PIP2 expression in calyceal presynaptic terminals was identified from its immunofluorescence intensity profiles (green) overlapping with that of synaptophysin (red) (Figure 6A). When we preincubated

slices (for 1h at RT) with Rp-cGMPS (3 μM), or PTIO (100 μM), immunofluorescence intensity of PIP2 in the calyceal terminal was reduced by ∼50% (Figures 6A and 6B). To further examine whether the NO-linked PKG activity upregulates the PIP2 level, we tested the effect of Rp-cGMPS and PTIO on the level Gemcitabine of PIP2 in whole-brainstem lysates using ELISA. In the brainstem tissue of rats after hearing (P13–P15), the PIP2 concentration was 68.9 ± 1.4 pmol/mg (n = 6). After incubation with Rp-cGMPS (3 μM, 1 hr at RT) the PIP2 concentration declined by 44% (to 38.7 ± 2.5 pmol/mg, n = 6; Figure 6C). Likewise, preincubation of brainstem lysate with PTIO (100 μM, 1h at RT) reduced the PIP2 concentration by 52% (to 32.8 ± 1.3 pmol/mg, n = 6). These results are consistent with those

of the immunocytochemical density Selleckchem LY294002 quantification of PIP2 in calyces (Figures 6A and 6B), suggesting that the retrograde NO-PKG mechanism might operate widely at many synapses in the brainstem. We further measured the PIP2 concentration in the brainstem lysate from P7–P9 rats using the same protocol. The PIP2 concentration of P7–P9 brainstem (63.2 ± 2.0 pmol/mg, n = 6; Figure 6C) was slightly lower than that of P13–P15 brainstem (p < 0.05). More importantly, Rp-cGMPS (3 μM, 1h incubation at RT) had no effect on the PIP2 level (58.6 ± 2.1 pmol/mg, n = 6) in P7–P9 brainstem (Figure 6C). These results suggest that the PKG-PIP2 linkage is established only after hearing onset. We next examined whether PKG expression level changes during the second postnatal week in the MNTB region and in the brainstem tissue Substrate-level phosphorylation using immunocytochemical (Figure 7A) and western blot (Figure 7B) analysis.

Immunocytochemical analysis showed that the immunoreactivity of PKG 1α in the MNTB neuron and calyceal terminals increased from P7 to P14. Densitometric quantification of immunoreactivity indicated a significant difference between two ages (p < 0.01, Figure 7A). We obtained similar results for PKG1β (data not shown). In western blot analyses, pan-PKG1 antibody revealed a strong immunoreactivity in the brainstem of P14 rats, whereas the expression level was much lower in P7 rat brainstem (Figure 7B). Densitometric analysis indicated 3.6-fold difference between two ages (n = 3, p < 0.01) (Figures 7B and S3). Strong PKG immunoreactivity was also found in the heart tissue, but with no significant difference between two ages. PKG immunoreactivity was not detected in the liver tissue. Thus, the developmental upregulation of PKG1 during the second postnatal week might be a brain-specific phenomenon.