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.

Activation of Basal Forebrain Astrocytes Induces Wakefulness without Compensatory Changes in Sleep Drive.

Mammalian sleep is regulated by a homeostatic process that increases sleep drive and intensity as a function of prior wake time. Sleep homeostasis has traditionally been thought to be a product of neurons, but recent findings demonstrate that this process is also modulated by glial astrocytes. The precise role of astrocytes in the accumulation and discharge of sleep drive is unknown. We investigated this question by selectively activating basal forebrain (BF) astrocytes using designer receptors exclusively activated by designer drugs (DREADDs) in male and female mice. DREADD activation of the Gq-protein-coupled pathway in BF astrocytes produced long and continuous periods of wakefulness that paradoxically did not cause the expected homeostatic response to sleep loss (e.g., increases in sleep time or intensity). Further investigations showed that this was not because of indirect effects of the ligand that activated DREADDs. These findings suggest that the need for sleep is not only driven by wakefulness per se, but also by specific neuronal-glial circuits that are differentially activated in wakefulness.SIGNIFICANCE STATEMENT Sleep drive is controlled by a homeostatic process that increases sleep duration and intensity based on prior time spent awake. Non-neuronal brain cells (e.g., glial astrocytes) influence this homeostatic process, but their precise role is unclear. We used a genetic technique to activate astrocytes in the basal forebrain (BF) of mice, a brain region important for sleep and wake expression and sleep homeostasis. Astroglial activation induced prolonged wakefulness without the expected homeostatic increase in sleep drive (i.e., sleep duration and intensity). These findings indicate that our need to sleep is also driven by non-neuronal cells, and not only by time spent awake.

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.

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.

Goodnight, astrocyte: waking up to astroglial mechanisms in sleep.

Astrocytes mediate many important aspects of neural homeostasis, but until recently, their role in sleep was largely unknown. The situation has dramatically changed in the last decade. The use of transgenic animals, optogenetics, chemogenetics, brain imaging and sophisticated molecular assays has led to exciting discoveries. Astrocytes dynamically change their activity across the sleep-wake cycle and may encode sleep need via changes in intracellular signalling pathways. Astrocytes also exocytose/secrete sleep-inducing molecules which modulate brain activity, sleep architecture and sleep regulation. Many of these observations have been made in mice and Drosophila melanogaster, indicating that astroglial sleep mechanisms are evolutionarily conserved. We review recent findings and discuss future directions.

Noradrenergic Signaling in Astrocytes Influences Mammalian Sleep Homeostasis.

Astrocytes influence sleep expression and regulation, but the cellular signaling pathways involved in these processes are poorly defined. We proposed that astrocytes detect and integrate a neuronal signal that accumulates during wakefulness, thereby leading to increased sleep drive. Noradrenaline (NA) satisfies several criteria for a waking signal integrated by astrocytes. We therefore investigated the role of NA signaling in astrocytes in mammalian sleep. We conditionally knocked out (cKO) ß2-adrenergic receptors (ß2-AR) selectively in astrocytes in mice and recorded electroencephalographic and electromyographic activity under baseline conditions and in response to sleep deprivation (SDep). cKO of astroglial ß2-ARs increased active phase siesta duration under baseline conditions and reduced homeostatic compensatory changes in sleep consolidation and non-rapid eye movement slow-wave activity (SWA) after SDep. Overall, astroglial NA ß2-ARs influence mammalian sleep homeostasis in a manner consistent with our proposed model of neuronal-astroglial interactions.

REM sleep promotes bidirectional plasticity in developing visual cortex in vivo.

Sleep is required for the full expression of plasticity during the visual critical period (CP). However, the precise role of rapid-eye-movement (REM) sleep in this process is undetermined. Previous studies in rodents indicate that REM sleep weakens cortical circuits following MD, but this has been explored in only one class of cortical neuron (layer 5 apical dendrites). We investigated the role of REM sleep in ocular dominance plasticity (ODP) in layer 2/3 neurons using 2-photon calcium imaging in awake CP mice. In contrast to findings in layer 5 neurons, we find that REM sleep promotes changes consistent with synaptic strengthening and weakening. This supports recent suggestions that the effects of sleep on plasticity are highly dependent upon the type of circuit and preceding waking experience.

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.

Renormalizing synapses in sleep: The clock is ticking.

Sleep has been hypothesized to renormalize synapses potentiated in wakefulness. This is proposed to lead to a net reduction in synaptic strength after sleep in brain areas like the cortex and hippocampus. Biological clocks, however, exert independent effects on these synapses that may explain some of the reported differences after wake and sleep. These include changes in synaptic morphology, molecules and efficacy. In this commentary, I discuss why no firm conclusions should be drawn concerning the role of sleep in synaptic renormalization until the role of circadian rhythms are isolated and determined.

Challenging sleep homeostasis.

In this commentary, I play the Devil's advocate and assume the title of High Contrarian. I intend to be provocative to challenge long-standing ideas about sleep. I blame all on Professor Craig Heller, who taught me to think this way as a graduate student in his laboratory. Scientists should fearlessly jump into the foaming edge of what we know, but also consider how safe are their intellectual harbors. There are many ideas we accept as 'known': that sleep is ubiquitous in the animal kingdom, that it serves vital functions, that it plays an essential role in brain plasticity. All of this could be wrong. As one example, I reexamine the idea that sleep is regulated by a mysterious 'homeostat' that determines sleep need based on prior wake time.

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.

REM sleep promotes experience-dependent dendritic spine elimination in the mouse cortex.

In many parts of the nervous system, experience-dependent refinement of neuronal circuits predominantly involves synapse elimination. The role of sleep in this process remains unknown. We investigated the role of sleep in experience-dependent dendritic spine elimination of layer 5 pyramidal neurons in the visual (V1) and frontal association cortex (FrA) of 1-month-old mice. We found that monocular deprivation (MD) or auditory-cued fear conditioning (FC) caused rapid spine elimination in V1 or FrA, respectively. MD- or FC-induced spine elimination was significantly reduced after total sleep or REM sleep deprivation. Total sleep or REM sleep deprivation also prevented MD- and FC-induced reduction of neuronal activity in response to visual or conditioned auditory stimuli. Furthermore, dendritic calcium spikes increased substantially during REM sleep, and the blockade of these calcium spikes prevented MD- and FC-induced spine elimination. These findings reveal an important role of REM sleep in experience-dependent synapse elimination and neuronal activity reduction.

Sleep, brain development, and autism spectrum disorders: Insights from animal models.

Sleep is an evolutionarily conserved and powerful drive, although its complete functions are still unknown. One possible function of sleep is that it promotes brain development. The amount of sleep is greatest during ages when the brain is rapidly developing, and sleep has been shown to influence critical period plasticity. This supports a role for sleep in brain development and suggests that abnormal sleep in early life may lead to abnormal development. Autism spectrum disorder (ASD) is the most prevalent neurodevelopmental disorder in the United States. It is estimated that insomnia affects 44%-86% of the ASD population, predicting the severity of ASD core symptoms and associated behavioral problems. Sleep problems impact the quality of life of both ASD individuals and their caregivers, thus it is important to understand why they are so prevalent. In this review, we explore the role of sleep in early life as a causal factor in ASD. First, we review fundamental steps in mammalian sleep ontogeny and regulation and how sleep influences brain development. Next, we summarize current knowledge gained from studying sleep in animal models of ASD. Ultimately, our goal is to highlight the importance of understanding the role of sleep in brain development and the use of animal models to provide mechanistic insight into the origin of sleep problems in ASD.

The Ontogenesis of Mammalian Sleep: Form and Function.

PURPOSE OF REVIEW: To present an up-to-date review and synthesis of findings about perinatal sleep development and function. I discuss landmark events in sleep ontogenesis, evidence that sleep promotes brain development and plasticity, and experimental considerations in this topic. RECENT FINDINGS: Mammalian sleep undergoes dramatic changes in expression and regulation during perinatal development. This includes a progressive decrease in rapid-eye-movement (REM) sleep time, corresponding increases in nonREM sleep and wake time, and the appearance of mature sleep regulatory processes (homeostatic and circadian). These developmental events coincide with periods of rapid brain maturation and heightened synaptic plasticity. The latter involve an initial experience-independent phase, when circuit development is guided by spontaneous activity, and later occurring critical periods, when these circuits are shaped by experience. SUMMARY: These ontogenetic changes suggest important interactions between sleep and brain development. More specifically, sleep may promote developmental programs of synaptogenesis and synaptic pruning and influence the opening and closing of critical periods of brain plasticity.

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.

The Function(s) of Sleep.

Sleep is a highly conserved phenomenon in endotherms, and therefore it must serve at least one basic function across this wide range of species. What that function is remains one of the biggest mysteries in neurobiology. By using the word neurobiology, we do not mean to exclude possible non-neural functions of sleep, but it is difficult to imagine why the brain must be taken offline if the basic function of sleep did not involve the nervous system. In this chapter we discuss several current hypotheses about sleep function. We divide these hypotheses into two categories: ones that propose higher-order cognitive functions and ones that focus on housekeeping or restorative processes. We also pose four aspects of sleep that any successful functional hypothesis has to account for: why do the properties of sleep change across the life span? Why and how is sleep homeostatically regulated? Why must the brain be taken offline to accomplish the proposed function? And, why are there two radically different stages of sleep?The higher-order cognitive function hypotheses we discuss are essential mechanisms of learning and memory and synaptic plasticity. These are not mutually exclusive hypotheses. Each focuses on specific mechanistic aspects of sleep, and higher-order cognitive processes are likely to involve components of all of these mechanisms. The restorative hypotheses are maintenance of brain energy metabolism, macromolecular biosynthesis, and removal of metabolic waste. Although these three hypotheses seem more different than those related to higher cognitive function, they may each contribute important components to a basic sleep function. Any sleep function will involve specific gene expression and macromolecular biosynthesis, and as we explain there may be important connections between brain energy metabolism and the need to remove metabolic wastes.A deeper understanding of sleep functions in endotherms will enable us to answer whether or not rest behaviors in species other than endotherms are homologous with mammalian and avian sleep. Currently comparisons across the animal kingdom depend on superficial and phenomenological features of rest states and sleep, but investigations of sleep functions would provide more insight into the evolutionary relationships between EEG-defined sleep in endotherms and rest states in ectotherms.

Primed to Sleep: The Dynamics of Synaptic Plasticity Across Brain States.

It is commonly accepted that brain plasticity occurs in wakefulness and sleep. However, how these different brain states work in concert to create long-lasting changes in brain circuitry is unclear. Considering that wakefulness and sleep are profoundly different brain states on multiple levels (e.g., cellular, molecular and network activation), it is unlikely that they operate exactly the same way. Rather it is probable that they engage different, but coordinated, mechanisms. In this article we discuss how plasticity may be divided across the sleep-wake cycle, and how synaptic changes in each brain state are linked. Our working model proposes that waking experience triggers short-lived synaptic events that are necessary for transient plastic changes and mark (i.e., 'prime') circuits and synapses for further processing in sleep. During sleep, synaptic protein synthesis at primed synapses leads to structural changes necessary for long-term information storage.

The Role of Glia in Sleep Regulation and Function.

The cellular mechanisms governing the expression, regulation, and function of sleep are not entirely understood. The traditional view is that these mechanisms are neuronal. An alternative view is that glial brain cells may play important roles in these processes. Their ubiquity in the central nervous system makes them well positioned to modulate neuronal circuits that gate sleep and wake. Their ability to respond to chemical neuronal signals suggests that they form feedback loops with neurons that may globally regulate neuronal activity. Their potential role in detoxifying the brain, regulating neuronal metabolism, and promoting synaptic plasticity raises the intriguing possibility that glia mediate important functions ascribed to sleep.