SNARE protein-dependent glutamate release from astrocytes

We investigated the cellular mechanisms underlying the Ca2+ dependent relea se of glutamate from cultured astrocytes isolated from rat hippocampus. Usi ng Ca2+ imaging and electrophysiological techniques, we analyzed the effect s of disrupting astrocytic vesicle proteins on the ability of astrocytes to release glutamate and to cause neuronal electrophysiological responses, i. e., a slow inward current (SIC) and/or an increase in the frequency of mini ature synaptic currents. We found that the Ca2+-dependent glutamate release from astrocytes is not caused by the reverse operation of glutamate transp orters, because the astrocyte-induced glutamate-mediated responses in neuro ns were affected neither by inhibitors of glutamate transporters (beta-thre o-hydroxyaspartate, dihydrokainate, and L-trans-pyrrolidine-2,4-dicarboxyla te) nor by replacement of extracellular sodium with lithium. We show that C a2+-dependent glutamate release from astrocytes requires an electrochemical gradient necessary for glutamate uptake in vesicles, because bafilomycin A (1), a vacuolar-type H+-ATPase inhibitor, reduced glutamate release from as trocytes. Injection of astrocytes with the light chain of the neurotoxin Bo tulinum B that selectively cleaves the vesicle-associated SNARE protein syn aptobrevin inhibited the astrocyte-induced glutamate response in neurons. T herefore, the Ca2+-dependent glutamate release from astrocytes is a SNARE p rotein-dependent process that requires the presence of functional vesicle-a ssociated proteins, suggesting that astrocytes store glutamate in vesicles and that it is released through an exocytotic pathway.