In contrast, neither the Munc13-1W464R mutation nor the infusion of the CaM-inhibitory peptide in WT or Munc13-1W464R calyces had any deleterious effect on the recovery time course of the slowly releasing SV pool (Figure 3E). A reduced recovery rate of SV pools in Munc13-1W464R calyces was also evident when monitoring SV fusion by means of presynaptic membrane capacitance measurements, although the effect was less prominent, since this method reports the sum of fast and slow components (Figure S2). As elevations of presynaptic [Ca2+]i, e.g., upon changes in Ca2+ influx, strongly influence the recovery

of releasable SV pools (Dittman and Regehr, 1998; Sakaba and Neher, 2001; Stevens and Wesseling, 1998; Wang and Kaczmarek, 1998), we compared amplitudes of presynaptic Selleck KU-55933 Ca2+ currents resulting from the depolarizing pulses between WT and Munc13-1W464R mutant calyces but found no significant differences (WT, 1238 ± 79 pA, n = 6; Munc13-1W464R, 1283 ± 56 pA, n = 6; p > 0.05). Likewise, no differences were observed in the recovery time course of presynaptic Histone Methyltransferase inhibitor Ca2+ currents, as the ratios between the Ca2+ current amplitudes triggered by the second versus the first depolarization stimulus were identical for all interstimulus intervals (Figure 3C). These data show that in young calyx synapses the W464R mutation in Munc13-1 selectively affects the recovery of the

fast releasing SV pool, much like CaM inhibitors do, indicating that the Ca2+-CaM effect on releasable SV pool refilling is mediated by Munc13-1. The lack of CaM binding to Munc13-1W464R in the KI mutant does not appear to affect presynaptic Ca2+

channels, which are known to bind CaM (DeMaria et al., 2001; Lee et al., 1999; Peterson et al., 1999; Zühlke et al., 1999). Calyx of Held synapses undergo a structural and functional many refinement during postnatal development that transforms these synapses into fast and reliable relays. These developmental modifications include changes in SV pools, release probability, postsynaptic receptor desensitization, and expression of Ca2+ binding proteins (Crins et al., 2011; Erazo-Fischer et al., 2007; Sonntag et al., 2011; Taschenberger et al., 2002; Taschenberger et al., 2005; Taschenberger and von Gersdorff, 2000; Wang et al., 2008). In light of these changes, we decided to study the recovery rates of the two SV pools and the dependency of the recovery rates on CaM in more mature WT and Munc13-1W464R calyces (P14–P17). We measured the recovery of the fast and slowly releasing SV pools following their depletion by a 50 ms depolarizing pulse in P14–P17 calyces under the same conditions as with P9–P11 calyces (see Figure 3). The cumulative release in WT calyces of P14–P17 animals exhibited two components (τ1 = 1.3 ± 0.5 ms, 60% of the total fit; τ2 = 11.