This Ca2+ dependent

increase in the SV priming rate has been attributed to direct or indirect effects of increased [Ca2+]i on Munc13 activity, involving Ca2+-CaM binding, diacylglyerol binding, and Ca2+/phospholipid binding to regulatory domains of Munc13s (Betz et al., 2001; Dimova et al., 2006, 2009; Junge et al., 2004; Rhee et al., 2002; Shin et al., 2010). However, the corresponding evidence was exclusively obtained in cultured neurons. As a result, the question as to whether Munc13s are important determinants of Ca2+-dependent RRP replenishment and STP in native synapses within intact neuronal circuits has remained a focus of substantial controversy. By employing a KI mutant mouse line in which the WT protein is replaced by a Ca2+-CaM insensitive Munc13-1W464R variant and by using the calyx of Held as a model synapse, selleck screening library we demonstrate that Ca2+-CaM binding to Munc13-1 regulates RRP recovery from depletion and the time course of recovery from STD (Figures 3 and 7). These functional changes are a specific consequence

of blocked Ca2+-CaM binding to Munc13-1 (Figure 1G) because Munc13-1 expression (Figure 1F), its interaction with its key target protein Syntaxin 1 (Figure 1G), and its presynaptic localization (Figure 2), as well as the expression ABT263 levels of Munc13-1 interactors and functionally related proteins (Figure S1) are not affected by the KI mutation. Our data show that Munc13-1 is an important Ca2+-CaM effector in the replenishment of the releasable SV pool in the calyx of

Held. The reduction of the replenishment rate of the fast releasing SV pool caused by the Munc13-1W464R mutation is similar in calyces before and after hearing onset (Figures 3 and 4), demonstrating that the SV release machinery depends upon the priming activity of Ca2+-CaM activated Munc13-1 throughout development. Strikingly, the effects of presynaptic introduction Wilson disease protein of CaM inhibitors on RRP replenishment rates precisely mirror the effects of the Munc13-1W464R mutation, and the Munc13-1W464R mutation occludes any further effects of CaM inhibition (Figures 3D, 3E, 4D, and 4E). This indicates that the previously reported effects of CaM inhibition on RRP refilling in the calyx of Held (Sakaba and Neher, 2001) are mainly due to a perturbation of Ca2+-CaM-Munc13-1 signaling, and that Munc13-1 is a major presynaptic target of Ca2+-CaM-signaling in RRP replenishment. Ca2+-dependent acceleration of the replenishment rate of releasable SV pools is thought to contribute profoundly to the rapid recovery from synaptic depression after high-frequency AP trains and to determine the SSD level during the train (Neher and Sakaba, 2008; Wang and Kaczmarek, 1998). We report a significant retardation of the recovery of EPSCs after 300 Hz trains in P9–P11 calyces, and after 100 and 300 Hz trains in P14–P17 calyces of Munc13-1W464R mice (Figure 5), which likely reflects a reduction in Ca2+-dependent RRP recovery.