Thus, RIM proteins have a so far unrecognized role in enriching voltage-gated Ca2+ channels at the presynaptic nerve terminal (see also Kaeser et al., 2011). We showed that conditional removal of RIM1/2 leads to a strong reduction of transmitter release (by ∼80%; Figure 1) and that presynaptic Ca2+ currents are strongly reduced (∼50%; Figure 2). Does the reduced transmitter release primarily reflect a reduced release probability caused by the much smaller presynaptic Ca2+ influx or are there other factors, like a reduced readily releasable pool of vesicles (Calakos et al., 2004), which

contribute to the decreased transmitter release? To investigate changes in pool size and release probability, we next used brief high-frequency trains of afferent fiber stimulation to measure the size

of the readily releasable Doxorubicin cell line pool (Figure 3; Schneggenburger et al., 1999). In control synapses, LDN-193189 mw the first EPSC was large (∼8 nA in Figure 3A), and subsequent EPSCs decreased in amplitude to a new steady-state value (Figures 3A and 3B, right). In contrast, RIM1/2 cDKO synapses had much smaller EPSCs (∼2 nA in Figure 3A), and depression usually only occurred after the third or fourth stimulus (Figures 3A and 3B, left; see also Figure 3D for the average of all cells). To quantify the onset of depression, we made line fits to relative depression curves in the range of the first to the sixth stimulus (Figure 3B, blue fit lines). This analysis gave slopes of −62% ± 26% per five stimuli (n = 8) and −27% ± 33% per five stimuli (n = 9) for control and RIM1/2 cDKO synapses, respectively (p < 0.05; see also Figure 3D, bottom, for line fits to the averaged data sets for each genotype). Thus, the onset of depression was significantly slowed in RIM1/2 cDKO synapses, which suggests a decreased release probability of any given readily releasable vesicle. We next analyzed

the apparent size of the readily releasable pool by using the method of cumulative EPSC amplitudes back-extrapolated to time zero (Schneggenburger et al., 1999; Figure 3C). The back-extrapolated cumulative EPSC amplitude Mephenoxalone was 51.1 ± 16.2 nA in control mice (corresponding to ∼1700 vesicles, assuming an average mEPSC amplitude of 30 pA; n = 8 cells), but only 11.9 ± 6.9 nA in RIM1/2 cDKO mice (∼400 vesicles; n = 10 cells). Thus, there was a clear pool size reduction in RIM1/2 cDKO synapses (p < 0.001; Figure 3E). The average release probability, calculated by dividing the first EPSC amplitude by the pool size estimate (Iwasaki and Takahashi, 2001) was 0.27 ± 0.09 (n = 8) and 0.19 ± 0.05 (n = 9) in control and RIM1/2 cDKO synapses, respectively (p = 0.04; Figure 3F). These experiments with high-frequency trains show that the release deficit is primarily caused by a decreased pool of readily releasable vesicles (reduction by ∼75%; Figure 3E).