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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 2. — Conditional ablation of the Vmat2 gene in NEX-Cre neurons, a model for spatially restricted DA deficiency. A, Breeding strategy for generation of mice gene-targeted for Vmat2 in VTA NEX-Cre neurons. NEX-Cre transgenic mice were mated to Vmat2^lox/lox mice to generate NEX-Cre-positive mice homozygous for Vmat2^lox/lox (Vmat2^{lox/lox;NEX-Cre-tg}: cKO mice) and littermate control mice homozygous for Vmat2^lox/lox and negative for the NEX-Cre transgene (Vmat2^{lox/lox;NEX-Cre-wt}: Ctrl mice). B, Two-probe approach for detection of Vmat2 mRNA by ISH. Probe 1 detects exons 6–15 and probe 2 detects exon 2 of the Vmat2 gene. Exon 2 is floxed in Vmat2^lox/lox mice leading to failure of probe 2-binding to Vmat2 mRNA in cKO neurons. C, Implementation of Vmat2 mRNA two-probe approach in Vmat2^{lox/lox;NEX-Cre-wt} (Ctrl, left panel) and Vmat2^{lox/lox;NEX-Cre-tg} (cKO, right panel) brains. Wild-type neurons are positive for both Vmat2 probes, while cKO neurons are only positive for probe 1 due to targeted deletion of exon 2 (detected by probe 2). Probe 1 detected in green and probe 2 detected in blue results in green-blue double-labeling in wild-type cells and green-only labeling in cKO cells. Green arrows point to green-only cells, i.e., VMAT2 cKO cells. D, Vmat2 mRNA two-probe ISH in additional monoaminergic areas. E, TH immunohistochemistry in Ctrl and cKO midbrain and striatum. LC, locus coeruleus; ROB, raphe nucleus obscurus; VMH, ventromedial hypothalamus; VTA, ventral tegmental area; SNc, substantia nigra pars compacta; DStr, dorsal striatum; NAc, nucleus accumbens; OT, olfactory tubercle. TH, Tyrosine hydroxylase; Vmat2/VMAT2, Vesicular monoamine transporter 2; Ctrl, control; cKO, conditional knockout.