Temporal characteristics of vesicular fusion in astrocytes: examination of synaptobrevin 2-laden vesicles at single vesicle resolution

Astrocytes can release various gliotransmitters in response to stimuli that cause increases in intracellular Ca2+ levels; this secretion occurs via a regulated exocytosis pathway. Indeed, astrocytes express protein components of the vesicular secretory apparatus. However, the detailed temporal characteristics of vesicular fusions in astrocytes are not well understood. In order to start addressing this issue, we used total internal reflection fluorescence microscopy (TIRFM) to visualize vesicular fusion events in astrocytes expressing the fluorescent synaptobrevin 2 derivative, synapto-pHluorin. Although our cultured astrocytes from visual cortex express synaptosome-associated protein of 23 kDa (SNAP23), but not of 25 kDa (SNAP25), these glial cells exhibited a slow burst of exocytosis under mechanical stimulation; the expression of SNAP25B did not affect bursting behaviour. The relative amount of two distinct types of events observed, transient and full fusions, depended on the applied stimulus. Expression of exogenous synaptotagmin 1 (Syt1) in astrocytes endogenously expressing Syt4, led to a greater proportion of transient fusions when astrocytes were stimulated with bradykinin, a stimulus otherwise resulting in more full fusions. Additionally, we studied the stability of the transient fusion pore by measuring its dwell time, relation to vesicular size, flickering and decay slope; all of these characteristics were secretagogue dependent. The expression of SNAP25B or Syt1 had complex effects on transient fusion pore stability in a stimulus-specific manner. SNAP25B obliterated the appearance of flickers and reduced the dwell time when astrocytes were mechanically stimulated, while astrocytes expressing SNAP25B and stimulated with bradykinin had a reduction in decay slope. Syt1 reduced the dwell time when astrocytes were stimulated either mechanically or with bradykinin. Our detailed study of temporal characteristics of astrocytic exocytosis will not only aid the general understanding of this process, but also the interpretation of the events at the tripartite synapse, both in health and disease.