We next tested the effect of cGMP loaded into the terminal at different concentrations (10–300 μM). Cyclic GMP loaded at 300 μM caused a significant acceleration in the time course of vesicle endocytosis (Figure 4C). E7080 In the presence of cGMP (100 μM) in the presynaptic whole-cell pipette, PTIO was no longer able to slow endocytosis (τ0.5, 10.1 ± 0.7 s, n = 6; Figure 4D). These results are consistent with the hypothesis that

NO upregulates presynaptic cGMP, thereby activating PKG for speeding vesicle endocytosis. PIP2 in the presynaptic terminal membrane is thought to play essential roles in both CME and vesicle exocytosis (Cremona and De Camilli, 2001 and Jung and Haucke, 2007). It is also reported to support voltage-gated Ca2+ channel activity (Suh et al., 2010). We investigated whether presynaptic PIP2 is involved in the regulation of ICa, exocytosis or endocytosis, by loading the PI4 kinase inhibitor PAO (1 μM) into P13–P14 calyces. PAO inhibits production of the PIP2 precursor PIP, thereby decreasing the PIP2 level (Khvotchev and Südhof, 1998 and Várnai and Balla, 1998). At P13–P14 calyces, PAO slowed the endocytic τ0.5 to 16.5 ± 2.1 s (n = 4, p < 0.05, Figure 5A). However, at

this synapse, PAO had no effect on exocytosis or ICa (Figure 5A). When Rp-cGMPS (3 μM) and PAO (1 μM) were coloaded into calyces, the endocytic τ0.5 (13.8 ± 1.5 s, Ferroptosis inhibitor drugs n = 4) was similar to that with PAO alone (Figure 5A), indicating that the slowing effects of Rp-cGMPS and PAO on vesicle endocytosis occluded to each other. Further, to assess the presynaptic role of PIP2, we loaded PIP2 (5 μM) directly into calyces (Figure 5B). PIP2 by itself had no effect on endocytic τ, exocytic ΔCm or ICa. However, when co-loaded with Rp-cGMPS (3 μM), it blocked the slowing

effect of the PKG inhibitor on endocytic τ0.5 shown in Figure 1A. This is probably because excess PIP2 (5 μM) loaded from Phosphatidylinositol diacylglycerol-lyase a whole-cell pipette into calyces counteracted a decrease of intra-terminal PIP2 by the PKG inhibitor. Thus, in calyces after hearing onset, by linking to presynaptic PKG, PIP2 upregulates the rate of vesicle endocytosis. Might PIP2 be involved in vesicle endocytosis or exocytosis at calyces before hearing? We addressed this question by loading PAO into calyces at P7–P9. At these calyces, PAO markedly slowed endocytosis with its τ0.5 increasing from 17.2 ± 2.0 s (n = 6) to 33.9 ± 4.1 s (n = 7; Figure 5C). Like at calyces after hearing, PAO had no significant effect on ΔCm or ICa. These results suggest that PIP2 in calyces before hearing is already involved in presynaptic vesicle endocytosis, but it is only after hearing onset when it is linked to PKG. Immunolocalization of PIP2 on neuronal membrane, including that of presynaptic terminals, has been reported in various preparations (Honda et al., 1999, Micheva et al., 2001 and Nakano-Kobayashi et al., 2007).