Microglia stabilize sleep homeostasis via adenosine A3 receptor signaling.

Sleep homeostasis maintains the sleep-wake balance through sleep pressure, a process partly orchestrated by the accumulation of extracellular adenosine (eADO). Microglial Ca2+ activity has been implicated in sleep regulation, but the mechanism whereby microglia sense sleep pressure remains unclear. Here, we show that microglia regulate sleep homeostasis through brain state-dependent calcium ion activity driven by adenosine A3 receptor (A3R) signaling. Using miniaturized two-photon microscopy in freely behaving mice, we demonstrate that microglial calcium ion activity is rapidly altered by brain-state transitions. Pharmacological experiments reveal that microglial calcium ion dynamics are predominantly mediated by A3R in response to brain state-dependent eADO oscillations. Microglia-specific deletion of A3R attenuates these state-dependent calcium ion dynamics, impairs microglial morphological plasticity across sleep-wake cycles, and leads to sleep fragmentation by increasing transitions between wakefulness and non-rapid eye movement (NREM) sleep. Together, these findings establish that microglia contribute to sleep homeostasis by stabilizing both wakefulness and NREM sleep, a process partly involving eADO-A3R signaling.