Astrocytes release ATP/ADP and glutamate in flashes via vesicular exocytosis

Astrocytes regulate brain functions through gliotransmitters like ATP/ADP and glutamate, but their release patterns and mechanisms remain controversial. Here, we visualized ATP/ADP and glutamate response following astrocyte activation and investigated their mechanisms in vivo. Employing cOpn5-mediated optogenetic stimulation, genetically encoded fluorescent sensors, and two-photon imaging, we observed ATP/ADP released as temporally prolonged and spatially extended flashes that later converted to adenosine. This release occurs via Ca²⁺ and VNUT-dependent vesicular exocytosis. Additionally, astrocytes also release glutamate in flashes through TeNT-sensitive exocytosis, independent of ATP/ADP release. ATP/ADP released by astrocytes triggers further ATP/ADP release from microglia through P2Y12- and VNUT-dependent mechanisms. VNUT in astrocytes and microglia also contributes to ATP/ADP release under LPS-induced brain inflammation. These findings establish Ca²⁺-dependent vesicular exocytosis as a key mode of action, reveal intricate astrocyte-microglia interactions, and suggest a role for gliotransmission in brain inflammation. Furthermore, the methodologies may provide valuable tools for deciphering glial physiology and pathophysiology.