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. 2018 Aug 22;9:3362. doi: 10.1038/s41467-018-05871-5

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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. 8 — Amph is required for postsynaptic organization and normal synaptic transmission. a Schematic diagram for calcium imaging experiments. BO-mediated synaptic transmission is received by LNv dendrites (dashed gray circle) and measured by the changes of GCaMP6s signals in the axonal terminal region of LNvs (dashed red circle). b Knocking-down Amph led to reductions in light-induced responses in LNvs. The changes in GCaMP signal (ΔF/F) were plotted. Data are presented as line graph showing Mean ± SEM. Arrow indicates the delivery of the light stimulation. c Quantification reveals reduced amplitude and delayed peak time of calcium responses in LNvs expressing Amph RNAi. n = 7, pdf > Dicer2; n = 8, pdf > Dicer2, Amph^RNAi. Statistical significance was assessed by two-tailed Student’s t-test. Amplitude: t₁₃ = 2.742, P = 0.017. Peak time: t₁₃ = 2.491, P = 0.027. d Disrupted dendritic Fas2 distribution in LNvs expressing Amph RNAi. While CD8:GFP (pdf > CD8:GFP, left) shows no change, the volume of dendritic extra-Fas2:YFP (pdf > extra-Fas2:YFP, right) is reduced in LNvs with Amph knockdown. Representative projected confocal images (green) and 3D reconstructions (gray) are shown. Scale bar, 5 μm. e Quantification of dendritic extra-Fas2:YFP based on 3D reconstructions. n = 10, pdf > Dicer2; n = 14, pdf > Dicer2, Amph^RNAi. Statistical significance was assessed by two-tailed unpaired Student’s t-test. t₂₂ = 3.969, P < 0.001. f–h Amph knockdown reduces the synaptic distribution of Fas2. f Representative projected confocal images of LNvs are shown. Scale bar, 5 μm. Genotypes are as indicated. g A representative single optic section image illustrating the dendritic Fas2 (extra-Fas2:YFP, green) contacting the presynaptic site marked by Brp (Rh5,6-Brp, magenta). Contacts are indicated by *, non-contacts by o. Scale bar, 2 μm. h Quantifications of the number of contacts between Brp puncta and extra-Fas2:YFP. Knocking-down Amph led to reduced number of Fas2-Brp contacts. n = 11, pdf > Dicer2; n = 12, pdf > Dicer2, Amph^RNAi. Statistical significance was assessed by two-tailed Student’s t-test. t₂₁ = 2.988, P = 0.007. Data are presented as box plot (box, 25–75%; center line, median) overlaid with dot plot (individual data points). ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001