Transcriptional dynamics of sleep deprivation and subsequent recovery sleep in the male mouse cortex.

Sleep is an essential, tightly regulated biological function. Sleep is also a homeostatic process, with the need to sleep increasing as a function of being awake. Acute sleep deprivation (SD) increases sleep need, and subsequent recovery sleep (RS) discharges it. SD is known to alter brain gene expression in rodents, but it remains unclear which changes are linked to sleep homeostasis, SD-related impairments, or non-sleep-specific effects. To investigate this question, we analyzed RNA-seq data from adult wild-type male mice subjected to 3 and 5-6 hours of SD and 2 and 6 hours of RS after SD. We hypothesized molecular changes associated with sleep homeostasis mirror sleep pressure dynamics as defined by brain electrical activity, peaking at 5-6 hours of SD, and are no longer differentially expressed after 2 hours of RS. We report 5-6 hours of SD produces the largest effect on gene expression, affecting approximately half of the cortical transcriptome, with most differentially expressed genes (DEGs) downregulated. The majority of DEGs normalize after 2 hours of RS and are involved in redox metabolism, chromatin regulation, and DNA damage/repair. Additionally, RS affects gene expression related to mitochondrial metabolism and Wnt-signaling, potentially contributing to its restorative effects. DEGs associated with cholesterol metabolism and stress response do not normalize within 6 hours and may be non-sleep-specific. Finally, DEGs involved in insulin signaling, MAPK signaling, and RNA-binding may mediate the impairing effects of SD. Overall, our results offer insight into the molecular mechanisms underlying sleep homeostasis and the broader effects of SD.

A global transcriptional atlas of the effect of acute sleep deprivation in the mouse frontal cortex.

Sleep deprivation (SD) has negative effects on brain and body function. Sleep problems are prevalent in a variety of disorders, including neurodevelopmental and psychiatric conditions. Thus, understanding the molecular consequences of SD is of fundamental importance in biology. In this study, we present the first simultaneous bulk and single-nuclear RNA sequencing characterization of the effects of SD in the male mouse frontal cortex. We show that SD predominantly affects glutamatergic neurons, specifically in layers 4 and 5, and produces isoform switching of over 1500 genes, particularly those involved in splicing and RNA binding. At both the global and cell-type specific level, SD has a large repressive effect on transcription, downregulating thousands of genes and transcripts. As a resource we provide extensive characterizations of cell-types, genes, transcripts, and pathways affected by SD. We also provide publicly available tutorials aimed at allowing readers adapt analyses performed in this study to their own datasets.

A Global Transcriptional Atlas of the Effect of Sleep Deprivation in the Mouse Frontal Cortex.

Sleep deprivation (SD) has negative effects on brain function. Sleep problems are prevalent in neurodevelopmental, neurodegenerative and psychiatric disorders. Thus, understanding the molecular consequences of SD is of fundamental importance in neuroscience. In this study, we present the first simultaneous bulk and single-nuclear (sn)RNA sequencing characterization of the effects of SD in the mouse frontal cortex. We show that SD predominantly affects glutamatergic neurons, specifically in layers 4 and 5, and produces isoform switching of thousands of transcripts. At both the global and cell-type specific level, SD has a large repressive effect on transcription, down-regulating thousands of genes and transcripts; underscoring the importance of accounting for the effects of sleep loss in transcriptome studies of brain function. As a resource we provide extensive characterizations of cell types, genes, transcripts and pathways affected by SD; as well as tutorials for data analysis.

Ontogenesis of the molecular response to sleep loss.

Sleep deprivation (SD) results in profound cellular and molecular changes in the adult mammalian brain. Some of these changes may result in, or aggravate, brain disease. However, little is known about how SD impacts gene expression in developing animals. We examined the transcriptional response in the prefrontal cortex (PFC) to SD across postnatal development in male mice. We used RNA sequencing to identify functional gene categories that were specifically impacted by SD. We find that SD has dramatically different effects on PFC genes depending on developmental age. Gene expression differences after SD fall into 3 categories: present at all ages (conserved), present when mature sleep homeostasis is first emerging, and those unique to certain ages. Developmentally conserved gene expression was limited to a few functional categories, including Wnt-signaling which suggests that this pathway is a core mechanism regulated by sleep. In younger ages, genes primarily related to growth and development are affected while changes in genes related to metabolism are specific to the effect of SD in adults.

Critical periods and Autism Spectrum Disorders, a role for sleep.

Brain development relies on both experience and genetically defined programs. Time windows where certain brain circuits are particularly receptive to external stimuli, resulting in heightened plasticity, are referred to as "critical periods". Sleep is thought to be essential for normal brain development. Importantly, studies have shown that sleep enhances critical period plasticity and promotes experience-dependent synaptic pruning in the developing mammalian brain. Therefore, normal plasticity during critical periods depends on sleep. Problems falling and staying asleep occur at a higher rate in Autism Spectrum Disorder (ASD) relative to typical development. In this review, we explore the potential link between sleep, critical period plasticity, and ASD. First, we review the importance of critical period plasticity in typical development and the role of sleep in this process. Next, we summarize the evidence linking ASD with deficits in synaptic plasticity in rodent models of high-confidence ASD gene candidates. We then show that the high-confidence rodent models of ASD that show sleep deficits also display plasticity deficits. Given how important sleep is for critical period plasticity, it is essential to understand the connections between synaptic plasticity, sleep, and brain development in ASD. However, studies investigating sleep or plasticity during critical periods in ASD mouse models are lacking. Therefore, we highlight an urgent need to consider developmental trajectory in studies of sleep and plasticity in neurodevelopmental disorders.

Ontogenesis of the molecular response to sleep loss.

Sleep deprivation (SD) results in profound cellular and molecular changes in the adult mammalian brain. Some of these changes may result in, or aggravate, brain disease. However, little is known about how SD impacts gene expression in developing animals. We examined the transcriptional response in the prefrontal cortex (PFC) to SD across postnatal development in male mice. We used RNA sequencing to identify functional gene categories that were specifically impacted by SD. We find that SD has dramatically different effects on PFC genes depending on developmental age. Gene expression differences after SD fall into 3 categories: present at all ages (conserved), present when mature sleep homeostasis is first emerging, and those unique to certain ages in adults. Developmentally conserved gene expression was limited to a few functional categories, including Wnt-signaling which suggests that this pathway is a core mechanism regulated by sleep. In younger ages, genes primarily related to growth and development are affected while changes in genes related to metabolism are specific to the effect of SD in adults.

Shank3 influences mammalian sleep development.

Sleep problems are prevalent in autism spectrum disorder (ASD), can be observed before diagnosis, and are associated with increased restricted and repetitive behaviors. Therefore, sleep abnormalities may be a core feature of the disorder, but the developmental trajectory remains unknown. Animal models provide a unique opportunity to understand sleep ontogenesis in ASD. Previously we showed that adult mice with a truncation in the high-confidence ASD gene Shank3 (Shank3∆C ) recapitulate the clinical sleep phenotype. In this study we used longitudinal electro-encephalographic (EEG) recordings to define, for the first time, changes in sleep from weaning to young adulthood in an ASD mouse model. We show that Shank3∆C male mice sleep less overall throughout their lifespan, have increased rapid eye movement (REM) sleep early in life despite significantly reduced non-rapid eye movement (NREM) sleep, and have abnormal responses to increased sleep pressure that emerge during a specific developmental period. We demonstrate that the ability to fall asleep quickly in response to sleep loss develops normally between 24 and 30 days in mice. However, mutants are unable to reduce sleep latency after periods of prolonged waking and maintain the same response to sleep loss regardless of age. This phenomenon seems independent of homeostatic NREM sleep slow-wave dynamics. Overall, our study recapitulates both preclinical models and clinical studies showing that reduced sleep is consistently associated with ASD and suggests that problems falling asleep may reflect abnormal development of sleep and arousal mechanisms.

A chemical-genetic investigation of BDNF-NtrkB signaling in mammalian sleep.

STUDY OBJECTIVES: The neurotrophin brain-derived neurotrophic factor (BDNF) is hypothesized to be a molecular mediator of mammalian sleep homeostasis. This hypothesis is supported by correlational findings and results obtained from pharmacology. BDNF binds with high affinity to the membrane-bound receptor Neurotrophin Tyrosine Kinase Receptor B (NtrkB), which triggers several intracellular signaling cascades. It is therefore possible that BDNF's role in sleep homeostasis is mediated via NtrkB. We examined this hypothesis using a chemical-genetic technique that allows for rapid and selective inhibition of NtrkB in vivo. METHODS: We used mutant mice bearing a point mutation in the NtrkB that allows for selective and reversible inactivation in the presence of a small binding molecule (1-NM-PP1). Using a crossover design, we determined the effects of NtrkB inhibition on baseline sleep architecture and sleep homeostasis. RESULTS: We find that NtrkB inhibition reduced rapid eye movement (REM) sleep time and changed state transitions but had no effect on sleep homeostasis. CONCLUSIONS: These findings suggest that BDNF-NtrkB receptor signaling has subtle roles in sleep architecture, but no role in sleep homeostasis.

A Role for Astroglial Calcium in Mammalian Sleep and Sleep Regulation.

Mammalian sleep expression and regulation have historically been thought to reflect the activity of neurons. Changes in other brain cells (glia) across the sleep-wake cycle and their role in sleep regulation are comparatively unexplored. We show that sleep and wakefulness are accompanied by state-dependent changes in astroglial activity. Using a miniature microscope in freely behaving mice and a two-photon microscope in head-fixed, unanesthetized mice, we show that astroglial calcium signals are highest in wake and lowest in sleep and are most pronounced in astroglial processes. We also find that astroglial calcium signals during non-rapid eye movement sleep change in proportion to sleep need. In contrast to neurons, astrocytes become less synchronized during non-rapid eye movement sleep after sleep deprivation at the network and single-cell level. Finally, we show that conditionally reducing intracellular calcium in astrocytes impairs the homeostatic response to sleep deprivation. Thus, astroglial calcium activity changes dynamically across vigilance states, is proportional to sleep need, and is a component of the sleep homeostat.

Shank3 modulates sleep and expression of circadian transcription factors.

Autism Spectrum Disorder (ASD) is the most prevalent neurodevelopmental disorder in the United States and often co-presents with sleep problems. Sleep problems in ASD predict the severity of ASD core diagnostic symptoms and have a considerable impact on the quality of life of caregivers. Little is known, however, about the underlying molecular mechanisms of sleep problems in ASD. We investigated the role of Shank3, a high confidence ASD gene candidate, in sleep architecture and regulation. We show that mice lacking exon 21 of Shank3 have problems falling asleep even when sleepy. Using RNA-seq we show that sleep deprivation increases the differences in prefrontal cortex gene expression between mutants and wild types, downregulating circadian transcription factors Per3, Bhlhe41, Hlf, Tef, and Nr1d1. Shank3 mutants also have trouble regulating wheel-running activity in constant darkness. Overall, our study shows that Shank3 is an important modulator of sleep and clock gene expression.

Aging and sleep deprivation induce the unfolded protein response in the pancreas: implications for metabolism.

Sleep disruption has detrimental effects on glucose metabolism through pathways that remain poorly defined. Although numerous studies have examined the consequences of sleep deprivation (SD) in the brain, few have directly tested its effects on peripheral organs. We examined several tissues in mice for induction of the unfolded protein response (UPR) following acute SD. In young animals, we found a robust induction of BiP in the pancreas, indicating an active UPR. At baseline, pancreata from aged animals exhibited a marked increase in a pro-apoptotic transcription factor, CHOP, that was amplified by SD, whereas BiP induction was not observed, suggesting a maladaptive response to cellular stress with age. Acute SD increased plasma glucose levels in both young and old animals. However, this change was not overtly related to stress in the pancreatic beta cells, as plasma insulin levels were not lower following acute SD. Accordingly, animals subjected to acute SD remained tolerant to a glucose challenge. In a chronic SD experiment, young mice were found to be sensitized to insulin and have improved glycemic control, whereas aged animals became hyperglycemic and failed to maintain appropriate plasma insulin concentrations. Our results show that both age and SD cooperate to induce the UPR in pancreatic tissue. While changes in insulin secretion are unlikely to play a major role in the acute effects of SD, CHOP induction in pancreatic tissues suggests that chronic SD may contribute to the loss or dysfunction of endocrine cells and that these effects may be exacerbated by normal aging.

Disease and Degeneration of Aging Neural Systems that Integrate Sleep Drive and Circadian Oscillations.

Sleep/wake and circadian rest-activity rhythms become irregular with age. Typical outcomes include fragmented sleep during the night, advanced sleep phase syndrome and increased daytime sleepiness. These changes lead to a reduction in the quality of life due to cognitive impairments and emotional stress. More importantly, severely disrupted sleep and circadian rhythms have been associated with an increase in disease susceptibility. Additionally, many of the same brain areas affected by neurodegenerative diseases include the sleep and wake promoting systems. Any advances in our knowledge of these sleep/wake and circadian networks are necessary to target neural areas or connections for therapy. This review will discuss research that uses molecular, behavioral, genetic and anatomical methods to further our understanding of the interaction of these systems.

Distribution of orexin/hypocretin immunoreactivity in the brain of a male songbird, the house finch, Carpodacus mexicanus.

Previous research has shown orexin/hypocretin immunoreactive (orexin-ir) neurons in domesticated Galliformes. However, these findings may not be representative of other birds and these studies did not include a distribution of orexin-ir projections throughout the brain. The present study was carried out in a wild-caught passerine, the house finch, Carpodacus mexicanus, and includes a detailed description of orexin-ir neurons and their projections. Orexin A and B-ir neurons were located in a single population centered on the paraventricular nucleus of the hypothalamus extending into the lateral hypothalamic area, consistent with other studies in birds. Orexin A and B-ir fibers were similarly visible across the brain, with the highest density within the preoptic area, hypothalamus and thalamus. Orexin-ir projections extended from the paraventricular nucleus rostrally to the preoptic area, laterally towards the medial striatum, nidopallium, and dorsally along the lateral ventricle towards the mesopallium. Caudally, the highest densities of orexin-ir fibers were found along the third ventricle. The periaqueductal grey, substantia nigra pars compacta and the locus coeruleus also showed a high density of orexin-ir fibers. This study showed a detailed fiber distribution previously unreported in birds and showed that orexin-ir neurons were located in similar areas regardless of phylogeny or domestication in birds. The apparently conserved neural distribution of orexins suggests that these peptides play similar roles among birds. The widespread distribution of the projections in brain areas serving various roles indicates the potential involvement of these peptides in multiple behavioral and physiological functions.

Distribution of orexin/hypocretin immunoreactivity in the nervous system of the green Treefrog, Hyla cinerea.

We examined the distribution of orexin/hypocretin immunoreactive neurons and projections throughout the brain of the green treefrog (Hyla cinerea). Orexin A and B neurons were located in a single population centered on the suprachiasmatic nucleus. Orexin A and B fibers were visible across the brain, with the highest density within the preoptic area and hypothalamus. Our data suggest different distributions of orexin neurons but not projections between families of amphibians.