A calibration stimulus of 50 APs at 20 Hz is followed by a 60 s recovery interval and the test stimulus of interest. Fluorescence transients were normalized to the calibration response amplitudes, providing a signal that is independent of initial release probability and spH expression level (Figure 1B). Since ongoing endocytosis during stimulation counteracts protein accumulation at

the plasma membrane, spH fluorescence decreases in between stimuli, causing reduction of peak values in response to a given number of stimuli at low frequencies (Figure S1A, available online). In order to compensate for endocytosis and to further characterize the role of stimulation frequency on release rates, we developed a deconvolution routine, in which the normalized calibration response was taken as a replica for the elementary event (ee Supplemental Experimental Procedures for details). To validate this method

we performed deconvolution Capmatinib in vitro on normalized fluorescence transients in Figure 1B. This analysis revealed stepwise increases in cumulative release rate during periods of stimulation and cumulative release was found to increase linearly with the number of APs for mild stimulation up to 200 APs at 5 Hz (Figure 1C), Entinostat cell line in agreement with previous studies using alkaline trapping (Ariel and Ryan, 2010 and Li et al., 2005). However, we also observed that for stronger and longer-lasting stimulation, time constants of fluorescence decay upon exocytosis

become larger, confirming earlier results regarding the limited capacity of endocytosis (Balaji and Ryan, 2007). This compromises the use of deconvolution, in which a time-invariant template is assumed. We, therefore, explored the range of constant decay rates (Figure 1D) and found that for a given number of APs the time constant is invariant up to a certain firing frequency, which was 5 Hz for 200 APs and 40 Hz for 50 APs (see Supplemental Experimental Procedures for details). Despite its narrow range of applicability, the deconvolution method found has an advantage over other methods, which either block compensatory endocytosis or prevent vesicular reacidification (alkaline-trapping), since it directly measures the rate of exocytosis without any perturbations. When applicable, it provides a better estimate for exocytosis, since it takes into account the contributions of reused SVs (Figure S2). In fact, comparing the results of deconvolution with those of using, e.g., alkaline-trapping should allow one to estimate the contribution of SV reuse. To do so, we next performed measurements with Folimycin (V-ATPase inhibitor) and Dynasore (dynamin GTPase inhibitor). The effects of these two inhibitors are schematically illustrated in Figure 1E. We found for 200 APs at 5 Hz the fluorescence response in the presence of 80 nM Folimycin to be strikingly similar to the deconvolved-integrated signal obtained in the absence of the proton pump inhibitor.