Deconvolution of EPSCs to calculate the rates of transmitter Autophagy inhibitor in vitro release was done as first described by Neher and Sakaba (2001), with routines written in IgorPro. The deconvolution analysis assumes that mEPSC with a double-exponential decay (Schneggenburger and Neher, 2000) add linearly to give rise to an evoked EPSC. The calculated release rates were corrected for a contribution

of a spillover glutamate current as described (Neher and Sakaba, 2001). Cumulative release traces were obtained by simple integration of the transmitter release traces without further correction for an assumed recovery process. Cumulative release traces were fitted with the following functions: single-exponential, exponential plus line, double-exponential, double-exponential Protein Tyrosine Kinase inhibitor plus line, and triple-exponential (Wölfel et al., 2007). The best-fit function was selected based on the Bayesian information criterion (BIC; Kochubey et al., 2009). Data are reported as average ± standard deviation (SD) values unless otherwise noted. Statistical significance was evaluated with Student’s t test, and accepted at p < 0.05. For the comparison of release rates, release delays, and fast release time constants between two data sets at various [Ca2+]i (Figures 4E–4H), the data sets were double-logarithmized and then assessed for statistical

significance by analysis of covariance (ANCOVA). The Ca2+-uncaging data were fitted by a five-site model of Ca2+ binding and vesicle fusion (Schneggenburger Oxymatrine and Neher, 2000). The following parameters were used for control/RIM1/2

cDKO synapses respectively: kon, 1.65 ∗ 108 / 1.05 ∗ 108 [M−1s−1]; koff, 7000/5000 [s−1]; pool size, 1390/315 vesicles. The remaining parameters were the same for both data sets (cooperativity factor b, 0.35; final fusion rate γ, 7000 s−1). Ca2+ uncaging was done with a DP-10 flash-lamp (Rapp Optoelektronik) according to standard procedures described before (Schneggenburger and Neher, 2000 and Wölfel et al., 2007); details are given in Supplemental Experimental Procedures. Transmission EM was performed in the MNTB area of a RIM1/2 cDKO mouse and its control Cre-negative littermate (both at P11) with standard fixation and resin embedding procedures (see Supplemental Experimental Procedures). Serial images were taken with a Philips CM10 TEM operated at 80 kV at a magnification of 16,000 times with 10–20 adjacent sections of 50 nm thickness. Only active zones that were completely contained in the series were analyzed. The image series were aligned and active zones, including vesicles and surrounding plasma membrane, were reconstructed in 3D with the Fiji software (http://pacific.mpi-cbg.de/wiki/index.php/Main_Page). The shortest distance from the vesicle membrane to the active zone membrane was then calculated in the 3D model, and all vesicles at distances of less than 300 nm were taken into account.