First we examined the shape of the mEPSC as an indicator of postsynaptic receptor subunit changes and found no difference in the amplitude to charge ratio of mEPSCs from VGLUT1-, VGLUT2-, or VGLUT3-expressing neurons (Figure 3D). Finally, we examined the rate of refilling of the RRP by depleting the RRP with one 500 mM sucrose application, followed by a second application 5 s later. There were no differences in the percentage of the RRP refilled after 5 s between any of the groups (Figure 3E). Finally we considered that a differential protein-protein interaction might see more underlie the

differences between VGLUT isoforms. The most striking difference between VGLUT1 and VGLUT2/3 that has been reported so far is the existence of the polyproline motif on the C terminus of VGLUT1 that mediates an interaction with endophilins (De Gois et al., 2006, Vinatier

et al., 2006 and Voglmaier et al., 2006), a protein family primarily known for its role in synaptic vesicle endocytosis (Farsad et al., 2001, Guichet et al., 2002, Hill et al., 2001, Ringstad et al., 1999, Schmidt selleck compound et al., 1999, Schuske et al., 2003 and Simpson et al., 1999). Both VGLUT2 and VGLUT3 lack this motif. We therefore expressed VGLUT1 containing a point mutation shown to disrupt endophilin binding (P554A) (Vinatier et al., 2006) in VGLUT1−/− hippocampal cells and repeated the measurements of Pvr, paired-pulse ratio, and 10 Hz stimulation with VGLUT1- and VGLUT2-rescued cells. We found that VGLUT1 P554A unless induced paired-pulse

depression comparable to that observed in VGLUT2-expressing neurons, while VGLUT1 neurons showed facilitation (Figures 4A and 4B). The release probability analysis revealed a 40% increase in Pvr in both VGLUT1 P554A- and VGLUT2-expressing neurons compared to VGLUT1 (Figure 4C). VGLUT1 P554A also increased the amount of steady-state depression in response to 10 Hz stimulation from VGLUT1 levels to VGLUT2 levels (Figure 4D). These changes were accompanied by an increase in the charge contained in the EPSC of VGLUT1 P554A-expressing neurons, while the size of the RRP was not different between the three groups (Figures 4E and 4F). mEPSC amplitudes and frequency of VGLUT1 P554A-expressing neurons were not significantly different from VGLUT1- or VGLUT2-expressing neurons (Figures 4G and 4H). Endophilin is a protein that has been implicated in endocytosis and vesicle cycling, so how the interaction of VGLUT1 with endophilin could lower the probability that a vesicle is released in response to an action potential is unclear. We considered two possible mechanisms. First, VGLUT and endophilin may work together to promote a lower Pvr for synaptic vesicles on which the complex is present.