Synaptic vesicle recycling in cultured cerebellar granule cells: Role of vesicular acidification and refilling

Michael A. Cousin, David G. Nicholls*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract / Description of output

The role of the transvesicular protonmotive force n 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 tureens were neutral and/or depleted of transmitter could still recycle n 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.

Original languageEnglish
Pages (from-to)1927-1935
Number of pages9
JournalJournal of Neurochemistry
Issue number5
Publication statusPublished - Nov 1997

Keywords / Materials (for Non-textual outputs)

  • D-[H]Aspartate
  • Exocytosis
  • FM1-43
  • Granule cell
  • Refilling
  • Vesicle


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