Microglia stabilize sleep homeostasis via adenosine A 3 receptor signaling

Sleep homeostasis maintains the sleep-wake balance through sleep pressure, a process partly orchestrated by the accumulation of extracellular adenosine (eADO). Microglial Ca 2+ 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 A 3 receptor (A 3 R) 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 A 3 R in response to brain state–dependent eADO oscillations. Microglia-specific deletion of A 3 R 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-A 3 R signaling.