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. 2018 Mar 8;9:998. doi: 10.1038/s41467-018-03334-5

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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. 5 — HCN2 channels rescue nicotine-induced brain morphology defects in Xenopus embryos. a Representative image of stage 45 tadpole with an example of a minor brain morphology defect showing smaller forebrain and nostril on the left (magenta arrowhead), normal midbrain (MB - yellow bracket) and normal hindbrain (HB - cyan bracket). b Quantification of stage 45 tadpoles for subtle changes in overall brain morphology with or without microinjecting Hcn2-WT (wild-type) mRNA (0.75 ng/injection) in both blastomeres at 2-cell stage as indicated in the illustrations. Hcn2-WT mRNA injections significantly suppress the minor brain defects seen in uninjected control embryos. Three independent experiments (n = 3) were conducted with N > 50 embryos per treatment group for each of those experiments, collected from multiple animals across independent clutches. Data were analyzed with t-test and graphed as mean ± SD, **p < 0.01. Representative images of stage 45 tadpoles: c control tadpole showing nostrils (blue arrowhead), forebrain (FB) indicated by the orange bracket, midbrain (MB) indicated by the yellow bracket, and hind brain (HB) indicated by the cyan bracket, d tadpole from embryos exposed to nicotine (0.1 mg/mL – stage 10–35) showing severe brain morphology defects as indicated by magenta arrowheads. e tadpole from embryos exposed to nicotine (0.1 mg/mL – stages 10–35) and microinjected with Hcn2-WT mRNA (0.75 ng/injection) in both blastomeres at 2-cell stage showing intact nostrils (blue arrowheads), forebrain (FB - orange brackets), midbrain (MB - yellow brackets), and hindbrain (HB - cyan brackets). f Quantification of stage 45 tadpoles for major brain morphology phenotypes in absence or presence of nicotine exposure (0.1 mg/mL – stage 10–35) with or without microinjection of Hcn2-WT or Hcn2-DN (dominant-negative) mRNA (0.75 ng/injection) in both blastomeres at 2-cell stage as indicated in the illustrations. A significantly high incidence of malformed brain was observed in embryos exposed to nicotine in comparison to controls. Hcn2-WT mRNA injection significantly reduced the incidence of malformed brain, while Hcn2-DN mRNA injection had no significant effect on nicotine exposure induced malformed brain. Three independent experiments (n = 3) were conducted with N > 50 embryos per treatment group for each of those experiments collected from multiple animals across independent clutches. Data were analyzed with one way ANOVA and Tukey’s post-test and graphed as mean ± SD,***p < 0.001, n.s. non-significant