Cerebral function depends on the transmission of chemical signals between neuronal elements at synapses. Over the last two decades, accumulating data have shown that this fundamental process is regulated by astrocytes. Among other functions, astrocytes participate in the clearance of ions and neurotransmitters. In addition, these glial cells release gliotransmitters to regulate synaptic functions such as basal synaptic transmission and long-term synaptic plasticity. Clearance and gliotransmission are not the only ways through which astrocytes influence neuronal communication. One interesting but under-investigated pathway would be through the release of extracellular vesicles (EVs). These vesicles are 30-1000 nm in diameter and contain proteins, lipids and nucleic acids. To understand the role of EVs during synaptic transmission, we first investigated whether acute stimulation of astrocytes with neurotransmitters can impact the quantity, size, and content of secreted EVs. We combine calcium imaging, EV quantification techniques, electronic microscopy and RNAseq analysis to address whether exposing primary mouse astrocyte cultures to 50 μM ATP, Glutamate or GABA for 30 min is sufficient to modify the number of EVs released and their miRNA content.