5 to 18.7 ± 3.4 s (n =

6; Figure 1A). This effect of Rp-cGMPS was concentration-dependent, with a 50% inhibition concentration (IC50) of 0.15 μM (see Figure S1 available online). This IC50 is 4 times higher than that estimated in biochemical assays (0.035 μM) (Butt et al., 1995), but ∼50 times lower than that estimated for the vasodilatation assay (7.2 μM) (Taylor et al., 2004). Another type of PKG-specific inhibitor KT5823 (10 μM) also slowed endocytic τ0.5 to 19.8 ± 3.0 s (n = 6; Figure 1A) like Rp-cGMPS (3 μM). Neither Rp-cGMPS nor KT5823 affected ΔCm or ICa (Figure 1A). As Rp-cGMPS is membrane permeable, preincubation of slices with check details Rp-cGMPS (3 μM) for 1 hr at room temperature (RT) also slowed endocytic τ0.5 to 18.8 ± 2.9 s (n = 3, data not shown), similar to its effect after direct loading into calyces (Figure 1A). These results

suggest that endogenous PKG normally upregulates the endocytic rate of synaptic vesicles at mature calyces of Held. In contrast to PKG inhibitors, the PKA inhibitor KT5720 (1 μM), loaded into calyces, had no effect on the time course of vesicle endocytosis induced by a 20 ms depolarizing pulse (data not PCI32765 shown). However, it has recently been reported that preincubation of slices of P9–P11 rats with KT5720 (2 μM) slows endocytic capacitance change, more effectively after longer pulse depolarization (Yao and Sakaba, 2012). It remains to be seen whether the slowing effect of the PKA inhibitor on endocytosis persists after hearing onset. We further examined whether the PKG-dependent speeding of vesicle endocytosis operates before hearing. At calyces before hearing onset (P7–P9), endocytosis elicited with our standard

stimulation protocol was much slower (τ0.5, 17.2 ± 2.0 s, n = 6; Figure 1B) than that at P13–P14 calyces, but similar to that in Rp-cGMPS-loaded P13-P14 calyces (Figure 1B). Furthermore, in contrast to P13–P14 calyces, direct loading of Rp-cGMPS (3 μM) into P7–P9 calyces had no effect on endocytic time course (τ0.5, 17.3 ± 2.7 s, n = 5). These results suggest that at calyceal synapses after hearing onset, a PKG-dependent mechanism supports the high rate of endocytosis. At the calyx of Held, vesicle endocytosis undergoes developmental speeding (Renden and von Gersdorff, 2007 and Yamashita et al., Tolmetin 2010). Our results suggest that maturation of the PKG-dependent mechanism underlies this development. At many synapses, including immature rodent calyces before hearing, the time required for endocytosis is proportional to the amount of exocytosis (von Gersdorff and Matthews, 1994, Wu and Betz, 1996, Sun et al., 2002, Yamashita et al., 2005 and Balaji et al., 2008). However, at calyces of Held after hearing, the endocytic time constant becomes constant and independent of the magnitude of exocytosis (Renden and von Gersdorff, 2007 and Yamashita et al., 2010).