Nuclear Receptor Nr4a1 Regulates Striatal Striosome Development and Dopamine D₁ Receptor Signaling

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.

Nr4a1 promotes the maturation of specific medium spiny neuronal phenotypes, including of Oprm1, a striosomal marker. a, qRT-PCR assay of Nr4a1, Ppp1r1b, and Oprm1 mRNAs on DIV7 WT and Nr4a1-eGFP primary striatal neurons treated with BDNF 25 ng/ml vs 0.1% BSA reveals that Nr4a1 is overexpressed in Nr4a1-eGFP neurons (F_(3,15) = 10.15, p = 0.007; WT vs Nr4a1-eGFP: t₍₁₅₎ = 3695, p = 0.0022) and is associated with an increase in Ppp1r1b (F_(3,27) = 10.04, p = 0.0001; WT vs Nr4a1-eGFP: t₍₂₇₎ = 2.910, p = 0.0072) and Oprm1 (F_(3,13) = 17.77, p < 0.0001; WT vs Nr4a1-eGFP t₍₁₃₎ = 3.338, p = 0.0053). BDNF treatment is additive for both Ppp1r1b (WT vs WT+BDNF: t₍₂₇₎ = 2.729, p = 0.011; WT vs Nr4a1-eGFP+BDNF: t₍₂₇₎ = 5.479, p = 0.0001; Nr4a1-eGFP vs Nr4a1-eGFP+BDNF: t₍₂₇₎ = 2.688, p = 0.0127) and Oprm1 (WT vs Nr4a1-eGFP+BDNF: t₍₁₃₎ = 5.944, p = 0.0001). n = 5 samples/genotype. One-way ANOVA corrected for multiple comparisons (Sidak’s test): *p < 0.05, **p < 0.01, ****p < 0.0001. Data are presented as the mean ± SEM. b, Representative Pp1r1b/DARPP-32 staining on DIV7 WT and Nr4a1-eGFP primary striatal neurons treated with BDNF (25 ng/ml) shows a relatively increased number of DARPP-32-immunopositive cells in Nr4a1-eGFP primary cultures. The effects of increased Nr4a1 and BDNF are additive. Scale bars, 50 µm. c, Representative DARPP-32 immunolabeling of WT primary striatal neurons 96 h after transduction with ADV-GFP CT vs ADV-Nr4a1-eGFP showing increase in DARPP-32-immunopositive cells in the cultures overexpressing Nr4a1. Scale bars, 50 µm. d, qRT-PCR assay shows increases in Ppp1r1b (t₍₆₎ = 2.551, p = 0.0434) and Oprm1 (t₍₆₎ = 5.505, p = 0.0015) mRNA levels in wild-type primary striatal neurons 96 h after transduction with ADV-Nr4a1-GFP. n = 5 samples/treatment, two-tailed unpaired t test: *p < 0.05, **p < 0.01. Data are presented as the mean ± SEM.

Nr4a1 promotes the differentiation of MSNs phenotypes in human iPSC-derived NPCs into medium spiny neurons. a, Immunolabeling of DARPP-32 and βIII tubulin in iPSC-derived NSCs, untreated, vs transduction with ADV-GFP or ADV-NR4A1-GFP shows that Nr4a1 induces the upregulation of DARPP-32 and βIII tubulin. Scale bar, 100 µm. b, qRT-PCR assays show an increase in Ppp1r1b (F_(2,6) = 7.173, p = 0.0256; NT vs Nr4a1-GFP: t₍₆₎ = 3.691, p = 0.0102), Oprm1 (F_(2,6) = 6.105, p = 0.0358; NT vs Nr4a1-GFP: t₍₆₎ = 3.234, p = 0.0178), Calb2 (F_(2,6) = 15.75, p = 0.0041; NT vs Nr4a1-GFP: t₍₆₎ = 5.461, p = 0.0016; GFP-CT vs Nr4a1-GFP: t₍₆₎ = 3.854, p = 0.008), Calb1 (F_(2,5) = 72.94, p = 0.0002; NT vs Nr4a1-GFP: t₍₅₎ = 11.63, p = 0.0001; GFP vs Nr4a1-GFP: t₍₅₎ = 4.357, p = 0.0073), and Bcl11b (F_(2,6) = 13.69, p = 0.0058; NT vs Nr4a1-GFP: t₍₆₎ = 4.848, p = 0.0029; GFP vs Nr4a1-GFP: t₍₆₎ = 4.132, p = 0.0068) mRNA levels in human iPSC-derived NSCs transduced with ADV-Nr4a1-GFP for 14 d. n = 3 samples for each group. One-way ANOVA corrected for multiple comparisons (Tukey’s test): *p < 0.05, **p < 0.01, ***p < 0.001. Data are presented as the mean ± SEM.

Nr4a1 overexpression reduces the induction of phosphorylation of ERK and c-fos after acute cocaine injection and impairs locomotor sensitization to chronic cocaine. a, Representative pERK immunolabeling indicating the dorsomedial region of interest and fixed area used for the quantification of pERK⁺ cells in the striatum of 4-month-old WT, Nr4a1-eGFP, and Nr4a1-null mice 10 min after a single intraperitoneal injection of cocaine (20 mg/kg). Scale bars: 200 and 50 µm. b, Quantification of a showing a relative reduced induction of pERK⁺ cells in Nr4a1-eGFP mice (F_(2,8) = 7.281, p = 0.0158; WT vs Nr4a1-eGFP: t₍₈₎ = 3.816, p = 0.0051). n = 4 mice/genotype; one-way ANOVA corrected for multiple comparisons (Bonferroni’s correction): *p < 0.05. Data are presented as the mean ± SEM. c, ERK 1/2 basal protein levels are equal in 4-month-old WT, Nr4a1-eGFP, and Nr4a1-null mice (F_(2,9) = 1.340, p = 0.3094). n = 8 mice/genotype. One-way ANOVA corrected for multiple comparisons (Sidak’s test). Data are presented as the mean ± SEM. d, Calbindin and pERK immunolabeling shows that the induction of pERK occurs predominantly in the matrix compartment after a single intraperitoneal injection of cocaine (20 mg/kg). Scale bars, 100 µm. The graph shows the quantification of pERK⁺ cells in matrix and striosomes in sections from bregma 0.86 mm. n =3 mice, unpaired t test: t_(2.003) = 4.702, *p = 0.0423. Data are presented as the mean ± SEM. e, c-fos and GFP immunolabeling in the dorsal striatum of Drd1-eGFP and Nr4a1-eGFP adult mice 1 h after a single intraperitoneal injection of cocaine (20 mg/kg), indicating relatively reduced c-fos induction in Nr4a1-eGFP mice. Arrows indicate c-fos labeling in the GFP⁺ striosomes in the Nr4a1-eGFP mice. Scale bars, 50 µm. f, Quantification of c-fos ⁺ cells in Drd1-eGFP and Nr4a1-eGFP adult mice shown in e. n = 3 mice/genotype and treatment, unpaired t test: t₍₄₎ = 6.721, **p = 0.0026. Data are presented as the mean ± SEM. g, pERK immunostaining in WT and Nr4a1-eGFP mice after the injection NMDA antagonist MK-801(0.1 mg/kg) followed 30 min later by a single injection of cocaine (20 mg/kg) shows the abolition of pERK induction. Scale bars, 50 µm. h, Schematic representation of treatments used for the induction of locomotor sensitization to cocaine. i, Nr4a1-eGFP mice show decreased locomotor sensitization to chronic cocaine use relative to WT mice. One-way ANOVA corrected for multiple comparisons (Bonferroni’s correction; F_(7,108) = 8.639, p < 0.0001; WT cocaine day 1 vs WT cocaine day 5: t₍₁₀₈₎ = 3.705, **p = 0.003; Nr4a1-eGFP cocaine day 1 vs Nr4a1-eGFP cocaine day 5: t₍₁₀₈₎ = 0.6920, p = 0.4904). n = 15 mice/genotype, One-way ANOVA corrected for multiple comparisons (Bonferroni’s correction). Data are presented as the mean ± SEM. ns = non-significant.

Characterization of electrophysiological properties of dorsomedial striosomal MSNs and the impact of Nr4a1 overexpression on striatal synaptic excitability and plasticity. a, Action potential sample traces from single cells derived from the dorsal striatum in WT mice and in the center of the striosomes for GFP⁺ and GFP^– neurons from Nr4a1-eGFP mice. b, Number of action potentials as a function of injected current intensity in WT, GFP⁺, and GFP^– neurons indicate that neuronal action potentials are increased in GFP⁺ neurons. Two-way ANOVA with genotype factor (F_(2,57) = 7.421, p = 0.0014). n ≥ 17/genotype and cell type; **p < 0.01. Data are presented as the mean ± SEM. c, Resting membrane potential is more depolarized in GFP⁺ neurons compared with WT (WT MSNs: average, −75.8 mV, n = 17 cells; GFP⁺ MSNs: average, −71.7mV, n = 22; GFP⁻ MSNs: average, −74.2 mV, n = 21). One-way ANOVA followed by Bonferroni’s multiple-comparisons test: F_(2,55) = 4.303, p = 0.0183, *p < 0.05. Data are presented as the mean ± SEM. d, Rheobase current is lower in GFP⁺ neurons relative to WT and GFP⁻ MSNs (WT MSNs: average, 171.1 pA, n = 17 cells; GFP⁺ MSNs: average, 134.1 pA, n = 22; GFP⁻ MSNs: average, 164.7 pA, n = 21). One-way ANOVA-Bonferroni’s multiple-comparison test, F_(2,51) = 4.342, p = 0.0181; *p < 0.05. Data presented as ± SEM. e, Membrane resistance recorded in voltage-clamp experiments is equal in the three cell types. WT, n = 10; GFP⁺ and GFP⁻, n = 16. One-way ANOVA-Bonferroni’s multiple-comparison test: F_(2,50) = 0.7388, p = 0.4828. Data are presented as the mean ± SEM. f, Spike threshold is equal in the three cell types. WT, n = 15; GFP⁺ and GFP⁻, n = 19. One-way ANOVA-Bonferroni’s multiple-comparison test: F_(2,49) = 0.5219, p = 0.5967. Data are presented as the mean ± SEM. g, Whole-cell patch-clamp recordings of long-term synaptic depression (LTD) induced by high-frequency stimulation in WT and GFP⁺ MSNs showing overlapping traces for both genotypes. Data are presented as the mean ± SEM. h, Bar graph representing the average of the last 5 min after LTD induction in WT and GFP⁺ MSNs indicating no significant differences between the two groups: WT, 10 recordings/7 mice; GFP⁺, 7 recordings/3 mice. Unpaired two-tailed t test, t₍₁₅₎ = 1.868, p = 0.0815. Data are presented as the mean ± SEM. i, LTP assayed in field recordings in WT and Nr4a1-eGFP shows reduced LTP in Nr4a1-eGFP mice after high-frequency stimulation. Data are presented as the mean ± SEM. j, Bar graph representing the average of the last 5 min after LTP induction in field recordings (7 recordings and 3 mice for both genotypes). Unpaired two-tailed t test: t₍₁₂₎ = 3.011, p = 0.0108; *p < 0.05; Data are presented as the mean ± SEM.