Optical methods to assess vesicular cycling at bead-induced presynapses. (A–C) Anti-synaptotagmin (syt) antibody feeding experiments to assess the cycling activity of presynapses. (A) Cartoon of the anti-syt feeding experiments. Living neurons are incubated with the syt antibody (orange) directed against an extracellular epitope of synaptotagmin (yellow), either for 60 min to measure constitutive cycling, or for 3 min in the presence of 50 mM KCl to measure stimulated cycling. Neurons are then fixed and the syt antibody is revealed with a secondary antibody (green). (B) Widefield fluorescence image of cultured neurons 2 d after bead seeding at 8 div, labeled for actin (green), synaptophysin (purple), map2 (gray), and feeding with anti-synaptotagmin antibody (syt) during a 3-min incubation with KCl (stimulated cycling). (C) Zooms corresponding to the natural synapse (NS), S+, and S− axon-bead contacts highlighted in B. Scale bars for B, 20 µm; for C, 2 µm. (D–G) FM1-43 dye loading/release experiment to assess the cycling activity of presynapses. (D) Cartoon of the FM1-43 experiment. Living neurons are first loaded with FM1-43 using a 3-min incubation in 50 mM KCl followed by 15 min of recovery, then images of the “loaded” time point are taken. FM1-43 is then released using a second 3-min incubation with 50 mM KCl and 15-min recovery, before images are taken of the “released” time point. (E) Widefield fluorescence image of cultured neurons 2 d after bead seeding at 8 div, labeled for actin using SiR (green) after loading with FM1-43 (orange). (F) Zooms corresponding to area highlighted in E, showing the images obtained after loading (left column) and release (right column) of FM1-43. Beads are indicated by dashed-line circles labeling the induced presynapses (S+) and axon-bead contact with no presynapse (S−). Scale bars for B, 20 µm; for C, 5 µm. (G) Quantification of the FM staining intensity after loading (orange) and release (yellow) in natural synapses (NS) and bead-induced presynapses (S+) as well as axon-bead contacts devoid of a presynapse (S−). Significance signs on the graph compare to the value for the same labeling condition in bead-induced presynapses (S+, normalized to 1.0 for the loading condition). See Data S1 file for detailed statistics.
Figure S2.

Optical methods to assess vesicular cycling at bead-induced presynapses. (A–C) Anti-synaptotagmin (syt) antibody feeding experiments to assess the cycling activity of presynapses. (A) Cartoon of the anti-syt feeding experiments. Living neurons are incubated with the syt antibody (orange) directed against an extracellular epitope of synaptotagmin (yellow), either for 60 min to measure constitutive cycling, or for 3 min in the presence of 50 mM KCl to measure stimulated cycling. Neurons are then fixed and the syt antibody is revealed with a secondary antibody (green). (B) Widefield fluorescence image of cultured neurons 2 d after bead seeding at 8 div, labeled for actin (green), synaptophysin (purple), map2 (gray), and feeding with anti-synaptotagmin antibody (syt) during a 3-min incubation with KCl (stimulated cycling). (C) Zooms corresponding to the natural synapse (NS), S+, and S− axon-bead contacts highlighted in B. Scale bars for B, 20 µm; for C, 2 µm. (D–G) FM1-43 dye loading/release experiment to assess the cycling activity of presynapses. (D) Cartoon of the FM1-43 experiment. Living neurons are first loaded with FM1-43 using a 3-min incubation in 50 mM KCl followed by 15 min of recovery, then images of the “loaded” time point are taken. FM1-43 is then released using a second 3-min incubation with 50 mM KCl and 15-min recovery, before images are taken of the “released” time point. (E) Widefield fluorescence image of cultured neurons 2 d after bead seeding at 8 div, labeled for actin using SiR (green) after loading with FM1-43 (orange). (F) Zooms corresponding to area highlighted in E, showing the images obtained after loading (left column) and release (right column) of FM1-43. Beads are indicated by dashed-line circles labeling the induced presynapses (S+) and axon-bead contact with no presynapse (S−). Scale bars for B, 20 µm; for C, 5 µm. (G) Quantification of the FM staining intensity after loading (orange) and release (yellow) in natural synapses (NS) and bead-induced presynapses (S+) as well as axon-bead contacts devoid of a presynapse (S−). Significance signs on the graph compare to the value for the same labeling condition in bead-induced presynapses (S+, normalized to 1.0 for the loading condition). See Data S1 file for detailed statistics.

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