Synaptic Vesicle Recycling in Cultured Cerebellar Granule Cells: Role of Vesicular Acidification and Refilling

Open Access
Publisher Website
Google Scholar
Abstract
The role of the transvesicular protonmotive force in synaptic vesicle recycling was investigated in cultured cerebellar granule cells. The vesicular V‐ATPase was inhibited by 1 µM bafilomycin A1; as an alternative, the pH component of the gradient was selectively collapsed by equilibration of the cells with 10 mM methylamine and monitored with the fluorescent probe Lysosensor Green. Electrical field‐evoked exocytosis of d‐[3H]aspartate was inhibited by bafilomycin A1 but not by methylamine, indicating that a transvesicular membrane potential rather than pH gradient is required for transmitter retention within vesicles. In contrast, neither compound affected the field‐evoked uptake, recycling, or destaining of the vesicle‐specific dye FM2‐10; thus, vesicles whose lumens were neutral and/or depleted of transmitter could still recycle in the nerve terminal. No exhaustion of d‐[3H]aspartate exocytosis was observed when cells were subjected to six consecutive trains of field stimuli (40 Hz/10 s separated by 10 s). In contrast, the release of preloaded FM2‐10 was reduced by ∼50%, with each stimulus indicating that unlabeled vesicles with accumulated d‐[3H]aspartate were competing with labeled vesicles for exocytosis. As d‐[3H]aspartate was accumulated rapidly across the vesicle membrane from the large cytoplasmic pool, the transmitter‐loaded but unlabelled vesicles may represent refilled recycling vesicles. FM2‐10 destaining and d‐[3H]aspartate exocytosis were reduced in parallel at low frequencies, challenging a role for transient vesicle fusion.

This publication has 0 references indexed in Scilit: