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.

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.

Sleep and circadian rhythms: pillars of health-a Keystone Symposia report.

The human circadian system consists of the master clock in the suprachiasmatic nuclei of the hypothalamus as well as in peripheral molecular clocks located in organs throughout the body. This system plays a major role in the temporal organization of biological and physiological processes, such as body temperature, blood pressure, hormone secretion, gene expression, and immune functions, which all manifest consistent diurnal patterns. Many facets of modern life, such as work schedules, travel, and social activities, can lead to sleep/wake and eating schedules that are misaligned relative to the biological clock. This misalignment can disrupt and impair physiological and psychological parameters that may ultimately put people at higher risk for chronic diseases like cancer, cardiovascular disease, and other metabolic disorders. Understanding the mechanisms that regulate sleep circadian rhythms may ultimately lead to insights on behavioral interventions that can lower the risk of these diseases. On February 25, 2021, experts in sleep, circadian rhythms, and chronobiology met virtually for the Keystone eSymposium "Sleep & Circadian Rhythms: Pillars of Health" to discuss the latest research for understanding the bidirectional relationships between sleep, circadian rhythms, and health and disease.

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.

Sleep and immunomodulatory responses to systemic lipopolysaccharide in mice selectively expressing interleukin-1 receptor 1 on neurons or astrocytes.

Sleep-wake behavior is altered in response to immune challenge. Although the precise mechanisms that govern sickness-induced changes in sleep are not fully understood, interleukin-1ß (IL-1) is one mediator of these responses. To better understand mechanisms underlying sleep and inflammatory responses to immune challenge, we used two transgenic mouse strains that express IL-1 receptor 1 (IL1R1) only in the central nervous system and selectively on neurons or astrocytes. Electroencephalographic recordings from transgenic and wild-type mice reveal that systemic challenge with lipopolysaccharide (LPS) fragments sleep, suppresses rapid eye movement sleep (REMS), increases non-REMS (NREMS), diminishes NREM delta power, and induces fever in all genotypes. However, the magnitude of REMS suppression is greater in mice expressing IL1R1 on astrocytes compared with mice in which IL1R1 is selectively expressed on neurons. Furthermore, there is a delayed increase in NREM delta power when IL1R1 is expressed on astrocytes. LPS-induced sleep fragmentation is reduced in mice expressing IL1R1 on neurons. Although LPS increases IL-1 and IL-6 in brain of all genotypes, this response is attenuated when IL1R1 is expressed selectively on neurons or on astrocytes. Collectively, these data suggest that in these transgenic mice under the conditions of this study it is neuronal IL1R1 that plays a greater role in LPS-induced suppression of REMS and NREM delta power, whereas astroglial IL1R1 is more important for sleep fragmentation after this immune challenge. Thus, aspects of central responses to LPS are modulated by IL1R1 in a cell type-specific manner.

Selective contributions of neuronal and astroglial interleukin-1 receptor 1 to the regulation of sleep.

Interactions between sleep and immune function are bidirectional. Although the mechanisms that govern these interactions are not fully elucidated, the pro-inflammatory cytokine, interleukin-1ß (IL-1), is a known regulator of sleep and mediator of immune responses. To further clarify the underlying substrates of sleep and immune interactions, we engineered two transgenic mouse lines that express interleukin-1 receptor 1 (IL1R1) only in the central nervous system (CNS) and selectively on neurons (NSE-IL1R1) or astrocytes (GFAP-IL1R1). During spontaneous sleep, compared to wild type (WT) animals, NSE-IL1R1 and GFAP-IL1R1 mice have more rapid eye movement sleep (REMS) that is characterized by reduced theta power in the electroencephalogram (EEG) spectra. The non-REM sleep (NREMS) EEG of each of the IL1R1 transgenic mouse strains also is characterized by enhanced power in the delta frequency band. In response to 6h of sleep deprivation, sleep of both IL1R1 transgenic mouse strains is more consolidated than that of WT animals. Additionally, the NREMS EEG of NSE-IL1R1 mice contains less delta power after sleep deprivation, suggesting astroglial IL1R1 activity may modulate sleep homeostasis. Intracerebroventricular injection of IL-1 fails to alter sleep or brain temperature of NSE-IL1R1 or GFAP-IL1R1 mice. These data suggest that selective IL1R1 expression on neurons or on astrocytes is not sufficient for centrally-administered IL-1 to induce sleep or fever. Lack of sleep and febrile responses to IL-1 in these IL1R1 transgenic mouse strains may be due to their inability to produce IL-6 in brain. Overall, these studies demonstrate, through the use of novel transgenic mice, that IL1R1 on neurons and astrocytes differentially mediates aspects of sleep under physiological conditions and in response to central IL-1 administration.

Sleep and immune function: glial contributions and consequences of aging.

The reciprocal interactions between sleep and immune function are well-studied. Insufficient sleep induces innate immune responses as evidenced by increased expression of pro-inflammatory mediators in the brain and periphery. Conversely, immune challenges upregulate immunomodulator expression, which alters central nervous system-mediated processes and behaviors, including sleep. Recent studies indicate that glial cells, namely microglia and astrocytes, are active contributors to sleep and immune system interactions. Evidence suggests glial regulation of these interactions is mediated, in part, by adenosine and adenosine 5'-triphosphate actions at purinergic type 1 and type 2 receptors. Furthermore, microglia and astrocytes may modulate declines in sleep-wake behavior and immunity observed in aging.

Sepsis-induced alterations in sleep of rats.

Sepsis is a systemic immune response to infection that may result in multiple organ failure and death. Polymicrobial infections remain a serious clinical problem, and in the hospital, sepsis is the number-one noncardiac killer. Although the central nervous system may be one of the first systems affected, relatively little effort has been made to determine the impact of sepsis on the brain. In this study, we used the cecal ligation and puncture (CLP) model to determine the extent to which sepsis alters sleep, the EEG, and brain temperature (Tbr) of rats. Sepsis increases the amount of time rats spend in non-rapid eye movement sleep (NREMS) during the dark period, but not during the light period. Rapid eye movements sleep (REMS) of septic rats is suppressed for about 24 h following CLP surgery, after which REMS increases during dark periods for at least three nights. The EEG is dramatically altered shortly after sepsis induction, as evidenced by reductions in slow-frequency components. Furthermore, sleep is fragmented, indicating that the quality of sleep is diminished. Effects on sleep, the EEG, and Tbr persist for at least 84 h after sepsis induction, the duration of our recording period. Immunohistochemical assays focused on brain stem mechanisms responsible for alterations in REMS, as little information is available concerning infection-induced suppression of this sleep stage. Our immunohistochemical data suggest that REMS suppression after sepsis onset may be mediated, in part, by the brain stem GABAergic system. This study demonstrates for the first time that sleep and EEG patterns are altered during CLP-induced sepsis. These data suggest that the EEG may serve as a biomarker for sepsis onset. These data also contribute to our knowledge of potential mechanisms, whereby infections alter sleep and other central nervous system functions.