INTRODUCTION: The original concept of the tripartite synapse assumes that gliotransmitters are released on a way of exocytosis. Despite years of research, the involvement of astrocytic regulated exocytosis in synaptic processes remains a matter of debate. For instance, there is still no clear link between Ca2+ signaling in astrocytes and gliotransmission.

AIM(S): We aim to verify if regulated exocytosis exists in astrocytes and what are the mechanisms regulating this process.

METHOD(S): We used mix hippocampal culture of neurons and astrocytes. To monitor exocytosis of small vesicles, we have used genetically encoded exocytosis sensor consisting of VAMP2 in fusion with pH-dependent GFP (Superecliptic pH-luorin). We have used Total Internal Reflection Fluorescence (TIRF) Microscopy in combination with electrical stimulation of the culture, to elucidate neuronal-activity dependence of astrocytic exocytosis.

RESULTS: Our results demonstrate that the rate of exocytosis is significantly lower in mix cultures compared to pure astrocytic cultures, which have been used in most previous studies. Furthermore, using electrical stimulation, we show that neuronal activity increases the frequency of exocytosis and that this process can be blocked by TTX. Our findings indicate that exocytosis in astrocytes is calcium-dependent, with the primary source of Ca2+ being extracellular rather than from the endoplasmic reticulum. Additionally, we highlight the involvement of AMPA receptors in the regulation of exocytosis.

CONCLUSIONS: Our findings provide insight into certain mechanisms of exocytosis regulation in astrocytes that warrant further investigation.

FINANCIAL SUPPORT: National Science Centre, Poland grants: 2017/26/D/NZ3/01017 and 2023/51/B/NZ4/02135