Alternatively, PKCs could be initially activated by the calcium signals during the train and then, because of positive cooperative binding, become sensitive to residual calcium. Once activated, PKC could phosphorylate proteins such as Munc18 to increase the probability of release (Wierda et al., 2007). Further studies are needed to determine if PKCα and PKCβ are indeed the calcium sensors in PTP, and if they influence release by phosphorylating Munc18. Tetanic stimulation increases the frequency of mEPSCs several-fold

at the www.selleckchem.com/products/MLN8237.html calyx of Held synapse and at other synapses (Figure 6) (Castillo and Katz, 1954, Eliot et al., 1994, Groffen et al., 2010, Habets and Borst, 2005, Korogod et al., 2005, Korogod et al., 2007 and Magleby, 1987). The increase in the frequency of spontaneous release and PTP are both dependent on presynaptic calcium increases (Bao et al., 1997, Korogod et al., 2005, Nussinovitch and Rahamimoff, 1988 and Zucker and Lara-Estrella, 1983), suggesting that they share a common mechanism. However, the elevation in mEPSC frequency does not last as long as the enhancement of evoked EPSCs (τ

∼ 12 s and 45 s, respectively) (see also MK-2206 chemical structure Korogod et al., 2007). In addition, pharmacological inhibitors of PKC that reduce the increase in evoked EPSC amplitude do not prevent the increase in mEPSC frequency at calyx of Held synapses (Korogod et al., 2007). Here, using a genetic approach, we also find that the frequency of mEPSC and the amplitude whatever of evoked EPSCs are regulated independently. Indeed, potentiation of evoked EPSCs is reduced by 80% in slices from PKCα−/−β−/− mice compared to controls (Figure 9A) whereas the increase in mEPSC frequency is largely

unaffected (Figure 9C). Therefore, the activity-dependent regulation of mEPSC frequency is not mediated by PKCs, and is likely regulated by other calcium-sensitive proteins in the presynaptic terminal, such as Doc2a and Doc2b (Groffen et al., 2010; but see Pang et al., 2011). Tetanic stimulation also results in increased mEPSC amplitude in slices from wild-type animals (Figure 7). Although modest, this increase has a time course (τ ∼ 47 s) that is similar to that of PTP (τ ∼ 45 s, compare Figure 2F and Figure 7F), and it is thought to contribute to PTP (He et al., 2009). The increase in mEPSC amplitude appears to reflect the fusion of vesicles with each other prior to ultimate fusion with the plasma membrane (He et al., 2009). We find that the increase in mEPSC amplitude persists in the absence of PKCα, PKCβ or both isoforms (Figure 7). This suggests that calcium-dependent isoforms of PKC do not regulate vesicle-to-vesicle fusion within the calyx of Held. The 10% increase in mEPSC amplitude that remains in PKCα/β double knockout animals could account for some of the remaining PTP observed in this group (Figure 9A).