Sleep has an essential role for optimal brain function, but the cellular substrates for sleep regulation are not fully understood. Microglia, the immune cells of the brain, have gained increasingly more attention over the last two decades for their important roles in various brain functions that extend beyond their well-known immune function, including brain development, neuronal protection, and synaptic plasticity. Here we review recent advances in understanding: i) morphological and phenotypic dynamics of microglia including process motility/growth and gene/protein expression, and ii) microglia-neuron interactions including phagocytosis and contact at synapses which alters neuronal circuit activity, both under physiological state in the adult brain. We discuss how the microglia-neuron interactions particularly at synapses could influence microglia and neuronal activities across circadian cycles and sleep/wake states. We also review recent findings on how microglia respond to sleep loss. We conclude by pointing out key questions and proposing suggestions for future research to better understand the role of microglia in sleep regulation, sleep homeostasis, and the function of sleep.

Chronic sleep restriction (CSR) is common in modern society, adversely affecting cognitive performance and health. Yet how it impacts neurons regulating sleep remains unclear. Several studies using mice reported substantial losses of wake‐active orexin/hypocretin and locus coeruleus (LC) noradrenergic neurons, but not rapid eye movement sleep‐active melanin‐concentrating hormone (MCH) neurons, following CSR. Here, we used immunohistochemistry and stereology to examine orexin, MCH and LC noradrenergic neurons in a rat model of CSR that uses programmed wheel rotation (3 h on/1 h off; ‘3/1’ protocol). Adult male Wistar rats underwent one or four cycles of the 4‐day 3/1 CSR protocol, with 2‐day recovery between cycles in home cages. Time‐matched control rats were housed in locked wheels/home cages. We found no significant differences in the numbers of orexin, MCH and LC noradrenergic neurons following either one‐ or four‐cycle CSR protocol compared to respective controls. Similarly, the four‐cycle CSR protocol had no effect on the densities of orexin axon terminals in the LC, noradrenergic dendrites in the LC and noradrenergic axon terminals in the frontal cortex. Body weights, however, decreased after one cycle of CSR and then increased with diminishing slope over the next three cycles. Thus, we found no evidence for loss of orexin or LC noradrenergic neurons following one and four cycles of the 4‐day 3/1 CSR protocol in rats. Differences in CSR protocols and/or possible species differences in neuronal vulnerability to sleep loss may account for the discrepancy between the current results in rats and previous findings in mice.

Chronic sleep insufficiency is common in our society and has negative cognitive and health impacts. It can also alter sleep regulation, yet whether it affects subsequent homeostatic responses to acute sleep loss is unclear. We assessed sleep and thermoregulatory responses to acute sleep deprivation before and after a '3/1' chronic sleep restriction protocol in adult male Wistar rats. The 3/1 protocol consisted of continuous cycles of wheel rotations (3 h on/1 h off) for 4 days. Sleep latency in a 2-h multiple sleep latency test starting 26 h post-3/1 was unchanged, whereas non-rapid eye movement sleep (NREMS) and associated electroencephalogram delta power (a measure of sleep need) over a 24-h period beginning 54 h post-3/1 were reduced, compared to respective pre-3/1 baseline levels. However, in response to acute sleep deprivation (6 h by 'gentle handling') starting 78 h post-3/1, the compensatory rebounds in NREMS and rapid eye movement sleep (REMS) amounts and NREMS delta power were unaltered. Body temperature increased progressively across the 3/1 protocol and returned to baseline levels on the second day post-3/1. The acute sleep deprivation also increased body temperature, followed by a decline below baseline levels, with no difference between before and after 3/1 sleep restriction. Non-sleep-restricted control rats showed responses to acute sleep deprivation similar to those observed in the sleep-restricted animals. These results suggest that the process of sleep homeostasis is altered on the third recovery day after a 4-day 3/1 sleep restriction protocol, whereas subsequent homeostatic sleep and temperature responses to brief sleep deprivation are not affected.

Sleep and circadian rhythm disruptions commonly occur in individuals with schizophrenia. Stable tubule only polypeptide (STOP) knockout (KO) mice show behavioral impairments resembling symptoms of schizophrenia. We previously reported that STOP KO mice slept less and had more fragmented sleep and waking than wild-type littermates under a light/dark (LD) cycle. Here, we assessed the circadian phenotype of male STOP KO mice by examining wheel-running activity rhythms and EEG/EMG-defined sleep/wake states under both LD and constant darkness (DD) conditions. Wheel-running activity rhythms in KO and wild-type mice were similarly entrained in LD, and had similar free-running periods in DD. The phase delay shift in response to a light pulse given early in the active phase under DD was preserved in KO mice. KO mice had markedly lower activity levels, lower amplitude activity rhythms, less stable activity onsets, and more fragmented activity than wild-type mice in both lighting conditions. KO mice also spent more time awake and less time in rapid eye movement sleep (REMS) and non-REMS (NREMS) in both LD and DD conditions, with the decrease in NREMS concentrated in the active phase. KO mice also showed altered EEG features and higher amplitude rhythms in wake and NREMS (but not REMS) amounts in both lighting conditions, with a longer free-running period in DD, compared to wild-type mice. These results indicate that the STOP null mutation in mice altered the regulation of sleep/wake physiology and activity rhythm expression, but did not grossly disrupt circadian mechanisms.

Chronic sleep restriction (CSR) negatively impacts brain functions. Whether microglia, the brain's resident immune cells, play any role is unknown. We studied microglia responses to CSR using a rat model featuring slowly rotating wheels (3 h on/1 h off), which was previously shown to induce both homeostatic and adaptive responses in sleep and attention. Adult male rats were sleep restricted for 27 or 99 h. Control rats were housed in locked wheels. After 27 and/or 99 h of CSR, the number of cells immunoreactive for the microglia marker ionized calcium-binding adaptor molecule-1 (Iba1) and the density of Iba1 immunoreactivity were increased in 4/10 brain regions involved in sleep/wake regulation and cognition, including the prelimbic cortex, central amygdala, perifornical lateral hypothalamic area, and dorsal raphe nucleus. CSR neither induced mitosis in microglia (assessed with bromodeoxyuridine) nor impaired blood-brain barrier permeability (assessed with Evans Blue). Microglia appeared ramified in all treatment groups and, when examined quantitatively in the prelimbic cortex, their morphology was not affected by CSR. After 27 h, but not 99 h, of CSR, mRNA levels of the anti-inflammatory cytokine interleukin-10 were increased in the frontal cortex. Pro-inflammatory cytokine mRNA levels (tumor necrosis factor-α, interleukin-1β, and interleukin-6) were unchanged. Furthermore, cortical microglia were not immunoreactive for several pro- and anti-inflammatory markers tested, but were immunoreactive for the purinergic P2Y12 receptor. These results suggest that microglia respond to CSR while remaining in a physiological state and may contribute to the previously reported homeostatic and adaptive responses to CSR.

Chronically insufficient sleep is common in our society and negatively impacts cognitive performance and health. Although there is a belief that one can adapt to chronic sleep restriction (CSR), there is evidence for negative consequences, including impaired cognitive performance, and increased risks for cardiovascular and metabolic diseases. This review briefly surveys the research using rodent models to evaluate the consequences of CSR on functions and systems ranging from sleep, cognitive performance, stress neuroendocrinology, metabolism, peripheral organs, and the brain. Many of these consequences are significant, and some are long-lasting. Future research is required to understand the dynamics of these various impacts over time and relative to each other and to identify biomarkers for the mechanisms that support adaptive responses to CSR. This information will help in developing strategies to reduce the impact of chronic sleep loss.

Sleep/wake states are controlled by sleep- and wake-promoting systems, and transitions between states are thought to be regulated by their reciprocal inhibition and homeostatic sleep need. Orexin neurons are known to promote wake maintenance and stabilize the sleep/wake switch. Thus, we asked whether orexin neurons are modulated by homeostatic sleep need. Rats were sleep deprived or left undisturbed to rest for 6 h, then acute brain slices were generated for patch clamp recordings. We found that sleep deprivation increased firing and reduced spike frequency adaptation in response to excitatory drive in orexin neurons. These changes were specific to D-type orexin neurons which, unlike H-type orexin neurons, lack A-type current. In D-type orexin neurons, sleep deprivation decreased afterhyperpolarizing potential, which was associated with increased gain, measured as the slope of the input-output relationship. These effects were mimicked by inhibition of SK channels. Furthermore, sleep deprivation resulted in presynaptic inhibition of excitatory inputs to both D-type and H-type orexin neurons, which preferentially affected sparse synaptic inputs while sparing high frequency synaptic activities. Taken together, our results indicate that sleep deprivation modulates the gain control and synaptic gating in orexin neurons. These pre-and postsynaptic changes would tune orexin neurons to strong wake-promoting excitatory signals, while dampening weak synaptic inputs to allow transition to sleep in the absence of such strong signals. These mechanisms are consistent with a role of orexin neurons not only as a key state stabilizer, but also as a homeostatic wake integrator in the sleep/wake switch. This article is part of the Special Issue entitled 'Hypothalamic Control of Homeostasis'.

The mesopontine tegmentum contains cholinergic neurons that project widely to the forebrain and brainstem. These neurons are distributed in the pedunculopontine tegmental (PPT) and laterodorsal tegmental (LDT) nuclei. Cholinergic neurons in the mesopontine tegmentum have long been thought to play a critical role in the generation of rapid eye movement sleep. Tegmental cholinergic neurons have long been thought to have an important role in behavioral wakefulness and cortical arousal as part of the ascending reticular activating system. Anatomically, cholinergic neurons in the mesopontine tegmentum are distributed in a continuum encompassing the PPT and LDT. Cholinergic neurons are not the only cell type found in the PPT and LDT, as non-cholinergic neurons are also present intermixed with cholinergic neurons. In conclusion, it is clear that non-cholinergic neurons vary in their morphology, projection, and neurotransmitter content.

We recently found evidence for microglia activation in the rat brain using a model of chronic sleep restriction (CSR) that elicits adaptive responses in sleep and attention. Specifically, we found that 1 and 4 days of CSR increased the number of Iba1-immunoreactive (ir) microglia in brain regions involved in sleep/wake and cognitive functions, including the frontal cortex. Here, we examined possible mechanisms underlying the CSR-induced increase in microglia and whether this increase is accompanied by changes in inflammatory cytokine expression. First, we found no effect of 4 days of CSR on the number of Iba1-ir microglia co-labelled with bromodeoxyuridine, a cell division marker, suggesting the absence of microglia proliferation. Second, we found no extravasation of the fluorescent tracer Evans blue into brain tissue after 4 days of CSR, suggesting the absence of peripheral macrophage recruitment through the blood-brain barrier. Third, using qRT-PCR, we found that anti-inflammatory interleukin-10 expression increased in the frontal cortex after 1 but not 4 days of CSR, while pro-inflammatory tumor necrosis factor-α and interleukin-1β expression remained unchanged. These results suggest that the observed increase in Iba1-ir microglia in response to CSR is likely due to increased Iba1 expression in resident microglia and that this accompanies alteration in inflammatory processes, suggesting a role of the central immune system in adaptive responses to CSR.

Glutamate transporter 1 (GLT1) is the main astrocytic transporter that shapes glutamatergic transmission in the brain. However, whether this transporter modulates sleep–wake regulatory neurons is unknown. Using quantitative immunohistochemical analysis, we assessed perisomatic GLT1 apposition with sleep–wake neurons in the male rat following 6 h sleep deprivation (SD) or following 6 h undisturbed conditions when animals were mostly asleep (Rest). We found that SD decreased perisomatic GLT1 apposition with wake-promoting orexin neurons in the lateral hypothalamus compared with Rest. Reduced GLT1 apposition was associated with tonic presynaptic inhibition of excitatory transmission to these neurons due to the activation of Group III metabotropic glutamate receptors, an effect mimicked by a GLT1 inhibitor in the Rest condition. In contrast, SD resulted in increased GLT1 apposition with sleep-promoting melanin-concentrating hormone (MCH) neurons in the lateral hypothalamus. Functionally, this decreased the postsynaptic response of MCH neurons to high-frequency synaptic activation without changing presynaptic glutamate release. The changes in GLT1 apposition with orexin and MCH neurons were reversed after 3 h of sleep opportunity following 6 h SD. These SD effects were specific to orexin and MCH neurons, as no change in GLT1 apposition was seen in basal forebrain cholinergic or parvalbumin-positive GABA neurons. Thus, within a single hypothalamic area, GLT1 differentially regulates excitatory transmission to wake- and sleep-promoting neurons depending on sleep history. These processes may constitute novel astrocyte-mediated homeostatic mechanisms controlling sleep–wake behavior. SIGNIFICANCE STATEMENT Sleep–wake cycles are regulated by the alternate activation of sleep- and wake-promoting neurons. Whether and how astrocytes can regulate this reciprocal neuronal activity are unclear. Here we report that, within the lateral hypothalamus, where functionally opposite wake-promoting orexin neurons and sleep-promoting melanin-concentrating hormone neurons codistribute, the glutamate transporter GLT1, mainly present on astrocytes, distinctly modulates excitatory transmission in a cell-type-specific manner and according to sleep history. Specifically, GLT1 is reduced around the somata of orexin neurons while increased around melanin-concentrating hormone neurons following sleep deprivation, resulting in different forms of synaptic plasticity. Thus, astrocytes can fine-tune the excitability of functionally discrete neurons via glutamate transport, which may represent novel regulatory mechanisms for sleep.

Chronic sleep restriction (CSR) is prevalent in today's 24/7 society and has profound consequences on health and cognition. Recent evidence indicates increased levels of inflammatory molecules following CSR; however, the role of microglia, the resident immune cells of the brain, is unknown. Using a rat model of CSR (4 days) that we developed in our lab, we previously found that CSR initiated an increase in the number and density of ionized calcium binding adaptor molecule-1 (Iba1)-immunoreactive (ir) microglia in brain regions involved in sleep/wake and cognitive functions; microglia morphology was unaffected. Here, we explored the mechanisms underlying the region-specific increases in microglia numbers after CSR. First, proliferating cells were immunohistochemically identified using bromodeoxyuridine (BrdU). We found that the number of double-labeled Iba1/BrdU-ir cells was not affected by CSR. Second, blood brain barrier permeability, assessed by examining extravasation of the fluorescent tracers sodium fluorescein and Evans blue into the brain tissue, was also unaffected by CSR. These results suggest that neither microglia proliferation nor the recruitment of peripheral macrophages across the blood-brain barrier contributed to the observed increase in Iba1-ir microglia after CSR, suggesting that increased Iba1 expression may be responsible for the effect. Preliminary results with qPCR indicate decreased mRNA expression (∼60%) of the pro-inflammatory interleukin-1β and tumor necrosis factor-α and increased expression (∼25%) of the anti-inflammatory IL-10 in the hippocampus after CSR.

Age-related changes in sleep and circadian regulation occur as early as the middle years of life. Research also suggests that sleep and circadian rhythms are regulated differently between women and men. However, does sleep and circadian rhythms regulation age similarly in men and women? In this review, we present the mechanisms underlying age-related differences in sleep and the current state of knowledge on how they interact with sex. We also address how testosterone, estrogens, and progesterone fluctuations across adulthood interact with sleep and circadian regulation. Finally, we will propose research avenues to unravel the mechanisms underlying sex differences in age-related effects on sleep.

Using a rat model of chronic sleep restriction (CSR) featuring periodic sleep deprivation with slowly rotating wheels (3 h on/1 h off), we previously observed that 99 h of this protocol induced both homeostatic and allostatic (adaptive) changes in physiological and behavioural measures. Notably, the initial changes in sleep intensity and attention performance gradually adapted during CSR despite accumulating sleep loss. To identify brain regions involved in these responses, we used FosB/ΔFosB immunohistochemistry as a marker of chronic neuronal activation. Adult male rats were housed in motorized activity wheels and underwent the 3/1 CSR protocol for 99 h, or 99 h followed by 6 or 12 days of recovery. Control rats were housed in home cages, locked activity wheels, or unlocked activity wheels that the animals could turn freely. Immunohistochemistry was conducted using an antibody that recognized both FosB and ΔFosB, and 24 brain regions involved in sleep/wake, autonomic, and limbic functions were examined. The number of darkly-stained FosB/ΔFosB-immunoreactive cells was increased immediately following 99 h of CSR in 8/24 brain regions, including the medial preoptic and perifornical lateral hypothalamic areas, dorsomedial and paraventricular hypothalamic nuclei, and paraventricular thalamic nucleus. FosB/ΔFosB labeling was at control levels in all 8 brain areas following 6 or 12 recovery days, suggesting that most of the immunoreactivity immediately after CSR reflected FosB, the more transient marker of chronic neuronal activation. This region-specific induction of FosB/ΔFosB following CSR may be involved in the mechanisms underlying the allostatic changes in behavioural and physiological responses to CSR.

The basal forebrain (BF) plays an important role in regulating cortical activity and sleep/wake states. Both cholinergic and non-cholinergic neurons of the BF project to the cerebral cortex and hippocampus, whereas the hypothalamus and brainstem nuclei are mostly innervated by non-cholinergic BF neurons. Neurons in the BF show various discharge profiles in relation to cortical activity and behavioral states and are differentially modulated by neurotransmitters of other sleep/wake regulatory neurons. Recent technical advances have made it possible to correlate discharge profiles of single BF neurons during sleep/wake states with their neurochemical phenotypes, and to make selective lesions of certain cell types. The goal of this review is to summarize the current knowledge of the anatomy and sleep/wake regulatory functions of cholinergic and non-cholinergic BF neurons. We will first review the anatomical heterogeneity of BF neurons, and then discuss recent evidence for the firing patterns of BF cholinergic and non-cholinergic neurons during natural sleep–wake patterns, and finally, discuss their roles in sleep homeostasis. It is proposed that through different neurotransmitters, projections, and state-regulated activity, the cholinergic and non-cholinergic BF neurons collectively and differently regulate cortical activity and sleep-wake states.

STUDY OBJECTIVESChronic sleep restriction (CSR) impairs sustained attention in humans, as commonly assessed with the psychomotor vigilance task (PVT). To further investigate the mechanisms underlying performance deficits during CSR, we examined the effect of CSR on performance on a rat version of PVT (rPVT).DESIGNAdult male rats were trained on a rPVT that required them to press a bar when they detected irregularly presented, brief light stimuli, and were then tested during CSR. CSR consisted of 100 or 148 h of continuous cycles of 3-h sleep deprivation (using slowly rotating wheels) alternating with a 1-h sleep opportunity (3/1 protocol).MEASUREMENTS AND RESULTSAfter 28 h of CSR, the latency of correct responses and the percentages of lapses and omissions increased, whereas the percentage of correct responses decreased. Over 52-148 h of CSR, all performance measures showed partial or nearly complete recovery, and were at baseline levels on the first or second day after CSR. There were large interindividual differences in the magnitude of performance impairment during CSR, suggesting differential vulnerability to the effects of sleep loss. Wheel-running controls showed no changes in performance.CONCLUSIONSA 28-h period of the 3/1 chronic sleep restriction (CSR) protocol disrupted performance on a sustained attention task in rats, as sleep deprivation does in humans. Performance improved after longer periods of CSR, suggesting allostatic adaptation, contrary to some reports of progressive deterioration in psychomotor vigilance task performance during CSR in humans. However, as observed in humans, there were individual differences among rats in the vulnerability of their attention performance to CSR.

Chronic sleep restriction (CSR) has various negative consequences on cognitive performance and health. Using a rat model of CSR that uses alternating cycles of 3h of sleep deprivation (using slowly rotating activity wheels) and 1h of sleep opportunity continuously for 4 days ('3/1' protocol), we previously observed not only homeostatic but also allostatic (adaptive) sleep responses to CSR. In particular, non-rapid eye movement sleep (NREMS) electroencephalogram (EEG) delta power, an index of sleep intensity, increased initially and then declined gradually during CSR, with no rebound during a 2-day recovery period. To study underlying mechanisms of these allostatic responses, we examined the levels of brain-derived neurotrophic factor (BDNF), which is known to regulate NREMS EEG delta activity, during the same CSR protocol. Mature BDNF protein levels were measured in the frontal cortex and basal forebrain, two brain regions involved in sleep and EEG regulation, and the hippocampus, using Western blot analysis. Adult male Wistar rats were housed in motorized activity wheels, and underwent the 3/1 CSR protocol for 27 h, for 99 h, or for 99 h followed by 24h of recovery. Additional rats were housed in either locked wheels (locked wheel controls [LWCs]) or unlocked wheels that rats could rotate freely (wheel-running controls [WRCs]). BDNF levels did not differ between WRC and LWC groups. BDNF levels were increased, compared to the control levels, in all three brain regions after 27 h, and were increased less strongly after 99 h, of CSR. After 24h of recovery, BDNF levels were at the control levels. This time course of BDNF levels parallels the previously reported changes in NREMS delta power during the same CSR protocol. Changes in BDNF protein levels in the cortex and basal forebrain may be part of the molecular mechanisms underlying allostatic sleep responses to CSR.

Sleep disturbances are commonly associated with menopause. Hormone replacement therapy is often used to treat various menopausal symptoms, but its efficacy for improving sleep is a matter of debate. We addressed this question by using a rodent model of ovarian hormone loss and replacement in midlife. Middle-aged female rats were ovariectomized and implanted with capsules containing estradiol with or without progesterone, or oil. After two weeks, sleep/wake states were recorded polygraphically during a 24-h baseline period, followed by 6h of sleep deprivation in the second half of the light phase, and a 24-h recovery period. During the baseline dark phase, hormone treatments increased wakefulness, and decreased non-rapid eye movement sleep (NREMS) by shortening NREMS episodes; however, NREMS EEG delta power or energy (cumulative power) was unaffected by combined hormones. Following sleep deprivation, all the groups showed NREMS and rapid eye movement sleep (REMS) rebounds, with similar relative increases from respective baseline levels. The increases in NREMS EEG delta power/energy during recovery were enhanced by combined hormones. These results from middle-aged ovariectomized rats indicate that replacement with estrogen with or without progesterone reduces baseline NREMS without affecting sleep intensity, particularly during the dark (active) phase, whereas following sleep deprivation the same hormone treatments do not affect the ability to increase NREMS or REMS, but treatment with both hormones, in particular, enhances the intensity of recovery sleep. These results support the usefulness of ovariectomized middle-aged rats as a model system to study the biological effects of hormone replacement on sleep regulation.

People often sleep deprive themselves voluntarily for social and lifestyle reasons. Animals also appear to stay awake longer as a result of their natural curiosity to explore novel environments and interact socially with conspecifics. Although multiple arousal systems in the brain are known to act jointly to promote and maintain wakefulness, it remains unclear whether these systems are similarly engaged during voluntary vs. forced wakefulness. Using c-Fos immunohistochemistry, we compared neuronal responses in rats deprived of sleep for 2 h by gentle sensory stimulation, exploration under social isolation, or exploration with social interaction, and rats under undisturbed control conditions. In many arousal, limbic, and autonomic nuclei examined (e.g., anterior cingulate cortex and locus coeruleus), the two sleep deprivation procedures involving exploration were similarly effective, and both were more effective than sleep deprivation with sensory stimulation, in increasing the number of c-Fos immunoreactive neurons. However, some nuclei (e.g., paraventricular hypothalamic nucleus and select amygdala nuclei) were more responsive to exploration with social interaction, while others (e.g., histaminergic tuberomammillary nucleus) responded more strongly to exploration in social isolation. In the rostral basal forebrain, cholinergic and GABAergic neurons responded preferentially to exploration with social interaction, whereas resident neurons in general responded most strongly to exploration without social interaction. These results indicate that voluntary exploration with/without social interaction is more effective than forced sleep deprivation with gentle sensory stimulation for inducing c-Fos in arousal and limbic/autonomic brain regions, and suggest that these nuclei participate in different aspects of arousal during sustained voluntary wakefulness.