Sleep loss changes microRNA levels in the brain : A possible mechanism for state-dependent translational regulation

MicroRNAs (miRNAs) are small (∼22 nucleotide) non-coding RNA strands that base pair with mRNA to degrade it or inhibit its translation. Because sleep and sleep loss induce changes in many mRNA species, we hypothesized that sleep loss would also affect miRNA levels in the brain. Rats were sleep-deprived for 8 h then decapitated; hippocampus, prefrontal and somatosensory cortices and hypothalamus tissues were harvested and frozen in liquid nitrogen. MiRNA was extracted and then characterized using microarrays. Several let-7 miRNA microarray results using hippocampus and prefrontal cortex samples were verified by PCR. From the array data it was determined that about fifty miRNA species were affected by sleep loss. For example, in the hippocampus of sleep-deprived rats, miRNA expression increased compared to cage control samples. In contrast, the majority of miRNA species in the somatosensory and prefrontal cortices decreased, while in the hypothalamus miRNA species were both up- and down-regulated after sleep deprivation. The number of miRNA species affected by sleep loss, their differential expression in separate brain structures and their predicted targets suggest that they have a role in site-specific sleep mechanisms. Current results are, to our knowledge, the first demonstration of the homeostatic process, sleep, altering brain miRNA levels

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

Attenuation of the influenza virus sickness behavior in mice deficient in Toll-like receptor 3

Certain sickness behaviors occur consistently in influenza-infected humans and mice. These include body temperature changes, somnolence, and anorexia. Several cytokines serve as mediators of the influenza acute phase response (APR), including these sickness behaviors, and one likely inducer of these cytokines is dsRNA produced during viral replication. TLR3 is known to be one of the host cellular components capable of recognizing dsRNA and activating cytokine synthesis. To determine the role of TLR3-detected viral dsRNA in the causation of viral symptoms, TLR3-deficient mice (TLR3 knockouts, or KOs) were infected with a marginally-lethal dose of mouse-adapted X-31 influenza virus. TLR3 KOs and their wild-type (WT) controls were monitored for baseline body temperature, locomotor activity, and sleep profiles prior to infection. Both mouse strains were then infected and monitored for changes in these sickness behaviors plus body weight changes and mortality for up to 14 days post-infection. Consistent with the observations that influenza pathology is reduced in TLR3 KOs, we showed that hypothermia after post-infection day 5 and the total loss of body weight were attenuated in the TLR3 KOs. Sleep changes characteristic of this infection model [particularly increased non-rapid-eye-movement sleep (NREMS)] were also attenuated in TLR3 KOs and returned to baseline values more rapidly. Locomotor activity suppression was similar in both strains. Therefore virus-associated dsRNA detected by TLR3 appears to play a substantial role in mediating several aspects of the influenza syndrome in mice

Olfactory Bulb and Hypothalamic Acute-Phase Responses to Influenza Virus: Effects of Immunization

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: Determine if immunization affects OB-localization of influenza or the molecular brain mechanisms regulating the 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 hour of viral challenge. However, it greatly attenuated OB viral RNA 6 days post-viral challenge and the APR including hypothermia and body weight loss responses. Within the OB, 24 hours after influenza challenge, prior immunization blocked virus-induced up-regulation of toll-like receptor 7 and interferon gamma (IFNγ) mRNAs. At this time, hypothalamic (HT) growth hormone releasing hormone receptor and tumor necrosis factor-alpha mRNAs were greatly enhanced in immunized but not in positive control mice. By 6 days post-viral challenge, OB and HT mRNAs returned towards baseline values. In lungs, mRNA up-regulations were greater than those in the brain and were maximized 6 days post-challenge. Lung IFNγ mRNA decreased at 24 hours but increased at 6 days post-challenge in the positive controls 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

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

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

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

Tumor necrosis factor-α (TNF-α) is associated with sleep regulation in health and disease. Previous studies assessed sleep in mice genetically deficient in the TNF-α 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-α 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-α 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-α is involved in physiological sleep regulation but plays a limited role in the acute-phase response induced by influenza virus