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. 2018 Jun 27;8:9741. doi: 10.1038/s41598-018-28090-w

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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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The effects of single and combined MIA and VIT_D treatments on the coronal positioning of mature mesencephalic dopamine neurons (mesDAs). (A) Schematic image of registered floor plate (FP) in a medial coronal mesencephalic (MES) section with mediolateral (ML, x) and dorsoventral (DV, y) positions of cells. The coordinates (x₀, y₀) were chosen as the most ventral point of ventricle (V) along the midline (*). The black dot represents the mean center positioning (x_n, y_n) of a representative mesDA nucleus (ellipse). The ML positioning (x) of mesDA cells was measured bilaterally as the absolute distance from the center of mesDA nucleus (x_n) to the coordinate (x₀) = ABS (x_n−x₀). The DV positioning (y) of mesDA cell was calculated as the distance from the center of mesDA nucleus (y_n) to the coordinate (y₀) = (y₀−y_n). (B) An example of mesDA positioning in the FP. Yellow dots represent mesDA progenitors (Lmx1a+Sox2+), and green dots represent post-mitotic (Lmx1a+Sox2−) mesDAs. (C) A representative FP section showing cells that were triple-labeled by DAPI (blue), Lmx1a (green) and Sox2 (red). No significant effects of MIA or VIT_D treatment were noticeable for the mean ML (D) or DV (E) positioning of mesDA progenitors (p’s > 0.05). (F) There were no significant effects of MIA or VIT_D treatments on the mean ML positioning of post-mitotic (Lmx1a+Sox2−) mesDAs (p’s > 0.05). (G) There were significant interactions between MIA or VIT_D treatments on the average DV positioning of post-mitotic (Lmx1a+Sox2−) (p < 0.05). MIA treatment (POL-VEH) reduced the DV positioning of post-mitotic mesDAs compared to its control (CON-VEH) (p < 0.05). (H) A representative FP of a coronal MES section showing cells that were triple-labeled by DAPI (blue), Nurr1 (magenta) and TH (green). There were no significant effects of MIA or VIT_D treatment for the mean ML (I) or DV (J) positioning of immature (Nurr1+TH−) mesDAs in the FP (p’s > 0.05). (K) MIA treatment (POL) reduced the mean ML positioning of mesDAs relative to CON (p < 0.05). (L) There were significant interactions between MIA x VIT_D treatments in the average DV positioning of mature (Nurr1+TH+) mesDAs (p < 0.05). Post hoc comparison revealed MIA treatment (POL-VEH) decreased the mean DV positioning relative to the control (CON-VEH) (p < 0.05). Additionally, the co-treatment of VIT_D restored the mean DV positioning of mature mesDAs in POL-VIT_D group compared to POL-VEH group (p < 0.05). All values were means ± SEM. ^*p < 0.05. n.s. represents not statistically significant. Scale bars: 50 µm.