title: Perturbation of synaptic vesicle delivery during neurotransmitter release triggered independently of calcium influx.
abstract: Although much evidence suggests that calcium (Ca(2+)) usually triggers synaptic vesicle exocytosis and neurotransmitter release, the role of Ca(2+) in vesicle endocytosis and in the delivery of fusion-competent vesicles (i.e. mobilisation and/or priming) in nerve terminals remains unclear. To address this issue, we have studied synaptic vesicle dynamics in cultured rat neurones under conditions where neurotransmitter release is triggered independently of Ca(2+) using the secretagogue Ruthenium Red (RR). Using a prolonged stimulation protocol, we find that RR causes a rapid increase in neurotransmitter release followed by a gradually decrementing response. In contrast, when release is triggered by moderate membrane depolarisation caused by saline containing 18 mM K(+), release is sustained. These observations suggest that when release is triggered independently of a rise in Ca(2+), endocytosis or vesicle mobilisation/priming are perturbed. Using FM2-10, a fluorescent indicator of synaptic vesicle cycling, we find that neurotransmitter release triggered by RR is accompanied by both uptake and release of this dye, thereby suggesting that vesicle endocytosis is not blocked. To evaluate whether synaptic vesicle mobilisation/priming is perturbed in the absence of a rise in Ca(2+), we compared the kinetics of FM2-10 loss during prolonged stimulation. While 18 mM K(+) induced gradual and continuous dye loss, RR only induced substantial dye loss during the first minute of stimulation. In the presence of low concentrations of the Ca(2+) ionophore ionomycin, release caused by RR was prolonged. Taken together, these results provide evidence suggesting that, although a rise in intraterminal Ca(2+) is not required for endocytosis, it is essential for the continuous delivery of fusion-competent vesicles and to maintain neurotransmitter release during prolonged stimulation.
authors.affiliation: Département de Pharmacologie, Centre de Recherche en Sciences Neurologiques and Centre de Recherche Fernand Seguin, Faculté de Médecine, Université de Montréal, Québec, Canada.
authors.forename: Patrice
authors.initials: P
authors.lastname: Congar
authors.forename: Louis-Eric
authors.initials: LE
authors.lastname: Trudeau
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004357:Drug Synergism; D004705:Endocytosis / Q000502:physiology; D019706:Excitatory Postsynaptic Potentials / Q000187:drug effects; D007425:Intracellular Membranes / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D009474:Neurons / Q000502:physiology; D018377:Neurotransmitter Agents / Q000378:metabolism*; D017729:Presynaptic Terminals / Q000378:metabolism; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D051381:Rats; D017207:Rats, Sprague-Dawley; D012430:Ruthenium Red / Q000494:pharmacology; D013572:Synaptic Vesicles / Q000502:physiology*
chemical_list: C086686:FM2 10; D007476:Ionophores; D018377:Neurotransmitter Agents; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D012430:Ruthenium Red; D015759:Ionomycin; D002118:Calcium