GSE137665
Single-cell transcriptomics and analysis for new molecular regulators of sleep
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. Overall design: To assess how 12-h of sleep deprivation affected the sleep/wake behaviours, 3 days baseline EEG/EMG were recorded after mice were acclimatized for a week. Mice were recorded from for next 3-4 days (which encompassed sleep deprivation and recovery phases). Sleep deprivation was initiated at lights on [Zeitgeber Time 0 (ZT0)] by switching on the motors, choosing the continuous sweeping mode. Post sleep deprivation, animals were allowed to recover for 24-h. Baseline recording 1-day before sleep deprivation was used as the control condition (normal sleep). Following sacrifice by cervical dislocation, whole brains were isolated at ZT12 (i.e. lights off) for ad libitum Normal Sleep (NS, ZT0-12), Sleep Deprivation (SD, ZT0-12) and 12-h sleep deprivation followed by 12-h of Recovery Sleep (RS, ZT12-12) groups. Brainstem, cortex and hypothalamus were quickly dissected and tissue collected for preparation of single cell suspensions. The single cell suspensions preparation procedure for all the three brain regions was adapted from Holt and colleagues. Briefly, tissues were dissociated with Papain in the manufacturer supplied medium followed by manual trituration using a fire polished silanized Pasteur pipette and filtration through 70µm cell strainer. Cells were pelleted at 300 x g, 10 min, the supernatant was discarded, and cells resuspended in a minimal volume of PBS with 0.5% of BSA. To reduce the debris, we incubated the cell suspensions with myelin removal beads and passed it through an LS Column with 70µm pre-separation filter. Flow-through containing single cell suspension were collected and estimated the yield and viability. Single cell suspension were diluted in PBS with 0.5% of BSA to obtain ~ 350 cells/ µl and followed the 10X Genomics Chromium Single Cell Kit Version 2 manual for the capture, cDNA synthesis, library preparation, and sequencing.