Endocytosis Assay for Synaptic Vesicle Recycling

The endocytosis assay to monitor SV recycling was performed using rabbit polyclonal antibodies directed against the intravesicular domain of synaptotagmin1 (Synaptic System), applied for 5 min at RT on the cultures, as described previously [30 (link)]. Incubations with the antibody (1:400) were performed in Tyrode solution containing 124 mM NaCl, 5 mM KCl, 2 mM MgCl₂, 30 mM glucose, 25 mM HEPES, pH 7.4 and 2 mM CaCl₂. After fixation and permeabilization, a synaptophysin counter staining with mouse anti synaptophysin, 1:400 (Sigma-Aldrich) visualized the totality of synaptic vesicles. Acquired images were processed and quantitatively analyzed with ImageJ software as previously described [47 (link)]. Briefly, cultures were infected at DIV4 with GPF expressing viruses and assayed at DIV14 as in [22 (link)]. GFP positive processes were manually tracked and the number of synaptotagmin and synaptophysin positive clusters and synaptophysin positive clusters present in the region of interest were automatically counted.