Figure 2. Constitutive upregulation or downregulation of Nr4a1 mRNA alters spatial development of the striosomal compartment and mRNA levels of its markers in vivo. a, Coronal section of adult Nr4a1-eGFP/Drd1-tdTomato showing the colocalization of Drd1-tdTomato and Nr4a1-eGFP. The ROI selection indicates the section represented in higher magnification. Single channels are shown in the miniatures. Scale bars: 200 and 50 µm. b, Quantification of tdTomato+, EGFP+, and tdTomato++/EGFP+ cells in striosomes and matrix shown in a. Percentage of each cell population was calculated relative to the total number of cells counted by DAPI immunofluorescence. c, Graphic representation of the percentage distribution of Drd1+0/Nr4a1−, Drd1+/Nr4a1+, Drd2+/Nr4a1+, and Drd2+/Nr4a1− in the striosomes. For this, the Drd1 (i.e., tdTomato−) cells were counted as Drd2+ cells. d, Representative OPRM1 immunolabeling on 30-µm-thick coronal sections from 4-month-old WT, Nr4a1-eGFP, and Nr4a1-null mice with superimposition of selected ROIs delineating the total striatal and striosomal areas in the bottom panel. Scale bars, 200 µm. e, Quantification of the striatal area, the percentage of the area occupied by the striosomes, and of the number of striosomes in 4-month-old and P3 WT, Nr4a1-eGFP, and Nr4a1-null mice shows a decrease in the percentage of the area occupied by the striosomes in Nr4a1-null mice at both ages. For both P3 and adult analysis, n = 6 mice/genotype. One-way ANOVA corrected for multiple comparisons (Sidak’s test). For adults: striatal area F(2,15) = 1.897, p = 0.1943; striosomal area: F(2,15) = 40.83, p < 0.0001; WT vs Nr4a1-eGFP: t(15) = 0.1803, p = 0,8593; WT vs Nr4a1-null: t(15) = 7.914, ***p = 0.0001; number of striosomes: F(2,15) = 2.007, p = 0.1689. For P3: striatal area: F(2,19) = 1,5481, p = 0.2383; striosomal area: F(2,19) = 12.87, p = 0.0003; WT vs Nr4a1-eGFP: t(19) = 0.3318, p = 0.7437; WT vs Nr4a1-KO: t(19) = 4.333, **p = 0.0004; number of striosomes: F(2,15) = 2.818, p = 0.0914. Data are presented as the mean ± SEM. f, mRNA levels of striosome and matrix marker as determined by qRT-PCR of striatal mRNA from 4-month-old and P3 WT, Nr4a1-eGFP, and Nr4a1-null mice reveals a positive correlation between Nr4a1 expression levels and striosomal markers Oprm1 (F(2,20) = 15.52, p < 0.0001; followed by Sidak’s multiple-comparisons test vs WT: t(20) = 4.732, p = 0.0001 for Nr4a1-eGFP; t(20) = 1.099, p = 0.2848 for Nr4a1-KO), Rasgrp1 (F(2,11) = 16.49, p = 0.0005; t(11) = 4.082, p = 0.0018; t(11) = 1.639, p = 0.1295), and Foxp2 (F(2,11) = 20.02, p = 0.0002; t(11) = 2.795, p = 0.0174; t(11) = 3.174, p = 0.0089) in the adult. In P3, Nr4a1-KO mice have reduced levels of Ppp1r1b (F(2,15) = 13.96, p = 0.0004; t(15) = 2.899, p = 0.011), Calb1 (F(2,12) = 10.77, p = 0.0021; t(12) = 2.721, p = 0.0186), and Rasgrp1 (F(2,8) = 4.657, p = 0.0456; t(8) = 2.378, p = 0.0447). n ≥ 5 mice/genotype, one-way ANOVA corrected for multiple comparisons (Sidak’s test). *p < 0.05, **p < 0.01, ***p < 0.001. Data are presented as the mean ± SEM.