期刊
JOURNAL OF NEUROSCIENCE
卷 26, 期 8, 页码 2313-2320出版社
SOC NEUROSCIENCE
DOI: 10.1523/JNEUROSCI.4425-05.2006
关键词
hippocampus; fluorescence microscopy; synaptic vesicle release; optical imaging; synaptic transmission; synaptic
资金
- NINDS NIH HHS [R01 NS036942] Funding Source: Medline
After exocytosis, synaptic vesicles are recycled locally in the synaptic terminal and are refilled with neurotransmitter via vesicular transporters. The biophysical mechanisms of refilling are poorly understood, but it is clear that the generation of a proton gradient across the vesicle membrane is crucial. To better understand the determinants of vesicle refilling, we developed a novel method to measure unambiguously the kinetics of synaptic vesicle reacidification at individual synaptic terminals. Hippocampal neurons transfected with synapto-pHluorin (SpH), a synaptic vesicle-targeted lumenal GFP ( green fluorescent protein), whose fluorescence is quenched when protonated (pK(a) similar to 7.1), were rapidly surface-quenched immediately after trains of repetitive electrical stimulation. The recently endocytosed alkaline pool of SpH is protected from such surface quenching, and its fluorescence decay reflects reacidification kinetics. These measurements indicate that, after compensatory endocytosis, synaptic vesicles reacidify with first-order kinetics (tau similar to 4 - 5 s) and that their rate of reacidification is subject to slowing by increased external buffer.
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