We next recorded mIPSCs from uninfected neurons or neurons expressing full-length vti1a- or ΔN vti1a-pHluorin. Representative traces are shown in Figure 6J, and cumulative probability histograms of the inter-event intervals are shown in Figure 6K. Buparlisib Significant increases were seen in mIPSC frequency in neurons expressing either wild-type or ΔN vti1a-pHluorin, and this difference was greatest at low inter-event intervals, consistent with the results of the vti1a KD studies (Figures 5E–5H). The effect of ΔN vti1a-pHluorin expression was greater than that of the wild-type

protein, consistent with the notion of an autoinhibitory function of the N-terminal portion of vti1a. No significant differences were seen in average mIPSC amplitude between wild-type neurons and those expressing vti1a- or ΔN vti1a-pHluorin (wild-type = 37.8 ± 5.3 pA, vti1a = 40.1 ± 3.3 pA, p = 0.72, ΔN vti1a = 38 ± 3.8 pA, p = 0.98). Similar results were seen with spontaneous excitatory transmission. Sample traces of recordings from uninfected neurons or neurons expressing full-length vti1a- or ΔN vti1a-pHluorin are shown in Figure 6L. Cumulative probability histograms of the inter-event intervals are shown in Figure 6M. Although expression of full-length vti1a has little effect on mEPSC frequency, expression of ΔN vti1a-pHluorin robustly increases the probability of high-frequency spontaneous events. Differences

AZD9291 cost seen in average mEPSC amplitudes between wild-type neurons and those expressing vti1a- or ΔN vti1a-pHluorin were significant (wild-type = 25.6 ± 1.4 pA, vti1a = 20.2 ± 0.9 pA, p = 0.005, ΔN vti1a = 35.06 ± 3.9 pA, p = 0.03), suggesting a potential postsynaptic effect of vti1a or a possible consequence of alterations in spontaneous glutamate release. These data complement the loss-of-function studies described above and support a specific role for vti1a in meditating spontaneous transmission. Additionally, the data suggest an autoinhibitory function for the N terminus of vti1a. Our current data suggest Methisazone a specific role for vti1a in spontaneous neurotransmission, as well as the presence

of this SNARE on a pool of vesicles distinct from those containing syb2. To address whether vti1a traffics independently of syb2, we monitored the spontaneous trafficking of vti1a-pHluorin in syb2 knockout (KO) neurons. As a control, syb2-pHluorin trafficking was monitored in separate cultures of syb2 KO neurons. An averaged time course from multiple experiments is shown in Figure 7A. No differences were found between syb2- and vti1a-pHluorin trafficking in the average slope values of the increase in fluorescence at rest (Figure 7B) or the percentage of total fluorescence generated during spontaneous activity normalized to the total protein levels visualized after NH4Cl treatment (Figure 7C). These findings strongly argue that vti1a is localized to a pool of vesicles distinct from those containing syb2, which is mobilized at rest.