Interleukin-1 receptor accessory proteins are required for normal homeostatic responses to sleep deprivation.

Interleukin-1ß (IL1) is a sleep regulatory substance. The IL1/IL1 type 1 receptor complex requires a receptor accessory protein (AcP) to signal. There are three isoforms of AcP. In the current experiments, mice lacking a neuron-specific isoform, called AcPb knockout (AcPb KO), or mice lacking AcP + AcPb isoforms (AcP KO) or wild-type (WT) mice were used. Spontaneous sleep and sleep responses to sleep deprivation (SD) between zeitgeber time (ZT) 20-ZT4 and ZT8-ZT16 were characterized. Furthermore, somatosensory cortical protein extracts were examined for phosphorylated (p) proto-oncogene tyrosine-protein kinase sarcoma (Src) and p38MAPK levels at ZT4 and ZT16 and after SD. Spontaneous sleep was similar in the three strains, except rapid eye movement sleep (REMS) duration between ZT12-ZT16 was greater in AcP KO than WT mice. After SD at ZT4, only WT mice had non-REMS (NREMS) rebounds. All mouse strains lacked an NREMS rebound after SD at ZT16. All strains after both SD periods had REMS rebounds. AcPb KO mice, but not AcP KO mice, had greater EEG delta wave (0.5-4 Hz) power during NREMS than WT mice. p-Src was very low at ZT16 but high at ZT4, whereas p-p38MAPK was low at ZT4 and high at ZT16. p-p38MAPK levels were not sensitive to SD. In contrast, p-Src levels were less after SD at the P = 0.08 level of significance in the strains lacking AcPb. We conclude that AcPb is required for NREMS responses to sleep loss, but not for SD-induced EEG delta wave or REMS responses.NEW & NOTEWORTHY Interleukin-1ß (IL1), a well-characterized sleep regulatory substance, requires an IL1 receptor accessory protein (AcP); one of its isoforms is neuron-specific (called AcPb). We showed that in mice, AcPb is required for nonrapid eye movement sleep responses following 8 h of sleep loss ending 4 h after daybreak but did not affect rapid eye movement sleep rebound. Sleep loss reduced phosphorylation of proto-oncogene tyrosine-protein kinase sarcoma but not of the less sensitive p38MAPK, downstream IL1 signaling molecules.

The neuron-specific interleukin-1 receptor accessory protein alters emergent network state properties in Vitro.

Small in vitro neuronal/glial networks exhibit sleep-like states. Sleep regulatory substance interleukin-1ß (IL1) signals via its type I receptor and a receptor accessory protein (AcP). AcP has a neuron-specific isoform called AcPb. After sleep deprivation, AcPb, but not AcP, upregulates in brain, and mice lacking AcPb lack sleep rebound. Herein we used action potentials (APs), AP burstiness, synchronization of electrical activity (SYN), and delta wave (0.5-3.75 Hz) power to characterize cortical culture network state. Homologous parameters are used in vivo to characterize sleep. Cortical cells from 1-2-day-old pups from AcP knockout (KO, lacking both AcP and AcPb), AcPb KO (lacking only AcPb), and wild type (WT) mice were cultured separately on multi-electrode arrays. Recordings of spontaneous activity were taken each day during days 4-14 in vitro. In addition, cultures were treated with IL1, or in separate experiments, stimulated electrically to determine evoked response potentials (ERPs). In AcP KO cells, the maturation of network properties accelerated compared to those from cells lacking only AcPb. In contrast, the lack of AcPb delayed spontaneous network emergence of sleep-linked properties. The addition of IL1 enhanced delta wave power in WT cells but not in AcP KO or AcPb KO cells. The ontology of electrically-induced ERPs was delayed in AcP KO cells. We conclude IL1 signaling has a critical role in the emergence of sleep-linked network behavior with AcP playing a dominant role in the slowing of development while AcPb enhances development rates of sleep-linked emergent network properties.

Local sleep.

The historic sleep regulatory paradigm invokes "top-down" imposition of sleep on the brain by sleep regulatory circuits. While remaining conceptually useful, many sleep phenomena are difficult to explain using that paradigm, including, unilateral sleep, sleep-walking, and poor performance after sleep deprivation. Further, all animals sleep after non-lethal brain lesions, regardless of whether the lesion includes sleep regulatory circuits, suggesting that sleep is a fundamental property of small viable neuronal/glial networks. That small areas of the brain can exhibit non-rapid eye movement sleep-like states is summarized. Further, sleep-like states in neuronal/glial cultures are described. The local sleep states, whether in vivo or in vitro, share electrophysiological properties and molecular regulatory components with whole animal sleep and exhibit sleep homeostasis. The molecular regulatory components of sleep are also involved in plasticity and inflammation. Like sleep, these processes, are initiated by local cell-activity dependent events, yet have at higher levels of tissue organization whole body functions. While there are large literatures dealing with local initiation and regulation of plasticity and inflammation, the literature surrounding local sleep is in its infancy and clinical applications of the local sleep concept are absent. Regardless, the local use-dependent sleep paradigm can advise and advance future research and clinical applications.

Tumor necrosis factor alpha in sleep regulation.

This review details tumor necrosis factor alpha (TNF) biology and its role in sleep, and describes how TNF medications influence sleep/wake activity. Substantial evidence from healthy young animals indicates acute enhancement or inhibition of endogenous brain TNF respectively promotes and inhibits sleep. In contrast, the role of TNF in sleep in most human studies involves pathological conditions associated with chronic elevations of systemic TNF and disrupted sleep. Normalization of TNF levels in such patients improves sleep. A few studies involving normal healthy humans and their TNF levels and sleep are consistent with the animal studies but are necessarily more limited in scope. TNF can act on established sleep regulatory circuits to promote sleep and on the cortex within small networks, such as cortical columns, to induce sleep-like states. TNF affects multiple synaptic functions, e.g., its role in synaptic scaling is firmly established. The TNF-plasticity actions, like its role in sleep, can be local network events suggesting that sleep and plasticity share biochemical regulatory mechanisms and thus may be inseparable from each other. We conclude that TNF is involved in sleep regulation acting within an extensive tightly orchestrated biochemical network to niche-adapt sleep in health and disease.

Interleukin 37 expression in mice alters sleep responses to inflammatory agents and influenza virus infection.

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-1ß 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-1ß 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.

P2X7 receptors in body temperature, locomotor activity, and brain mRNA and lncRNA responses to sleep deprivation.

The ionotropic purine type 2X7 receptor (P2X7R) is a nonspecific cation channel implicated in sleep regulation and brain cytokine release. Many endogenous rhythms covary with sleep, including locomotor activity and core body temperature. Furthermore, brain-hypothalamic cytokines and purines play a role in the regulation of these physiological parameters as well as sleep. We hypothesized that these parameters are also affected by the absence of the P2X7 receptor. Herein, we determine spontaneous expression of body temperature and locomotor activity in wild-type (WT) and P2X7R knockout (KO) mice and how they are affected by sleep deprivation (SD). We also compare hypothalamic, hippocampal, and cortical cytokine- and purine-related receptor and enzyme mRNA expressions before and after SD in WT and P2X7RKO mice. Next, in a hypothesis-generating survey of hypothalamic long noncoding (lnc) RNAs, we compare lncRNA expression levels between strains and after SD. During baseline conditions, P2X7RKO mice had attenuated temperature rhythms compared with WT mice, although locomotor activity patterns were similar in both strains. After 6 h of SD, body temperature and locomotion were enhanced to a greater extent in P2X7RKO mice than in WT mice during the initial 2-3 h after SD. Baseline mRNA levels of cortical TNF-α and P2X4R were higher in the KO mice than WT mice. In response to SD, the KO mice failed to increase hypothalamic adenosine deaminase and P2X4R mRNAs. Further, hypothalamic lncRNA expressions varied by strain, and with SD. Current data are consistent with a role for the P2X7R in thermoregulation and lncRNA involvement in purinergic signaling.

Tumor necrosis factor enhances the sleep-like state and electrical stimulation induces a wake-like state in co-cultures of neurons and glia.

We characterise sleep-like states in cultured neurons and glia during development in vitro as well as after electrical stimulation, the addition of tumor necrosis factor alpha (TNF), and the combination of TNF plus electrical stimulation. We also characterise optogenetic stimulation-induced ATP release and neuronal interleukin-1 and TNF expression in vitro demonstrating the activity dependence of these putative sleep-regulatory substances. Action potential (AP) burstiness, expressed as the burstiness index (BI), synchronization of slow electrical potentials between recording electrodes (SYN), and slow wave (SW) power (0.25-3.75 Hz) determined using fast Fourier analyses emerged as network properties, maturing after 2 weeks in culture. Homologous in vivo measures are used to characterise sleep. Electrical stimulation reduced the BI, SYN and SW power values during and/or after the stimulus period. One day later, homeostasis was evident from rebounds of SYN and SW power values to above baseline levels; the magnitude of the rebound was stimulus pattern-dependent. The addition of TNF enhanced BI, SYN and SW power values, suggesting the induction of a deeper sleep-like state. Electrical stimulation reversed these TNF effects, suggesting the network state was more wake-like. The day after TNF plus electrical stimulation, the changes in SYN and SW power values were dependent upon the stimulus patterns the cells received the day before. We conclude that sleep and wake states in cultured in vitro networks can be controlled and they share molecular regulatory mechanisms with local in vivo networks. Further, sleep is an activity-dependent emergent local network property.

The neuron-specific interleukin-1 receptor accessory protein is required for homeostatic sleep and sleep responses to influenza viral challenge in mice.

Interleukin-1ß (IL1) is involved in sleep regulation and sleep responses induced by influenza virus. The IL1 receptor accessory protein (AcP) and an alternatively spliced isoform of AcP found primarily in neurons, AcPb, form part of the IL1 signaling complex. IL1-induced sleep responses depend on injection time. In rat cortex, both IL1 mRNA and AcPb mRNA peak at Zeitgeber Time (ZT) 0 then decline over the daylight hours. Sleep deprivation enhances cortical IL1 mRNA and AcPb mRNA levels, but not AcP mRNA. We used wild type (WT) and AcPb knockout (KO) mice and performed sleep deprivation between ZT10 and 20 or between ZT22 and 8 based on the time of day expression profiles of AcPb and IL1. We hypothesized that the magnitude of the responses to sleep loss would be strain- and time of day-dependent. In WT mice, NREMS and REMS rebounds occurred regardless of when they were deprived of sleep. In contrast, when AcPbKO mice were sleep deprived from ZT10 to 20 NREMS and REMS rebounds were absent. The AcPbKO mice expressed sleep rebound if sleep loss occurred from ZT22 to 8 although the NREMS responses were not as robust as those that occurred in WT mice. We also challenged mice with intranasal H1N1 influenza virus. WT mice exhibited the expected enhanced sleep responses. In contrast, the AcPbKO mice had less sleep after influenza challenge compared to their own baseline values and compared to WT mice. Body temperature and locomotor activity responses after viral challenge were lower and mortality was higher in AcPbKO than in WT mice. We conclude that neuron-specific AcPb plays a critical role in host defenses and sleep homeostasis.

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.

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.

Brain-specific interleukin-1 receptor accessory protein in sleep regulation.

Interleukin (IL)-1ß 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-1ß/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-1ß. 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-1ß, 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.

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.

MicroRNA 132 alters sleep and varies with time in brain.

MicroRNA (miRNA) levels in brain are altered by sleep deprivation; however, the direct effects of any miRNA on sleep have not heretofore been described. We report herein that intracerebroventricular application of a miRNA-132 mimetic (preMIR-132) decreased duration of non-rapid-eye-movement sleep (NREMS) while simultaneously increasing duration of rapid eye movement sleep (REMS) during the light phase. Further, preMIR-132 decreased electroencephalographic (EEG) slow-wave activity (SWA) during NREMS, an index of sleep intensity. In separate experiments unilateral supracortical application of preMIR-132 ipsilaterally decreased EEG SWA during NREMS but did not alter global sleep duration. In addition, after ventricular or supracortical injections of preMIR-132, the mimetic-induced effects were state specific, occurring only during NREMS. After local supracortical injections of the mimetic, cortical miRNA-132 levels were higher at the time sleep-related EEG effects were manifest. We also report that spontaneous cortical levels of miRNA-132 were lower at the end of the sleep-dominant light period compared with at the end of the dark period in rats. Results suggest that miRNAs play a regulatory role in sleep and provide a new tool for investigating sleep regulation.

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.

Localized suppression of cortical growth hormone-releasing hormone receptors state-specifically attenuates electroencephalographic delta waves.

Growth hormone-releasing hormone (GHRH) promotes non-rapid eye movement sleep (NREMS), in part via a well characterized hypothalamic sleep-promoting site. However, GHRH may also act in the cortex to influence sleep. Application of GHRH to the surface of the cortex changes electroencephalographic (EEG) delta power. GHRH and the GHRH receptor (GHRHR) mRNAs are detectable in the rat cortex, and the expression of cortical GHRHR is activity dependent. Here, we microinjected a GHRH antagonist or GHRHR small interfering RNA (siGHRHR) onto the somatosensory cortex surface in rats. The unilateral application of the GHRH antagonist ipsilaterally decreased EEG delta wave power during NREMS, but not wakefulness, during the initial 40 min after injection. Similarly, the injection of siGHRHR reduced cortical expression of GHRHR and suppressed NREMS EEG delta wave power during 20-24 h after injection. Using the fura-2 calcium imaging technique, cultured cortical cells responded to GHRH by increasing intracellular calcium. Approximately 18% of the GHRH-responsive cells were GABAergic as illustrated by glutamic acid decarboxylase-67 (GAD67) immunostaining. Double labeling for GAD67 and GHRHR in vitro and in vivo indicated that only a minority of cortical GHRHR-containing cells were GABAergic. Our data suggest that endogenous cortical GHRH activates local cortical cells to affect EEG delta wave power state-specifically. Results are also consistent with the hypothesis that GHRH contributes to local network state regulation.

Whisker stimulation increases expression of nerve growth factor- and interleukin-1beta-immunoreactivity in the rat somatosensory cortex.

Activity-dependent changes in cortical protein expression may mediate long-term physiological processes such as sleep and neural connectivity. In this study we determined the number of nerve growth factor (NGF)- and interleukin-1beta (IL1beta)-immunoreactive (IR) cells in the somatosensory cortex (Sctx) in response to 2 h of mystacial whisker stimulation. Manual whisker stimulation for 2 h increased the number of NGF-IR cells within layers II-V in activated Sctx columns, identified by enhanced Fos-IR. IL1beta-IR neurons increased within layers II-III and V-VI in these activated columns and IL1beta-IR astrocytes increased in layers I, II-III and V as well as the external capsule beneath the activated columns. These whisker-stimulated increases in the Sctx did not occur in the auditory cortex. These data demonstrate that expression of NGF or IL1beta in Sctx neurons and IL1beta in Sctx astrocytes is, in part, afferent input-dependent.

Influenza virus- and cytokine-immunoreactive cells in the murine olfactory and central autonomic nervous systems before and after illness onset.

Influenza virus invades the olfactory bulb (OB) and enhances cytokine mRNAs therein at the time of illness onset. Here we show that viral antigen immunoreactivity co-localized with glial markers in the OB but could not be detected in other brain areas. Interleukin 1beta- and tumor necrosis factor alpha-immunoreactivity co-localized with neuronal markers in olfactory and central autonomic systems, and the number of cytokine-immunoreactive neurons increased at the time of illness onset [15 h post-inoculation (PI)] but not before (10 h PI). These results suggest that the OB virus influences the brain cytokines and therefore the onset of illness.

Spontaneous and influenza virus-induced sleep are altered in TNF-alpha double-receptor deficient mice.

Tumor necrosis factor-alpha (TNF-alpha) is associated with sleep regulation in health and disease. Previous studies assessed sleep in mice genetically deficient in the TNF-alpha 55-kDa receptor. In this study, spontaneous and influenza virus-induced sleep profiles were assessed in mice deficient in both the 55-kDa and 75-kDa TNF-alpha receptors [TNF-2R knockouts (KO)] and wild-type (WT) strain controls. Under baseline conditions the TNF-2R KO mice had less non-rapid eye movement sleep (NREMS) than WTs during the nighttime and more rapid eye movement sleep (REMS) than controls during the daytime. The differences between nighttime maximum and daytime minimum values of electroencephalogram (EEG) delta power during NREMS were greater in the TNF-2R KO mice than in WTs. Viral challenge (mouse-adapted influenza X-31) enhanced NREMS and decreased REMS in both strains roughly to the same extent. EEG delta power responses to viral challenge differed substantially between strains; the WT animals increased, whereas the TNF-2R KO mice decreased their EEG delta wave power during NREMS. There were no differences between strains in body temperatures or locomotor activity in uninfected mice or after viral challenge. Analyses of cortical mRNAs confirmed that the TNF-2R KO mice lacked both TNF-alpha receptors; these mice also had higher levels of orexin mRNA and reduced levels of the purine P2X7 receptor compared with WTs. Results reinforce the hypothesis that TNF-alpha is involved in physiological sleep regulation but plays a limited role in the acute-phase response induced by influenza virus.

Cytokine mRNA induction by interleukin-1beta or tumor necrosis factor alpha in vitro and in vivo.

Hypothalamic and cortical mRNA levels for cytokines such as interleukin-1beta (IL1beta), tumor necrosis factor alpha (TNFalpha), nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) are impacted by systemic treatments of IL1beta and TNFalpha. To investigate the time course of the effects of IL1beta and TNFalpha on hypothalamic and cortical cytokine gene expression, we measured mRNA levels for IL1beta, TNFalpha, interleukin-6 (IL-6), interleukin-10 (IL-10), IL1 receptor 1, BDNF, NGF, and glutamate decarboxylase-67 in vitro using hypothalamic and cortical primary cultures. IL1beta and TNFalpha mRNA levels increased significantly in a dose-dependent fashion after exposure to either IL1beta or TNFalpha. IL1beta increased IL1beta mRNA in both the hypothalamic and cortical cultures after 2-6 h while TNFalpha mRNA increased significantly within 30 min and continued to rise up to 2-6 h. Most of the other mRNAs showed significant changes independent of dose in vitro. In vivo, intracerebroventricular (icv) injection of IL1beta or TNFalpha also significantly increased IL1beta, TNFalpha and IL6 mRNA levels in the hypothalamus and cortex. IL1beta icv, but not TNFalpha, increased NGF mRNA levels in both these areas. Results support the hypothesis that centrally active doses of IL1beta and TNFalpha enhance their own mRNA levels as well as affect mRNA levels for other neuronal growth factors.