Fig. 2.

Background miniature activity does not significantly affect the estimation of p_r by FM1-43. (A) Experimental scheme for comparing miniature spontaneous activity and evoked release. In the first session, FM1-43 was loaded by spontaneous activity, whereas in the second session FM1-43 was loaded by 30 APs at 1 Hz. Fluorescence signals from spontaneous miniature and evoked events were obtained by subtracting the unloaded image: a–b (spontaneous activity) or c–d (evoked response). (B) Scatter plot showing FM1-43 fluorescence intensities loaded by field stimulation (evoked) and spontaneous miniature activity (mini). The gray line is a linear regression fit with a slope of 0.142, and the black line has a slope of 1. Data from 107 regions of interest from three coverslips are shown. (C) Synapses are classified by their p_r, and the distribution of the miniature endocytotic event frequency is shown for each group. FM1-43 fluorescence intensity shown in B is converted to p_r for evoked events. Higher p_r synapses tend to show higher miniature synaptic activity (r = 0.65, P < 0.0001, Spearman correlation), although the overall contribution of miniature activity is small. (D) Ratio of the number of miniature release events to that of evoked release at individual synapses as a function of p_r (evoked). On average, the uptake of FM1-43 by miniature synaptic activity contributes to ≈7% of our estimation of p_r. Data are shown as mean ± SEM.