Extended Data Fig. 6: Chelating residual Ca²⁺ blocks fusion intermediates at 11 ms but not 5 ms after an action potential.

a-b, Example transmission electron micrographs of synapses from neurons pre-treated with a 0.25% DMSO or b 25 μM EGTA-AM and frozen either without stimulation, 5 ms after stimulation, or 11 ms after stimulation (these are other examples from the same experiments shown in Fig. 4). Scale bar: 100 nm. PSD: post-synaptic density. AP: action potential. Arrows indicate “pits” in the active zone (opposite the post-synaptic density), which are presumed to be vesicles fusing with the plasma membrane. c, Size of active zones from data in Fig. 4. Tukey boxplot shown; center: median, lower bound of box: 25^th percentile, upper bound of box: 75^th percentile, lower whisker: 25^th percentile minus 1.5x interquartile range, upper whisker: 75^th percentile plus 1.5x interquartile range; dots indicate values outside this range. See Supplementary Table 1 for full pairwise comparisons and summary statistics. d, Distances of synaptic vesicles from the plasma membrane at the active zone, including both vesicles that were annotated as docked and not docked, from data in Fig. 3. No stim, n = 274; 5 ms, n = 315; 8 ms, n = 343; 11 ms, n = 192; 14 ms, n = 211; TTX, no stim, n = 121; and TTX, 5 ms, n = 255 synaptic profiles. Distances are binned in 2-nm increments, except for “0”, which indicates vesicles ~0 nm from the active zone membrane (“2” indicates vesicles 0.1-2 nm from the membrane, “4” indicates 3-4 nm, etc.). Error bars indicate standard error of the mean. e, Number of pits in the active zone per 100 nm of active zone, from data in Fig. 4. f, Number of docked per 100 nm of active zone, from data in Fig. 4. All data from the experiments described in Fig. 4 are from 4 experiments for no stim and 5 ms time points, 3 experiments for 11 ms, and 2 experiments for 14 ms, from separate cultures frozen on different days (See Supplementary Table 2 for count data from each experiment).

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