Neuroinflammation, Sleep, and Circadian Rhythms.

Molecules involved in innate immunity affect sleep and circadian oscillators and vice versa. Sleep-inducing inflammatory molecules are activated by increased waking activity and pathogens. Pathologies that alter inflammatory molecules, such as traumatic brain injury, cancer, cardiovascular disease, and stroke often are associated with disturbed sleep and electroencephalogram power spectra. Moreover, sleep disorders, such as insomnia and sleep disordered breathing, are associated with increased dysregulation of inflammatory processes. Inflammatory molecules in both the central nervous system and periphery can alter sleep. Inflammation can also modulate cerebral vascular hemodynamics which is associated with alterations in electroencephalogram power spectra. However, further research is needed to determine the interactions of sleep regulatory inflammatory molecules and circadian clocks. The purpose of this review is to: 1) describe the role of the inflammatory cytokines interleukin-1 beta and tumor necrosis factor-alpha and nucleotide-binding domain and leucine-rich repeat protein-3 inflammasomes in sleep regulation, 2) to discuss the relationship between the vagus nerve in translating inflammatory signals between the periphery and central nervous system to alter sleep, and 3) to present information about the relationship between cerebral vascular hemodynamics and the electroencephalogram during sleep.

Sleep State Dependence of Optogenetically evoked Responses in Neuronal Nitric Oxide Synthase-positive Cells of the Cerebral Cortex.

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.

Chronic sleep restriction elevates brain interleukin-1 beta and tumor necrosis factor-alpha and attenuates brain-derived neurotrophic factor expression.

Acute sleep loss increases pro-inflammatory and synaptic plasticity-related molecules in the brain, including interleukin-1 beta (IL-1ß), 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 18h per day and allowed 6h of ad libitum sleep opportunity for 1 (SR1), 3 (SR3), or 5 (SR5) successive days (i.e., CSR). IL-1ß, TNF-α, and BDNF mRNA levels were determined in the somatosensory cortex, frontal cortex, hippocampus, and basal forebrain. Largely, brain IL-1ß 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-1ß expression was significantly reduced. The chronic elevations of IL-1ß 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.

Vagotomy attenuates brain cytokines and sleep induced by peripherally administered tumor necrosis factor-α and lipopolysaccharide in mice.

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-1ß) 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-1ß 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.

Olfactory bulb and hypothalamic acute-phase responses to influenza virus: effects of immunization.

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.

Influence of chronic moderate sleep restriction and exercise training on anxiety, spatial memory, and associated neurobiological measures in mice.

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 (∼4h loss/day) vs. ad libitum sleep] × 2 [exercise (1h/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.

A novel telemetric system to measure polysomnographic biopotentials in freely moving animals.

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.

5'-Ectonucleotidase-knockout mice lack non-REM sleep responses to sleep deprivation.

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 (IL1ß) 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.

Influence of chronic moderate sleep restriction and exercise on inflammation and carcinogenesis in mice.

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.

Biochemical regulation of sleep and sleep biomarkers.

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 ß (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.

Involvement of cytokines in slow wave sleep.

Cytokines such as tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL1ß) play a role in sleep regulation in health and disease. TNFα or IL1ß injection enhances non-rapid eye movement sleep. Inhibition of TNFα or IL1ß reduces spontaneous sleep. Mice lacking TNFα or IL1ß 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 IL1ß. IL1ß 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 IL1ß 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 IL1ß 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.

Sleep and innate immunity.

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.

ATP and the purine type 2 X7 receptor affect 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.

No effect of 8-week time in bed restriction on glucose tolerance in older long sleepers.

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.

Exercise delays allogeneic tumor growth and reduces intratumoral inflammation and vascularization.

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 x 10(6) 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 x 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.