VAMP7 also localizes to endosomal and lysosomal membranes at the cell body and dendrites, but it has been found to reside only on synaptic vesicles at boutons (Muzerelle et al., 2003, Salazar et al., 2006 and Scheuber et al., 2006), where we analyzed the data. We further confirm the localization of endogenous VAMP7 to membranes that behave like synaptic vesicles by both gradient fractionation and immunoisolation (Figure S4A–S4C). However, it remains possible that overexpression

may result in mislocalization of VAMP7 to endosomes or lysosomes at presynaptic sites, and hence an apparent reduction in recycling pool size. To address this possibility, we expressed VAMP7 and VAMP2 as control fused at their lumenal C-termini to horseradish peroxidase (HRP) (Leal-Ortiz et al., 2008). Localization of HRP within vesicles prevents diffusion of the HRP reaction product KU-57788 research buy and thus unambiguously labels the VAMP7+ or VAMP2+ population. Both VAMP7- and VAMP2-HRP indeed label only

a subset BMS-907351 in vitro of synaptic vesicles that intermingle with unlabeled vesicles (Figure 4A). In both cases, the HRP reaction product accumulates in small, round vesicles with the same diameter as unlabeled vesicles (Figures 4B and 4C), and not within any other compartment at or adjacent to the nerve terminal. Thus, VAMP7 localizes to membranes morphologically indistinguishable from synaptic vesicles. Resting pool vesicles do not apparently respond to field stimulation, but do they have the capacity for exocytosis under other circumstances? Previous work has suggested that evoked and spontaneous release may derive from distinct vesicle populations (Chung et al., 2010, Fredj and Burrone, 2009 and Sara et al., 2005) (but see also (Groemer and Klingauf, 2007, Hua et al., 2010 and Wilhelm et al., 2010). This predicts that as a protein enriched in the resting (and

hence unresponsive) pool of synaptic vesicles, VAMP7 may undergo more spontaneous release than VGLUT1. To test this possibility, we used an optical assay for spontaneous release. Like bafilomycin, the H+-ATPase inhibitor until folimycin prevents the reacidification of vesicles that have undergone exocytosis. However, folimycin is less cell permeant than bafilomycin, reducing its access to intracellular vesicles that have not undergone exocytosis (Atasoy et al., 2008). In the presence of folimycin and tetrodotoxin (TTX), the increase in fluorescence of VGLUT1- and VAMP7-pHluorin should thus reflect spontaneous release (Figure 5A). This increase in fluorescence is not due to leakage of folimycin into the neurons and alkalinization of vesicles that have not undergone exocytosis because it is greatly reduced by the high-affinity, cell-permeant calcium chelator BAPTA-AM (Figure S5A), consistent with the known calcium dependence of spontaneous release (Wasser and Kavalali, 2009).