Experimentally, in most case, we have loaded drugs and molecules directly into presynaptic terminals, whereas in the previous study in hippocampal culture (Micheva et al., 2003) they used membrane permeable reagents, which can affect both presynaptic and postsynaptic mechanisms. Finally, our occlusion experiments have revealed that individual molecular cascades are connected in series for endocytic acceleration mechanism. In membrane capacitance measurements from the calyx of Held after hearing

onset, intraterminal loadings of PKG inhibitors slowed endocytic time course (Figures 1 and 2). Bath-applied PKG blocker also slowed endocytosis (data not shown) and reduced PIP2 level in calyces or brainstem tissues (Figure 6). However, these effects were absent before hearing onset. Such a developmental change can be explained by the findings that PKG Cobimetinib concentration level in brainstem tissue and MNTB region increases by >2-fold during the second postnatal week (Figure 7). As animals mature, PKG activity is upregulated in the nerve terminal, thereby speeding HA-1077 cost the endocytic rate. In paired AP recordings from presynaptic and postsynaptic elements, intraterminal loading of PKG inhibitor lowered the fidelity of synaptic transmission during sustained high-frequency stimulation (Figure 8). Thus, maturation of the PKG-dependent mechanism

accelerates endocytic rate thereby likely enhancing vesicle reuse for the maintenance of high frequency synaptic transmission at this fast glutamatergic synapse. At many synapses, such as frog neuromuscular junction (NMJ) (Wu and Betz, 1996), hippocampal synapses in culture (Balaji et al., 2008), goldfish bipolar cell terminal second (von Gersdorff and Matthews, 1994) and

calyces of Held before hearing onset (Sun et al., 2002 and Yamashita et al., 2005), endocytic time constants, assessed by capacitance measurements, or by vesicle imaging, become longer in relation to the magnitude of exocytosis, whereas no such correlation exists in Drosophila NMJ ( Poskanzer et al., 2006) and at calyces of Held of rodents after hearing onset ( Renden and von Gersdorff, 2007 and Yamashita et al., 2010). This positive correlation is interpreted as a saturation of endocytic machinery and ensuing accumulation of unretrieved vesicles ( Sun et al., 2002 and Balaji et al., 2008). Clearly, such a positive correlation is unfavorable with respect to the exoendocytic balance of synaptic vesicles. In the present study, we show that this positive correlation can be reproduced at P13–P14 calyces by pharmacological block of presynaptic PKG ( Figure 2A), suggesting that maturation of the PKG-dependent mechanism underlie the developmental loss of the positive correlation between ΔCm and endocytic time constant. It remains to be seen whether this mechanism generally apply to other type of synapses. During synaptic transmission at high rate, synaptic vesicles are recycled and reused.