Neurone-to-astrocyte communication by endogenous ATP in mixed culture of rat hippocampal neurones and astrocytes

ATP is recognized as an important intercellular signaling molecule in the peripheral and CNS. Glutamate is reported to be an important neurone-to-glia mediator being released from neurones and astrocytes that activates astrocytic and neuronal Ca²⁺ responses, respectively. We demonstrate here that endogenous ATP could be an extracellular molecule for neurone-to-astrocyte communication in cocultured rat hippocampal neurones and astrocytes. Hippocampal neurones reveal synchronized Ca²⁺ oscillation, which was due to glutamatergic synaptic transmission. When analyzed in a fura-2 method, a slight and very slow increase in intracellular Ca²⁺ concentration ([Ca²⁺]i) elevation was observed in some population of astrocytes. Such astrocytic [Ca²⁺]i elevation was dramatically inhibited by apyrase, though apyrase itself had no effect on neuronal Ca²⁺ oscillation. For a detail analysis, we investigated changes in [Ca²⁺]i in cells using a confocal microscopy. When cocultured hippocampal neurones and astrocytes were depolarized electronically in the presence of glutamate-receptor antagonists, a transient elevation in [Ca2+]i was observed in neurones, which was followed by a slowly initiated and small rise in [Ca²⁺]i in astrocytes. Apyrase or P2 receptor antagonists almost abolished the [Ca2+]i rises in astrocytes, suggesting that depolarization-evoked ATP release from neurones should produce astrocytic [Ca2+]i elevation via P2 receptors. Using a luciferin-luciferase bioluminescence assay, we found that neurones could release ATP in an activity-dependent manner. These findings suggest that endogenous ATP should be an important intercellular mediator between neurones and astrocytes and that functions of these cells should be finetuned by endogenously released ATP in situ.