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. 2019 Jun 7;6(3):ENEURO.0066-19.2019. doi: 10.1523/ENEURO.0066-19.2019

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Copyright © 2019 Bimpisidis et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Figure 1. — NeuroD6 mRNA is found in a modest population of the VTA, co-localizes with dopaminergic markers and partially with a glutamatergic marker. A–G, Double FISH in the ventral midbrain of adult wild-type mice detecting the following mRNAs. A, A’, NeuroD6 (red). B, B’, Th (green). C, C’, NeuroD6 (red) and Th (green). Th/NeuroD6 mRNA overlap shown in yellow. Low magnification to the left; close-ups to the right. Schematic outline shows borders for SNc and subregions of VTA: PN, PIF, PBP, IF, RLi. D, Quantification of percentage of NeuroD6-positive cells among all Th VTA cells; all NeuroD6 cells are positive for Th mRNA. E, NeuroD6 (red) and Dat (green), inset with high magnification of Dat/NeuroD6 mRNA overlap (yellow). F, NeuroD6 (red) and Vglut2 (green). G, NeuroD6 (red) and Viaat (green), inset with high magnification of Viaat-negative/NeuroD6-positive (red). H–P, Triple-labeling FISH in the ventral midbrain of adult wild-type mice detecting: (H) Th (blue); (I) NeuroD6 (red); (J) Vglut2 (green) mRNAs and their co-localization: (K) NeuroD6/Th; (L) NeuroD6/Vglut2; (M) Th/NeuroD6/Vglut2. Cellular closeups: (N) NeuroD6/Th (top), NeuroD6/Vglut2 (middle), Th/NeuroD6/Vglut2 (bottom). Arrows point to NeuroD6 mRNA-positive cells. O, Quantification of percentage of NeuroD6+/Th+/Vglut2+ and NeuroD6+/Th+/Vglut2− neurons of the VTA. P, Schematic illustration of distribution pattern of NeuroD6+/Th+/Vglut2+ and NeuroD6+/Th+/Vglut2− neurons within the VTA (same as shown with experimental data in M). NeuroD6+/Th+/Vglut2- cells in magenta; NeuroD6+/Th+/Vglut2+ cells in cyan. VTA, ventral tegmental area; SNc, substantia nigra pars compacta; PBP, parabrachial pigmented nucleus; PN, paranigral nucleus; PIF, parainterfascicular nucleus; RLi, rostral linear nucleus; IF, interfascicular nucleus. FISH, fluorescent in situ; Dat, Dopamine transporter; Th, Tyrosine hydroxylase; Vglut2, Vesicular glutamate transporter 2; Viaat, Vesicular inhibitory amino acid transporter.