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.

星形胶质细胞通过 ATP/ADP 和谷氨酸等神经胶质递质调节大脑功能，但它们的释放模式和机制仍然存在争议。在这里，我们可视化了星形胶质细胞活化后的 ATP/ADP 和谷氨酸反应，并在体内研究了它们的机制。采用 cOpn5 介导的光遗传学刺激、基因编码的荧光传感器和双光子成像，我们观察到 ATP/ADP 以时间延长和空间扩展闪光的形式释放，随后转化为腺苷。这种释放通过 Ca²⁺ 和 VNUT 依赖性囊泡胞吐作用发生。此外，星形胶质细胞还通过 TeNT 敏感的胞吐作用快速释放谷氨酸，与 ATP/ADP 释放无关。星形胶质细胞释放的 ATP/ADP 通过 P2Y12 和 VNUT 依赖性机制触发 ATP/ADP 从小胶质细胞进一步释放。星形胶质细胞和小胶质细胞中的 VNUT 也有助于在 LPS 诱导的脑炎症下释放 ATP/ADP。这些发现确定了 Ca²⁺ 依赖性囊泡胞吐作用是一种关键作用模式，揭示了错综复杂的星形胶质细胞-小胶质细胞相互作用，并表明神经胶质细胞传递在脑部炎症中的作用。此外，这些方法可能为破译神经胶质生理学和病理生理学提供有价值的工具。