses revealed that amyloid-ß accumulation in the pontine tegmental area and ventral medulla followed a course similar to that of the REMS reduction. These findings support the notion that changes in REMS are an early marker of AD and provide a starting point to address the mechanism of sleep deficits in AD and the effects on cognition.

Widely Distributed Neurotensinergic Neurons in the Brainstem Regulate NREM Sleep in Mice.

Classical transection studies suggest that, in addition to the hypothalamus, the brainstem is essential for non-rapid eye movement (NREM) sleep. The circuits underlying this function, however, have remained largely unknown. We identified a circuit distributed in the midbrain, pons, and medulla that promotes NREM sleep in mice. We focused on the sublaterodorsal tegmentum, an area implicated in dual regulation of REM and NREM sleep. Transcriptomic and genetic analyses revealed that neurons positive for the neuropeptide neurotensin promote NREM sleep. Further analyses identified downstream NREM sleep-promoting neurons in the dorsal deep mesencephalic nucleus, the lateral part of the periaqueductal gray, and the medial vestibular nucleus that were also neurotensinergic. Infusion of neurotensin into the fourth ventricle induced NREM sleep-like cortical activity, whereas mice deficient for neurotensin exhibited increased REM sleep, implicating the involvement of the neuropeptide itself. These findings identify a widely distributed NREM sleep-regulating circuit in the brainstem with a common molecular property.

Effects of 3 Weeks of Water Immersion and Restraint Stress on Sleep in Mice.

Repeated stress is a risk factor for mental disorders and can also lead to sleep disturbances. Although the effects of stress on sleep architecture have been investigated in rodents, the length of the stress exposure period in most studies has been limited to about 10 days, and few studies have analyzed the effects of chronic stress over a longer period. Here we investigated how sleep is affected in a mouse model of depression induced by 3 weeks of daily water immersion and restraint stress (WIRS). Sleep was recorded after 1, 2, and 3 weeks of stress exposure. Some stress-induced changes in several sleep measures were maintained across the 3 weeks, whereas other changes were most prominent during the 1st week. The total amount of non-rapid eye movement sleep (NREMS) was increased and the total amount of time spent awake was decreased across all 3 weeks. On the other hand, the amount of REMS during the dark phase was significantly increased in the 1st week compared with that at baseline or the 2nd and 3rd weeks. Electroencephalogram (EEG) power in the delta range was decreased during NREMS, although the total amount of NREMS was increased. These findings indicate that repeated WIRS, which eventually leads to a depression-like phenotype, differentially affects sleep between the early and subsequent periods. The increase in the amount of REMS during the dark phase in the 1st week significantly correlated with changes in body weight. Our results show how sleep changes throughout a long period of chronic stress in a mouse model of depression.

Life Without Dreams: Muscarinic Receptors Are Required to Regulate REM Sleep in Mice.

Mammalian sleep comprises REM and NREM stages, but the regulation mechanisms are unclear. In this issue of Cell Reports, Niwa et al. (2018) comprehensively knocked out cholinergic receptors in mice and found that muscarinic signaling is crucial for REM sleep and possibly important for NREM sleep.

[The Function of REM Sleep: Implications from Transgenic Mouse Models].

Our sleep is composed of rapid eye movement (REM) sleep and non-REM (NREM) sleep. REM sleep is the major source of dreams, whereas synchronous cortical oscillations, called slow waves, are observed during NREM sleep. Both stages are unique to certain vertebrate species, and therefore, REM and NREM sleep are thought to be involved in higher-order brain functions. While several studies have revealed the importance of NREM sleep in growth hormone secretion, memory consolidation and brain metabolite clearance, the functions of REM sleep are currently almost totally unknown. REM sleep functions cannot be easily indicated from classical REM sleep deprivation experiments, where animals are forced to wake up whenever they enter REM sleep, because such experiments produce extreme stress due to the stimuli and because REM sleep is under strong homeostatic regulation. To overcome these issues, we developed a novel transgenic mouse model in which REM sleep can be manipulated. Using these mice, we found that REM sleep enhances slow wave activity during the subsequent NREM sleep. Slow wave activity is known to contribute to memory consolidation and synaptic plasticity. Thus, REM sleep might be involved in higher-order brain functions through its role in enhancing slow wave activity.

Cells of a common developmental origin regulate REM/non-REM sleep and wakefulness in mice.

Mammalian sleep comprises rapid eye movement (REM) sleep and non-REM (NREM) sleep. To functionally isolate from the complex mixture of neurons populating the brainstem pons those involved in switching between REM and NREM sleep, we chemogenetically manipulated neurons of a specific embryonic cell lineage in mice. We identified excitatory glutamatergic neurons that inhibit REM sleep and promote NREM sleep. These neurons shared a common developmental origin with neurons promoting wakefulness; both derived from a pool of proneural hindbrain cells expressing Atoh1 at embryonic day 10.5. We also identified inhibitory γ-aminobutyric acid-releasing neurons that act downstream to inhibit REM sleep. Artificial reduction or prolongation of REM sleep in turn affected slow-wave activity during subsequent NREM sleep, implicating REM sleep in the regulation of NREM sleep.