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. 2020 Aug 7;11:3935. doi: 10.1038/s41467-020-17824-y

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© The Author(s) 2020

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. 4 — a Confocal image of KCC2 staining in the rat SDH. b CGRP (blue), IB4 (green), and KCC2 (red) staining in the rat SDH (dorsal white matter border, solid line; barycentric origin of IB4, dashed line). Boxed areas represent regions of interest (ROIs) surrounding LI (small dash) and LII neurons (big dash). c CGRP/IB4/KCC2 mean fluorescence intensities in the SDH as a function of the position towards the IB4 barycentric origin (n = 8 rats). d Membrane KCC2 intensity (arrows) as a function of distance from the dorsal white matter (n = 6). Insets, ROIs of neurons located superficially (1) and deeper (2) in the SDH (boxed areas). The slope of KCC2 intensity across SDH is significantly different from 0 (F = 15.5, P = 0.008); one-way-RM-ANOVA, n = 6, F = 18.5, P < 0.001. e Membrane analysis of sub-cellular profile intensities (MASC-π). On the left, KCC2-expressing neurons (arrows) in LI and LII from ROIs in b. The color-coded distance map illustrate the shift in KCC2 intensity with the distance to the membrane profile. The graphs in the insets quantify the KCC2 intensity as a function of distance to the membrane profile. On the right, pooled KCC2 values per rat as a function of distance to the membrane profile for all LI (n = 8) and LII (n = 8). The pictogram on the top right is a schematic representation of the method (f). Membrane analysis by global index (MAGI). On the left, ROIs containing LI and LII, delineated based on IB4 staining (in green). The global intensity index of the membrane KCC2 intensity per pixel (M, red) in each lamina is measured by automated subtraction of intracellular intensity (I, white) from the total intensity (T). Top right, demonstrative LI and II ROIs. Bottom right, quantification of membrane KCC2 signal in LI and LII by global intensity index. i.u. intensity units, CGRP calcitonin gene-related peptide, IB4 isolectin B4. Data are shown as mean ± S.E.M. *P < 0.05, **P < 0.01.