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IntroductionAbnormalities in electroencephalographic (EEG) biomarkers occur in patients with schizophrenia and those clinically at high risk for transition to psychosis and are associated with cognitive impairment. While the pathophysiology of schizophrenia remains poorly understood, converging evidence suggests N-methyl-D-aspartate receptor (NMDAR) hypofunction plays a central role and likely contributes to biomarker impairments. Thus, the characterization of such biomarkers is of significant interest for both the early diagnosis of schizophrenia and the development of novel treatments.MethodsWe utilized an established model of chronic NMDAR hypofunction, serine racemase knockout (SRKO) mice. In vivo EEG recording and behavioral analyses were performed on adult male and female SRKO mice and wild-type littermates to determine the impact of chronic NMDAR hypofunction on a battery of translationally-relevant electrophysiological biomarkers.ResultsSRKO mice displayed impairments in investigation-elicited gamma power that corresponded with reduced short-term social recognition. This impairment was associated with enhanced background (pre-investigation) broadband gamma activity that only appeared during social task performance. Additionally, SRKO mice exhibited sensory gating impairments, in both gamma power and event-related potential amplitude. However, other biomarkers such as the auditory steady-state response, sleep spindles, and state-specific power spectral density were generally neurotypical.ConclusionsSRKO mice provide a useful model to understand how chronic NMDAR hypofunction contributes to deficits in a subset of translationally-relevant EEG biomarkers that are altered in schizophrenia. Importantly, our gamma band findings support the hypothesis that an aberrant signal-to-noise ratio impairing cognition occurs with NMDAR hypofunction, which may be tied to impaired taskdependent alteration in functional connectivity.

Slow-wave activity (SWA) in the electroencephalogram during slow-wave sleep (SWS) varies as a function of sleep-wake history. A putative sleep-active population of neuronal nitric oxide synthase (nNOS)-containing interneurons in the cerebral cortex, defined as such by the expression of Fos in animals euthanized after protracted deep sleep, may be a local regulator of SWA. We investigated whether electrophysiological responses to activation of these cells are consistent with their role of a local regulator of SWA. Using a Cre/loxP strategy, we targeted the population of nNOS interneurons to express the light-activated cation channel Channelrhodopsin2 and the histological marker tdTomato in mice. We then performed histochemical and optogenetic studies in these transgenic mice. Our studies provided histochemical evidence of transgene expression and electrophysiological evidence that the cerebral cortex was responsive to optogenetic manipulation of these cells in both anesthetized and behaving mice. Optogenetic stimulation of the cerebral cortex of animals expressing Channelrhodopsin2 in nNOS interneurons triggered an acute positive deflection of the local field potential that was followed by protracted oscillatory events only during quiet wake and slow wave sleep. The response during wake was maximal when the electroencephalogram (EEG) was in a negative polarization state and abolished when the EEG was in a positive polarization state. Since the polarization state of the EEG is a manifestation of slow-wave oscillations in the activity of underlying pyramidal neurons between the depolarized (LFP negative) and hyperpolarized (LFP positive) states, these data indicate that sleep-active cortical neurons expressing nNOS function in sleep slow-wave physiology.

Introduction Most traumatic brain injuries (TBI) are mild to moderate and can cause persistent dysregulated sleep, although the mechanisms are not well understood. Interleukin-1 beta (IL-1β) is a pro-inflammatory molecule that is activated in the cortex after waking activity and pathogenic challenge and alters non-rapid eye movement (NREM) sleep and electroencephalogram (EEG) delta power. Nucleotide-binding domain leucine rich family pyrin containing 3 (NLRP3) inflammasomes sense changes in their local environment to stimulate caspase-1 to activate IL-1β into its mature form. We previously found that NLRP3 inflammasomes are increased in the cortex after acute sleep loss and contribute to increased NREM sleep and EEG delta power after sleep loss and toxin challenge. We aimed to determine if NLRP3 inflammasomes contribute to the persistent dysregulated sleep caused by TBI. Methods Using 2-3-month-old mice lacking NLRP3 and C57BL/6J wild-type control mice, we assessed sleep states and sleep state episode durations and frequencies prior to TBI, and 24 h, 2-weeks, and 2 months after mild or moderate TBI using polysomnography. TBI occurred in the frontal cortex from a controlled cortical impact device. Additional mice received identical treatments serving as a TBI procedural control but received a craniectomy without the TBI. Results Similar sleep findings were observed between the craniectomy control group and baseline measures. However, when compared to baseline values, mice lacking NLRP3 had attenuations in the significant increased amounts of NREM sleep and EEG delta power occurring 24 h after TBI and the significant reductions in NREM sleep and EEG delta power seen 2 months after TBI that were observed in wild-type mice. These effects were similar in moderate and mild TBI groups. Mice lacking NLRP3 were not found to exhibit the fragmented sleep after mild or moderate TBI that was found to persist from 24 h to 2 months post-TBI in the wild-type mice. These effects were evident by significant increased frequencies of waking episodes induced by the TBIs. Conclusion Our findings suggest that NLRP3 inflammasomes contribute to dysregulated sleep occurring acutely or more persistently after TBI. Support (if any) Career Development Award IBX002823 (MZ) and Merit Review Award I01RX001144 (GK) from the United States Department of Veterans Affairs

Evidence indicates that the neuropeptide substance P (SP) can act through neurokinin receptors to alter sleep and/or non-rapid eye movement (NREM) sleep slow-wave activity. Consequently, drugs acting on SP receptors could potentially be used as a novel treatment for sleep-related disorders. In the present study, we used SP conjugated with cholera toxin A subunit (SP-CTA), which enhances its duration of activity on SP receptor-expressing cells, to determine the effects of selectively activating SP receptor-expressing brain cells on sleep regulation in mice. Herein, we found that intracerebroventricular administration of SP-CTA enhanced amounts of NREM sleep which was highly fragmented. This result suggests that the activation of SP receptor-expressing cells in the brain can produce not only arousal effects as shown in previous studies but also sleep-inducing effects.

Introduction Increased cerebral blood flow (CBF) is positively associated with non-rapid eye movement (NREM) sleep electroencephalogram (EEG) delta power, also known as slow-wave activity (SWA). The pro-inflammatory somnogenic cytokine interleukin-1 beta (IL-1β) can induce vasodilation and increase CBF. Nucleotide leucine-rich protein complex-3 (NLRP3) inflammasomes, which activate IL-1β, are increased in the cortex after sleep deprivation and increase SWA. We aimed to determine the relationship of NLRP3 inflammasomes on sleep loss-induced alterations in vasohemodynamics and SWA. Methods NLRP3 knock-out (KO) and wild-type (WT) mice underwent ad libitum sleep, 6 hours of sleep deprivation, or were given 10 ng of IL-1β or the vehicle intracerebroventricularly. SWA and CBF, blood velocity, and blood volume were determined concurrently during sleep/wake states using polysomnography and laser doppler flowmetry. Regional brain changes in CBF were determined using transponders, spectrophotometry, and fluorescent microspheres. Results SWA and CBF were significantly increased during the first 6 hours after sleep deprivation in WT but not NLRP3 KO mice. SWA and CBF significantly increased in the first 6 hours after IL-1β in both NLRP3 KO and WT mice. Additionally, alterations in cerebral blood velocity and volume demonstrated state specific changes that varied significantly during the transitions between states. SWA and CBF were significantly positively correlated during both ad libitum sleep and sleep after sleep deprivation in WT mice, although this relationship was not observed in NLRP3 KO mice. We also found significant phase-amplitude frequency coupling between SWA and CBF. Cortical changes CBF were significantly enhanced after sleep deprivation and IL-1β administration in WT mice, although were attenuated in the hypothalamus. NLRP3 KO mice showed these same regional effects in CBF after IL-1β but not sleep deprivation. Conclusion Our findings indicate that NLRP3 inflammasomes are involved in neurovascular coupling involving SWA. Support Department of Veterans Affairs IBX002823A (MRZ)

Slow-wave activity (SWA) is an oscillatory neocortical activity occurring in the electroencephalogram delta (δ) frequency range (~0.5–4 Hz) during nonrapid eye movement sleep. SWA is a reliable indicator of sleep homeostasis after acute sleep loss and is involved in memory processes. Evidence suggests that cortical neuronal nitric oxide synthase (nNOS) expressing neurons that coexpress somatostatin (SST) play a key role in regulating SWA. However, previous studies lacked selectivity in targeting specific types of neurons that coexpress nNOS—cells which are activated in the cortex after sleep loss. We produced a mouse model that knocks out nNOS expression in neurons that coexpress SST throughout the cortex. Mice lacking nNOS expression in SST positive neurons exhibited significant impairments in both homeostatic low-δ frequency range SWA production and a recognition memory task that relies on cortical input. These results highlight that SST+/nNOS+ neurons are involved in the SWA homeostatic response and cortex-dependent recognition memory.

We previously found that NLRP3 inflammasome activity is a critical mechanism regulating sleep and slow-waves. Microglia and astrocytes play a major role in sleep regulation and altering slow-waves. We investigated the contribution of NLRP3 inflammasome activation in microglia and astrocytes on sleep and slow-waves after sleep loss. For this study, C57BL6/J mice were sleep deprived or allowed to sleep ad libitum. NLRP3 inflammasome-activity related molecules (inflammatory cytokines - interleukin (IL)-1 beta (IL-1β) and IL-18) from the cortex were determined in microglia and astrocytes using flow-cytometry. Additionally, NLRP3 floxed mice were crossed with CX3CR1- or GFAP-CRE mice to target microglia or astrocytes, and sleep and slow-waves were assessed by polysomnography. In CD11b+ cells (i.e., microglia), a significant enhancement (p

Sleep is a complex physiological process that is regulated globally, regionally, and locally by both cellular and molecular mechanisms. It occurs to some extent in all animals, although sleep expression in lower animals may be co-extensive with rest. Sleep regulation plays an intrinsic part in many behavioral and physiological functions. Currently, all researchers agree there is no single physiological role sleep serves. Nevertheless, it is quite evident that sleep is essential for many vital functions including development, energy conservation, brain waste clearance, modulation of immune responses, cognition, performance, vigilance, disease, and psychological state. This review details the physiological processes involved in sleep regulation and the possible functions that sleep may serve. This description of the brain circuitry, cell types, and molecules involved in sleep regulation is intended to further the reader’s understanding of the functions of sleep.

Non-rapid eye movement (NREM) sleep electroencephalographic (EEG) delta power (~0.5-4 Hz), also known as slow wave activity (SWA), is typically enhanced after acute sleep deprivation (SD) but not after chronic sleep restriction (CSR). Recently, sleep-active cortical neurons expressing neuronal nitric oxide synthase (nNOS) were identified and associated with enhanced SWA after short acute bouts of SD (i.e., 6h). However, the relationship between cortical nNOS neuronal activity and SWA during CSR is unknown. We compared the activity of cortical neurons expressing nNOS (via c-Fos and nNOS immuno-reactivity, respectively) and sleep in rats in three conditions: (1) after 18-h of acute SD; (2) after five consecutive days of sleep restriction (SR) (18-h SD per day with 6h ad libitum sleep opportunity per day); (3) and time-of-day matched ad libitum sleep controls. Cortical nNOS neuronal activity was enhanced during sleep after both 18-h SD and 5 days of SR treatments compared to control treatments. SWA and NREM sleep delta energy (the product of NREM sleep duration and SWA) were positively correlated with enhanced cortical nNOS neuronal activity after 18-h SD but not 5days of SR. That neurons expressing nNOS were active after longer amounts of acute SD (18h vs. 6h reported in the literature) and were correlated with SWA further suggest that these cells might regulate SWA. However, since these neurons were active after CSR when SWA was not enhanced, these findings suggest that mechanisms downstream of their activation are altered during CSR.

Sleep deprivation can have deleterious effects on cognitive function and mental health. Moderate exercise training has myriad beneficial effects on cognition and mental health. However, physiological and behavioral effects of chronic moderate sleep restriction and its interaction with common activities, such as moderate exercise training, have received little investigation. The aims of this study were to examine the effects of chronic moderate sleep restriction and moderate exercise training on anxiety-related behavior, spatial memory, and neurobiological correlates in mice. Male mice were randomized to one of four 11-week treatments in a 2 [sleep restriction (~4 h loss/day) vs. ad libitum sleep] × 2 [exercise (1 h/day/6 d/wk) vs. sedentary activity] experimental design. Anxiety-related behavior was assessed with the elevated-plus maze, and spatial learning and memory were assessed with the Morris water maze. Chronic moderate sleep restriction did not alter anxiety-related behavior, but exercise training significantly attenuated anxiety-related behavior. Spatial learning and recall, hippocampal cell activity (i.e., number of c-Fos positive cells), and brain derived neurotrophic factor were significantly lower after chronic moderate sleep restriction, but higher after exercise training. Further, the benefit of exercise training for some memory variables was evident under normal sleep, but not chronic moderate sleep restriction conditions. These data indicate clear detrimental effects of chronic moderate sleep restriction on spatial memory and that the benefits of exercise training were impaired after chronic moderate sleep restriction.

Study objective Systemic tumor necrosis factor-α (TNF-α) is linked to sleep and sleep altering pathologies in humans. Evidence from animals indicates that systemic and brain TNF-α have a role in regulating sleep. In animals, TNF-α or lipopolysaccharide (LPS) enhance brain pro-inflammatory cytokine expression and sleep after central or peripheral administration. Vagotomy blocks enhanced sleep induced by systemic TNF-α and LPS in rats, suggesting that vagal afferent stimulation by TNF-α enhances pro-inflammatory cytokines in sleep-related brain areas. However, the effects of systemic TNF-α on brain cytokine expression and mouse sleep remain unknown. Design We investigated the role of vagal afferents on brain cytokines and sleep after systemically applied TNF-α or LPS in mice. Measurements and results Spontaneous sleep was similar in vagotomized and sham-operated controls. Vagotomy attenuated TNF-α- and LPS-enhanced non-rapid eye movement sleep (NREMS); these effects were more evident after lower doses of these substances. Vagotomy did not affect rapid eye movement sleep responses to these substances. NREMS electroencephalogram delta power (0.5-4 Hz range) was suppressed after peripheral TNF-α or LPS injections, although vagotomy did not affect these responses. Compared to sham-operated controls, vagotomy did not affect liver cytokines. However, vagotomy attenuated interleukin-1 beta (IL-1β) and TNF-α mRNA brain levels after TNF-α, but not after LPS, compared to the sham-operated controls. Conclusions We conclude that vagal afferents mediate peripheral TNF-α-induced brain TNF-α and IL-1β mRNA expressions to affect sleep. We also conclude that vagal afferents alter sleep induced by peripheral pro-inflammatory stimuli in mice similar to those occurring in other species.

Multiple interactions between the immune system and sleep are known, including the effects of microbial challenge on sleep or the effects of sleep loss on facets of the immune response. Cytokines regulate, in part, sleep and immune responses. Here we examine the role of an anti-inflammatory cytokine, interleukin-37 (IL-37) on sleep in a mouse strain that expresses human IL-37b (IL37tg mice). Constitutive expression of the IL-37 gene in the brains of these mice under resting conditions is low; however, upon an inflammatory stimulus, expression increases dramatically. We measured sleep in three conditions; (a) under baseline conditions and after 6 h of sleep loss, (b) after bolus intraperitoneal administration of lipopolysaccharide (LPS) or IL-1β and (c) after intranasal influenza virus challenge. Under baseline conditions, the IL37tg mice had 7% more spontaneous non-rapid eye movement sleep (NREMS) during the light period than wild-type (WT) mice. After sleep deprivation both WT mice and IL37tg mice slept an extra 21% and 12%, respectively, during the first 6 h of recovery. NREMS responses after sleep deprivation did not significantly differ between WT mice and IL37tg mice. However, in response to either IL-1β or LPS, the increases in time spent in NREMS were about four-fold greater in the WT mice than in the IL37tg mice. In contrast, in response to a low dose of mouse-adapted H1N1 influenza virus, sleep responses developed slowly over the 6 day recording period. By day 6, NREMS increased by 10% and REMS increased by 18% in the IL37tg mice compared to the WT mice. Further, by day 4 IL37tg mice lost less weight, remained more active, and retained their body temperatures closer to baseline values than WT mice. We conclude that conditions that promote IL-37 expression attenuate morbidity to severe inflammatory challenge. Keywords: Sleep deprivation, Interleukin-1β, IL-37, Influenza virus H1N1, Lipopolysaccharide, IL-1F7, Body weight

Background: Within hours of intranasal challenge, mouse-adapted H1N1 A/Puerto Rico/8/34 (PR8) influenza genomic RNA is found in the olfactory bulb (OB) and OB pro-inflammatory cytokines are up-regulated. Severing the olfactory tract delays the acute-phase response (APR) and the APR is attenuated by immunization. Objectives: To determine if immunization affects OB localization of influenza or the molecular brain mechanisms regulating APR. Methods: Male mice were immunized with PR8 influenza, then OB viral RNA, APR, and influenza-related cytokine responses were determined after homologous viral challenge. Results: Immunization did not prevent influenza OB viral invasion within 24 h of viral challenge. However, it greatly attenuated OB viral RNA 6 days after viral challenge and the APR including hypothermia and body weight loss responses. Within the OB, 24 h after influenza challenge, prior immunization blocked virus-induced up-regulation of toll-like receptor 7 and interferon (IFN) γ mRNAs. At this time, hypothalamic (HT) growth hormone-releasing hormone receptor and tumor necrosis factor-α mRNAs were greatly enhanced in immunized but not in positive control mice. By 6 days after viral challenge, OB and HT mRNAs returned towards baseline values. In the lung, mRNA up-regulation was greater than that in the brain and maximized 6 days after challenge. Lung IFNγ mRNA decreased at 24 h but increased 6 days after challenge in the positive compared to negative controls. Immunization prevented the up-regulation of most of the flu-related mRNAs measured in lungs. Conclusion: Collectively, these data suggest a role for OB and HT involvement in immunization protection against influenza infection.

Both sleep loss and pathogens can enhance brain inflammation, sleep, and sleep intensity as indicated by electroencephalogram delta (δ) power. The pro-inflammatory cytokine interleukin-1 beta (IL-1β) is increased in the cortex after sleep deprivation (SD) and in response to the Gram-negative bacterial cell-wall component lipopolysaccharide (LPS), although the exact mechanisms governing these effects are unknown. The nucleotide-binding domain and leucine-rich repeat protein-3 (NLRP3) inflammasome protein complex forms in response to changes in the local environment and, in turn, activates caspase-1 to convert IL-1β into its active form. SD enhances the cortical expression of the somnogenic cytokine IL-1β, although the underlying mechanism is, as yet, unidentified. Using NLRP3-gene knockout (KO) mice, we provide evidence that NLRP3 inflammasome activation is a crucial mechanism for the downstream pathway leading to increased IL-1β-enhanced sleep. NLRP3 KO mice exhibited reduced non-rapid eye movement (NREM) sleep during the light period. We also found that sleep amount and intensity (δ activity) were drastically attenuated in NLRP3 KO mice following SD (homeostatic sleep response), as well as after LPS administration, although they were enhanced by central administration of IL-1β. NLRP3, ASC, and IL1β mRNA, IL-1β protein, and caspase-1 activity were greater in the somatosensory cortex at the end of the wake-active period when sleep propensity was high and after SD in wild-type but not NLRP3 KO mice. Thus, our novel and converging findings suggest that the activation of the NLRP3 inflammasome can modulate sleep induced by both increased wakefulness and a bacterial component in the brain.

Adenosine and extracellular adenosine triphosphate (ATP) have multiple physiological central nervous system actions including regulation of cerebral blood flow, inflammation and sleep. However, their exact sleep regulatory mechanisms remain unknown. Extracellular ATP and adenosine diphosphate are converted to adenosine monophosphate (AMP) by the enzyme ectonucleoside triphosphate diphosphohydrolase 1, also known as CD39, and extracellular AMP is in turn converted to adenosine by the 5'-ectonuleotidase enzyme CD73. We investigated the role of CD73 in sleep regulation. Duration of spontaneous non-rapid eye movement sleep (NREMS) was greater in CD73-knockout (KO) mice than in C57BL/6 controls whether determined in our laboratory or by others. After sleep deprivation (SD), NREMS was enhanced in controls but not CD73-KO mice. Interleukin-1 beta (IL1β) enhanced NREMS in both strains, indicating that the CD73-KO mice were capable of sleep responses. Electroencephalographic power spectra during NREMS in the 1.0-2.5 Hz frequency range was significantly enhanced after SD in both CD73-KO and WT mice; the increases were significantly greater in the WT mice than in the CD73-KO mice. Rapid eye movement sleep did not differ between strains in any of the experimental conditions. With the exception of CD73 mRNA, the effects of SD on various adenosine-related mRNAs were small and similar in the two strains. These data suggest that sleep is regulated, in part, by extracellular adenosine derived from the actions of CD73.

Interleukin (IL)-1β is involved in several brain functions, including sleep regulation. It promotes non-rapid eye movement (NREM) sleep via the IL-1 type I receptor. IL-1β/IL-1 receptor complex signaling requires adaptor proteins, e.g., the IL-1 receptor brain-specific accessory protein (AcPb). We have cloned and characterized rat AcPb, which shares substantial homologies with mouse AcPb and, compared with AcP, is preferentially expressed in the brain. Furthermore, rat somatosensory cortex AcPb mRNA varied across the day with sleep propensity, increased after sleep deprivation, and was induced by somnogenic doses of IL-1β. Duration of NREM sleep was slightly shorter and duration of REM sleep was slightly longer in AcPb knockout than wild-type mice. In response to lipopolysaccharide, which is used to induce IL-1β, sleep responses were exaggerated in AcPb knockout mice, suggesting that, in normal mice, inflammation-mediated sleep responses are attenuated by AcPb. We conclude that AcPb has a role in sleep responses to inflammatory stimuli and, possibly, in physiological sleep regulation.

Background: Available treatments for anxiety have limitations and/or side effects. The aim of this study was to examine the influence of bright light exposure as a treatment in high-anxious young adults. Methods: In an acute exposure study, participants (n = 33) were randomly assigned to 45 min of (1) bright light or (2) placebo. Participants then performed a 5-week study (n = 29). Following a 1-week baseline, participants were randomly assigned to 4 weeks of daily exposure to either (1) bright light (45 min/day) or (2) placebo treatment, initiated ≤1 hr after awakening. Before and after the experiment, clinical ratings were conducted with the Hamilton Anxiety Scale (HAM-A), Hamilton Depression Scale, and Clinical Global Impressions scale. Following each week, blood pressure, anxiety (Spielberger State–Trait Anxiety Inventory Y1), depression, mood, sleep, and side effects were assessed. Results: No significant treatment effect was found in the acute exposure study. Likewise, in the 5-week study, no significant treatment effect was found. However, bright light elicited marginally greater reductions in psychic symptoms of the HAM-A (P = .06) and other measures. Conclusions: This pilot study provides little compelling evidence for an anxiolytic effect of bright light in high-anxious young adults. Depression and Anxiety, 2011. © 2011 Wiley-Liss, Inc.

Many pro-inflammatory molecules, such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) are somnogenic, while many anti-inflammatory molecules inhibit sleep. Sleep loss increases the production/release of these sleep regulatory pro-inflammatory molecules. Further, sleep changes occurring during various pathologies are mediated by these inflammatory substances in response to pathogen recognition and subsequent inflammatory cellular pathways. This review summarizes information and concepts regarding inflammatory mechanisms of the innate immune system that mediate sleep. Further, we discuss sleep-immune interactions in regards to sleep in general, pathologies, and sleep as a local phenomenon including the central role that extracellular ATP plays in the initiation of sleep.

Sleep is dependent upon prior brain activities, e.g., after prolonged wakefulness sleep rebound occurs. These effects are mediated, in part, by humoral sleep regulatory substances such as cytokines. However, the property of wakefulness activity that initiates production and release of such substances and thereby provides a signal for indexing prior waking activity is unknown. We propose that extracellular ATP, released during neuro- and gliotransmission and acting via purine type 2 (P2) receptors, is such a signal. ATP induces cytokine release from glia. Cytokines in turn affect sleep. We show here that a P2 receptor agonist, 2′(3′)- O-(4-benzoylbenzoyl)adenosine 5′-triphosphate (BzATP), increased non-rapid eye movement sleep (NREMS) and electroencephalographic (EEG) delta power while two different P2 receptor antagonists, acting by different inhibitory mechanisms, reduced spontaneous NREMS in rats. Rat P2X7 receptor protein varied in the somatosensory cortex with time of day, and P2X7 mRNA was altered by interleukin-1 treatment, by sleep deprivation, and with time of day in the hypothalamus and somatosensory cortex. Mice lacking functional P2X7 receptors had attenuated NREMS and EEG delta power responses to sleep deprivation but not to interleukin-1 treatment compared with wild-type mice. Data are consistent with the hypothesis that extracellular ATP, released as a consequence of cell activity and acting via P2 receptors to release cytokines and other sleep regulatory substances, provides a mechanism by which the brain could monitor prior activity and translate it into sleep.

THERE IS ACCUMULATING EPIDEMIOLOGIC EVIDENCE ASSOCIATING BOTH SHORT (≤ 6 H) AND LONG (≥ 8 H) SELF-REPORTED SLEEP DURATIONS WITH INCREASED mortality1–7 and morbidity,8–12 with the lowest risks generally associated with 7 to 8 hours of sleep. The largest of these studies,1 which included a sample size of 1.1 million adults, found that long sleep (≥ 8 h) was more than twice as common as short sleep (< 7 h) with higher associated hazard ratios, implying approximately 2.5 times greater population attributable mortality for long sleep. Though the risks of short sleep have been the topic of extensive experimental investigation, the potential risks of long sleep (or long time in bed [TIB]) have received scarce attention. Indeed, the notion that long sleep could be hazardous has seemed counterintuitive, particularly since 8 hours of sleep is often said to be ideal for health and well-being. Critics have argued that the associations of long sleep with mortality and morbidity must be attributable to confounding biologic factors such as sleep apnea or depression, to psychosocial factors such as unemployment or retirement, or to a reverse pathway in which long sleep might simply reflect a moribund state.13,14 However, the epidemiologic associations of long sleep with mortality and morbidity have persisted following statistical control for such potential confounds. Most of the evidence demonstrating risks of long sleep has come from studies using self reports of sleep duration, which might more accurately reflect TIB than total sleep time (TST). However, recent actigraphic data are available associating long actigraphically measured sleep with morbidity.11,15 Whether time in bed or time asleep is the primary risk factor has not yet been determined, since the two measures are generally highly correlated. Studies that have discriminated between self-reported TIB and TST have found similar mortality associations with these measures.16 For a behavioral intervention, TIB is more easily measured, and more practical to alter, as many people may not have accurate awareness and control over their sleep durations. Hypothetically, if long sleep (or long TIB) causes mortality or morbidity, then moderate TIB restriction might prove a simple and effective way to improve health and longevity for long sleepers.17 However, before large-scale, randomized, risk-prevention trials to test this hypothesis can be justified, research must first establish whether long sleepers can tolerate moderate TIB restriction without negative consequences. If long sleep reflects a normal variation in sleep need, then detrimental effects of sleep loss might be evident for long sleepers as well as normal sleepers, perhaps, even more so, considering evidence that sleep restriction elicits greater homeostatic sleep pressure in long versus short sleepers.18 On the other hand, if long sleep reflects “optional”,19 unnecessary sleep, then moderate restriction of TIB might be well tolerated, or even beneficial, for long sleepers.17 TIB restriction is one of the most effective treatments for primary insomnia,20 which is often exacerbated by compensatory increases in TIB. The benefits of TIB-restriction treatment have been particularly well documented for older adults,21 who have more sleep fragmentation and tend to spend more TIB, but sleep less, than young adults.22 Indeed, mild restriction of TIB (30 min/night) has been found to improve sleep consolidation while preserving TST, even in older adults with average sleep duration and no sleep complaints.23 Thus, it is plausible that older self-reported long sleepers, who have even greater TIB and sleep fragmentation than average, might tolerate restriction of TIB greater than 30 minutes. The aim of this investigation was to examine the influence of chronic 90-minute TIB restriction on several indexes of tolerance, including sleepiness, depression, quality of life, and neurobehavioral performance in older adults. Based on previous research indicating good tolerance of 30- to 60-minute TIB restriction in older adults who do not report sleep disturbances, 23,24 we hypothesized that there would be no clinically significant detrimental effects of TIB restriction on these variables.

The aim of this study was to investigate the effects of 8 weeks of moderate restriction of time in bed (TIB) on glucose tolerance and insulin sensitivity in healthy older self-reported long sleepers. Forty-two older adults (ages 50-70 years) who reported average sleep durations of >or=8.5 h per night were assessed. Following a 2-week baseline, participants were randomly assigned to two 8-week treatments: either (i) TIB restriction (n = 22), which involved following a fixed sleep schedule in which time in bed was reduced by 90 min compared with baseline; (ii) a control (n = 18), which involved following a fixed sleep schedule but no imposed change of TIB. Sleep was monitored continuously via wrist actigraphy recordings, supplemented with a daily diary. Glucose tolerance and insulin sensitivity were assessed before and following the treatments. Compared with the control treatment, TIB restriction resulted in a significantly greater reduction of nocturnal TIB (1.39 +/- 0.40 h versus 0.14 +/- 0.26 h), nocturnal total sleep time (TST) (1.03 +/- 0.53 h versus 0.40 +/- 0.42 h), and 24-h TST (1.03 +/- 0.53 h versus 0.33 +/- 0.43 h) from baseline values. However, no significant effect of TIB restriction was found for glucose tolerance or insulin sensitivity. These results suggest that healthy older long sleepers can tolerate 8 weeks of moderate TIB restriction without impairments in glucose tolerance or insulin sensitivity.

Downhill running is associated with fiber damage, inflammation, delayed-onset muscle soreness, and various functional deficits. Curcumin, a constituent of the Indian spice turmeric has been investigated for its anti-inflammatory activity and may offset some of the damage and functional deficits associated with downhill running. This study examined the effects of curcumin on inflammation and recovery of running performance following downhill running in mice. Male mice were assigned to downhill placebo (Down-Plac), downhill curcumin (Down-Cur), uphill placebo (Up-Plac), or uphill curcumin (Up-Cur) groups and run on a treadmill at 22 m/min at −14% or +14% grade, for 150 min. At 48 h or 72 h after the up/downhill run, mice ( experiment 1) underwent a treadmill performance run to fatigue. Another subset of mice was placed in voluntary activity wheel cages following the up/downhill run ( experiment 2) and their voluntary activity (distance, time and peak speed) was recorded. Additional mice ( experiment 3) were killed at 24 h and 48 h following the up/downhill run, and the soleus muscle was harvested for analysis of inflammatory cytokines (IL-1β, IL-6, and TNF-α), and plasma was collected for creatine kinase analysis. Downhill running decreased both treadmill run time to fatigue (48 h and 72 h) and voluntary activity (24 h) ( P < 0.05), and curcumin feedings offset these effects on running performance. Downhill running was also associated with an increase in inflammatory cytokines (24 h and 48 h) and creatine kinase (24 h) ( P < 0.05) that were blunted by curcumin feedings. These results support the hypothesis that curcumin can reduce inflammation and offset some of the performance deficits associated with eccentric exercise-induced muscle damage.

This investigation determined whether daily strenuous exercise would alter the progression and regression of an allogeneic lymphoid tumor in mice. We also determined whether exercise would alter the cellular composition and vascularity of the tumor. Female BALB/c mice (age 6–8 wk) were randomly assigned to sedentary control (Con) or daily exercised groups (EXH). EXH mice ran on a treadmill at incremental speeds (20–40 m/min) for 3 h or until fatigue. Each mouse was subcutaneously injected with 20 × 106 EL-4 lymphoma cells immediately after the first exercise bout ( day 1) and run daily. Tumor volume was measured daily with calipers. In some experiments, mice were euthanized on days 5–10, 12, and 14. Tumors were excised and stained with hematoxylin and eosin or for Factor VIII-associated antigen using immunohistochemistry and analyzed in a blinded fashion under a light microscope. There was no significant treatment main effect found for tumor volumes. Interestingly, a significant treatment × time interaction was found, such that there was a 2-day delay in peak tumor volume and a more rapid tumor regression in EXH. Tumors isolated from Con exhibited significantly higher numbers of apoptotic bodies, blood vessels, macrophages, and neutrophils when compared with EXH. Intratumoral lymphocytes were higher in Con early in tumor growth but higher in EXH at peak tumor size. These data indicate that daily strenuous exercise may influence tumor growth by affecting the microenvironment of the tumor, resulting in a delay in tumor growth and a more rapid regression.

The neuropeptide substance P is an excitatory neurotransmitter produced by various cells including neurons and microglia that is involved in regulating inflammation and cerebral blood flow--functions that affect sleep and slow-wave activity (SWA). Substance P is the major ligand for the neurokinin-1 receptor (NK-1R), which is found throughout the brain including the cortex. The NK-1R is found on sleep-active cortical neurons expressing neuronal nitric oxide synthase whose activity is associated with SWA. We determined the effects of local cortical administration of a NK-1R agonist (substance P-fragment 1, 7) and a NK-1R antagonist (CP96345) on sleep and SWA in mice. The NK-1R agonist significantly enhanced SWA for several hours when applied locally to the cortex of the ipsilateral hemisphere as the electroencephalogram (EEG) electrode but not after application to the contralateral hemisphere when compared to saline vehicle control injections. In addition, a significant compensatory reduction in SWA was found after the NK-1R agonist-induced enhancements in SWA. Conversely, injections of the NK-1R antagonist into the cortex of the ipsilateral hemisphere of the EEG electrode attenuated SWA compared to vehicle injections but this effect was not found after injections of the NK-1R antagonist into contralateral hemisphere as the EEG electrode. Non-rapid eye movement sleep and rapid eye movement sleep duration responses after NK-1R agonist and antagonist injections were not significantly different from the responses to the vehicle. Our findings indicate that the substance P and the NK-1R are involved in regulating SWA locally.

Acute sleep loss increases pro-inflammatory and synaptic plasticity-related molecules in the brain, including interleukin-1beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and brain-derived neurotrophic factor (BDNF). These molecules enhance non-rapid eye movement sleep slow wave activity (SWA), also known as electroencephalogram delta power, and modulate neurocognitive performance. Evidence suggests that chronic sleep restriction (CSR), a condition prevalent in today's society, does not elicit the enhanced SWA that is seen after acute sleep loss, although it cumulatively impairs neurocognitive functioning. Rats were continuously sleep deprived for 18 h per day and allowed 6 h of ad libitum sleep opportunity for 1 (SR1), 3 (SR3), or 5 (SR5) successive days (i.e., CSR). IL-1β, TNF-α, and BDNF mRNA levels were determined in the somatosensory cortex, frontal cortex, hippocampus, and basal forebrain. Largely, brain IL-1β and TNF-α expression were significantly enhanced throughout CSR. In contrast, BDNF mRNA levels were similar to baseline values in the cortex after 1 day of SR and significantly lower than baseline values in the hippocampus after 5 days of SR. In the basal forebrain, BDNF expression remained elevated throughout the 5 days of CSR, although IL-1β expression was significantly reduced. The chronic elevations of IL-1β and TNF-α and inhibition of BDNF might contribute to the reported lack of SWA responses reported after CSR. Further, the CSR-induced enhancements in brain inflammatory molecules and attenuations in hippocampal BDNF might contribute to neurocognitive and vigilance detriments that occur from CSR.

Mice are by far the most widely used species for scientific research and have been used in many studies involving biopotentials, such as the electroencephalogram (EEG) and electromyogram (EMG) signals monitored for sleep analysis. Unfortunately, current methods for the analysis of these signals involve either tethered systems that are restrictive and heavy for the animal or wireless systems that use transponders that are large relative to the animal and require invasive surgery for implantation; as a result, natural behavior/activity is altered. Here, we propose a novel and inexpensive system for measuring electroencephalographic signals and other biopotentials in mice that allows for natural movement. We also evaluate the new system for the analysis of sleep architecture and EEG power during both spontaneous sleep and the sleep that follows sleep deprivation in mice. Using our new system, vigilance states including non-rapid eye movement sleep (NREMS), rapid eye movement sleep (REMS), and wakefulness, as well as EEG power and NREMS EEG delta power in the 0.5-4 Hz range (an indicator of sleep intensity) showed the diurnal rhythms typically found in mice. These values were also similar to values obtained in mice using telemetry transponders. Mice that used the new system also demonstrated enhanced NREMS EEG delta power responses that are typical following sleep deprivation and few signal artifacts. Moreover, similar movement activity counts were found when using the new system compared to a wireless system. This novel system for measuring biopotentials can be used for polysomnography, infusion, microdialysis, and optogenetic studies, reduces artifacts, and allows for a more natural moving environment and a more accurate investigation of biological systems and pharmaceutical development.

The effects of chronic moderate sleep restriction and exercise training on carcinogenesis were examined in adenomatous polyposis coli multiple intestinal neoplasma (APC Min(+/-)) mice, a genetic strain which is predisposed to developing adenomatous polyposis. The mice were randomized to one of four 11 week treatments in a 2×2 design involving sleep restriction (by 4 h/day) vs. normal sleep and exercise training (1h/day) vs. sedentary control. Wild-type control mice underwent identical experimental treatments. Compared with the wild-type mice, APC Min(+/-) mice had disrupted hematology and enhanced pro-inflammatory cytokine production from peritoneal exudate cells. Among the APC Min(+/-) mice, consistent interactions of sleep loss and exercise were found for measures of polyp formation, inflammation, and hematology. Sleep loss had little effect on these variables under sedentary conditions, but sleep loss had clear detrimental effects under exercise conditions. Exercise training resulted in improvements in these measures under normal sleep conditions, but exercise tended to elicit no effect or to exacerbate the effects of sleep restriction. Significant correlations of inflammation with polyp burden were observed. Among wild-type mice, similar, but less consistent interactions of sleep restriction and exercise were found. These data suggest that the benefits of exercise on carcinogenesis and immune function were impaired by chronic moderate sleep restriction, and that harmful effects of sleep restriction were generally realized only in the presence of exercise.

Symptoms commonly associated with sleep loss and chronic inflammation include sleepiness, fatigue, poor cognition, enhanced sensitivity to pain and kindling stimuli, excess sleep and increases in circulating levels of tumor necrosis factor α (TNF) in humans and brain levels of interleukin-1 β (IL1) and TNF in animals. Cytokines including IL1 and TNF partake in non-rapid eye movement sleep (NREMS) regulation under physiological and inflammatory conditions. Administration of exogenous IL1 or TNF mimics the accumulation of these cytokines occurring during sleep loss to the extent that it induces the aforementioned symptoms. Extracellular ATP associated with neuro- and glio-transmission, acting via purine type 2 receptors, e.g., the P2X7 receptor, has a role in glia release of IL1 and TNF. These substances in turn act on neurons to change their intrinsic membrane properties and sensitivities to neurotransmitters and neuromodulators such as adenosine, glutamate and GABA. These actions change the network input-output properties, i.e., a state shift for the network. State oscillations occur locally within cortical columns and are defined using evoked response potentials. One such state, so defined, shares properties with whole animal sleep in that it is dependent on prior cellular activity—it shows homeostasis. The cortical column sleep-like state is induced by TNF and is associated with experimental performance detriments. ATP released extracellularly as a consequence of cellular activity is posited to initiate a mechanism by which the brain tracks its prior sleep-state history to induce/prohibit sleep. Thus, sleep is an emergent property of populations of local neural networks undergoing state transitions. Specific neuronal groups participating in sleep depend upon prior network use driving local network state changes via the ATP-cytokine-adenosine mechanism. Such considerations add complexity to finding biochemical markers for sleepiness. Citation: Clinton JM; Davis CJ; Zielinski MR; Jewett KA; Krueger JM. Biochemical regulation of sleep and sleep biomarkers. J Clin Sleep Med 2011;7(5):Supplement S38-S42.

Cytokines such as tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL1β) play a role in sleep regulation in health and disease. TNFα or IL1β injection enhances non-rapid eye movement sleep. Inhibition of TNFα or IL1β reduces spontaneous sleep. Mice lacking TNFα or IL1β receptors sleep less. In normal humans and in multiple disease states, plasma levels of TNFα covary with EEG slow wave activity (SWA) and sleep propensity. Many of the symptoms induced by sleep loss, for example, sleepiness, fatigue, poor cognition, enhanced sensitivity to pain, are elicited by injection of exogenous TNFα or IL1β. IL1β or TNFα applied unilaterally to the surface of the cortex induces state-dependent enhancement of EEG SWA ipsilaterally, suggesting greater regional sleep intensity. Interventions such as unilateral somatosensory stimulation enhance localized sleep EEG SWA, blood flow, and somatosensory cortical expression of IL1β and TNFα. State oscillations occur within cortical columns. One such state shares properties with whole animal sleep in that it is dependent on prior cellular activity, shows homeostasis, and is induced by TNFα. Extracellular ATP released during neuro- and gliotransmission enhances cytokine release via purine type 2 receptors. An ATP agonist enhances sleep, while ATP antagonists inhibit sleep. Mice lacking the P2X7 receptor have attenuated sleep rebound responses after sleep loss. TNFα and IL1β alter neuron sensitivity by changing neuromodulator/neurotransmitter receptor expression, allowing the neuron to scale its activity to the presynaptic neurons. TNFα's role in synaptic scaling is well characterized. Because the sensitivity of the postsynaptic neuron is changed, the same input will result in a different network output signal and this is a state change. The top-down paradigm of sleep regulation requires intentional action from sleep/wake regulatory brain circuits to initiate whole-organism sleep. This raises unresolved questions as to how such purposeful action might itself be initiated. In the new paradigm, sleep is initiated within networks and local sleep is a direct consequence of prior local cell activity. Whole-organism sleep is a bottom-up, self-organizing, and emergent property of the collective states of networks throughout the brain.

Although self-reported long sleep is associated with increased morbidity and mortality, little is known about the objective sleep patterns and daytime functioning of long sleepers, particularly those aged ≥50 years. Our primary aim was to compare the objective and subjective sleep patterns of a sample (n = 35) of middle- to older-aged adults who reported sleeping ≥8.5 h per night. A secondary aim was to characterize the mood and functioning of the sample. Over a 2-week period, sleep was recorded via actigraphy and a daily diary. Sleepiness was assessed daily. At the conclusion of the 2-week period, daytime sleepiness, mood, and quality of life were assessed. Measures of sleep and functioning were compared with available representative data. In the sample, actigraphic total sleep time (TST; 7.35 ± 0.97 h) was approximately 60 min greater than age-related representative values but substantially less than diary-assessed TST (8.59 ± 0.74 h) and survey-assessed TST (8.92 ± 0.78 h). Survey and diary-based subjective TST assessments agreed more closely with actigraphic time in bed (TIB; 9.11 ± 0.72 h) than TST, and correlations between subjective TST and actigraphic TIB were similar to those between subjective and actigraphic TST. Measures of mood, sleepiness, and daytime functioning were similar to population-representative values. These results suggest that, among middle- to older-aged adults, self-reported long sleep is primarily indicative of long TIB, but it also represents long objective sleep duration, particularly in comparison to age-matched data. Findings of little functional impairment corroborated previous descriptions of older long sleepers.

Previous findings of time-of-day differences in athletic performance could be confounded by diurnal fluctuations in environmental and behavioral “masking” factors (e.g., sleep, ambient temperature, and energy intake). The purpose of this study was to examine whether there is a circadian rhythm in swim performance that is independent of these masking factors. Experienced swimmers ( n = 25) were assessed for 50–55 consecutive hours in the laboratory. The swimmers followed a 3-h “ultra-short” sleep-wake cycle, involving 1 h of sleep in darkness and 2 h of wakefulness in dim light, that was repeated throughout the observation. The protocol distributes behavioral and environmental masking factors equally across the 24-h period. Each swimmer was scheduled to perform six maximal-effort 200-m swim trials that were distributed equally across eight times of day ( n = 147 trials). Each trial was separated by 9 h. A cosine fit of intra-aural temperature data established the time of the lowest body temperature (Tmin). Swim performances were z-transformed and compared across the eight times of day and across twelve 2-h intervals relative to Tmin. Analysis of covariance, controlling for trial number, revealed a significant ( P < 0.001) pattern in swim performance relative to environmental and circadian times of day. Performance peaked 5–7 h before Tmin(∼2300) and was worst from 1 h before to 1 h after Tmin(∼0500). Mean swim performance was 169.5 s; circadian variation from peak to worst performance was 5.8 s. These data suggest a circadian rhythm in athletic performance independent of environmental and behavioral masking effects.