title: Synaptic vesicle recycling in cultured cerebellar granule cells: role of vesicular acidification and refilling.
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 microM 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 approximately 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.
authors.affiliation: Neurosciences Institute, Department of Pharmacology, Ninewells Medical School, University of Dundee, Scotland, U.K.
authors.forename: M A
authors.initials: MA
authors.lastname: Cousin
authors.forename: D G
authors.initials: DG
authors.lastname: Nicholls
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001224:Aspartic Acid / Q000378:metabolism*; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005089:Exocytosis; D005456:Fluorescent Dyes; D018942:Macrolides; D008561:Membrane Fusion; D008744:Methylamines / Q000494:pharmacology; D009411:Nerve Endings / Q000502:physiology; D016501:Neurites / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D009474:Neurons / Q000145:classification / Q000166:cytology / Q000502:physiology*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D051381:Rats; D017208:Rats, Wistar; D013572:Synaptic Vesicles / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D025262:Vacuolar Proton-Translocating ATPases
chemical_list: D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; C073804:FM1 43; D005456:Fluorescent Dyes; D018942:Macrolides; D008744:Methylamines; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D001224:Aspartic Acid; C040929:bafilomycin A1; D000255:Adenosine Triphosphate; C027451:methylamine; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases