, 2010), Kavalali and coworkers (Chung et al., 2010) along with Fredj and Burrone (Fredj and Burrone, 2009) have concluded that both types of neurotransmission are fueled by distinct SV populations. Furthermore, the molecular interactions driving spontaneous versus evoked release seem to differ (Deák et al., 2006 and Xu et al., 2009). Based on the premise that distinct SV pools may differentially contribute to various forms of synaptic transmission, Ramirez et al. (2012) reasoned that spontaneously fusing SVs may in fact be distinguished by molecular composition from their counterparts involved in AP-driven exocytosis. SVs are known Selleck RG7204 to contain a number of SNARE proteins besides the canonical SNARE synaptobrevin 2 (Syb2) (Takamori

et al., 2006), a crucial E7080 order factor for evoked release that acts via complex formation with its cognate SNAREs syntaxin and SNAP-25 on the plasma membrane. Among the noncanonical SNAREs found on SVs are the endosomal proteins Vti1a and vesicle-associated membrane protein 7 (VAMP7, also called Ti-VAMP) (Takamori et al., 2006). Vti1a, by associating with VAMP4, syntaxin 6, and syntaxin 13, regulates fusion of early endosomes. An isoform of Vti1a is enriched on SVs and has been postulated to form a distinct SNARE complex with unclear function (Antonin et al., 2000). VAMP7 mediates exocytosis of lysosome-related organelles and

may regulate neurotransmission at hippocampal mossy fiber synapses (Chaineau et al., Mannose-binding protein-associated serine protease 2009). To investigate whether these noncanonical SNAREs differentially regulate evoked versus spontaneous release, Ramirez et al. (2012) first characterized the exo-endocytic trafficking of Vti1a and VAMP7 tagged with a pH-sensitive GFP moiety commonly referred to as pHluorin. In hippocampal neuronal cultures, pHluorin-tagged Vti1a and VAMP7 appear to be localized to synapses where they reside in acidic compartments, most likely SVs, although a fraction of them may be targeted to endosomes.

When Kavalali and coworkers compared the exocytic behavior of their various SNARE-pHluorin chimeras they noted surprising differences: AP stimulation at 20 Hz caused the exocytic fusion of a sizeable fraction of Syb2-pHluorin as expected, whereas Vti1a-pHluorin and VAMP7-pHluorin were only reluctantly mobilized. By contrast, when they probed spontaneous neurotransmission using the v-ATPase blocker folimycin to prevent postfusion reacidification of SVs, they observed that Vti1a-pHluorin underwent substantial fusion detectable by fluorescence dequenching. VAMP7-pHluorin showed only modest levels of exocytosis both in the presence or absence of electrical stimulation. These data suggest that Vti1a preferentially traffics at rest. As differential behavior of pHluorin-tagged SNAREs could originate from different synapses (Kavalali et al., 2011), the authors produced spectrally shifted pHluorin variants tagged with the DsRed derivative mOrange, which also exhibits pH-dependent changes in fluorescence.