Abstract
Using pHluorin-tagged synaptic vesicle proteins we have examined the partitioning of these probes into recycling and nonrecycling pools at hippocampal nerve terminals in cell culture. Our studies show that for three of the major synaptic vesicle components, vGlut-1, VAMP-2, and Synaptotagmin I, approximately 50-60% of the tagged protein appears in a recycling pool that responds readily to sustained action potential stimulation by mobilizing and fusing with the plasma membrane, while the remainder is targeted to a nonrecycling, acidic compartment. The fraction of recycling and nonrecycling (or resting) pools varied significantly across boutons within an individual axon, from 100% resting (silent) to 100% recycling. Single-bouton bleaching studies show that recycling and resting pools are dynamic and exchange between synaptic boutons. The quantitative parameters that can be extracted with the approaches outlined here should help elucidate the potential functional role of the resting vesicle pool.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Action Potentials / physiology
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Animals
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Animals, Newborn
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Cells, Cultured
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Electric Stimulation / methods
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Electrophysiology / methods
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Green Fluorescent Proteins / genetics
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Green Fluorescent Proteins / metabolism*
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Hippocampus / cytology
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Membrane Fusion / physiology
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Microscopy, Confocal / methods
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Microscopy, Fluorescence / methods
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Neurons / cytology
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Neurons / metabolism
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Neurons / physiology*
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Presynaptic Terminals / metabolism
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Presynaptic Terminals / physiology
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Rats
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Rats, Sprague-Dawley
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Synapses / metabolism
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Synapses / physiology*
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Synaptic Transmission / physiology*
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Synaptic Vesicles / metabolism
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Synaptic Vesicles / physiology
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Synaptotagmin I / genetics
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Synaptotagmin I / metabolism
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Vesicle-Associated Membrane Protein 2 / genetics
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Vesicle-Associated Membrane Protein 2 / metabolism
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Vesicular Glutamate Transport Protein 1 / genetics
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Vesicular Glutamate Transport Protein 1 / metabolism
Substances
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PHluorin
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Slc17a7 protein, rat
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Synaptotagmin I
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Vesicle-Associated Membrane Protein 2
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Vesicular Glutamate Transport Protein 1
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Green Fluorescent Proteins