Recycled Synaptic Vesicles Contain Vesicle but Not Plasma Membrane Marker, Newly Synthesized Acetylcholine, and a Sample of Extracellular Medium

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Abstract
To monitor the fate of the synaptic vesicle membrane compartment, synaptic vesicles were isolated under varying experimental conditions from blocks of perfused Torpedo electric organ. In accordance with previous results, after low‐frequency stimulation (0.1 Hz, 1,800 pulses) of perfused blocks of electric organ, a population of vesicles (VP2 type) can be separated by density gradient centrifugation and chromatography on porous glass beads that is denser and smaller than resting vesicles (VP1 type). By simultaneous application of fluorescein isothiocyanate‐dextran as extracellular volume marker and [3H]acetate as precursor of vesicular acetylcholine, and by identifying the vesicular membrane compartment with an antibody against the synaptic vesicle transmembrane glycoprotein SV2, we can show that the membrane compartment of part of the synaptic vesicles becomes recycled during the stimulation period. It then contains both newly synthesized acetylcholine and a sample of extra cellular medium. Recycled vesicles have not incorporated the presynaptic plasma membrane marker acetylcholinesterase. Cisternae or vacuoles are presumably not involved in vesicle recycling. After a subsequent period of recovery (18 h), all vesicular membrane compartments behave like VP1 vesicles on subcellular fractionation and still retain both volume markers. Our results imply that on low‐frequency stimulation, synaptic vesicles are directly recycled, equilibrating their luminal contents with the extracellular medium and retaining their membrane identity and capability to accumulate acetylcholine.