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. 2018 Aug 24;9:3421. doi: 10.1038/s41467-018-05699-z

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© The Author(s) 2018

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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Fig. 1 — Vti1a/b-deficient neurons have shorter neurites and contain less synapses and DCVs. a Representative examples of single DIV-14 neurons immunostained for MAP2 and Synaptophysin-1. b Lower synapse density in DKO neurons (DHZ: 0.24 ± 0.01, n = 57; DKO: 0.15 ± 0.01, n = 32; DKO + Vti1a: 0.20 ± 0.01, n = 60; DKO + Vti1b: 0.22 ± 0.01 synapses/µm dendrite, n = 43; Kruskal–Wallis). c Reduced Synaptophysin-1 puncta intensity in DKO neurons (DHZ: 151.71 ± 8.82, n = 57; DKO: 106.96 ± 5.16, n = 32; DKO + Vti1a: 133.47 ± 5.24, n = 60; DKO + Vt1b: 137.75 ± 8.09 AU, n = 43; ANOVA). d Histogram showing higher proportion of low-intensity Synaptophysin-1 puncta in DKO neurons. e Sholl analysis showing similar synapse distribution, with a non-significant increase at 5 µm in DKO (6.40 ± 0.46) compared to DHZ (5.01 ± 0.33), DKO + Vti1a (5.29 ± 0.34) and DKO + Vti1a (4.76 ± 0.29%; Kruskal–Wallis). f Representative stretches of single DIV-14 neurons expressing NPY-pHluorin and immunostained for MAP2. g Reduced NPY-pHluorin puncta density in DKO neurons (DHZ: 0.38 ± 0.01, n = 42; DKO: 0.32 ± 0.02, n = 37, DKO + Vti1a: 0.39 ± 0.01, n = 59; DKO + Vti1b: 0.37 ± 0.01 puncta/µm neurite, n = 34; Kruskal–Wallis). h Reduced NPY-pHluorin puncta intensity in DKO neurons (DHZ: 49.8 ± 12.0, n = 42; DKO: 24.9 ± 4.4, n = 37, DKO + Vti1a: 51.0 ± 15.7, n = 59; DKO + Vti1b: 38.3 ± 9.7 AU, n = 34; Kruskal–Wallis). i Histogram showing reduced NPY-pHluorin puncta intensity in DKO neurons. j Sholl analysis showing similar distribution of NPY-pHluorin puncta between groups. k Representative synaptic micrographs of single DIV-14 neurons. Arrowhead points at DCV. l Similar amount of SVs per synapse between DHZ (113.53 ± 7.71, n = 122) and DKO neurons (129.28 ± 8.11, n = 119; Mann–Whitney). m Similar active zone length between DHZ (527.17 ± 18.67, n = 122) and DKO neurons (534.20 ± 25.26 nm, n = 119; Mann–Whitney). n Histogram of DCVs per synapse in DCV-containing synapses showing more DCVs in DHZ (2.11 ± 0.22) than DKO neurons (1.35 ± 0.17). o Representative examples of DCVs. p Similar DCV diameter between DHZ (67.11 ± 0.48, n = 626) and DKO neurons (66.17 ± 0.50 nm, n = 502; Mann–Whitney). Bars show mean ± SEM. Scatterplots and columns represent individual neurons and litters, respectively.*p < 0.05; **p < 0.01; ***p < 0.001. Scale bar = 40 µm (a, overview), 2 µm (a, zoom), 5 µm (h), 100 nm (k), 20 nm (o). Intensity color-code used for Synaptophysin-1 zooms and NPY-pHluorin