The peak release rates were strongly reduced in RIM1/2 cDKO synapses (Figure 5D) and the width of the transmitter release at half-maximal amplitudes was longer in RIM1/2 cDKO synapses (5.1 ± 1.8 ms, n = 6) than in control (2.3 ± 1.1 ms, n = 5; p < 0.05).

The integrated release rate traces were fitted with a series of single- and double-exponential function with or without line component to determine the best fit function (see Experimental Procedures). In both genotypes, cumulative release was best fitted by functions that contained at least two exponential components (Figure 5C, blue fit lines), indicating a fast and a slow release component (Sakaba and Neher, 2001, Wadel et al., 2007 and Wölfel et al., 2007). In RIM1/2 cDKO synapses, the fast release time mTOR inhibitor constant was significantly slower (5.2 ± 1.7 ms, n = 6) than in control synapses (1.8 ± 0.8 ms, n = 5; Figure 5E; p = 0.002), but it was significantly faster than the slow release time constant in control synapses, find more which was 23 ± 3.7 ms (n = 5; Figure 5F; p < 0.001). Similarly, when cumulative release

traces were arbitrarily fitted with monoexponential functions, the resulting time constant in RIM1/2 cDKO synapses (9.3 ± 1.1 ms; n = 6) was still significantly faster than the slow release time constant in wild-type cells (p < 0.001). Both of these comparisons show that the FRP is not simply missing completely but rather that release from the remaining FRP is slowed in the RIM1/2 cDKO synapses. Figures 5E and 5F show further parameters extracted from the kinetic analysis of transmitter release for each genotype. PDK4 Overall, the analysis shows a strongly reduced number of readily releasable vesicles in both the FRP and the SRP, as well as a significant, ∼2.5-fold slowing of the fast release component. The kinetics of transmitter release in response to Ca2+ influx depends on the intrinsic speed of release, as well as on the “local” [Ca2+]i that builds up close to the readily releasable vesicles, which, in turn, is a function of the distance between Ca2+ channels and vesicles (Neher,

1998 and Wadel et al., 2007). The Ca2+ uncaging experiments showed that the intrinsic Ca2+ sensitivity is reduced in the absence of RIM1/2 (Figure 4). To ask whether the spatial coupling between Ca2+ channels and vesicles was impaired as well, we back-calculated the local [Ca2+]i that was necessary to reproduce the kinetics of the fast release component in response to depolarizations (Figures 5B and 5C, gray traces; Schneggenburger and Neher, 2000). This was done by using the specific sets of kinetic parameters that describe the intracellular Ca2+ sensitivities of transmitter release of RIM1/2 cDKO and control synapses (Figure 4). In the examples of Figure 5C, a step-like local [Ca2+]i signal of 7.