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The Journal of Neuroscience, February 22, 2006, 26(8):2313-2320; doi:10.1523/JNEUROSCI.4425-05.2006

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Cellular/Molecular
The Kinetics of Synaptic Vesicle Reacidification at Hippocampal Nerve Terminals

Pradeep P. Atluri and Timothy A. Ryan

Department of Biochemistry, Weill Medical College of Cornell University, New York, New York 10021

Correspondence should be addressed to Timothy A. Ryan, Department of Biochemistry, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021. Email: taryan{at}med.cornell.edu

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 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 ( 4–5 s) and that their rate of reacidification is subject to slowing by increased external buffer.

Key words: hippocampus; fluorescence microscopy; synaptic vesicle release; optical imaging; synaptic transmission; synaptic

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Received Oct. 17, 2005; revised Jan. 11, 2006; accepted Jan. 12, 2006.

Correspondence should be addressed to Timothy A. Ryan, Department of Biochemistry, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021. Email: taryan{at}med.cornell.edu

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