Synaptic Vesicle Populations in Saccular Hair Cells Reconstructed by Electron Tomography

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Abstract
We used electron tomography to map the three-dimensional architecture of the ribbon-class afferent synapses in frog saccular hair cells. The synaptic body (SB) at each synapse was nearly spherical (468 ± 65 nm diameter; mean ± SD) and was covered by a monolayer of synaptic vesicles (34.3 nm diameter; 8.8% coefficient of variation), many of them tethered to it by ∼20-nm-long filaments, at an average density of 55% of close-packed (376 ± 133 vesicles per SB). These vesicles could support ∼900 msec of exocytosis at the reported maximal rate, which the cells can sustain for at least 2 sec, suggesting that replenishment of vesicles on the SB is not rate limiting. Consistent with this interpretation, prolonged K+depolarization did not deplete vesicles on the SB. The monolayer of SB-associated vesicles remained after cell lysis in the presence of 4 mmCa2+, indicating that the association is tight and Ca2+-resistant. The space between the SB and the plasma membrane contained numerous vesicles, many of which (∼32 per synapse) were in contact with the plasma membrane. This number of docked vesicles could support maximal exocytosis for at most ∼70 msec. Additional docked vesicles were seen within a few hundred nanometers of the synapse and occasionally at greater distances. The presence of omega profiles on the plasma membrane around active zones, in the same locations as coated pits and coated vesicles labeled with an extracellular marker, suggests that local membrane recycling may contribute to the three- to 14-fold greater abundance of vesicles in the cytoplasm (not associated with the SB) near synapses than in nonsynaptic regions.