BAPTA significantly slowed release as compared to EGTA or perforated patch. The perforated-patch recordings suggest an endogenous buffer capacity less than 1 mM BAPTA, but more than 1 mM EGTA, similar to

that suggested previously (Moser and Beutner, 2000) and consistent with a release mechanism located near the source of Ca2+ influx. A comparison of initial release rates against Ca2+ load for individual cells is presented in Figure S6B illustrating how BAPTA reduces release rates. As compared to EGTA, BAPTA also increased the duration of the plateau between the first release component and the onset of the superlinear component, represented by the Ca2+ load required for the onset of the superlinear release (Figure 6G). In this instance perforated-patch responses suggest endogenous buffering was stronger than either BAPTA or EGTA. This might indicate that the site of action is further removed from the Ca2+ source where concentration INCB024360 supplier rather than kinetics is more relevant (Naraghi and Neher, 1997). In contrast, changes in the rate of the superlinear

component (Figure 6E) suggest the perforated-patch response was less efficacious than EGTA or BAPTA, supporting the contention that endogenous buffer kinetics are slow. Finally, the difference LGK-974 in vitro in Ca2+ load required to initiate the superlinear component of release led to an increase in the magnitude of the first component plateau (Figure 6F), supporting the conclusion that vesicle movement to release sites was rapid and Ca2+ dependent. The delay between release components also supports this conclusion, demonstrating that despite the presence of Ca2+ to drive release, depletion persisted for longer periods of time when Ca2+ buffering was increased because trafficking was slowed. An alternative

possibility is that vesicle position at the synapse is Ca2+ dependent and that greater buffer efficacy Non-specific serine/threonine protein kinase leads to diffusion of vesicles away from the synapse. We tested this hypothesis by recording cells with 10 mM BAPTA internally, blocking release at all but maximal stimulus levels, and then increasing Ca2+ load by using Bay K (10 μM), which prolongs Ca2+ channel open time (Figure 6H). Capacitance changes evoked by Bay K treatment included both linear and superlinear components supporting the hypothesis that vesicle movement toward the synapse is Ca2+ dependent and largely unidirectional. It is possible that additional sources of Ca2+, for example Ca2+ stores, enhance release during longer stimulations (Lelli et al., 2003). We tested this hypothesis with high-speed confocal Ca2+ imaging. Labeling ribbons with a rhodamine-tagged ctbp2-terminal binding peptide in the patch electrode (Zenisek et al., 2003) allowed synapses to be localized during simultaneous Ca2+ imaging and capacitance measurements (Figure 7A). The capacitance changes show a typical response with both release components (Figures 7C–7E). The fluorescent signal, however, is quite complex and not simply the integral of the current, as might be predicted.