Astrocytes and Neurons cell-specific transcriptomics

Sleep regulation follows a homeostatic pattern. The mammalian cerebral cortex is the repository of homeostatic sleep drive and neurons and astrocytes of the cortex are principal responders of sleep need. The molecular mechanisms by which these two cell types respond to sleep loss are not yet clearly understood. By combining cell-type specific transcriptomics and nuclear proteomics we investigated how sleep loss affects the cellular composition and molecular profiles of these two cell types in a focused approach. The results indicate that sleep deprivation regulates gene expression and nuclear protein abundance in a cell-type-specific manner. Our integrated multi-omics analysis suggests that this distinction arises because neurons and astrocytes employ different gene regulatory strategies under accumulated sleep pressure. These findings provide a comprehensive view of the effects of sleep deprivation on gene regulation in neurons and astrocytes. Overall design: Experimental design and sleep deprivation: Animals were divided into ad libitum Normal Sleep (NS) and Sleep Deprived (SD) groups. The NS group was left undisturbed SD group was sleep deprived by using a device that applied tactile stimulus with a horizontal bar sweeping just above the cage floor (bedding), as described previously 13. Once the sweeper was on, animals needed to step over it to continue their normal activities. Sleep deprivation began at the start of the light cycle (Zeitgeber Time 0 (ZT0)) and lasted for 12 h, with continuous sweeping mode (approximately 7.5 s cycle time). We made additional attempts to maintain wakefulness during the second half of the sleep deprivation period by occasionally tapping on the cage or gently touching the animals with a brush. Following sacrifice by cervical dislocation, whole brains were isolated at ZT12 (i.e. lights off). Isolated whole brains were placed in ice-cold Hibernate EB (BrainBits LLC, HEB) media, and the cerebral cortex was quickly dissected for the preparation of single-cell suspensions.