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. 2017 Oct 23;7:13843. doi: 10.1038/s41598-017-13764-8

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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The synaptic vesicle recycling study of connections between EGFP-ScNs and CN neurons. (a) EGFP-ScNs and CN neurons were co-cultured for 4–6 days, followed by FM4-64 synaptic vesicle recycling study. In initial exposure to FM4-64 for 1 min, no obvious staining was found in 3 groups. After high potassium stimulation, obvious staining was found in the soma and neurite outgrowths in the ACM and TSP1 groups. In the recovery stage, significant vesicle staining was observed in soma and nerve terminals of ACM and TSP1 groups. During rinse stages, the staining of vesicles remained distinct in the soma and neurite outgrowths. (b) The puncta at the recovery stage was used for the quantitative study. It was observed that relative number and area of FM4-64 vesicle along connections were lower in the control group, whereas they were significantly higher in ACM and TSP1 groups (mean ± standard error shown in the figure; **indicates P < 0.01, ANOVA followed by Tukey post hoc test). Scale bar: 25 µm in (a).