Extended Data Figure 5. pHuse reveals differences between evoked and spontaneous fusion site areas.

a. Comparison of spontaneous frequency measured presynaptically using vGpH (n = 77/22) and postsynaptically using GCaMP6f ⁴⁵(n = 61/5), t = 1.02, n.s. b. Average bouton areas across groups, t = 0.87, n.s. c. Cumulative distributions of fusion areas for spontaneous and evoked release (K–S test, D = 0.23*) d. Cumulative distributions of normalized fusion areas for 1 AP evoked fusion excluding events with photon counts > mean + 2SD of spontaneous events (n = 91/27) compared to all evoked events (n = 104/28, K–S test, D = 0.05, n.s.) and spontaneous events (n = 77/22, K–S test, D = 0.25*) e–f. Interestingly, while evoked Pr was significantly positively correlated with Syn1a area, as reported previously⁵⁰, spontaneous event frequency showed no relationship with Syn1a area (e, linear fit, evoked R = 0.30**, spontaneous R = 0.12, n.s.). On the other hand, both spontaneous event frequency and evoked Pr significantly positively correlated with pHuse area (f, linear fit, evoked R = 0.64***, spontaneous R = 0.60***). This suggests that pHuse area may be a better approximate for AZ area and the functional parameters of a synapse than bouton area. g. Normalized pHuse area as a function of cell age shows no significant correlation (evoked R = 0.03, n.s., spont R = 0.004, n.s.). e–g: n_evoked = 104/28, n_spont = 77/22. h. Normalized pHuse area was not significantly different at RT (n_evoked = 51/10, n_spont = 32/7) vs physiological temperature (n_evoked = 35/9, n_spont = 34/4) within modes of release but still significantly different between modes of release. i. Normalized pHuse area was not significantly different at different thresholds for Syn1a within modes of release but still significantly different between modes of release (n = 51/10). j. Both numbers of events and mode of release are significant factors for pHuse area, but they do not have a significant interaction n_evoked = 155/38, n_spont = 109/29. (For i–j, see Supplementary Tables for statistics.) n given in synapses/experiments, n.s. = not significant, *p < 0.05, **p < 0.01, ***p < 0.001.