Figure 1.
Nr4a1-eGFP expression in the basal ganglia.
Nr4a1-eGFP expression (A1–A4) in the striatum and striatal projection areas compared to Drd1-eGFP (B1–B4) and Drd2-eGFP (C1–C4) expression in the mature mouse. The DLS is shown in row 1, ventral striatum in row 2, globus pallidus in row 3 and SN/VTA in row 4. The scale bar, 200 µm, applies to all images.
Figure 2.
Serial sections through the striatum and extended amygdala of a mature Nr4a1-eGFP mouse.
Striosomes appear brighter than the surrounding matrix, particularly the subcallosal streak (rows 1–2) and lateral striatal streak (rows 3–5), while a laminar organization is apparent in the ventromedial striatum (rows 1–4). Regions of intense expression are also present within the BNST (rows 4 and 5), the striatal fundus (IPAC, row 5) and within the intercalated cells of the amygdala (row 6). A uniform background level was subtracted from these images using Image J. When sections were missing or damaged, adjacent or contralateral sections were substituted. Intervals - range from 80–120 µm. Due to differences in mouse strain of the reference atlas (Allen Brain Atlas, C57/Bl6J) and tissue shrinkage the coordinates are approximate.
Figure 3.
Nr4a1-eGFP expression in the striatum compared to the matrix marker calbindin 28K and striosome markers calretinin and mu-OR.
eGFP is shown in the left column. Calbindin expression in the dorsolateral striatum (A2) and globus pallidus/caudal striatum (B2) is merged with eGFP in A3 and B3. Calretinin immunoreactivity in the dorsomedial striatum is shown in C2 and merged with eGFP in C3. Mu-OR activity (D2) is merged with eGFP in D3. The scale bar in A1 (100 µm) applies to all panels.
Figure 4.
Nr4a1-eGFP expression compared with striosomal markers in the extended striatum and amygdala.
Nr4a1-driven eGFP expression is present in the NAc (A1), BNST (B1) and amygdala (C1, D1). Robust colocalization with mu-OR is observed in the NAc core but not the shell (A2, A3). The BNST shows heterogeneous eGFP expression (B1) that overlaps with mu-OR immunoreactivity in most subregions (B2, B3). Mu-OR immunoreactivity in the amygdala (C2) overlaps with Nr4a1-eGFP expression in the ITC (C3, arrows) but not in the CeA. TH immunoreactivity is not uniform with greater innervation of the medial CeA (CeAM) than lateral CeA (CeAL) at this level (D2). TH+ fibers innervating the ITC were faint compared to the medial CeA but overlapped with the ITC (D2, D3). Drd1-eGFP expression (E1, E3 merge) and Drd1 immunoreactivity (E2, E3) in the amygdala are shown for comparison. Scale bars in A3 and B3 are 200 µm. Scale bar in E3 is 100 µm and applies to panels C–E.
Figure 5.
Immunolocalization of endogenous Nr4a1 in the developing and adult striatum.
Dorsolateral striatum from a neonatal (PN3/4, A, B) and mature mouse (PN30, C, D). Higher power images indicate colocalization of eGFP and nuclear Nr4a1 in the developing dopamine islands (A1–A3) that is present throughout the structure at low power (B1–B3). In contrast, there is little overlap between nuclear Nr4a1 expression and eGFP in the mature striatum (C, D). High power images detected a speckled distribution (C2) in addition to nuclear localization. The lack of correlation between eGFP levels and Nr4a1 is shown in low power images of the mature dorsolateral striatum (D1–D3). The scale bars in A and C are 50 µm, 100 µm for B and 200 µm for D.
Figure 6.
Developmental expression of Nr4a1 in striatonigral projections compared to Drd1, TH and mu-OR in horizontal sections.
eGFP expression (A1, B1, C1) overlaps with Drd1 immunoreactivity (A2, A3, A5 merged with the DAPI channel) and TH immunoreactivity (B2, B3, B5 merged with the DAPI channel) Mu-OR immunoreactivity (C2) colocalized with Nr4a1-eGFP is shown in C3 and in C5 merged with the DAPI channel. DAPI staining is less intense in the striosomes (A4, B4, C4), indicating that the striosome-like distribution is not due to increased cell density. The scale bar in H (200 µm) applies to all images. Panels A and B were taken with a Zeiss Axiovert microscope. Panels in C were taken with a Zeiss Lumar stereomicroscope.
Figure 7.
TrkB and Nr4a1 are co-expressed in developing striosomes.
TrkB expression (A1, B1) and Nr4a1-eGFP (A2, B2) merged (A3, B3) in the developing striosomes at PN3/4. Dorsolateral (A) and ventrolateral striatum (B) are shown. Arrow (B1) indicates the lateral striatal streak. Scale bar in J (100 µm) applies to all images.
Figure 8.
Phosphorylation of CREB and ERK in the neonatal Nr4a1-eGFP striatum.
Phospho-CREB immunoreactivity is lower in the striosomes than in the surrounding matrix at PN3/4 (A1–A3) and PN7 (B1–B3). ERK phosphorylation shows the opposite pattern of immunoreactivity at both PN3/4 (C1–C3) but the distribution of pERK changes during development with nuclear and process localization at PN7 (C1 compared to D1). Nr4a1 and active ERK and CREB were also detected in the vasculature. Scale bar in A3 (50 µm) applies to all images.
Figure 9.
eGFP expression in mice administered 5 mg/kg of methylphenidate.
Sections of similar striatal regions are shown for comparison. Images were pseudo-colored using image J (16-bit spectrum). Fluorescence intensity indicates increased levels in the more intense (striosomal, red) regions but also an increase in expression within the matrix (green to yellow shades). Scale bar is 100 µm.
Figure 10.
Native fluorescence and fluorometric detection of stimulated eGFP expression in primary cultures of medium spiny neurons stimulated in vitro.
Pseudocolored images of control (A), 5 ng/mL BDNF (B), 1 µM forskolin (C), 1 µM SKF-83822 (D) or 30 mM KCl (E) reveal eGFP induction after 20 hrs. eGFP expression was also measured fluorometrically in lysates at 3, 8 and 20 hrs after exposure (F). Native fluorescence is representative of 4 replicate fields taken at the same exposure time (set for linear detection in control cultures). Fluorometry data in F are the mean +/− SE of 16 replicates for each time point and treatment. Data were analyzed by ANOVA (* p<0.05). Scale bar in E (20 µm) applies to all panels.
Figure 11.
Time course for the in vitro induction of native Nr4a1 mRNA and protein compared to Nr4a1 promoter-driven eGFP expression in MSNs.
Semi-quantitative PCR detection of Nr4a1, eGFP and actin mRNA after exposure to 30 mM KCl for 30 min to 8 hrs is shown in A. Western blot detection of eGFP, Nr4a1 and actin induced by 30 mM KCl or forskolin (1 hr to 8 hr) is shown in panel B. Immunofluorescent detection Nr4a1 (red) and eGFP in DAPI stained MSN cultures is shown in C1–C4 after 2 hrs of treatment with 30 mM KCl (C1, C2) or 8 hrs (C3, C4). Arrows in C1 indicate nuclear expression in brightly fluorescent cells after 2 hrs of exposure to 30 mM KCl. DAPI and Nr4a1 are shown together in C2 for this time point. Cells treated for 8 hrs with 30 mM KCl are shown in C3,C4. Arrows (C3) indicate cells with perinuclear eGFP and Nr4a1 immunoreactivity. The DAPI and Nr4a1 channels are shown merged in C4. Arrows (C4) indicate the absence of Nr4a1 immunoreactivity in the nuclei of brightly eGFP-expressing cells at 8 hrs. Scale bar in C1 (10 µm) applies to all images.
Figure 12.
Immunodetection of Drd1, met-enkephalin and eGFP in cultured MSNs.
eGFP (A) and Drd1 immunoreactivity (B) are shown merged in panel C. eGFP (D) and met-enkephalin (E) immunoreactivity was detected together in ∼15% of cells expressing high levels of eGFP (merge in F). Cells were treated for 12 hr with 30 mM KCl prior to immunostaining. Scale bar in C (20 µm) and applies to all images.