Microglia stabilize sleep homeostasis via adenosine A 3 receptor signaling

Abstract Sleep homeostasis maintains the sleep-wake balance through sleep pressure, a process 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 Ca 2+ activity driven by adenosine A 3 receptor (A 3 R) signaling. Using miniaturized two-photon microscopy (mTPM) in freely behaving mice, we demonstrate that microglial Ca²⁺ activity is rapidly altered by brain-state transitions. Pharmacological experiments reveal that microglial Ca 2+ 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 Ca 2+ 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 regulate sleep homeostasis by stabilizing both wakefulness and NREM sleep, a process critically dependent on eADO-A 3 R signaling.