Journal: Molecular Neurodegeneration

Article Title: Developmental dopamine loss rewires striatal circuits to promote locomotion

doi: 10.1186/s13024-025-00920-2

Figure Lengend Snippet: Preferential loss of ALDH1A1⁺ SNc DANs and hyperlocomotion in Pitx3 ak/ak mice. a Representative image showing ALDH1A1 (magenta), TH (green), and DAPI (blue) immunolabeling in the midbrain of Pitx3 +/ak and Pitx3 ak/ak mice. Dashed lines outline the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA). Scale bar, 500 μm. b Percentage of ALDH1A1⁺ cells among TH⁺ cells in the SNc and VTA. Two-way ANOVA followed by Sidak’s post hoc correction: Genotype, F (1, 6) = 116.6, **** p < 0.0001; Region, F (1, 6) = 49.49, *** p = 0.0004; Interaction, F (1, 6) = 116.6, **** p < 0.0001. Multiple comparisons: SNc, **** p < 0.0001; VTA, p = 0.6346. N = 4 mice per group ( b – e ). c Representative images of ALDH1A1 (magenta), TH (green), and DAPI (blue) staining in the dorsal striatum. Dashed lines indicate the boundaries of the dorsal striatum. Scale bar, 1 mm. d Quantification of ALDH1A1⁺ and TH⁺ signal as a percentage of total striatal area across multiple bregma levels ( n = 4 mice per genotype). e Group averages of the percentage of ALDH1A1⁺ and TH⁺ areas relative to the total striatum area from ( d ). Unpaired two-tailed t-test: TH, t (6) = 14.57, **** p < 0.0001; ALDH1A1, t (6) = 57.21, **** p < 0.0001. f Locomotor velocity over time during a 30-min open-field test. Two-way ANOVA followed by Sidak’s post hoc correction: Genotype, F (1, 22) = 13.02, ** p = 0.0016; Time, F (2.42, 53.27) = 75.72, **** p < 0.0001; Interaction, F (5, 110) = 28.59, **** p < 0.0001. Multiple comparisons: 5 min, *** p = 0.0003; 10 min, * p = 0.042. g Average velocity across the 30-min session from (f). Unpaired two-tailed t-test, t (22) = 3.608, ** p = 0.0016. h Time spent in surround and center zones of the open field. Two-way ANOVA followed by Sidak’s post hoc correction: Genotype, F (1, 22) = 0.000, p > 0.9999; Area, F (1,22) = 347.8, **** p < 0.0001; Interaction, F (1, 22) = 26.77, **** p < 0.0001. Multiple comparisons: Surround, **** p < 0.0001; Center, **** p < 0.0001. i Distance traveled in the surround and center zones. Two-way ANOVA followed by Sidak’s post hoc correction: Genotype, F (1, 22) = 13.02, ** p = 0.0016; Area, F (1, 22) = 176.0, **** p < 0.0001; Interaction, F (1, 22) = 33.20, **** p < 0.0001. Multiple comparisons: Surround, **** p < 0.0001; Center, p = 0.3426. Sample sizes: Pitx3 +/ak , n = 7 males, 6 females; Pitx3 ak/ak , n = 4 males, 7 females ( f – i ). Data are presented as mean ± SEM

Article Snippet: The following strains were obtained from The Jackson Laboratory: Pitx3 ak/ak (Stock No: 000942) [ ], Drd1– tdTomato (Stock No: 016204) [ ], Pdyn IRES−Cre (Stock No: 027958) [ ], and Ai14 (Stock No: 007908) [ ].

Techniques: Immunolabeling, Staining, Two Tailed Test

Journal: Molecular Neurodegeneration

Article Title: Developmental dopamine loss rewires striatal circuits to promote locomotion

doi: 10.1186/s13024-025-00920-2

Figure Lengend Snippet: Altered numbers of Kremen1 + dSPNs and iSPNs in Pitx3 ak/ak mice. a , b Representative confocal images showing RNAscope labeling of Kremen1 (green), Drd1 (blue), Drd2 (red), and DAPI (gray) in the dorsal striatum (dStr) of Pitx3 +/ak and Pitx3 ak/ak mice. Right panels show high-magnification views of boxed regions from the left panels. Scale bars: 500 μm (left), 50 μm (right). c Quantification of total dStr area. Unpaired two-tailed t-test: t (4) = 3.239, * p = 0.032. d , e Quantification of spatial patch area (d) and patch number (e) in Pitx3 +/ak and Pitx3 ak/ak mice. Unpaired t-test, patch area: t (4) = 1.379, p = 0.24; patch number: t (4) = 1.000, p = 0.37. Spatial patches were defined as clusters of ≥ 5 Kremen1⁺ SPNs with local density ≥ 200 cells/mm². f Total numbers of dSPNs and iSPNs in the dStr. Two-way ANOVA followed by Sidak’s post hoc correction: Genotype, F (1, 4) = 4.000, p = 0.1161; Subtype, F (1, 4) = 269.7, **** p < 0.0001; Interaction, F (1, 4) = 0.5594, p = 0.4961. Multiple comparisons: dSPN, p = 0.1274; iSPN, p = 0.2355. g Numbers of Kremen1 ⁺ dSPNs and iSPNs in the dStr. Two-way ANOVA followed by Sidak’s post hoc correction: Genotype, F (1, 4) = 16.35, * p = 0.0156; Subtype, F (1, 4) = 20.01, * p = 0.0110; Interaction, F (1, 4) = 232.9, *** p = 0.0001. Multiple comparisons: dSPN, **** p < 0.0001; iSPN, p = 0.5309. Unpaired two-tailed t-test was used for the analysis of Kremen1 ⁺ total SPNs: t (4) = 4.044, * p = 0.0156. h Proportion of Kremen1 ⁺ dSPNs among total dSPNs, and Kremen1 ⁺ iSPNs among total iSPNs. Two-way ANOVA followed by Sidak’s post hoc correction: Genotype, F (1, 4) = 7.755, * p = 0.0496; Subtype, F (1, 4) = 8.646, * p = 0.0424; Interaction, F (1, 4) = 180.8, *** p = 0.0002. Multiple comparisons: dSPN, **** p < 0.0001; iSPN, * p = 0.0201. i Ratio of dSPNs to iSPNs and of Kremen1 ⁺ dSPNs to Kremen1 ⁺ iSPNs. Two-way ANOVA followed by Sidak’s post hoc correction: Genotype, F (1, 4) = 29.59, ** p = 0.0055; Subtype, F (1, 4) = 29.77, ** p = 0.0055; Interaction, F (1, 4) = 69.92, ** p = 0.0011. Multiple comparisons: Total SPN, p = 0.8421; K + SPN, **** p < 0.0001. j Ratio of Kremen1 ⁺ dSPNs to Kremen1 ⁺ iSPNs in the dorsal lateral striatum (DLS) and dorsal medial striatum (DMS). Two-way ANOVA followed by Sidak’s post hoc correction: Genotype, F (1, 4) = 23.38, ** p = 0.0084; Region, F (1, 4) = 0.2594, p = 0.6374; Interaction, F (1, 4) = 8.837, * p = 0.0410. Multiple comparisons: DLS, *** p = 0.0009; DMS, p = 0.0630. k Ratio of Kremen1 ⁺ dSPNs to Kremen1 ⁺ iSPNs in the patch and exo-patch region. Two-way ANOVA followed by Sidak’s post hoc correction: Genotype, F (1, 4) = 65.34, ** p = 0.0013; Compartment, F (1, 4) = 14.89, * p = 0.0182; Interaction, F (1, 4) = 9.326, * p = 0.0379. Multiple comparisons: Patch, *** p = 0.0006; Exo-patch, **** p < 0.0001. Data are presented as mean ± SEM. N = 3 mice per group

Article Snippet: The following strains were obtained from The Jackson Laboratory: Pitx3 ak/ak (Stock No: 000942) [ ], Drd1– tdTomato (Stock No: 016204) [ ], Pdyn IRES−Cre (Stock No: 027958) [ ], and Ai14 (Stock No: 007908) [ ].

Techniques: RNAscope, Labeling, Two Tailed Test

Journal: Molecular Neurodegeneration

Article Title: Developmental dopamine loss rewires striatal circuits to promote locomotion

doi: 10.1186/s13024-025-00920-2

Figure Lengend Snippet: Altered numbers of Nr4a1 -eGFP + dSPNs and iSPNs in Pitx3 ak/ak mice. a Representative coronal sections of the dorsal striatum from Nr4a1 -eGFP; Drd1 -tdT mice on Pitx3 +/ak and Pitx3 ak/ak backgrounds, stained for GFP (green), tdT (red) and CTIP2 (blue). Row 2 and 4 show magnified views of the dotted rectangles in row 1 and 3, respectively. Patch regions are outlined in row 2 and 4. Scale bars: 500 μm. b Quantification of average numbers of dSPNs and iSPNs in the hemi-dorsal striatum of Pitx3 +/ak and Pitx3 ak/ak mice. Two-way ANOVA followed by Sidak’s post hoc correction: Genotype, F (1, 4) = 0.9691, p = 0.3806; Subtype, F (1, 4) = 35.92, ** p = 0.0039; Interaction, F (1, 4) = 3.788, p = 0.1235. Multiple comparisons: dSPN, p = 0.1445; iSPN, p = 0.8103. c Quantification of average numbers of Nr4a1 -eGFP + dSPNs and iSPNs in the hemi-dorsal striatum. Two-way ANOVA followed by Sidak’s post hoc correction: Genotype, F (1, 4) = 2.438, p = 0.1934; Subtype, F (1, 4) = 74.90, *** p = 0.0010; Interaction, F (1, 4) = 277.6, **** p < 0.0001. Multiple comparisons: dSPN, ** p = 0.0022; iSPN, p = 0.1784. d Percentage of Nr4a1 -eGFP + SPNs among total SPNs in the dorsal striatum. Two-way ANOVA followed by Sidak’s post hoc correction: Genotype, F (1, 4) = 1.264, p = 0.3238; Subtype, F (1, 4) = 32.56, ** p = 0.0047; Interaction, F (1, 4) = 96.43, *** p = 0.0006. Multiple comparisons: dSPN, ** p = 0.0024; iSPN, * p = 0.0439. e Ratio of dSPNs to iSPNs and Nr4a1 -eGFP + dSPNs to Nr4a1 -eGFP + iSPNs in the dorsal striatum. Two-way ANOVA followed by Sidak’s post hoc correction: Genotype, F (1, 4) = 29.05, ** p = 0.0057; Subtype, F (1, 4) = 149.1, *** p = 0.0003; Interaction, F (1, 4) = 23.72, ** p = 0.0082. Multiple comparisons: Total SPN, p = 0.0502; GFP + SPN, *** p = 0.0002. Data are presented as mean ± SEM. N = 3 mice per group

Article Snippet: The following strains were obtained from The Jackson Laboratory: Pitx3 ak/ak (Stock No: 000942) [ ], Drd1– tdTomato (Stock No: 016204) [ ], Pdyn IRES−Cre (Stock No: 027958) [ ], and Ai14 (Stock No: 007908) [ ].

Techniques: Staining

Journal: Molecular Neurodegeneration

Article Title: Developmental dopamine loss rewires striatal circuits to promote locomotion

doi: 10.1186/s13024-025-00920-2

Figure Lengend Snippet: Altered distribution and projection patterns of Kremen1 ⁺ SPNs in the dorsal striatum of Pitx3 ak/ak mice. a Representative sagittal brain sections from Kremen1 2A − Cre ; Ai14 mice on Pitx3 +/ak and Pitx3 ak/ak backgrounds, stained for TH (green), tdT (red), and DAPI (blue). Scale bar, 1 mm. Abbreviations: dStr, dorsal striatum; GPe, globus pallidus externus; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; tdT, tdTomato. b Coronal sections showing altered tdT signal distribution in the dorsal striatum of Pitx3 ak/ak mice. Right panel shows a magnified view of the dashed region in the left panel. Arrow points to the dispersed tdT + neurons in DLS. Scale bar, 500 μm. Abbreviations: DLS, dorsolateral striatum. c Representative coronal sections showing increased tdT signal in the dorsal GPe of Pitx3 ak/ak mice. Right panel shows a magnified view of the dashed area. Scale bar, 200 μm. d Reduced tdT signal in the SNr of Pitx3 ak/ak mice. Arrows indicate dendron-bouquet structures. Scale bar, 200 μm. e Quantification of tdT integrated signal density in the GPe and SNr. Two-way ANOVA followed by Sidak’s post hoc correction: Genotype, F (1, 4) = 0.1004, p = 0.7672; Projection, F (1, 4) = 32.26, ** p = 0.0047; Interaction, F (1, 4) = 33.81, ** p = 0.0044. Multiple comparisons: GPe, * p = 0.0319; SNr, * p = 0.0147. f Percentage of tdT-positive area in the GPe and SNr. Two-way ANOVA followed by Sidak’s post hoc correction: Genotype, F (1, 4) = 0.005926, p = 0.9423; Projection, F (1, 4) = 19.23, * p = 0.0118; Interaction, F (1, 4) = 33.69, *** p = 0.0004. Multiple comparisons: GPe, ** p = 0.0062; SNr, ** p = 0.0076. Data are presented as mean ± SEM. N = 3 mice per group

Article Snippet: The following strains were obtained from The Jackson Laboratory: Pitx3 ak/ak (Stock No: 000942) [ ], Drd1– tdTomato (Stock No: 016204) [ ], Pdyn IRES−Cre (Stock No: 027958) [ ], and Ai14 (Stock No: 007908) [ ].

Techniques: Staining

Journal: Molecular Neurodegeneration

Article Title: Developmental dopamine loss rewires striatal circuits to promote locomotion

doi: 10.1186/s13024-025-00920-2

Figure Lengend Snippet: Altered distribution and projection patterns of patchy SPNs in Nr4a1 -eGFP mice on a Pix3 ak/ak background. a Representative coronal sections from rostral to caudal levels of Nr4a1 -eGFP mice on Pitx3 +/ak and Pitx3 ak/ak backgrounds, stained for TH (magenta). Note the altered GFP signal distribution in the dorsal striatum and dorsal GPe of Pitx3 ak/ak mice. Scale bar, 1 mm. Abbreviations: dStr, dorsal striatum; GPe, globus pallidus externus. b GFP signal distribution in the GPe of Pitx3 ak/ak mice merged with PV (red) expression in the GPe. Dotted lines outline the GPe. Solid lines indicate the regions used for intensity analysis in e . Scale bar, 250 μm. c Quantification of integrated GFP signal density in the GPe and SNr of Pitx3 +/ak and Pitx3 ak/ak mice ( n = 3 mice per genotype). Two-way ANOVA followed by Sidak’s post hoc correction: Genotype, F (1, 4) = 17.13, * p = 0.0144; Projection, F (1, 4) = 133.1, *** p = 0.0003; Interaction, F (1, 4) = 120.7, *** p = 0.0004. Multiple comparisons: GPe, * p = 0.00408; SNr, **** p < 0.0001. d Percentage of high GFP-expressing area within the GPe and SNr (Drd1 + area) in Pitx3 +/ak and Pitx3 ak/ak mice ( n = 3 mice per each genotype). Two-way ANOVA followed by Sidak’s post hoc correction: Genotype, F (1, 4) = 6.848, p = 0.0590; Projection, F (1, 4) = 315.0, **** p < 0.0001; Interaction, F (1, 4) = 338.8, **** p < 0.0001. Multiple comparisons: GPe, **** p < 0.0001; SNr, **** p < 0.0001. e Intensity profile analysis of GFP signals along the solid lines in b . GFP signal intensity is higher in the dorsal GPe of Pitx3 ak/ak mice (brown) compared to the controls (blue). f Colocalization analysis of GFP and PV expression in the GPe of Pitx3 ak/ak mice ( n = 3 mice). M1 = 0.97 ± 0.0007; M2 = 0.92 ± 0.04. M1: the proportion of PV signal overlapping with GFP. M2: the proportion of GFP signal overlapping with PV. g Representative coronal SNr sections from Nr4a1 -eGFP mice on Pitx3 +/ak and Pitx3 ak/ak backgrounds, stained for TH (magenta) and DRD1 (red). DRD1 was used to outline the SNr. Arrows indicate dendron-bouquet structure in Pitx3 +/ak mice; arrowhead indicates reduced GFP signal in the ventral SNr. Solid lines indicate the regions used for intensity analysis in h. h Intensity profile analysis of GFP signals along the solid lines in the SNr from Pitx3 +/ak (top panel) and Pitx3 ak/ak (bottom panel) mice in g. Data are presented as mean ± SEM. N = 3 mice per group

Article Snippet: The following strains were obtained from The Jackson Laboratory: Pitx3 ak/ak (Stock No: 000942) [ ], Drd1– tdTomato (Stock No: 016204) [ ], Pdyn IRES−Cre (Stock No: 027958) [ ], and Ai14 (Stock No: 007908) [ ].

Techniques: Staining, Expressing

Journal: Molecular Neurodegeneration

Article Title: Developmental dopamine loss rewires striatal circuits to promote locomotion

doi: 10.1186/s13024-025-00920-2

Figure Lengend Snippet: Altered distribution and projection patterns of patchy SPNs in the dorsal striatum of Pdyn IRES−Cre ; Ai14 mice on a Pix3 ak/ak background. a Representative coronal sections from rostral to caudal levels of Pdyn IRES − Cre ; Ai14 mice on Pitx3 +/ak and Pitx3 ak/ak backgrounds, stained for TH (green) and DAPI (blue). Scale bar, 1 mm. Abbreviations: dStr, dorsal striatum; GPe, globus pallidus externus. b Representative coronal SNr sections from Pdyn IRES − Cre ; Ai14 mice on Pitx3 +/ak and Pitx3 ak/ak backgrounds, stained for TH (magenta) and DAPI (blue). Arrows indicate dendron-bouquet structures in Pitx3 +/ak mice. Scale bar, 200 μm. c Quantification of integrated tdT signal density in the GPe and SNr of Pitx3 +/ak and Pitx3 ak/ak mice. Two-way ANOVA followed by Sidak’s post hoc correction: Genotype, F (1, 4) = 4.816, p = 0.0932; Projection, F (1, 4) = 280.9, **** p < 0.0001; interaction, F (1, 4) = 5.445, p = 0.0799. Multiple comparisons: GPe, p = 0.9870; SNr, * p = 0.0256. d Percentage of tdT-expressing area within the GPe and SNr in Pitx3 +/ak and Pitx3 ak/ak mice. Two-way ANOVA followed by Sidak’s post hoc correction: Genotype, F (1, 4) = 29.24, ** p = 0.0057; Projection, F (1, 4) = 880.1, **** p < 0.0001; interaction, F (1, 4) = 29.22, *** p = 0.0057. Multiple comparisons: GPe, p > 0.9999; SNr, *** p = 0.0001. Data are presented as mean ± SEM. N = 3 mice per genotype

Article Snippet: The following strains were obtained from The Jackson Laboratory: Pitx3 ak/ak (Stock No: 000942) [ ], Drd1– tdTomato (Stock No: 016204) [ ], Pdyn IRES−Cre (Stock No: 027958) [ ], and Ai14 (Stock No: 007908) [ ].

Techniques: Staining, Expressing

Journal: Molecular Neurodegeneration

Article Title: Developmental dopamine loss rewires striatal circuits to promote locomotion

doi: 10.1186/s13024-025-00920-2

Figure Lengend Snippet: Optogenetic activation of Kremen1 + SPNs in the dorsal striatum promote locomotion in Pitx3 ak/ak mice. a Schematic illustrating AAV-FLEX-ChR2 vector injection and optical fiber implantation for selective activation of Kremen1 + SPNs in the dorsal striatum or the SN in Pitx3 +/ak and Pitx3 ak/ak mice. b Representative coronal images showing ChR2 (green), TH (magenta) and DAPI (blue) expression in the dStr. Fiber implant locations in the dStr are marked. Scale bars: 1 mm. c Instantaneous velocity aligned to optogenetics stimulations (10s, blue shaded area) of Kremen1 + SPNs in the dorsal striatum. d Average velocity during pre-stimulation (Pre), stimulation (Stim, blue shaded area), and post-stimulation (Post) in Pitx3 +/ak mice (left) and Pitx3 ak/ak mice (right) from panel c . For Pitx3 +/ak mice: Pre = 5.14 ± 0.21 cm/s, Stim = 2.96 ± 0.31 cm/s, Post = 4.86 ± 0.29 cm/s, n = 7 mice; one-way ANOVA with multiple comparisons, Pre vs. Stim, *** p = 0.0009, Stim vs. Post, ** p = 0.0038. For Pitx3 ak/ak mice: Pre = 5.54 ± 0.47 cm/s, Stim = 6.29 ± 0.46 cm/s, Post = 5.33 ± 0.53 cm/s, n = 7 mice; one-way ANOVA with multiple comparisons, Pre vs. Stim, * p = 0.0169, Stim vs. Post, * p = 0.0365. Error bars represent mean ± SEM. e Instantaneous velocity aligned to optogenetics stimulations (10s, blue shaded area) of Kremen1 + dSPN axon terminals in the SN. f Average velocity during pre-stimulation (Pre), stimulation (Stim, blue shaded area), and post-stimulation (Post) in Pitx3 +/ak mice (left) and Pitx3 ak/ak mice (right) from panel e . For Pitx3 +/ak mice: Pre = 4.04 ± 0.64 cm/s, Stim = 2.09 ± 0.20 cm/s, Post = 4.12 ± 0.51 cm/s, n = 7 mice; one-way ANOVA with multiple comparisons, Pre vs. Stim, ** p = 0.0065, Stim vs. Post, ** p = 0.0017. For Pitx3 ak/ak mice: Pre = 3.69 ± 0.80 cm/s, Stim = 3.51 ± 0.80 cm/s, Post = 3.92 ± 0.67 cm/s, n = 7 mice; one-way ANOVA with multiple comparisons, Pre vs. Stim, p = 0.70, Stim vs. Post, p = 0.44. Error bars represent mean ± SEM

Article Snippet: The following strains were obtained from The Jackson Laboratory: Pitx3 ak/ak (Stock No: 000942) [ ], Drd1– tdTomato (Stock No: 016204) [ ], Pdyn IRES−Cre (Stock No: 027958) [ ], and Ai14 (Stock No: 007908) [ ].

Techniques: Activation Assay, Plasmid Preparation, Injection, Expressing, Optogenetics

Journal: Molecular Neurodegeneration

Article Title: Developmental dopamine loss rewires striatal circuits to promote locomotion

doi: 10.1186/s13024-025-00920-2

Figure Lengend Snippet: Model of patchy SPN reorganization in Pitx3 ak/ak mice. This schematic illustrates a selective reorganization of patchy SPNs in Pitx3 ak/ak mice, characterized by a marked reduction in the ratio of patchy dSPNs to patchy iSPNs, and a reversal of their net effect on motor output due to early dopaminergic depletion. The loss of SNc-projecting patchy dSPNs may abolish their locomotor-suppressing effect, whereas the increased projections of patchy iSPNs to the GPe may enhance inhibition of Arky neurons, thereby weakening their inhibitory feedback to striatal SPNs, including matrix dSPNs. This resulting disinhibition of matrix dSPNs likely contribute to the hyperlocomotion observed in Pitx3 ak/ak mice, particularly under conditions of heightened patchy iSPN activity. dStr: dorsal striatum, SNc: Substantia nigra pars compacta , SNr: Substantia nigra pars reticulata , GPe: Globus pallidus externa , SPN: Striatal projection neuron, dSPN: Direct-pathway striatal projection neuron, iSPN: Indirect-pathway striatal projection neuron, P-dSPN: patch dSPN, P-iSPN: patch iSPN, M-dSPN: matrix dSPN, M-iSPN: matrix iSPN, Arky: arkypallidal neuron

Article Snippet: The following strains were obtained from The Jackson Laboratory: Pitx3 ak/ak (Stock No: 000942) [ ], Drd1– tdTomato (Stock No: 016204) [ ], Pdyn IRES−Cre (Stock No: 027958) [ ], and Ai14 (Stock No: 007908) [ ].

Techniques: Inhibition, Activity Assay