File:A-dynamin-1--dynamin-3--and-clathrin-independent-pathway-of-synaptic-vesicle-recycling-mediated-by-elife01621f011.jpg

English: (A) Western blotting of extracts of Dyn1/3 DKO neuronal cultures showing depletion of CHC by transduction with lentivirus encoding CHC shRNAmiR but not by lentivirus encoding control shRNAmiR. (B) Drastic reduction in CCP number in axon terminals of Dyn1/3 DKO neurons maintained under spontaneous network activity upon CHC KD. (C–F) Impact of CHC KD on the ultrastructure of Dyn1/3 DKO neurons preincubated overnight in TTX, stimulated with high K+ for 90 s in the presence of soluble HRP and then allowed to recover for 30 min in HRP-free medium. (C) Electron micrographs showing that the KD of CHC does not prevent the reformation of SVs after stimulation in Dyn1/3 DKO neurons. (D) High K+ stimulation induces an increase of CCPs number in Dyn1/3 DKO neurons that express control shRNAmiR but not in neurons that express CHC shRNAmiR (CCPs had already been reduced to very low level also in the control treated cultures before stimulation by the overnight treatment in TTX). (E) The recovery of SV number is similar in control and CHC KD conditions. (F) The reduction of bulk endosomes during recovery at 30 min is not affected by CHC KD. The values of SVs, CCPs and bulk endosomes per μm2 were calculated by averaging the corresponding values calculated at individual synapses. Scale bar = 200 nm. ****, **, * indicate p-values of <0.0001, <0.01 and <0.05, respectively. n.s., ‘not significant’. Black asterisks refer to comparisons between total vesicles, colored asterisks to comparisons between HRP-labeled vesicles. Bars: standard error of the mean.