We showed that in the absence of RIM1/2, the number of presynaptic Ca2+ channels was reduced by ∼50% (Figure 2) and the readily releasable pool was strongly reduced,

together with a small reduction of the release probability (Figure 3). To study the reduced readily releasable pool size more directly, we used Ca2+ uncaging to strongly stimulate release independent of Ca2+ channel opening. In addition, Ca2+ uncaging will allow us to address the possibility of a reduced intrinsic Ca2+ sensitivity of release in RIM1/2 cDKO synapses, which could underlie the reduced release probability. In RIM1/2 cDKO synapses, Ca2+ uncaging stimuli evoked EPSCs with small amplitudes (∼6 nA in the example of C59 wnt nmr Figure 4B, left) and flashes that elevated [Ca2+]i above ∼15 μM did not evoke EPSCs with larger amplitudes in RIM1/2 cDKO synapses (Figure 4B), showing directly the limited pool of readily releasable vesicles in RIM1/2 cDKO synapses. The maximal EPSC amplitudes at 10–15 μM [Ca2+]i were much LY294002 smaller in RIM1/2 cDKO synapses (5.7 ± 4.9 nA, n = 7) than in control synapses (14.2 ± 7.8 nA, n = 6; p < 0.01), indicating a reduced pool size. To analyze the number of releasable vesicles and their release kinetics in more detail, we

deconvolved the Ca2+ uncaging-evoked EPSCs to reveal rates of transmitter release (Figure 4B, inset) and cumulative release rates (Figure 4C). These were best fitted by the sum of at least two exponential functions (see Experimental Procedures), indicating a fast and a slow release component in response to Ca2+ uncaging (Wölfel et al., 2007 and Kochubey et al., 2009). Thus, the pool of readily releasable vesicles can be kinetically subdivided into a fast releasable pool (FRP) and a slowly releasable pool (SRP) (Sakaba and Neher, 2001 and Wölfel

et al., 2007). To estimate the size of the FRP, we analyzed the number of vesicles released in the fast release component in response to [Ca2+]i steps to 10–15 μM. The FRP was strongly reduced, Sodium butyrate from 1091 ± 396 vesicles in control synapses to 271 ± 178 vesicles in RIM1/2 cDKO synapses (n = 6 and 7 respectively; Figure 4D). In addition, the amplitude of the slow-release component was also reduced, from 833 ± 658 to 211 ± 205 vesicles in control and RIM1/2 cDKO synapses, respectively (p = 0.036). We next plotted the absolute peak release rates as a function of the presynaptic [Ca2+]i step amplitude (Figure 4E). This plot showed that the peak release rates were significantly smaller at all [Ca2+]i in RIM1/2 cDKO synapses as compared to control (p < 0.001; analysis of covariance, ANCOVA). However, the absolute peak release rates at any given [Ca2+]i are a function of the intrinsic Ca2+ sensitivity of release, as well as of the absolute pool size.