Other mechanisms that additionally contribute to Ca2+-dependent RRP recovery include, for example, CaM independent signaling to the priming machinery, e.g., via the C1 and C2 domains of Munc13s (Rhee et al., 2002; Shin et al., 2010), or facilitation of the release of reluctant Everolimus molecular weight vesicles following elevation of [Ca2+]i (Wu and Borst,

1999). The SSD levels during high-frequency synaptic activity are thought to be defined by a balance between SV release and replenishment (Dittman and Regehr, 1998; Saviane and Silver, 2006; Wang and Kaczmarek, 1998). We therefore expected that the reduction of RRP replenishment rates seen in Munc13-1W464R KI calyces (Figures 3 and 4) would result in lower SSD levels. However, a reduction of SSD levels was

only found in calyces of more mature KI animals, whereas in WT and Munc13-1W464R calyces at P9–P11 SSD was similar at all frequencies tested (Figure 6). This is surprising in view of the findings that acute application of CaM inhibitors causes lower SSD levels in the rat calyx of Held at P9–P11 (Hosoi et al., 2007; Lee et al., 2012; Sun et al., 2006) and that cultured hippocampal neurons expressing only Munc13-1W464R find more from a viral rescue construct show an increased STD and lower SSD levels (Junge et al., 2004). At least four scenarios may account for this unexpected finding. First, basal, Ca2+-independent activity of Munc13-1 (Basu et al., 2005) in the Munc13-1W464R mutant might be sufficient to maintain normal SSD levels during phases of moderate to strong synaptic activity, but not upon complete RRP depletion by sustained presynaptic depolarization. Second, the priming activity of Munc13-1W464R can still be strongly potentiated via the C1 domain or the C2B domain (Rhee

et al., 2002; Shin et al., 2010). Third, it is possible that the regulation of Munc13-1 activity by CaM in the calyx of Held in vivo is mainly relevant at Bacterial neuraminidase rather high [Ca2+]i. Indeed, the dual pulse protocol we used to assess the replenishment of the fast and slowly releasable SV pools (Figures 3 and 4) involves long depolarizations, during which global presynaptic Ca2+-concentrations are expected to reach higher levels than during AP trains (Hosoi et al., 2007). In addition, an effect of the Munc13-1W464R mutation on the evoked synaptic responses was seen during recovery from synaptic depression after high-frequency stimulation trains, which likely cause a strong and long-lasting rise in [Ca2+]i (Figures 5A–5D). The notion that the Ca2+-CaM-Munc13-1 signaling may be only operational at rather high [Ca2+]i in intact cells is supported by a recent study on the calyx of Held (Lee et al.