Transcriptional Responses to Sleep in Peripheral Tissues (Heart)
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ABSTRACT: Molecular profiles in sleep and sleep deprivation in peripheral tissues using microarrays Time point study. Mice were sacrificed by cervical dislocaton following 3, 6, 9, and 12 h of total sleep deprivation (n = 8 or 9 at each time point). Deprivation was initiated at lights-on and performed through gentle handling.
Project description:Molecular profiles in sleep and sleep deprivation in peripheral tissues using microarrays Time point study. Mice were sacrificed by cervical dislocaton following 3, 6, 9, and 12 h of total sleep deprivation (n = 8 or 9 at each time point). Deprivation was initiated at lights-on and performed through gentle handling.
Project description:These studies address temporal changes in gene expression during spontaneous sleep and extended wakefulness in the mouse cerebral cortex, a neuronal target for processes that control sleep; and the hypothalamus, an important site of sleep regulatory processes. We determined these changes by comparing expression in sleeping animals sacrificed at different times during the lights on period, to that in animals sleep deprived and sacrificed at the same diurnal time. Experiment Overall Design: Experiments were performed on male mice (C57/BL6), 10 weeks of age ±1 week. Animals were housed in a light/dark cycle of 12 hrs, in a pathogen free, temperature- and humidity-controlled room (22°C and 45-55%, respectively) with water available ad libitum. Food was accessible for 12 hrs only during the active period. Animals were subjected to 14 days of acclimatization during which a nighttime feeding pattern was established. This was done to avoid differential food intake between mice that were subsequently sleep deprived during the lights on period and those allowed to sleep. Mice were sacrificed following 3, 6, 9 and 12 hrs of total sleep deprivation (n=5 at each time point). Deprivation was initiated at lights-on, and performed through gentle handling. Sleeping animals, which were left undisturbed, were sacrificed at the same diurnal time points as sleep deprived mice (n=5 at each time point). An additional control group of mice were sacrificed at time zero, i.e., at the time of lights-on (n=5). All mice were behaviorally monitored using the AccuScan infrared monitoring system that detects movement when the mouse crosses electronic beams (Columbus Instruments). Mice were sacrificed by cervical dislocation. Brain sectioning was performed according to the mouse brain atlas of Franklin and Paxinos . The primary and secondary motor areas (M1 and M2) of the cerebral cortex and broadly defined regions and zones of the hypothalamus were sampled. RNA was isolated with Trizol (Invitrogen) and further purified using RNeasy columns (Qiagen) as per the manufacturer's instructions.
Project description:We hybridzed cRNA from epididymal white adipose tissue collected at ZT18 of control animals and TSR animals (TSR: these mice were sleep restricted for 6 hours every day by gentle handling for 5 consecutive days and killed on the last day at ZT18) mice used in this study were C57BL/6 control mice were compared to timed sleep restriction mice (TSR: these mice were sleep restricted for 6 hours every day by gentle handling for 5 consecutive days and killed on the last day at ZT18)
Project description:To gain insight into the molecular changes of sleep need, this study addresses gene expression changes in a subpopulation of neurons selectively activated by sleep deprivation. Whole brain expression analyses after 6h sleep deprivation clearly indicate that Homer1a is the best index of sleep need, consistently in all mouse strains analyzed. Transgenic mice expressing a FLAG-tagged poly(A)-binding protein (PABP) under the control of Homer1a promoter were generated. Because PABP binds the poly(A) tails of mRNA, affinity purification of FLAG-tagged PABP proteins from whole brain lysates, is expected to co-precipitate all mRNAs from neurons expressing Homer1a. Three other activity-induced genes (Ptgs2, Jph3, and Nptx2) were identified by this technique to be over-expressed after sleep loss. All four genes play a role in recovery from glutamate-induced neuronal hyperactivity. The consistent activation of Homer1a suggests a role for sleep in intracellular calcium homeostasis for protecting and recovering from the neuronal activation imposed by wakefulness. Experiment Overall Design: Experiments were performed on male mice, 12 weeks of age +/- 1 week. Animals were housed in polycarbonate cages (31x18x18cm) in an experimental room with an ambient temperature varying from 23° to 25°C under a 12:12 hrs light/dark cycle. Food and water were available ad libitum. At light onset mice were either sleep deprived by gentle handling (n=10) or left undisturbed (n=10) for 6 hrs. Animals were then randomly sacrificed by cervical dislocation. Total RNA from the whole brain was isolated for control (n=4) and sleep deprived (n=4) using a commercial RNA extraction kit (RNeasy Lipid Tissue Kit, Quiagen). Specific Homer1a-expressing cells polyA RNAs were immunoprecipitated following the total brain crosslinking (1% formaldehyde perfusion) for sleep deprived (n=6) and control (n=6) animals. The total RNA from the pull-down supernatants were also harvested (n=4). To test for transcriptional changes after sleep deprivation Homer1a-expressing cells, we proceeded in 2 steps: (1) identify probe sets enriched in the pull-down extracts, (2) among those probe sets compare sleep deprivation to control condition in both pull-down (6 vs. 6 chip comparison) and whole-brain (4 vs. 4 chip comparison) extracts. 4728 probe sets were significantly enriched at 5% FDR when pull-downs were compared to both supernatant and whole-brain extracts.
Project description:Why we sleep is still one of the most perplexing mysteries in biology. Strong evidence, however, indicates that sleep is necessary for normal brain function and that the need to sleep is a tightly regulated process. Surprisingly molecular mechanisms that determine the need to sleep are incompletely described. Moreover, very little is known about transcriptional changes that specifically accompany the accumulation and discharge of sleep need. In this study we present an integrated 2 cross-laboratory analysis of the effects of sleep deprivation (SD) in gene expression in the mouse cortex. We also evaluate changes in gene expression genome-wide following various lengths of subsequent recovery sleep. (RS). We demonstrate that changes in gene expression specifically linked to SD or RS, and not to technical factors (e.g. the assay used), requires a novel analysis methodology not previously utilized in the field of sleep research. Cortical samples from mice were analyzed, from groups that were sleep deprived, sleep deprived and allowed to recover for 1, 2, 3, or 6 hours, and circadian control animals that were not sleep deprived. The experimental protocol began at lights on (ZT0) with 13 mice: 1 sacrificed, 4 control mice left undisturbed, and 8 mice kept awake with gentle brushing when attempting to sleep. After 5 hours of sleep deprivation the mice were randomly assigned to recovery sleep or continued sleep deprivation, and at fixed intervals the mice were sacrificed, dissected and the left cortex retained. The experimental protocol was repeated 7 times, one animal per timepoint per experimental day, so that 7 independent experiments are represented for each timepoint. All animals were acclimated to the brushing and tapping on cages used during sleep deprivationfor 6 days, and dissections and tissue collection were performed by a single experimenter.
Project description:Galanin neurons at ventrolateral preoptic nuleus (VLPO) have been hypothesized to be sleep-active neurons. With the use of laser capture microdissection (LCM), we specifically isolated single galanin neurons, and our goal was to assess expression changes in VLPO galanin cells between sleeping mice and mice kept awake using gentle handling.
Project description:We hybridzed cRNA from epididymal white adipose tissue collected at ZT18 of control animals and TSR animals (TSR: these mice were sleep restricted for 6 hours every day by gentle handling for 5 consecutive days and killed on the last day at ZT18) mice used in this study were C57BL/6
Project description:Sleep deprivation (SD) performed before stroke induces an ischemic tolerance state as observed in other forms of preconditioning. As the mechanisms underlying this effect are not well understood we used DNA oligonucleotide microarray analysis, to identify the genes and the gene-pathways underlying SD preconditioning. Gene expression was analyzed 3 days after stroke surgery in four experimental groups: i) SD performed before focal cerebral ischemia induction; ii) SD performed before Sham surgery; iii) IS without SD; and iv) Sham surgery without SD. SD was performed by gentle handling during the last 6h of the light period and ischemia was induced immediately after. Stroke induced a massive alteration in gene expression both in sleep deprived and non-sleep deprived animals. However, compared to animals that underwent ischemia alone, SD induced a general reduction in transcriptional changes with a reduction in the upregulation of genes involved in cell cycle regulation and immune response. Moreover an upregulation of a new neuroendocrine pathway which included melanin concentrating hormone, glycoprotein hormones-M-kM-1-polypeptide and hypocretin was observed exclusively in rats sleep deprived before stroke. Our data indicate that SD before stroke reprogrammed the signaling response to injury. The inhibition of cell cycle regulation and inflammation are neuroprotective mechanisms reported also for other forms of preconditioning treatment whereas the implication of the neuroendocrine function is novel and has never been described before. These results therefore provide new insights into neuroprotective mechanisms involved in ischemic tolerance mechanisms.
Project description:Analysis of the effects of sleep deprivation, recovery sleep, and three time-of-day controls on seven brain regions laser microdissected from mouse brain. The regions include the locus coeruleus, suprachiasmatic nucleus, hypocretin area, tuberomammillary nucleus, orbital cortex, posteromedial cortical amygdala, and entorhinal cortex. In this study, 7 brain regions were collected by laser microdissection from brain tissue of mice from 5 different treatment groups and used for microarray experiments. Four biological replicates were generated for each regionxcondition. Conditions are: SD, sleep deprivation for 6 hours from ZT0 - 6; SDC, time-of-day control for SD at ZT6; RS, recovery sleep for 4 hours following SD; RSC, time-of-day control for RS at ZT10; W, spontaneous waking at ZT18.
Project description:Sleep loss regulates gene expression throughout the brain and impacts learning and memory. However, the molecular consequences of sleep deprivation and the ability of subsequent sleep (recovery sleep) to restore baseline gene expression remain underexplored. Our goal here is to overview transcriptional changes at the gene level in the cortex of adult male wildtype mice in response to sleep loss and recovery sleep. This dataset constitutes an integration of novel data with two publicly available RNA-seq studies and contains 3, 5, and 6 hours of sleep deprivation and 2 and 6 hours of recovery sleep time points.