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. 2018 Feb 14;8:2996. doi: 10.1038/s41598-018-20448-4

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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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Repetitive stimulation does not induce presynaptic failure in VPS35 KD neurons. (a) Representative synaptopHluorin fluorescence images of neurites during the base line, the first stimulation, the first recovery period, the exposure to NH₄Cl and the exposure to pH = 5.5. Scale bar = 40 µm. (b) Time course of synaptopHluorin fluorescence during the imaging protocol, plotted as ΔF/Fmax. The grey boxes indicate the electrical stimulation (100 AP, 40 Hz, 30 mV each), the black box the duration of the exposure to NH₄Cl and the white box the duration of the exposure to pH = 5.5. (n = 24 ± 2 fields of view, N = 3 animals). (c) Maximum response amplitude during the electrical stimulation plotted as ΔF/Fmax. (d) Percentage of responsive synapses during the stimulation. (e) Maximum synaptopHluorin levels during exposure to NH₄Cl. (f) Average fluorescence of synaptopHluorin during baseline recordings. (g) Ratio of the maximum synaptopHluorin fluorescence amplitude between the first and the second electrical stimulation. (h) Minimum response to the exposure to pH = 5.5. Detailed information (average, SEM, n and statistics) is available in Supplementary Table S1.