This compound prevents reacidification of endocytosed vesicles (Dröse and Altendorf, 1997) and, therefore, permits continued visualization of fused vesicles. We found that vti1a does not participate in rapid SV endocytosis during stimulation because folimycin treatment did not reveal any increase in vti1a-pHluorin fluorescence compared to the same synapses imaged prior to treatment www.selleckchem.com/products/E7080.html (Figure S1 available online). Next, we measured trafficking of vesicles containing pHluorin-tagged

syb2, vti1a, or VAMP7 during both spontaneous and evoked transmission using folimycin. Treatment with this compound allows the small cumulative increases in fluorescence due to spontaneous transmission or mild evoked stimulation to be detected (Atasoy et al., 2008). As shown in Figure 2A, Ferroptosis activation in 2 mM external Ca2+, syb2- and VAMP7-pHluorin fluorescence signals increased minimally at rest, indicating that these proteins show only modest levels of spontaneous trafficking. These data are consistent

with previous work showing that syb2 mediates a portion of spontaneous release (Schoch et al., 2001). In contrast to its insensitivity to folimycin treatment during stimulation, vti1a exhibited a substantial fluorescence increase at rest, suggesting that spontaneously released vesicles contain abundant vti1a. After 10 min of imaging at rest, the neurons were subjected to 300 APs at 1 Hz in the same external solution. As expected, the rate of syb2 fluorescence change increased dramatically due to the stimulation-dependent exocytosis of syb2 containing vesicles. In contrast, the slope of fluorescence increase for vti1a decreased slightly during stimulation compared to the measurements at rest, whereas the slope for VAMP7 of increased only slightly, suggesting little evoked release of vesicles containing vti1a and/or VAMP7. The slope of fluorescence

increase at rest for vti1a is significantly higher than that of syb2, whereas the slope during 1 Hz stimulation for syb2 is significantly higher than that of vti1a (Figure 2B). The percentage of total fluorescence increase at rest for vti1a was also significantly higher than that of syb2 (Figure 2C). These results confirm that little vti1a is trafficked during evoked transmission and suggest that vti1a is preferentially trafficked at rest. While these experiments utilized a full-length pHluorin-tagged version of vti1a, a longer alternative splice variant of the protein, designated vti1a-β, is reportedly enriched in synaptic terminals (Antonin et al., 2000b). We confirmed the subcellular localization and trafficking behaviors of vti1a-β at rest and with stimulation and found no differences between vti1a-β and full-length vti1a (Figure S2). Spontaneous trafficking of vti1a was also observed under similar conditions in the presence of tetrodotoxin (TTX) to block APs (Figure S3).