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

• Published in: The Journal of physiology 2011-09, Vol.589 (17), p.4271-4300
• Main Authors: Malarkey, Erik B., Parpura, Vladimir
• Format: Article
• Language: English
• Subjects: Astrocytes; Astrocytes - metabolism; Bradykinin; Calcium (intracellular); Cells, Cultured; Cortex (visual); Exocytosis; Glial cells; Mechanical stimuli; Membrane Fusion; Neuroscience: Cellular/Molecular; Plasma membranes; Proteins; Secretion; Secretory Vesicles - metabolism; SNAP-25 protein; Synapses; Synaptobrevin; Synaptotagmin; Vesicle fusion; Vesicle-Associated Membrane Protein 2 - metabolism
• ISSN: 0022-3751; 1469-7793
• DOI: 10.1113/jphysiol.2011.210435

Non‐technical summary  Astrocytes have been shown to release transmitters by vesicle fusion, in a manner similar to that of neuronal exocytosis. The details of this process in astrocytes are not well understood, so we used a fluorescently labelled vesicle protein, synapto‐pHluorin (spH), to track how these fusions occurred. When astrocytes were mechanically stimulated we saw a slow burst of fusions, while other stimuli caused a relatively even sustained rate of fusion. We observed two distinct types of events, transient and full fusions, the proportion of which was stimulus dependent. Similarly, stability of the vesicle fusion pore with the plasma membrane varied with the stimulus. We describe the effects on fusion events resulting from expressing variants of exocytotic proteins, synaptotagmin 1 and SNAP25B. Studying the characteristics of astrocytic exocytosis will aid in the general understanding of this process and also events at the tripartite synapse, both in health and disease.   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 h