To analyze the functional consequences of APD application on synaptic vesicle recycling, we used rat hippocampal neurons transfected with spH, an established reporter of exo- and endocytosis (Figure 4A) (Sankaranarayanan et al., 2000). Our experimental setup consisted of 18 electrical stimulations with 50 AP (10 Hz)

every 1.5 min. During min 6–18 of the experiment, the cells were B-Raf assay perfused with either APDs or vehicle (Figure 4B). We observed dose-dependent reductions of the evoked exocytosis fluorescence responses upon administration of APDs in contrast to vehicle control (Figure 4C). This reduction was reversible for all four APDs. Compensatory endocytosis of synaptic vesicles was not affected by HAL, CPZ, CLO, and RSP (Figure S4). We conclude that APDs inhibit electrically stimulated synaptic vesicle exocytosis in a dose-dependent manner. Action potential propagation along the axon results in the influx of Ca2+ through voltage-gated ion channels into synaptic boutons, which triggers fast synaptic vesicle exocytosis. Because APDs have been

described to have diverse effects on the ion channels involved in this process (Ogata et al., 1989; Sah and Bean, 1994; Wakamori et al., 1989; Yang and Wang, 2005), we next tested whether the observed inhibition of exocytosis is directly linked to ion channel modulation or is the result of effects on the Farnesyltransferase multitude of proteins involved in the assembly and function of the presynaptic vesicle release machinery itself. We first analyzed the role of calcium channels because Ca2+ is directly linked to vesicle Anti-cancer Compound Library release via the Ca2+-sensor synaptotagmin. We measured

stimulation-dependent changes in fluo-4-fluorescence while blocking postsynaptic Ca2+ influx by AP5 (Koester and Johnston, 2005; Oertner et al., 2002; Schiller et al., 1998). Fluo-4 showed a strong fluorescence increase upon electrical stimulation and Ca2+ influx (Figure 5A) (Gee et al., 2000). Synaptic boutons were identified by their activity-dependent FM styryl dye uptake and release (Groemer and Klingauf, 2007), and fluo-4 fluorescence was quantified in these regions (Figure 5A). Similar to the spH experiments (Figure 4), the vehicle control did not alter the presynaptic Ca2+ influx evoked by subsequent trains of 50 AP, whereas CPZ, HAL, CLO, and RSP significantly reduced the Ca2+ influx (Figures 5B–5D). This inhibition of Ca2+ influx was reversible upon drug washout for all of the APDs (Figures 5C and 5D). When comparing the APD-induced amplitude reductions in the evoked fluo-4 and spH fluorescence increases, we found a strong positive correlation (Figure 5E) (R = 0.86; p < 0.001) between the two parameters. Thus, the inhibition of presynaptic Ca2+ influx correlated strongly with the inhibition of synaptic vesicle exocytosis for all of the APDs.