Spontaneous release

is quite heterogeneous across different boutons but does not correlate with the level of reporter expression (Figure S5B), further excluding a role for mislocalization of overexpressed proteins. Since VAMP7 exhibits higher spontaneous but less evoked release than VGLUT1, spontaneous release cannot simply reflect the size of the recycling pool, suggesting that spontaneous release might derive from the resting pool. Since the pHluorin is large and might interfere with membrane trafficking, we have also used an alternative approach to monitor evoked and spontaneous exocytosis. There buy Cyclopamine are no available antibodies that recognize the lumenal domain of either VGLUT1 or VAMP7, so we fused a short (ten residue) peptide containing the HA epitope to the lumenal domain

of both proteins. Twelve days after transfection, hippocampal cultures were incubated with unlabeled anti-HA antibody to block HA-tagged protein already at the cell surface, then with HA antibody directly conjugated to Alexa 488 to detect protein newly delivered to the plasma membrane (Figure 5C). As anticipated, field stimulation at 10 Hz for 2 min greatly increases surface exposure of both VGLUT1- and VAMP7-HA (Figure 5D). However, incubation for 20 min in the absence of stimulation also enables detection of spontaneously delivered vesicles containing both VGLUT1 and VAMP7. To assess the total amount of reporter

expressed at boutons, find protocol we immunostained for HA after fixation and permeabilization, using a secondary antibody conjugated to Alex 635 (Figures 5C and 5D). Normalized to total HA-tagged reporter, VGLUT1 shows a strong response to stimulation (Figure 5E), consistent with targeting to the recycling pool. In contrast, VAMP7-HA exhibits considerably less response to stimulation. However, both proteins show similar levels of spontaneous release, with spontaneous release of VAMP7 over 20 min approaching that observed after stimulation for 2 min (Figure 5F). The analysis by antibody labeling thus supports the preferential Phosphoprotein phosphatase targeting of VAMP7 to the resting pool, which undergoes spontaneous release. Although indistinguishable from typical synaptic vesicles by morphology and standard fractionation, the VAMP7+ membranes and by inference the resting pool thus behave like constitutive secretory vesicles. To characterize the sources of spontaneous release, we determined whether spontaneous release can occlude the effects of stimulation. Using the pHluorin-based reporters, we first measured recycling pool size by stimulation alone (experiment 1).