Actin-enriched induced presynapses have a higher vesicular cycling than non-enriched induced presynapses. (A) 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 constitutive cycling (orange). (B) Zooms corresponding to the A+, A−, S− axon-bead contacts and natural synapses (NS) highlighted in B. (C) Widefield fluorescence image of cultured neurons 2 d after bead seeding at 8 div, labeled for actin (green), synaptophysin (purple), and map2 (gray) and feeding with anti-synaptotagmin antibody (syt) during KCl-stimulated vesicular cycling (orange). (D) Zooms corresponding to the A+, A−, S− axon-bead contacts and natural synapses (NS) highlighted in C. Scale bars in A and C, 20 µm; B and D, 2 µm. (E) Quantification of the labeling intensity for actin (green), synaptophysin (purple), and after syt feeding for constitutive (orange) and stimulated (yellow) vesicular cycling at actin-enriched presynapses (A+), induced presynapses with no actin enrichment (A−), and axon-bead contacts devoid of presynapse (S−), normalized to the intensity at A+ presynapses. (F) Zooms on axon-bead contacts of living neurons stained with SiR-actin (green) and loaded with FM1-43 (orange, left image) before release (right image). Left two images show an A+ presynapse, right images show an A− presynapse. Scale bar, 2 µm. (G) Quantification of the FM staining intensity after loading (orange) and release (yellow) in A+ and A− induced presynapses as well as axon-bead contacts devoid of a presynapse (S−). Significance signs on graphs compare to the value for the same labeling condition in actin-enriched presynapses (normalized to 1.0 for A+ in E, normalized to 1.0 for the A+ in loading condition in G). See Data S1 file for detailed statistics.
Figure 3.

Actin-enriched induced presynapses have a higher vesicular cycling than non-enriched induced presynapses. (A) 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 constitutive cycling (orange). (B) Zooms corresponding to the A+, A−, S− axon-bead contacts and natural synapses (NS) highlighted in B. (C) Widefield fluorescence image of cultured neurons 2 d after bead seeding at 8 div, labeled for actin (green), synaptophysin (purple), and map2 (gray) and feeding with anti-synaptotagmin antibody (syt) during KCl-stimulated vesicular cycling (orange). (D) Zooms corresponding to the A+, A−, S− axon-bead contacts and natural synapses (NS) highlighted in C. Scale bars in A and C, 20 µm; B and D, 2 µm. (E) Quantification of the labeling intensity for actin (green), synaptophysin (purple), and after syt feeding for constitutive (orange) and stimulated (yellow) vesicular cycling at actin-enriched presynapses (A+), induced presynapses with no actin enrichment (A−), and axon-bead contacts devoid of presynapse (S−), normalized to the intensity at A+ presynapses. (F) Zooms on axon-bead contacts of living neurons stained with SiR-actin (green) and loaded with FM1-43 (orange, left image) before release (right image). Left two images show an A+ presynapse, right images show an A− presynapse. Scale bar, 2 µm. (G) Quantification of the FM staining intensity after loading (orange) and release (yellow) in A+ and A− induced presynapses as well as axon-bead contacts devoid of a presynapse (S−). Significance signs on graphs compare to the value for the same labeling condition in actin-enriched presynapses (normalized to 1.0 for A+ in E, normalized to 1.0 for the A+ in loading condition in G). See Data S1 file for detailed statistics.

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