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.

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.

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.

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.

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.

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.

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.

TNFalpha siRNA reduces brain TNF and EEG delta wave activity in rats.

Tumor necrosis factor alpha (TNFalpha) is a pleiotropic cytokine with several CNS physiological and pathophysiological actions including sleep, memory, thermal and appetite regulation. Short interfering RNAs (siRNA) targeting TNFalpha were incubated with cortical cell cultures and microinjected into the primary somatosensory cortex (SSctx) of rats. The TNFalpha siRNA treatment specifically reduced TNFalpha mRNA by 45% in vitro without affecting interleukin-6 or gluR1-4 mRNA levels. In vivo the TNFalpha siRNAalpha reduced TNFalpha mRNA, interleukin-6 mRNA and gluR1 mRNA levels compared to treatment with a scrambled control siRNA. After in vivo microinjection, the density of TNFalpha-immunoreactive cells in layer V of the SSctx was also reduced. Electroencephalogram (EEG) delta wave power was decreased on days 2 and 3 on the side of the brain that received the TNFalpha siRNA microinjection relative to the side receiving the control siRNA. These findings support the hypothesis that TNFalpha siRNA attenuates TNFalpha mRNA and TNFalpha protein in the rat cortex and that those reductions reduce cortical EEG delta power. Results also are consistent with the notion that TNFalpha is involved in CNS physiology including sleep regulation.

Influenza virus-induced glucocorticoid and hypothalamic and lung cytokine mRNA responses in dwarf lit/lit mice.

Influenza virus infection up-regulates cytokines such as interleukin-1beta (IL-1beta) and activates the somatotropic axis and the hypothalamic-pituitary axis. Mice with deficits in growth hormone releasing hormone (GHRH) signaling (lit/lit mice) respond to influenza virus challenge with a progressive decrease in sleep and lower survival rates. Current experiments characterize plasma glucocorticoid responses and hypothalamic and lung mRNA expression of sleep-related genes in lit/lit mice and their heterozygous controls after influenza virus challenge. lit/lit mice had higher basal and post-infection plasma corticosterone levels compared to controls. In contrast, the heterozygous mice increased hypothalamic GHRH-receptor, CRH-type 2 receptor, IL-1beta, and tumor necrosis factor-alpha (TNF-alpha) mRNAs after virus treatment while the lit/lit mice failed to up-regulate these substances. In contrast, lung levels of IL-1beta and TNF-alpha mRNAs were greater in the lit/lit mice. These data are consistent with the hypothesis that the sleep response to influenza infection is mediated, in part, by an up-regulation of hypothalamic sleep-related transcripts and they also show that a primary deficit in GHRH signaling is associated with enhanced corticosterone secretion and attenuated hypothalamic cytokine response to infection.

Brain distribution of cytokine mRNA induced by systemic administration of interleukin-1beta or tumor necrosis factor alpha.

Brain cytokine mRNA levels are impacted by systemic cytokines. For example, systemic interleukin-1beta (IL1beta) increases brain IL1beta mRNA; subdiaphragmatic vagotomy blocks this effect. To localize which brain regions respond to intraperitoneal cytokines, we measured mRNA levels in selected brain regions for a variety of cytokines and growth factors, IL1beta, TNFalpha, interleukin-6 (IL-6), interleukin-10 (IL10), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Relative to saline administration, IL1beta increased IL1beta, TNFalpha and IL6 mRNAs in the nucleus tractus solitarius (NTS), hypothalamus, hippocampus and somatosensory cortex (SSctx), but did not induce any changes in IL10. TNFalpha also increased TNFalpha and IL1beta mRNAs in the hypothalamus, hippocampus and SSctx. TNFalpha increased TNFalpha, IL1beta and IL10 mRNAs in the NTS, but did not induce any changes in IL-6 mRNA. In the amygdala, IL1beta enhanced IL6 mRNA and TNFalpha increased IL1beta mRNAs. In the insular cortex, IL1beta enhanced IL6 mRNA and TNFalpha increased IL1beta mRNA. TNFalpha administration increased NGF mRNA in the SSctx but decreased NGF and BDNF mRNA levels in the insular cortex. Both IL1beta and TNFalpha decreased BDNF mRNA in the amygdala. We also verified the IL1beta-induced increases in TNFalpha mRNA within the NTS using in situ hybridization. These results support the hypothesis that somnogenic doses of IL1beta and TNFalpha enhance their own mRNA levels as well as affect mRNA levels for other sleep-promoting substances.

Rapid eye movement sleep is reduced in prolactin-deficient mice.

Prolactin (PRL) is implicated in the modulation of spontaneous rapid eye movement sleep (REMS). Previous models of hypoprolactinemic animals were characterized by changes in REMS, although associated deficits made it difficult to ascribe changes in REMS to reduced PRL. In the current studies, male PRL knock-out (KO) mice were used; these mice lack functional PRL but have no known additional deficits. Spontaneous REMS was reduced in the PRL KO mice compared with wild-type or heterozygous littermates. Infusion of PRL for 11-12 d into PRL KO mice restored their REMS to that occurring in wild-type or heterozygous controls. Six hours of sleep deprivation induced a non-REMS and a REMS rebound in both PRL KO mice and heterozygous littermates, although the REMS rebound in the KOs was substantially less. Vasoactive intestinal peptide (VIP) induced REMS responses in heterozygous mice but not in KO mice. Similarly, an ether stressor failed to enhance REMS in the PRL KOs but did in heterozygous littermates. Finally, hypothalamic mRNA levels for PRL, VIP, neural nitric oxide synthase (NOS), inducible NOS, and the interferon type I receptor were similar in KO and heterozygous mice. In contrast, tyrosine hydroxylase mRNA was lower in the PRL KO mice than in heterozygous controls and was restored to control values by infusion of PRL, suggesting a functioning short-loop negative feedback regulation in PRL KO mice. Data support the notion that PRL is involved in REMS regulation.

Brainstem prolactin mRNA is enhanced in mice with suppressed neuronal nitric oxide synthase activity.

Prolactin (PRL) and vasoactive intestinal polypeptide (VIP) mRNA levels were elevated in the brainstem of neuronal nitric oxide synthase (nNOS) gene knockout (KO) mice compared to the levels in nNOS control mice. In addition, PRL mRNA levels increased in the hypothalamus and the brainstem of nNOS control mice after administration of 7-nitro-indazole (7-NI), a relatively selective nNOS inhibitor. The results suggest that NO inhibits PRL. No differences in the genes measured were observed in inducible NOS KO mice.

Influenza virus-induced sleep responses in mice with targeted disruptions in neuronal or inducible nitric oxide synthases.

Influenza viral infection induces increases in non-rapid eye movement sleep and decreases in rapid eye movement sleep in normal mice. An array of cytokines is produced during the infection, and some of them, such as IL-1beta and TNF-alpha, are well-defined somnogenic substances. It is suggested that nitric oxide (NO) may mediate the sleep-promoting effects of these cytokines. In this study, we use mice with targeted disruptions of either the neuronal NO synthase (nNOS) or the inducible NO synthase (iNOS) gene, commonly referred to as nNOS or iNOS knockouts (KOs), to investigate sleep changes after influenza viral challenge. We report that the magnitude of viral-induced non-rapid eye movement sleep responses in both nNOS KOs and iNOS KOs was less than that of their respective controls. In addition, the duration of rapid eye movement sleep in nNOS KO mice did not decrease compared with baseline values. All strains of mice had similar viral titers and cytokine gene expression profiles in the lungs. Virus was not isolated from the brains of any strain. However, gene expression in the brain stem differed between nNOS KOs and their controls: mRNA for the interferon-induced gene 2',5'-oligoadenylate synthase 1a was elevated in nNOS KOs relative to their controls at 15 h, and IL-1beta mRNA was elevated in nNOS KOs relative to their controls at 48 h. Our results suggest that NO synthesized by both nNOS and iNOS plays a role in virus-induced sleep changes and that nNOS may modulate cytokine expression in the brain.

The role of nitric oxide synthases in the sleep responses to tumor necrosis factor-alpha.

It is well established that cytokines such as tumor necrosis factor-alpha (TNFalpha) and interleukin-1beta (IL-1beta) are involved in physiological sleep regulation, yet their downstream somnogenic mechanisms remain largely uninvestigated. Nitric oxide (NO) is an effector molecule for some TNFalpha actions. Neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) gene knockout (KO) mice sleep differently than their respective controls. In this study, we tested the hypothesis that NO mediates TNFalpha-induced sleep using iNOS and nNOS KO mice and their corresponding wild-type controls. Systemic administration of TNFalpha increased non-rapid eye movement sleep (NREMS) in the two control strains and in the iNOS KO mice during the first 4 h post-injection but failed to increase NREMS in nNOS KO mice. Rapid eye movement sleep (REMS) was suppressed by TNFalpha in nNOS controls but not in the other strains examined. The results suggest that TNFalpha affects sleep, in part, through nNOS.

Sleep in mice with nonfunctional growth hormone-releasing hormone receptors.

The role of the somatotropic axis in sleep regulation was studied by using the lit/lit mouse with nonfunctional growth hormone (GH)-releasing hormone (GHRH) receptors (GHRH-Rs) and control heterozygous C57BL/6J mice, which have a normal phenotype. During the light period, the lit/lit mice displayed significantly less spontaneous rapid eye movement sleep (REMS) and non-REMS (NREMS) than the controls. Intraperitoneal injection of GHRH (50 microg/kg) failed to promote sleep in the lit/lit mice, whereas it enhanced NREMS in the heterozygous mice. Subcutaneous infusion of GH replacement stimulated weight gain, increased the concentration of plasma insulin-like growth factor-1 (IGF-1), and normalized REMS, but failed to restore normal NREMS in the lit/lit mice. The NREMS response to a 4-h sleep deprivation was attenuated in the lit/lit mice. In control mice, intraperitoneal injection of ghrelin (400 microg/kg) elicited GH secretion and promoted NREMS, and intraperitoneal administration of the somatostatin analog octretotide (Oct, 200 microg/kg) inhibited sleep. In contrast, these responses were missing in the lit/lit mice. The results suggest that GH promotes REMS whereas GHRH stimulates NREMS via central GHRH-Rs and that GHRH is involved in the mediation of the sleep effects of ghrelin and somatostatin.