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.

调控睡眠的分子机制在很大程度上仍未明确。本研究结合单细胞RNA测序（single-cell RNA sequencing）技术，探究睡眠的分子与功能基础。睡眠相关的三个关键脑区（脑干、大脑皮层、下丘脑）内的不同细胞类型，对睡眠需求展现出相似的转录响应，且在恢复睡眠阶段有大量细胞发生表达改变。与之相对，睡眠剥夺对各脑区的功能基因表达调控存在特异性差异：脑干、大脑皮层及下丘脑分别调控抗原加工、突触传递与细胞代谢相关基因的表达。当睡眠需求升高时，脑干内的转录因子Sox2、Mafb及Zic1的表达会上调；大脑皮层内的Hlf、Cebpb与Sox9，以及下丘脑内的Atf3、Fosb及Mef2c的表达亦会升高。这些转录因子可在睡眠剥夺与恢复阶段调控下游基因的表达。在大脑皮层中，本研究还探究了两种主要细胞类型（神经元与星形胶质细胞）的蛋白质组。研究意外发现，两种细胞内介导囊泡与神经递质转运的蛋白质存在功能重叠；而其他功能则呈现细胞类型特异性。整体实验设计：为评估12小时睡眠剥夺对小鼠睡眠-觉醒行为的影响，研究先让小鼠适应环境一周，随后记录3天的脑电图（EEG）与肌电图（EMG）。后续连续记录3~4天，覆盖睡眠剥夺与恢复阶段。睡眠剥夺于光照开启时[授时因子时间0（Zeitgeber Time 0, ZT0）]启动，通过开启电机并选择持续清扫模式实现。睡眠剥夺结束后，允许小鼠恢复睡眠24小时。睡眠剥夺前1天的基础记录作为对照组（正常睡眠组）。随后通过颈椎脱臼法处死小鼠，于ZT12（即关灯时刻）分离全脑，分别获取自由正常睡眠组（NS, ZT0-12）、睡眠剥夺组（SD, ZT0-12）以及12小时睡眠剥夺后伴随12小时恢复睡眠组（RS, ZT12-12）的脑组织。快速剥离脑干、大脑皮层与下丘脑组织，收集后用于制备单细胞悬液。三个脑区的单细胞悬液制备流程均参考Holt及其同事的方法。具体而言，组织先于厂商提供的培养基中用木瓜蛋白酶解离，随后使用经火焰抛光的硅化巴斯德吸管进行手动吹打，再通过70μm细胞滤器过滤。以300×g离心10分钟沉淀细胞，弃去上清后，用含0.5%牛血清白蛋白（BSA）的少量磷酸盐缓冲液（PBS）重悬细胞。为减少细胞碎片，将细胞悬液与髓鞘去除磁珠共孵育，随后通过带有70μm预分离滤器的LS分选柱。收集流出的单细胞悬液，并对细胞产量与活性进行评估。将单细胞悬液用含0.5% BSA的PBS稀释至约350个细胞/微升，随后遵循10X Genomics Chromium单细胞试剂盒V2的操作手册进行细胞捕获、cDNA合成、文库制备与测序。