Fusion Properties of Gliotransmitter Vesicles in Cultured Astrocytes

Abstract

Astrocytes actively participate in brain signalling by releasing a plethora of gliotransmitters which can modulate synaptic transmission. Vesicle-based mechanisms mediate the release of gliotransmitters. However, the anatomy and nature of exocytotic vesicle interaction with the plasma membrane is unclear. Using STED and SIM super-resolution microscopies, we studied the morphology of distinct gliotransmitter vesicles, whereas the interaction between a single vesicle with the plasma membrane was monitored by measurements of membrane capacitance (Cm), a parameter linearly related to the surface area of the plasma membrane. Immunolabelling of vesicles containing D-serine, glutamate, atrial natriuretic peptide (ANP) and brain derived neurotrophic factor (BDNF) yielded their diameter to be ∼70 nm, whereas ATP was found in larger vesicles (∼200 nm diameter). Cell-attached measurements have shown the predominant reversible unitary exocytotic events exhibiting transient fusion were found in two different populations, the first corresponding to smaller vesicles with diameters around 70 nm and the second corresponding to bigger vesicles with the median at 200 nm, consistent with the STED and SIM measurements. Upon stimulation with ATP, which increases intracellular Ca2+ concentration, the smaller vesicles persisted in transient fusion, whereas the bigger vesicles proceeded to full fusion. This was interpreted previously to be due to different SNARE protein densities in different sized vesicles and was tested here by astrocyte treatment with botulinum neurotoxin D (BotD), which significantly reduced the occurrence of unitary exocytotic events for both vesicle types. Additionally, when we expressed dominant-negative SNARE in astrocytes, the fusion-pore diameter was narrower in both vesicle types. Taken together, this work shows that vesicle content discharge is modulated by vesicle size, however independently of the functional integrity of the SNARE proteins, indicating that SNARE-dependent mechanisms determining vesicle merger with the plasmalemma are not strictly part of those modulating fusion-pore geometry.