, 2006 and Xu et al., 2008) and peripheral synapses (Maeno-Hikichi et al., 2011 and Zhang et al., 2010) without reporting toxicity and using vehicle solution as negative control. We applied stimulation trains (10 s at 10, 30, and 100 Hz), before and after incubation in dynasore (160 μM, 15 min). After the incubation, the stimulus evoked a higher fluorescence response with respect to control conditions (0.05% DMSO) in WT NMJs (Figures 6A and S4) because spH was not retrieved likely due to the acute dynamin1 inhibition (Chung et al., 2010). In contrast to WT synapses, the spH fluorescence in mutant synapses changed rather little with dynasore (Figure 6B). The occlusion of dynasore effect suggested an

impairment of dynamin1-dependent endocytosis at the CSP-α KO junctions. The endocytic efficiency (Nicholson-Tomishima and Ryan, 2004) (subtraction of normalized ΔF control from normalized ΔF in dynasore) was 71.3 ± Doxorubicin chemical structure 17.8% for WT and 30.2 ± 16 for CSP-α KO synapses (Figure 6C). Similar results were obtained at 10 and 100 Hz stimulation frequencies (Figures S4A–S4F), indicating that decreased dynasore-sensitive endocytosis

in the mutants was not GSK1210151A nmr due to lower exocytic load. Curiously, the recovery of fluorescence after the stimulation train, in WT and mutant junctions, was almost insensitive to dynasore, suggesting that post-stimulus endocytosis was less dependent on dynamin1 than endocytosis during the stimulus. Next, we investigated endocytosis by

challenging the terminals with longer stimulation trains (180 s at 30 Hz) (Figure 6D). Under these conditions, through the fluorescence recovery was fitted to a single exponential, that was longer in the mutants (τwt = 81.7 ± 4.7 s, n = 14 junctions; τko = 172.9 ± 36.5 s, n = 13 junctions, p = 0.016 Student’s t test) (Figure 6E), as expected for a slowing down in the time-course of endocytosis. Interestingly, endocytosis became more sensitive to dynasore for both WT and CSP-α KO after longer stimulation trains (Figure 6F). This observation indicated that, in contrast to short, longer stimulation favors the dynamin1 recruitment for post-stimulus endocytosis. Also under these conditions, dynasore-sensitive endocytosis during the stimulus, although evident, was lower in the mutant (Figure 6F). Next, we analyzed the electrophysiological recordings and found that in the WT, although the EPPs amplitude decreased at the end of the train, the terminals maintained a significant level of neurotransmitter release (Figure 6G, upper panels) due to rapid vesicle recycling (Maeno-Hikichi et al., 2011). In contrast, mutant terminals underwent strong synaptic depression that almost abolished EPPs before the train ended (Figure 6G, lower panels). Remarkably, the effect of dynasore at the WT terminals was a phenocopy of the CSP-α KO phenotype (Figure 6G). We compared the time course of the cumulative quantal content (ΣQC) released during the train in control conditions and in dynasore.