Counting Released Vesicles and Ca2+ Channels at the Cone Ribbon Synapse Reveals That Few Channel Openings Regulate Exocytosis of Each Synaptic Vesicle

Abstract

Purpose: : We tested the hypothesis that the cone ribbon synapse ensures reliable communication by tightly coupling Ca²⁺ channel opening with vesicular release.

Methods: : We used paired whole cell recordings in the tiger salamander retina to record cone-driven excitatory post-synaptic currents (EPSCs) from horizontal cells.

Results: : By comparing exocytotic capacitance measurements from cones with simultaneously recorded EPSCs, we found that the entire cone terminal releases 7 times more vesicles than in the individual EPSC. This indicates that, out of the 12.4 ribbons per cone, an average of 1.8 ribbons synapse onto each horizontal cell. In a second approach, we analyzed the distribution of EPSC amplitudes using a multiple Gaussian fit and achieved the best fit with components of 26 vesicles apiece giving an average of 1.9 ribbons per EPSC. By dividing the charge transfer of the EPSC by the charge transfer of the average miniature EPSC (mEPSC), we determined that the fast component of the EPSC is due to release of 46 vesicles distributed over an average of 1.85 ribbons. Therefore, each ribbon releases 25 vesicles in the initial burst of exocytosis, similar to the number of vesicles at the base of the synaptic ribbon. Using variance-mean analysis of cone I_Ca tail currents, we determined there were ≤83 channels per ribbon suggesting ~3 channels are associated with each vesicle in the rapidly releasable pool. We calculate that 15 vesicles fuse within the first 2.7 ms of depolarization. By comparing the whole cell and single channel current amplitudes determined from the mean-variance relationship, we found a peak mean Ca²⁺ channel open probability of 0.3. Since each channel remains open for ~1 ms, these results suggest ~4 Ca²⁺ channel openings accompany fusion of each vesicle during the first 2.7 ms of a depolarizing test step.

Conclusions: : These results are consistent with findings at other ribbon synapses that demonstrate few channel openings accompanying the fusion of a vesicle and show an efficient coupling between Ca²⁺ channel opening and exocytosis at the cone ribbon synapse.

Keywords: synapse • calcium • photoreceptors