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Single-cell transcriptomics and analysis for new molecular regulators of sleep

ABSTRACT: The molecular mechanisms governing sleep are largely unknown. Here, we used a combination of single-cell RNA sequencing to interrogate the molecular and functional underpinnings of sleep. Different cell types in three important brain regions for sleep (brainstem, cortex and hypothalamus) had a similar transcriptional response to sleep need, with a large proportion of cells changing during recovery sleep. In contrast, sleep deprivation regulated expression of different functions in each brain region. This includes antigen processing, synaptic transmission and cellular metabolism in brainstem, cortex and hypothalamus, respectively. Increased sleep need enhances expression of the transcription factors Sox2, Mafb, and Zic1 in brainstem; Hlf, Cebpb and Sox9 in cortex, and Atf3, Fosb and Mef2c in hypothalamus. In turn, these transcription factors regulate downstream gene expression during sleep deprivation and recovery. In cortex, we also interrogated the proteome of two major cell types: neurons and astrocytes. We found surprising functional overlap of proteins that mediate vesicle and neurotransmitter transport in both cell types. In contrast, other functions were specific to each cell type.

ORGANISM(S): Mus musculus

PROVIDER: GSE137665 | GEO | 2022/08/24

REPOSITORIES: GEO

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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 Â±1 week. Animals were housed in a light/dark cycle of 12 hrs, in a pathogen free, temperature- and humidity-controlled room (22Â°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.

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Single-cell transcriptomics and analysis for new molecular regulators of sleep

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