, 2005, Schuske et al., 2003 and Verstreken et al., 2003). To assess the potential occurrence of an endocytic

delay, as expected if endophilin was involved in CCP fission, we performed dynamic assays of endocytosis using a synaptopHluorin-based strategy (Sankaranarayanan and Ryan, 2000). In TKO cells, the time constant of endocytic recovery following a 10 Hz stimulus for 30 s was approximately 2.5-fold slower in TKO (71.4 ± 15.8 s) than in WT (29.3 ± 5.2 s) (Figure 4A). Given sufficient time, however, the signal recovered and synapses could sustain multiple rounds of exo/endocytosis. Similar results were obtained with vGLUT1-pHluorin, this website a chimera of the vesicular glutamate transporter vGLUT1 with pHluorin (Voglmaier et al., 2006) (26.6.5 ± 6.7 s in WT and 82.2 ± 12 s in TKO) (Figure 4B). Thus, although the SH3 domains of endophilin 1 and 3 interact with vGLUT1 (Voglmaier et al., 2006), the defect in the compensatory endocytic recapture of this protein in endophilin TKO cells is not significantly more severe that the defect in the reinternalization of synaptobrevin. In principle, the delayed poststimulus

recovery could be due to a delay in the acidification of the newly formed vesicles. However, a brief exposure to acid medium during the recovery (Sankaranarayanan and Ryan, 2000) demonstrated that the pHluorin responsible for the increased signal remained buy EPZ5676 cell-surface exposed, thus suggesting a bona fide endocytic delay (Figure 4F). The slower kinetics of endocytosis in TKO neurons could be fully rescued by transfection with endophilin

1 (Figures 4B–4E). In contrast, a mutant endophilin 1 construct that contains the BAR domain but that lacks the SH3 domain produced a limited rescue of the endocytic defect, and primarily during the late phase of the recovery (Figures 4B–4E). Because the BAR domain of endophilin for alone is recruited to the CCP neck, a possible interpretation of this partial rescue of endocytosis is a facilitatory and/or stabilizing effect of the overexpressed BAR domain on the vesicle neck. To gain direct insight into whether the endocytic delay observed in TKO cultures was due to a block in fission, we performed electron microscopy (EM). TKO synapses revealed a strikingly different phenotype relative to controls: a reduced number of SVs and a strong accumulation of clathrin-coated vesicular profiles (Figures 5A–5C). Surprisingly, no accumulation of CCPs was observed. In sections of some nerve terminals, nearly the entire pool of SVs had been replaced by clathrin-coated profiles (Figure 5B). Quantification of EM micrographs showed that the mean number of SVs per synapse was substantially lower (39.8%) in TKO than in controls, whereas the number of CCVs had increased more than 31 times (Figures 5F–5I). Similar, but less severe, changes were observed at synapses of DKO neurons (Figures 5F–5I).