Spontaneous and multifaceted ATP release from astrocytes at the scale of hundreds of synapses

Astrocytes participate in information processing by releasing neuroactive substances termed gliotransmitters, including ATP. Individual astrocytes come into contact with thousands of synapses with their ramified structure, but the spatiotemporal dynamics of ATP gliotransmission remains unclear, especially in physiological brain tissue. Using a genetically encoded fluorescent sensor, GRAB_ATP1.0, we discovered that extracellular ATP increased locally and transiently in absence of stimuli in neuron–glia co-cultures, cortical slices, and the anesthetized mouse brain. Spontaneous ATP release events were tetrodotoxin-insensitive but suppressed by gliotoxin, fluorocitrate, and typically spread over 50–250 μm² area at concentrations capable of activating purinergic receptors. Besides, most ATP events did not coincide with Ca²⁺ transients, and intracellular Ca²⁺ buffering with BAPTA-AM did not affect ATP event frequency. Clustering analysis revealed that these events followed multiple distinct kinetics, and blockade of exocytosis only decreased a minor group of slow events. Overall, astrocytes spontaneously release ATP through multiple mechanisms, mainly in non-vesicular and Ca²⁺-independent manners, thus potentially regulating hundreds of synapses all together.